BRPI0601390B1 - USE OF BAUHINIA BAUHIOIOES PROTEASE INHIBITORS AND / OR ENTEROLOBIUM CONTORTISILIQUUM - Google Patents

Abstract

use of protease inhibitors of bauhinia bauhinioides and / or enterolobium contortisiliquum. The present invention relates to the use of protease inhibitors isolated from bauhinia bauhinioides and / or enterolobium contortisiliquum for the treatment of cancer, as well as a pharmaceutical composition comprising them.

Description

(54) Title: USE OF BAUHINIA PROTEASE INHIBITORS BAUHINIOIDS AND / OR

ENTEROLOBIUM CONTORTISILIQUUM (51) Int.CI .: A61K 38/56; A61P 35/00; C07K 14/81; C07K 14/815 (73) Holder (s): UNIVERSIDADE FEDERAL DE SÃO PAULO - UNIFESP. SÃO PAULO STATE RESEARCH FOUNDATION - FAPESP (72) Inventor (s): MARIA LUIZA VILELA OLIVA; ADRIANA MITI NAKAHATA; MISAKO UEMURA SAMPAIO; MÍRIAM GALVONAS JASIULIONIS

Descriptive report of the invention patent for: USE OF

PROTEASE INHIBITORS OF BAUHINIA BAUHINIOIDES AND / OR

BNTBROLOBIÜM CONTORTISILIQUUM.

FIELD OF THE INVENTION

The present invention relates to the use of protease inhibitors isolated from Bauhinia bauhinioides and / or Enterolobium contortisiliquwn for the treatment of cancer, as well as a pharmaceutical composition comprising them.

BACKGROUND OF THE INVENTION

In the first half of the 20th century, infectious diseases were the biggest cause of human suffering and deaths, however, with the advance of research in vaccines and antibiotics, the number of infectious diseases has been reduced. Thus diseases of the immune system and cancer have attracted the attention of

researchers ( Tuchs & Matzinger, 1996). 0 cancer not is a unique disease, but the name employed for an wide variety of malignant tumors. We can say what cancer occurs when some cells

escape the mechanisms of intra and extracellular surveillance. These cells acquire the ability to invade tissues and colonize organs at a distance, characterizing the processes of invasion and metastasis (Chammas, 1998), the main

2/108 contributing factor to cancer morbidity and mortality (Stetler-Stevenson & Yu, 2001, Wollina et. Al.,

2001). Knowledge of the molecular, genetic and biological bases of tumor dissemination and angiogenesis has increased considerably in the past two decades (Stetler-Stevenson & Yu, 2001).

The progression of the tumor depends on a number of important processes. These include the continuous growth of tumor cells (autonomous growth), their ability to prevent programmed cell death (apoptosis) and inhibitory growth signaling, as well as developing a continuous angiogenic response, locally invading surrounding tissues and distant organs (metastases) (Hanahan, 2000).

Tumor invasion and metastasis are considered to be phenomena with multiple phases, involving proteolytic degradation of the basement membrane and extracellular matrix (ECM), alteration of cell adhesion and physical movements of tumor cells (Curran & Murray, 2000). During the invasion, tumor cells disengage from the primary tumor, migrate and cross structural barriers, including the basement membrane and the surrounding stroma of the extracellular matrix that consists mainly of collagen fibrils, in addition to fibronectin, laminin and several

3/108 proteoglycans. In addition, proteolytic degradation of the extracellular matrix is considered essential in the induction of tumor angiogenesis (Káhári í Saarialho-Kere, 1999).

Degradation of the basement membrane and extracellular matrix components by proteolytic enzymes is essential for tumor progression (Wollina et al., 2001). Local proteolysis is facilitated by proteases outside the tumor cell, perhaps attached to the surface and / or secreted by the tumor cell. More recent data suggest that tumor cell proteases also participate in phagocytosis proteolysis of the extracellular matrix (Koblinski et al., 2000;

Wolf et al. , 2001).

Proteases are normally synthesized as latent forms, known as zymogens, which can be converted into their active forms. The interactions of several proteases converge to the degradation of extracellular matrix components. In this way, overexpression or activation of a protease provides a proteolytically active environment that surrounds the tumor.

Currently, it is already established that there is a direct correlation with the increase in expression, activity and the change in the location of many proteases and the progression of the tumor (Koblinski et al., 2000).

Proteases can generate fragments of proteins from

4/108 matrix, influence release, activation and biocapacity »zi

V of growth factors, and consequently modulate cell growth, invasion, apoptosis and angiogenesis.

Additionally, proteases, their receptors and inhibitors may be directly involved in cell migration and in the processing or shedding of cell surface proteins (Noel et al., 1997). Many proteases have an important action in this degradation process, including serinoproteases, cysteine proteases, aspartic proteases and metalloproteases (Polette & Birembaut, 1998; Bode et al.,

2000).

It is worth noting that tumor cells overexpress proteases to influence the local blood supply, extravasation through the vessels and migration through the extracellular matrix during metastasis (Wolf et al.,

01), and it is for this reason that protease inhibitors present themselves as potent candidates for controlling these events.

Isolated inhibitors of B. bauhinioides legumes, and

Enterolobium contortisiliquum were isolated and characterized chemically and biochemically. In particular, the action of inhibitors has been studied on various blood coagulation enzymes such as plasma kallikrein, factor Xlla, factor Xa and thrombin, in addition to proteases that have

5/108 crucial participation in tumor development such as plasmin and metalloproteases. According to the indications we have, these inhibitors, all highly homologous to the soybean trypsin inhibitor, present small structural differences possibly responsible for this ability to discriminate in connection with enzymes (Oliva et al., 2001 a, b) .

The crucial role of proteases in the metastatic process is attributed to the proteolytic degradation of the extracellular matrix, 10 which is initiated by specific proteases, secreted by different cell types, participating in the invasion of tumor cells and increasing the expression or activity of many other enzymes that are involved in the process of invasion and malignancy of tumor cells.

The actions of inhibitors of protelolytic enzymes, which differ in specificity of action, allow one to explore these effects and thus block the action of proteases in cancer. For this reason, specific protease inhibitors have potential therapeutic value in cancer.

More specifically, in the area of oncology, protease inhibitors are being studied mainly, in models that involve the metastatic process, such as adhesion, migration, cycle,

6/108 metabolism, cell proliferation and angiogenesis using models that analyze tumor development in vitro and in vivo. Thus, inhibitors isolated from plants, as well as peptides derived from their structures, may have an important role in the control of tumor processes. BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are part of this specification and are included here to illustrate certain aspects of the invention. The object of the present invention can be better understood by reference to one or more of these figures in combination with the detailed description of the preferred embodiment presented here.

Figure 1 shows the inhibition of HNE by EcTI. HNE (17.0 nM) and increasing doses of inhibitor were pre-incubated for 10 minutes at 37 ° C. Inhibitory activity was determined by decreasing the activity of the enzyme, using MeO-Suc-Ala-Ala-ProVal-pNan as substrate (11.0 mM).

Figure 2 shows the activation analysis of pro-MMP-9 in MMP-9 and pro-MMP-2 in MMP-2 induced by plasminogen and / or PMA in HT1080 cells, treated or not with BbCI, BbKI,

EcTI and aprotinin.

Figure 3 shows the action of (a) BbCI and (b) rBbCI in the adhesion of melan-a cells to collagen IV, fibronectin and

7/108 vitronectin. The melan-a cells and increasing concentrations of BbCI and rBbCI were pre-incubated for 15 minutes at room temperature and added to plates previously adsorbed with collagen IV or fibronectin or vitronectin (5 gg / 100 μΐ / well} or BSA, as described here .

Figure 4 shows the action of (a) BbCI and (b) rBbCI in the adhesion of Tm5 cells to collagen IV, fibronectin and vitronectin. Tm5 cells and increasing concentrations of

BbCI and rBbCI were pre-incubated for 15 minutes at room temperature and added to plates previously adsorbed with collagen IV or fibronectin or vitronectin (5 gg / 100 μΐ / well) or BSA, as described here.

Figure 5 shows the action of (a) BbKI and (b) rBbKI in the adhesion of melan-a cells to collagen IV, fibronectin and vitronectin. Cells and concentrations of BbKI and rBbKI were pre-incubated for 15 minutes at room temperature and added to plates previously adsorbed with collagen IV or fibronectin or vitronectin (5 gg / 100 μΐ / well) or BSA, as described here.

Figure 6 shows the action of (a) BbKI and (b) rBbKI in the adhesion of Tm5 cells to collagen IV, fibronectin and vitronectin. Tm5 cells and increasing concentrations of

BbKI and rBbKI were pre-incubated for 15 minutes at

8/108 at room temperature and added to plates previously adsorbed with collagen IV or fibronectin or vitronectin (5 pg / 100 μΐ / well) or BSA, as described here.

Figure 7 shows the action of EcTI on the adhesion of (a) melan-a (b) Tm5 cells to collagen IV, fibronectin and vitronectin. Melan-a or Tm5 cells and increasing concentrations of EcTI were pre-incubated for 15 minutes at room temperature and added to plates previously adsorbed with collagen IV or fibronectin or vitronectin (5 μg / 100 μΐ / well) or BSA, as described here .

Figure 8 shows the action of (a) BbCI and (b) rBbCI in the proliferation of melan-a cells. The melan-a cells and increasing concentrations of BbCI and rBbCI were pre-incubated for 15 minutes at room temperature, at different incubation times (2h, 24h, 48h and 72h).

Figure 9 shows the action of (a) BbCI and (b) rBbCI in the proliferation of Tm5 cells. The Tm5 cells and increasing concentrations of BbCI and rBbCI were pre-incubated for 15 minutes at room temperature, added to 96-well plates and analyzed at different incubation times (2h, 24h, 48h and 72h).

Figure 10 shows the action of (a) BbKI and (b) rBbKI on

9/108 \ <

proliferation of melan-a cells. The melan-a cells and increasing concentrations of BbKI and rBbKI were pre-incubated for 15 minutes at room temperature, added to 96-well plates and analyzed at different incubation times (2h, 24h, 48h and 72h).

Figure 11 shows the action of (a) BbKI and (b) rBbKI in the proliferation of Tm5 cells. Tm5 cells and increasing concentrations of BbKI and rBbKI were pre-incubated for 15 minutes at room temperature, added in 96-well plates and analyzed at different incubation times (2h, 24h, 48h and 72h).

Figure 12 shows the action of EcTI on the proliferation of (a) melan-a and (b) Tm5 cells. The cells and increasing concentrations of EcTI were pre-incubated for 15 minutes at room temperature, added in 96-well plates and

I analyzed at different incubation times (2h, 24h, 48h and 72h).

Figure 13 shows the cellular metabolism of Tm5 treated with 5-FU, rBbCI, rBbKI and EcTI. Tm5 cells (8x10 ³ / 100pl / well) were incubated for 24 hours and after this period increasing concentrations of 5-FU, rBbCI, rBbKI and

EcTI were added to the plates and kept incubated for (a) 24 hours and (b) 48 hours.

Figure 14 shows the cell cycle of melan-a with the

10/108 rBbCI, rBbKI and EcTI inhibitors. (a) melan-a, (b) melan-a treated with rBbCI, (c) melan-a treated with rBbKI, (d) melan-a treated with EcTI, after 24 hours of incubation as described in methods. The phases Ml to M4 indicate the phases of the cell cycle, Ml = fragmented cells, M2 = GO ^ G1,

M3 = S, M4 = G2— »M.

Figure 15 shows the Tm5 cell cycle with the inhibitors rBbCI, rBbKI and EcTI. (a) Tm5, (b) Tm5 treated with rBbCI, (c) Tm5 treated with rBbKI, (d) Tm5 treated with

EcTI, after 24 hours of incubation as described in methods. The phases Ml to M4 indicate the phases of the cell cycle, Ml = fragmented cells, M2 = G0—> G1, M3 = S,

M4 = G2—> M.

Figure 16 shows the migration of melan-a and

Tm5. The melan-a and Tm5 cells were added to the plates, previously adsorbed with fibronectin and kept incubated for 1, 2 and 3 hours.

Figure 17 shows the action of rBbCI in the migration of melan-a and Tm5 cells. The melan-a or Tm5 cells and increasing concentrations of rBbCI were pre-incubated for minutes at room temperature, and later added to the plates, previously adsorbed with fibronectin.

11/108 ο

Figure 18 shows the action of rBbKI in the migration of melan-a and Tm5 cells. The melan-a or Tm5 cells and increasing concentrations of rBbKI were pre-incubated for minutes at room temperature, and later added to the plates, previously adsorbed with fibronectin.

Figure 19 shows the action of EcTI in the migration of melan-a and Tm5 cells. The melan-a or Tm5 cells and increasing concentrations of EcTI were pre-incubated for 15 minutes at room temperature, and later added to the plates, previously adsorbed fibronectin.

Figure 20 shows the effect of BbCl and rBbCI on Tm5-induced tumorigenesis. Tumorigenesis was assessed 15 dependent on the tumor volume (mm ³ ). C57B1 / 6 mice (n = 5) received subcutaneous injection with Tm5 and daily treatment with the inhibitor, (a) BbCX (2mg / kg / day / animal), in detail pictures of the tumor of the control group and the group that received BbCI; (b) rBbCI (2mg / kg / day / animal).

0 Figure 21 shows the effect of BbKI and rBbKI on Tm5-induced tumorigenesis. Tumorigenesis was assessed depending on the tumor volume (mm ³ ). C57B1 / 6 mice (n = 5) received subcutaneous injection with Tm5 and daily treatment with the inhibitor, (a) BbKI (2mg / kg / day / animal), in the

12/108 detail photos of the tumor of the control group and the group that received / BbKI; (b) rBbKI (2mg / kg / day / animal).

Figure 22 shows the effect of EcTI on Tm5-induced tumorigenesis. Tumorigenesis was assessed depending on the tumor volume (mm ³ ). C57B1 / 6 mice (n = 5) received subcutaneous injection with Tm5 and daily treatment with the inhibitor, (a) EcTI (1mg / kg / day / animal) in detail tumor pictures of the control group and the group that received EcTI; (B)

EcTI (2mg / kg / day / animal).

Figure 23 shows the reverse phase chromatography of the material with affinity for rBbCI-Sepharose. Sample: 20 μ9 (Α ₂ ιε) from rBbCISepharose affinity chromatography (fraction c) was applied to column C ₄ (4.6 mm x 15 cm) equilibrated with 0.1% TFA in water. Elution by linear gradient of acetonitrile in tfa o, i% in water from 0 to 100% in 60 minutes. The arrows indicate the materials subsequently subjected to sequencing. In detail rBbCI-Sepharose affinity chromatography. RBbCI-Sepharose affinity column (2.0 ml) balanced with buffer

0.1 M Tris / HCl, pH 8.0. (a) non-adsorbed material; (b) material eluted with Tris / HCl pH 8.0, containing 0.15 M NaCl;

(c) Elution with 0.5 M KC1 / HCl, pH 2.0, followed by immediate neutralization with 1.0 M Tris / HCl solution; (d) material eluted with 0.5 M glucose; (e) material eluted with

10/13

7 /

Tris / HCl pH 8.0, containing 1 M urea. ^u

Figure 24 shows the reverse phase chromatography of the material with affinity for rBbKI-Sepharose. Sample; 20 gg (A ₂ is) from affinity chromatography rBbKI5 Sepharose (fraction c) was applied to column C ₄ (4.6 mm x 15 cm) equilibrated with 0.1% TFA in water. Elution by linear gradient of acetonitrile in 0.1% TFA in water from 0 to 100% in 60 minutes. The arrows indicate the materials subsequently submitted to sequencing. In detail rBbKI-Sepharose affinity chromatography. RBbKI-Sepharose affinity column (2.0 ml) balanced with buffer

0.1 M Tris / HCl, pH 8.0. (a) non-adsorbed material; (b) material eluted with Tris / HCl pH 8.0, containing 0.15 M NaCl;

(c) Elution by 0.5 M KC1 / HCl, pH 2.0, followed by immediate neutralization with 1.0 M Tris / HCl solution; (d) material eluted with 0.5 M glucose; (e) material eluted with

Tris / HCl pH 8.0, containing 1 M urea. The arrow indicates the fraction analyzed in sequence.

Figure 25 shows the reverse phase chromatography of the material with affinity for EcTI-Sepharose. Sample: 20 gg (A ₂ is) from EcTISepharose affinity chromatography (fraction c) was applied to column C ₄ (4.6 mm x 15 cm) equilibrated with 0.1% TFA in water. Elution by

14/108 linear gradient of acetonitrile in 0.1% trifluoroacetic acid (TFA) in water from 0 to 100% in 60 minutes. The arrows indicate the materials subsequently submitted to sequencing. In detail, EcTI5 Sepharose affinity chromatography. EcTI-Sepharose affinity column (2.0 ml) balanced with 0.1 M Tris / HCl buffer, pH 8.0. (a) non-adsorbed material; (b) material eluted with Tris / HCl pH 8.0, containing 0.15 M NaCl; (c) Elution by 0.5 M KC1 / HCl, pH

2.0, followed by immediate neutralization with solution

1.0 M Tris / HCl; (d) material eluted with 0.5 M glucose; (e) material eluted with Tris / HCl pH 8.0, containing 1 M urea.

Figure 26 shows the sequence of the aminoterminal region of the material eluted with 38 ', in reverse-phase chromatography, referring to the material with affinity to rBbCI15 Sepharose, and similarity. The indication shows the sequence of the amino-terminal region. Below, other proteins that showed similarity to this sequence, obtained through analysis in the NCBIBlast2 primary structure database (www.ebi.ac.uk/blastall). Highlighted, the name of the protein that shows similarity and residues with identity.

Figure 27 shows the sequence of the aminoterminal region of the material eluted with 40 ', in reverse phase chromatography, referring to the material with affinity to rBbCI15 / 108

Sepharose, and similarity. The indication shows sequence of the amino-terminal region. Below, other proteins that showed similarity to this sequence, obtained through analysis in the NCBI5 Blast2 primary structure database (www.ebi, ac.uk / blastall). Highlighted, the name of the protein that shows similarity and residues with identity.

Figure 28 shows the sequence of the aminoterminal region of the material eluted with 35 ', in reverse-phase chromatography, referring to the material with affinity to rBbKISepharose, and similarity. The indication shows the sequence of the amino-terminal region. Below, other proteins that showed similarity to this sequence, obtained through analysis in NCBI15 Blast2 primary structure database (www.ebi.ac.uk/blastall). Highlighted. the name of the protein that shows similarity and residues with identity.

Figure 29 shows the sequence of the aminoterminal region of the material eluted with 37 ', in reverse-phase chromatography, referring to the material with affinity to rBbKISepharose, and similarity. The indication demonstrates the sequence of the amino-terminal region. Below, other proteins that showed similarity to this sequence, obtained through analysis in a structure database

10/168

primary NCBI-Blast2 (www.ebi.ac.uk/blastall). Highlighted, the name of the protein that shows similarity and residues with identity.

Figure 30 shows the sequence of the aminoterminal region of the material eluted with 31 ', in reverse-phase chromatography, referring to materials with affinity to EcTISepharose, and similarity. The indication demonstrates the sequence of the amino-terminal region. Below, other proteins that showed similarity to this sequence, obtained through analysis in the NCBI-Blast2 primary structure database (www.ebi.ac.uk/blastall). Highlighted, the name of the protein that shows similarity and residues with identity.

Figure 31 shows the sequence of the aminoterminal region of the material eluted with 35 ', in reverse-phase chromatography, referring to the material with affinity to EcTISepharose, and similarity. The indication demonstrates the sequence of the amino-terminal region. Below, other proteins that showed similarity to this sequence, obtained through analysis in the NCBI-Blast2 primary structure database (www.ebi.ac.uk/blastall). Highlighted, the name of the protein that shows similarity and residues with identity.

Figure 32 shows the cellular metabolism of MKN28

17/108 (gastric cancer - less invasive lineage) treated with 5FU, rBbCI, rBbKI and EcTI. The MKN28 cells (8 x 10 ^3/100 μΐ / well) were incubated for 24 hours and after this period increasing concentrations of 5-FU rBbCI, rBbKI and ECTI were added to the plates and maintained incubated for (a) 24 hours and ( b) 48 hours.

Figure 33 Cellular metabolism of Hs746T (gastric cancer - more invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. The Hs746T cells (8 x 10 ^3/100 μΐ / well) were 10 incubated for 24 hours and after this period increasing concentrations of 5-FU rBbCI, rBbKI and ECTI were added to the plates and maintained incubated for (a) 24 hours and (b) 48 hours.

Figure 34 shows the cellular metabolism of HT-2 9 (colon cancer 1 less invasive swelling) effected with 5FU, rBbCI, rBbKI and EcTI. The HT-29 cells (8x10 ³ / 100μl / ρ well) were incubated for 24 hours and after this period increasing concentrations of 5-FU, rBbCI, rBbKI and

EcTI were added to the plates and kept incubated for (a) 24 hours and (b) 48 hours.

Figure 35 shows the cellular metabolism of HCT 116 (colon cancer - most invasive strain) treated with 5FU, rBbCI, rBbKI and EcTI. HCT 116 cells (8 x 10 ^3/100

10/188

μΐ / well) were incubated for 24 hours and after this period increasing concentrations of 5-FU, rBbCI, rBbKI and EcTI were added to the plates and kept incubated for (a) 24 hours and (b) 48 hours.

Figure 36 shows the cellular metabolism of MCF-7 (breast cancer - less invasive lineage) treated with 5FU, rBbCI, rBbKI and EcTI. MCF-7 cells (8 x 10 ^3/100 μΐ / well) were incubated for 24 hours and after this period increasing concentrations of 5-FU rBbCI, rBbKI and ECTI were added to the plates and maintained incubated for (a) 24 hours and (b) 48 hours.

Figure 37 shows the cellular metabolism of SKBR-3 (breast cancer - most invasive strain) treated with 5-FU, rBbCI, rBbKI and EcTI. The SKBR-3 cells (4 x 10 ^3/100 μΐ / well) fõreuu incubated for 24 hours and after this period increasing concentrations of 5-FU rBbCI, rBbKI and ECTI were added to the plates and maintained incubated for (a) 24 hours and (b) 48 hours.

Figure 38 shows the cellular metabolism of K562 (leukemic cell line) treated with 5-FU, rBbCI, rBbKI and EcTI. K562 cells (8 x 10 ^3/100 μΐ / well) were incubated for 24 hours and after this period increasing concentrations of 5-FU rBbCI, rBbKI were added and ECTI

19/108 on the plates and kept incubated for (a) 24 hours and (b) 48 hours.

Figure 39 shows the cellular metabolism of THP-1 (monocyte leukemic cell line) treated with 5 5-FU, rBbCI, rBbKI and EcTI. THP-1 cells (2 x 10 ^4/100 μΐ / well) were incubated for 24 hours and after this period increasing concentrations of 5-FU rBbCI, rBbKI and ECTI were added to the plates and maintained incubated for (a) 24 hours and (b) 48 hours.

Figure 40 shows the cellular metabolism of MKN28 (gastric cancer - less invasive lineage) treatment with 5-FU, EcTI and 5-FU + EcTI. The MKN28 cells (8 x 10 ^3/100 μΐ / well) were incubated for 24 hours and after this period increasing concentrations of 5-FU, ECTI and 5-FU + ECTI were added in maintained c plates incubated for (a) 24 hours and (b) 48 hours.

Figure 41 shows the cellular metabolism of Hs746T (gastric cancer - most invasive lineage) treatment with

5-FU, EcTI and 5-FU + EcTI. At Hs746T cells (8 x 10 ^3/100 20 μΐ / well) were incubated per 24 hours and after This one period concentrations rising in 5-FU, EcTI and 5- FU + EcTI were

added to the plates and kept incubated for (a) 24 hours and (b) 48 hours.

10/20

Figure 42 shows the cellular metabolism of MKN28 (gastric cancer - less invasive lineage) treatment with carboplatin, EcTI and carboplatin + EcTI. The MKN28 cells (8 x 10 ^3/100 μΐ / well) were incubated for 24 hours and after 5 this period increasing concentrations of carboplatin, ECTI and carboplatin + ECTI were added to the plates and maintained incubated for (a) 24 hours and (b ) 48 hours.

Figure 43 shows the cellular metabolism of Hs746T (gastric cancer - more invasive lineage) treatment with carboplatin, EcTI and carboplatin + EcTI. The Hs746T cells (8 x 10 ^3/100 μΐ / well) were incubated for 24 hours and after this period increasing concentrations of carboplatin, ECTI and carboplatin + ECTI were added to the plates and maintained incubated for (a) 24 hours and (b) 48 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of isolated protease inhibitors, preferably from the seeds of the

Bauhinia bauhinioides and Enterolobium contortísiliquum for the treatment of cancer.

The specificity of inhibition on different proteases, presented by compounds isolated from seeds of Bauhinia and Enterolobium implied a potent action in cancer, whose metastatic process involves the action of

10/21

different proteases. These products were able to block tumor development in an in vivo model without apparent toxicity. In human cancer cell lines, there is an important inhibition of cell proliferation.

Combined therapy of a protease inhibitor according to the present invention and a chemotherapeutic agent considerably reduces the amount of chemotherapeutic agent needed to inhibit tumor cell proliferation. In this regard, inhibitors can reduce the side effects presented by these chemotherapeutic agents.

The present invention also includes pharmaceutical compositions comprising an effective amount of at least one of the inhibitors of the invention and at least one pharmaceutically acceptable carrier.

According to another embodiment, the compositions of the present invention may further comprise at least one chemotherapeutic agent. Such chemotherapeutic agents include, for example, 5-Fluorouracil (5-FU) and 20 carboplatin. When a second chemotherapy is used, the second chemotherapy can be administered as a much smaller amount, in addition to increasing the effectiveness of the composition.

Typically, the pharmaceutical compositions of the present

22/108 .n was an invention comprises at least one inhibitor according to the present invention that can be administered in combination with at least one chemotherapeutic agent and at least one pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. The pH and the exact concentration of various components of the composition can be adjusted according to standard practice.

It will also be understood that a specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration.

excretion rate, drug combination and the doctor's judgment and the severity of the disease being treated in particular. The amount of active ingredients will also depend on the particular compound and the other therapeutic agent, if present, in the composition.

In the following, the present invention will be explained in more detail with the help of the description of its preferred modality.

The inhibitors were purified using techniques

10/23

Usual chromatographic chromatography consisting of ion exchange chromatography, affinity chromatography, gel filtration chromatography, reverse phase chromatography and according to previously described methods such as, for example, by Oliva et al. , 1999; Oliveira et al. , 2001b, Batista et al. , 1996 and the recombinant inhibitors obtained according to the method described, for example, by Hansen, 2004.

Enzymatic Dosages and Determination of Constants of

Inhibition

Chromogenic substrates, peptides derived from pnitroanilide, were used mainly for the high sensitivity of photometric detection (in A ₄ os) of pnitroaniline released after enzymatic hydrolysis (Erlanger et al., 1961), in Packard spectrophotometer (model

SpectraCount).

The substrates used in each experiment were those that showed the best specificity for the enzymes tested. The substrates derived from p-nitroanilide were initially diluted in dimethylsulfoxide (DMSO) and further dilutions were performed in an appropriate buffer for each assay.

Fluorogenic substrates, peptides derived from aminomethylcoumarin (AMC), were also used in some dosages, as they are more sensitive than

24/108 chromogenic substrates derived from p-nitroanilide. Hydrolysis was monitored at 3 80 nm (excitation) and 460 nm (emission) wavelengths, using Packard spectrofluorimeter (FluoroCount model). These substrates were diluted in dimethylformamide (DMF).

Assays using dim fluorescence substrates (ortho aminobenzoic acid [Abz] -X-2,4 ethylene diaminodinitropheni [EDDnp]) were performed on a Hitachi spectrofluorimeter (model F-2000), and detection of hydrolysis at 320 nm wavelengths (excitation) and 420 nm (emission) and diluted in dimethylformamide and water 1 ·. 1 (v / v).

The inhibition constants were determined by the value of the dissociation constant of the inhibitory enzyme complex (K ±). The determinations were carried out using the Morrison model (1989), whose final equation was defined by Knight, 1986 adapted to a program for graphs of enzymatic kinetics on a computer, the value being calculated using the GraFit® program (version 3.0).

Hydrolysis of substrates by proteases and determination of inhibitory activities

The enzymatic activities of the proteases were carried out on specific substrates, using enzyme [M] that was pre-incubated at 37 ° C, with different

25/108 / ⁷ concentrations of the inhibitor, in an appropriate buffer, and after 10 minutes, substrate [M] (Table I) was added, in a final volume of 250 μΐ, and incubation continued, varying between 20 - 30 minutes (time specific for each enzyme) at 3 7 ⁰ C, stopping the reaction with 40 0 μΐ of 40% acetic acid.

The hydrolysis of the substrate by the enzyme was monitored through the photometric reading (in A ₄₀₅ ) of the released p-nitroaniline or the hydrolysis of the substrate (AMC) was monitored by detecting fluorescence at 380 nm (excitation) and 460 nm ( issue). The inhibitory activity was calculated by determining the residual enzyme activity in the assays. Inhibitor concentrations were calculated by assuming '-K stoichiometry.

'This method was also used to locate the inhibitory activity during the purification processes of the inhibitors. The experiments were carried out in triplicate.

Table I shows the conditions used to determine the activities of the studied inhibitors on the proteases that are involved in cellular activity.

Table I: Enzyme, substrate and buffer used for

10/26

I verify hydrolysis of substrates by proteases and determination of inhibitory activities.

Enz Fridge Magnet Substrate Plug trypsin (20 μΐ, 0.43 μΜ) * p-nitrophenyl- p'-guanidino benzoate (NPGB) Bz-Arg-pNan (25 μΐ, 10.0 mM) 0.1 M Tris / HCl, pH 8.0, 0.02% (v / v) CaCl ₂ HNE (20 μΐ, 0.21 μΜ) (♦ αι- anti-trypsin) MeO-Suc-Ala-Ala-Pro- Val -pNan (11.0 mM, 25 μΐ) Tris / HCl 0.1 M pH 7.0, 0.5 M NaCl kallikrein human plasmatic (HuPK) (20 μΐ, 0.84 μΜ) (* EcTI) H-D-prolil-L- phenylalanyl-L- arginine-p- nitroanilide (H-D- Pro-Phe-Arg-pNan) (5 mM, 25 μΐ) 0.05 M Tris / HCl, pH 8.0 kallikrein pancreatic porcine (PoPK) (30 μΐ, 0.16 nM) (* aprotinin) H-D-prolil-L- phenylalanyl-L- arginine- aminomethylcoumarinic (H-D-Pro-Phe-Arg- AMC) (30 μΐ, 5 mM) 0.1 M Tris / HCl, pH 9.0 containing 0.1% albumin (v / v) chymotrypsin (40 μΐ, 0.88 μΜ) (* EcTI) succinyl-L- phenylalanine-p- nitroanilide (Suc-Phe-pNan) (20 μΐ, 20 mM) 0.1 M Tris / HCl, pH 8.0, 0.02% (v / v) CaCl ₂

10/278

Enzyme Substrate Plug plasmin (25 μΐ, 0.028 μΜ) (* inhibitor of trypsin from Bauhinia variegata [BvTI]) H-D-valyl-L-leucyl- L-lysine-p- nitroanilide (H-D- Va1-Leu-Lys-pNan) (20 μΐ, 9.0 mM) 0.1 M Tris / HCl, pH 7.4, 0.2 M NaCl thrombin (10 μΐ, 0.267 μΜ) (* rhodinina) H-D-phenylalanyl-L- pipecolil-L- arginine-p- nitroanilide (H-D- Phe-L-Pip-L-Arg- pNan) (20 μΐ, 2.0 mM) 0.05 M Tris / HCl, pH 8.0 factor Xa (30 μΐ, 0.467 μΜ) (* inhibitor of Xa factor of Bauhinia ungulata [BuXI]) tert-butyl- oxycarbonyl-L- isoleucil-L- glutamyl-L-glycyl-L- arginine- aminomethylcoumarinic (Boc-Ile-Glu-Gly- Arg-AMC) (60 μΐ, 6 mM) 0.05 M Tris / HCl, pH 8.0 cathepsin G (30 μΐ, 0.25 μΜ) (* <Xi-anti- trypsin) N-Suc-Ala-Ala-Pro- Phe-pNan (25 μΐ, 1 mM) 0.05 M Tris / HCl, pH 7.0, 0.5 M NaCl

* inhibitor used for enzyme titration.

Hydrolysis of Z-Phe-Arg-AMC by cysteine proteases and determination of inhibitory activities

10/28 ι!

The concentration of cysteine proteases was obtained /

through titrations with egg cystatin. The inhibitory activity of cathepsin L or cruzipain or cruzain was determined through the residual activity of the enzyme on the substrate Z-phenylalanyl-arginine-aminomethylcoumarinic (ZPhe-Arg-AMC). The enzymes were activated in Na ₂ HPO ₄ buffer

0.1 M, pH 6.3 containing 10 mM EDTA, 400 mM NaCl and 2 mM DTT, maintained 10 min at 37 ° C. In typical experiments, performed in the same activation buffer, cathepsin L (18 nM) or cruzipain (18 nM) or cruzain (3.2 nM), was pre-incubated with increasing concentrations of the preparation of the purified inhibitor, for 10 minutes at 37 ° C, and then Z-Phe-Arg-AMC (0.3 mM) was added.

The substrate hydrolysis was monitored by detecting fluorescence at 380 nm (excitation) and 460 nm (emission) wavelengths, in a spectrofluorimeter

Hitachi (model F-2000). The increase in fluorescence was continuously recorded for 10 minutes. The experiments were carried out in triplicate.

Residual activity was determined by comparing the enzyme hydrolysis curves, in the presence and absence of the inhibitor.

Determination of the concentration of BbKI, rBbKI and EcTl

Different concentrations of kallikrein inhibitor

29/108 human plasma from Bauhinia bauhinioides (BbKI), recombinant inhibitor of human plasma kallikrein from Bauhinia bauhinioides (rBbKI) and trypsin inhibitor from Enterolobium contortisiliquum (EcTI) were pre-incubated at 37 ° C for 10 minutes in Tris / buffer 0.05 M HCI, containing 0.02% (v / v) CaCl ₂ , pH 8.0, with trypsin of known concentration. To the pre-incubation, 25 μΐ of the substrate H-DBenzoyl-L-arginine-p-nitroanilide (Bz-Arg-pNan) was added 10.0 mM in a final volume of 250 μΐ. The hydrolysis of the substrate by the enzyme was monitored by photometric reading (in A405) of the released p-nitroaniline, as previously described.

Inhibitor concentrations were calculated using 1: 1 reaction stoichiometry.

5 Determination of the concentration of BbCI and rBbCI

Different concentrations of cruzipain inhibitor of

Bauhinia bauhinioides (BbCI) and recombinant Bauhinia bauhinioides cruzipain inhibitor (rBbCI) were pre-incubated at 37 ° C for 10 minutes in 0.1 M Tris / HCI buffer pH 7.0, containing 0.5 M NaCl with neutrophil elastase (HNE) (17 nM), determined as previously described. To the pre-incubated, 25 μΐ of the methyl ester-L-succinyl-L-alanyl-L-alanyl-L30 / 108 prolyl-L-valine-p-nitroaniiide substrate (MeO-Suc-Ala-Ala-Pro-VaipNan) was added (11.0 mM), in a final volume of 250 μΐ. The hydrolysis of the substrate by the enzyme was monitored through the photometric reading (in A ₄ os) of the released p-nitroaniline.

Inhibitor concentrations were calculated using 1: 1 reaction stoichiometry.

Polyacrylamide Gel Electrofprese

The electrophoretic analysis of the protein was carried out according to Laemmli, 1970. Glass plates (8.0 x 10.0 cm) were previously washed, rinsed with distilled water and cleaned with acetone, to be used for the polymerization of the gel using acrylamide solution 15 %.

For the analyzes, 15 μ9 of lyophilized proteins were used, resuspended in an appropriate volume of distilled water 15 in an equal volume of 0.12 M Tria / KCl buffer containing 4% SDS (w / v) and 20% glycerol (v / v) ; when indicated, proteins were reduced by adding an equal volume of sample buffer containing dithiothreitol (DTT) (200 mg / ml). All samples were heated to

100 ° C for 7 min, centrifuged, and 1 μΐ of 0.02% bromophenol blue solution (w / v) containing SDS was added

0.1% and 10% glycerol in 0.1 M phosphate buffer, pH 6.8.

Proteins used as molecular weight standards

10/318 were phosphorylase b (94 kDa), bovine albumin (66 kDa), ovoalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (20 kDa) and a-lactalbumin (14 kDa).

Electrophoresis was performed in 0.025 M Tris buffer and 0.18 M glycine, containing 0.1% SDS. After the electrophoretic run, the gel was immersed in a 10% trichloroacetic acid (TCA) solution (v / v) for 30 min for protein precipitation. Subsequently washed with water, the gel was stained by immersion for a period of 1 hour in the Coomassie Briliant Blue R 250 0.5% (w / v) solution in a methanol / water / acetic acid solution (4.35: 4, 65: 1, v / v / v), in which the gel was immersed for 30 min, at room temperature. The excess dye was removed by washing in a solution of ethanol, water and acetic acid (4.35: 4.65: 1, v / v / v) until the appearance of several protein bands. The gels were maintained in 1.0% acetic acid.

Zymography

Human fibrosarcoma cells (HT1080) (2 x 10 ⁵ cells / well) were cultured in 24-well plates for 48 hours and then the cells were treated with inhibitors (BbKI, BbCI, EcTI and aprotinin), plasminogen and or PMA. In the control samples, none of the inhibitors were added, while plasminogen and PMA were added in the absence of the inhibitors, and maintained for 48 hours at 37 ° C

10/32. under tension of 5% CO ₂ . After this period, the conditioned medium of the cells, treated or not, was collected, centrifuged and submitted to electrophoretic analysis in% acrylamide gel containing 1 mg / ml of gelatin, as described by Baramova et al. , 1997. After the run, the gel was washed in 2% Triton X-100 for 1 hour and then incubated for 16 hours in the Tris / HCl 0.05 activation buffer

M, pH 7.4 containing 0.2 M NaCl, 5 mM CaCl ₂ and 0.02% NaN ₃ . The gel was stained by immersion for a period of 1 hour in the Coomassie Briliant Blue R 250 0.5% (w / v) solution in methanol / water / acetic acid (4.35: 4.65: 1, v / v / v), in which the gel was immersed for 30 min, at room temperature. The excess dye was removed by washing in a solution of ethanol, water and acetic acid (4.35: 4.65: 1, v / v / v) until the appearance of the protein bands. The gel was maintained in 1.0% acetic acid.

This trial was carried out in collaboration with Dr.

Christian Ries from Ludwing Maximilians University

Munich, Germany.

Cell culture

Cultivation conditions of Tm5 and melan-a cell lines

The murine cell lines of mice melanocytes (melan-a) and murine melanoma (Tm5) (Corrêa et

10/338

al. , 2005) were maintained in cell culture medium (RPMI) pH 6.9 enriched with 5% fetal bovine serum (SFB) and penicillin / streptomycin. The melan-a strain needs phorbol esters (PMA) for growth.

PMA maintains all tested characteristics of normal melanocytes except senescence (Bennet et al., 1987).

The cells were maintained at 37 ° C under a tension of 5%

CO ₂ . The subculture was carried out weekly, as described

Next. To the maintenance of stocks cell phones, 10 initially the middle was removed of the plate of cells confluent that went posteriorly washed with solution of

PBS pH 7.4. Then, the cells were incubated with a trypsin solution (2.5%) (1 ml in each 60 x 10 mm plate), keeping the solution for approximately 1 minute in the plate, time necessary for the cells to detach the board. Then, they were carefully resuspended several times with a Pasteur pipette and transferred (approximately 1 x 10 ⁵ cells) to a new plate containing RPMI medium enriched with 5% SFB, previously equilibrated at 37 ° C. The medium was replaced the day after the subculture, and every 3 days. For the melan-a strain, 200 nM PMA was added.

When observing the cells under a microscope, reaching a confluence of approximately 80-90%, the tests were

34/108 started.

Cultivation conditions for human cell lines

Human gastric cancer cell lines (MKN28) and human gastric cancer cells (Hs746T), human colon cancer cells (HT-29) and human colon cancer cells (HCT 116) _t human breast cancer cells (SKBR-3) and human breast cancer cells (MCF-7), leukemic cells (K562) and monocyte leukemic cells (THP-1) were maintained in RPMI medium pH 7.4 enriched with 7.5% serum fetal bovine (SFB) and penicillin / streptomycin.

The HT1080 strain (human fibrosarcoma) was maintained in DMEM pH 7.4 medium enriched with 10% fetal bovine serum (SFB) and penicillin / streptomycin.

The cells were maintained at 37 ° C under a tension of 5%

CO ₂ . The subculture was carried out as described in the previous item.

Cell adhesion assays

Cell adhesion tests were performed for melan-a and Tm5, by modifying the method described by Solimene et al. , 2001.

96-well plates were previously adsorbed with extracellular matrix proteins

35/108 (fibronectin / vitronectin / collagen IV) diluted in PBS (13 7 mM NaCl, 2.7 mM KC1, 8.1 mM Na ₂ HPO ₄ and 1.5 mM KH ₂ PO ₄ ) pH

7.4, sterile, at a concentration of 5 μ9 / 100 μΐ / well, incubated at 37 ° C under tension of 5% CO ₂ , for two hours or overnight at 4 ° C.

The plate wells were blocked with bovine albumin (BSA) 1% diluted in sterile PBS (100 μΐ / well) for 1 hour at 37 ° C. After incubation, excess BSA was discarded and the plates were washed three times with PBS pH

7.4.

The cells were removed from the culture flask using trypsin solution (2.5%), washed with PBS pH

7.4 sterile, centrifuged and suspended in RPMI pH 6.9, then they were counted in a Neubauer chamber.

We used a concentration of 5 x 10 ⁴¹ cells / 50 μΐ / well.

Cells and inhibitors (1.00-25.00 μΜ) diluted in

Sterile RPMI pH 6.9 (100 μΐ / well) were pre-incubated for minutes at room temperature and then added simultaneously to the plates and incubated at 37 ° C under

5% CO ₂ , for a period of 2 hours.

At the end of the incubation, non-adherent cells were removed through three washes with PBS, pH 7.4. 100% methanol was added to the wells for 5 minutes to

36/108 fix the adhered cells. Subsequently, the plates were washed three times with PBS, pH 7.4 and stained with 1% toluidine blue solution in 1% borax for 5 minutes. The wells were washed four times with PBS, pH 7.4 and the pigment retained by the cell was released by a 1% SDS solution.

The absorbance is detected at A ₅₇ o in the spectrophotometer

Packard (SpectraCount model).

The tests were carried out in triplicate for each concentration of inhibitors and the experiments were carried out at least twice.

Cell proliferation assays

The cells were removed from the culture flask using trypsin solution (2.5%), washed with PBS pH

7.4 sterile, centrifuged and suspended in RPMI pH 6.9 plus 5% SFB, then they were counted in a Neubauer chamber. We used a concentration of 5 x 10 ³ cells / 100 μΐ / well.

Inhibitors (1.00-12.50 μΜ) diluted in RPMI pH 6.9 plus 5% SFB and previously filtered in

Millipore (0.22 μτη) were pre-incubated with the cells for minutes at room temperature and added simultaneously in 96-well plates, incubated at 37 ° C under 5% CO ₂ tension for a period of 2, 24, 48 and 72 hours.

37/108

At the end of each incubation period, · was added. </ μΐ (3 - (4,5-dimethylthiazol-2-yl) - 2,5diphenyl tetrazolium (MTT) bromide (5 mg / ml) in each well and cells incubated for 2 hours at 37 ° C under tension 5%

CO ₂ . After this period, the medium was removed and isopropanol added and maintained for 20 minutes at 37 ° C. Finally, the absorbance was detected at A ₆₂ o in the spectrophotometer

Packard (SpectraCount model).

The tests were carried out in triplicate for each concentration of inhibitors and the experiments were carried out at least twice.

Cell metabolism assays Cell line metabolism was analyzed using MTS, a salt (tetrazolium) that changes to the color caused by the bioreduction of MTS in a water-soluble form called formazan, which accompanies the active dehydrogenase enzymes found in the mitochondria and the reaction it occurs only in living cells.

Cell metabolism assays were performed for the Tm5, MKN28, Hs746T, HT-29, HCT116, SKBR3, MCF-7, K562 and THP-1 lines, through modifications of the method described by Mayer et al. (2001).

The cells 4 x 10 ^3/100 μΐ / well (SKBR-3), 2 x 10 ^4/100

38/108 μΐ / well (THP-1) and 8 χ ΙΟ ³ cells / 100 μΐ / well (for the other strains used in this study) were incubated at ° C under 5% CO ₂ tension, for a period of 24 hours for metastatic strains and 48 hours for non-metastatic strains in an appropriate medium plus SFB, to allow adherence.

The inhibitors (1.00-25.00 μΜ) diluted in appropriate medium for each strain, plus SFB, previously filtered in Millipore (0.22 μτη) were added to the plates with a volume of 100 μΐ, incubated at 37 ° C under % CO ₂ voltage for 24 and 48 hours. In addition, cell metabolism assays, combined therapy of protease inhibitors with 5-FU or carboplatin, were performed at the same concentrations performed with the isolated therapy.

At the end of each incubation period, 5 μΐ of MTS (2 mg / ml; pH 6.5) was added and 3-mM PMS were dissolved in

PBS pH 7.4, separately and sterilized in a 0.22 μιτι filter. These solutions were stored at -20 ° C protected from

0 light. 1-methoxy (PMS) was added to 3 - (4,5-dimethylthiazol2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2Htetrazolium] (MTS) immediately before use (MTS: PMS ,

1:20), 20 μΐ was added to each well and incubated at 37 ° C

39/108, under tension of 5% C0 ₂ , for a period of 2 hours. Absorbance was detected at A ₄₉₀ .

The assays were performed in triplicate for each concentration of inhibitors, the experiments were performed at least twice and cell viability was controlled by Trypan blue staining at the beginning of all assays, and 0.1% of non-viable cells were found.

Cell Migration Assays

Transwell modified Boyden chambers (Costar,

Cambridge, USA) with polycarbonate membranes containing pores of 8 gm in diameter were sensitized on their lower faces with 10 gg of fibronectin / collagen IV, for 2 h at 37 ° C. The melan-a cells or Tm5 2 x 10 ^5/100 gl were pre-incubated with 100 gl rBbCI or rBbKI (1,0012,50 GM) or ECTI (1.00 gm) in RPMI pH 6.9 and finally the inner face of the insert was filled with

200 gL of the cell suspension in the presence of the inhibitor, incubated for 2 hours at 37 ° C in a humid oven containing 5%

C0 ₂ , Then, the membranes were washed carefully with PBS, fixed for 5 min in 10% methanol, washed with water, and stained with 1% toluidine blue solution in 1% borax for 15 minutes. The cells that remained on the face

The upper 40/108 were carefully removed with cotton, leaving only those that migrated to the underside of the membrane. The dye was eluted with 1% (w / v) of SDS in water, applying 200 μβ per chamber and incubating for 30 'at 37 ° C. Absorbance was detected in

The ₅₇₀ in the Packard spectrophotometer (SpectraCount model). Controls for non-specific migration towards 1% (w / v) of BSA in PBS have been discounted. All experiments were performed in duplicate.

Cell Cycle Assays

The melan-a and Tm5 cells (2 x 10 ⁵ cells / ml) were diluted in RPMI pH 6.9 plus 5% SFB and cultured in plates (60 x 10 mm) for 24 hours. After this period the cells were washed with PBS pH 7.4 and maintained with ODMT nU £ Q

---- ------ j--. -, - After 24 hours, the inhibitors (2.50 μΜ) previously diluted in RPMI pH 6.9 medium were added to the plates, which were previously washed with PBS pH 7.4 and incubated for 24 hours. Then the medium was transferred to a 15 ml tube and the cells were washed with PBS, removed from the bottle, and collected in the same tube in which the medium was transferred and centrifuged at 2,000 rpm for 5 min.

20 ° C.

41/108

The supernatant was discarded, 5 ml of PBS pH 7.4 was added to the pellet and centrifuged again. 0

PBS pH 7.4 was discarded, the cells resuspended in 0.5 ml of PBS, transferred to an eppendorf and 1 ml of ethanol

100% was added slowly under constant stirring and stored at 4 ° C for 16 hours.

After this period the materials were centrifuged at 2,000 rpm for 5 min, at 20 ° C, the ethanol was removed carefully, the cells were washed with 1 ml of PBS pH

7.4 and again centrifuged at 2,000 rpm for 5 minutes.

The PBS pH 7.4 was removed, the cells resuspended in 100 μΐ of PBS pH 7.4 and 100 μΐ of RNase 100 pg / ml remaining for 5 minutes at room temperature.

After this period, 4 00 μΐ of propidium iodide 50 pg / ml was added at 4 ° C for 30 minutes without the presence of light. Then the samples were analyzed using a flow cytometer (FACS; Becton-Dickinson, San Diego, CA).

Tumorigenesis in vivo

To study the effect of inhibitors on tumor growth in vivo, C57BL / 6 syngeneic mice were inoculated subcutaneously on the left flank with 2 x 10 ⁵ melanoma cells (Tm5) / 100 μΐ.

The inhibitors were injected subcutaneously close to the region where the cells were inoculated. The animals were

42/108 treated, at the concentration (2 mg / 100 μΐ / kg / mouse / day).

Tumors were measured daily with the aid of a caliper. After the tumors reached a volume of approximately 1500 mm ³ or after 20 days of experiment, the animals were sacrificed and examined both for local invasion and spontaneous metastasis in different organs.

The volume was calculated using the formula described below: Volume = (smaller diameter) ² x larger diameter

2

EcTI-Sepharose, rBbCI-Sepharose and rBbKI-Sepharose affinity chromatography

Tumors in the control group were excised and homogenized in 1 mM Tris / HCl pH 8.0 containing 0.02% Triton

X-100 maintained for 20 minutes in an ice bath, then the cells were lysed by sonication for 2 pulses of 30 seconds each, in an ice bath at 30 second intervals.

Then, they were chromatographed in an affinity column where the recombinant inhibitors of cruzipain and kallikrein isolated from Bauhinia bauhinioides (rBbKI and rBbCI) and trypsin inhibitor isolated from Enterolobium contortisiliquum (EcTI) coupled to the Sepharose resin according to the methodology described by Oliva, 1988. As sequence listings for these inhibitors are indicated in SEQ. IDs. we. 1, 2 and 3.

In columns (2.0 ml of resin) rBbCI-Sepharose, rBbKISepharose and EcTI-Sepharose, previously equilibrated in 0.1 M Tris / HCl buffer, pH 8.0.

The non-retained material was collected and the resin was washed with the buffer

Petition 870170051966, of 7/24/2017, p. 5/12

43/108 of equilibrium until the drop in absorbance in 280 of the effluent to values less than 0.03. The resin was then washed with 0.1 M Tris / HCl buffer, pH 8.0, containing NaCl

0.15 M. The retained inhibitor was eluted from the column by acidification with 0.5 M KC1 / HCl, pH 2.0 were immediately neutralized by the addition of 1.0 M Tris / HCl buffer, pH 8.0.

The materials were subsequently eluted with 0.5 glucose

M, then with 0.1 M Tris / HCl pH 8.0 containing 1 M urea. Protein elution was followed by photometric reading at 215 nm. Fractions eluted with 0.5 M KC1 / HCl, pH 2.0 were pooled, lyophilized and chromatographed in reverse phase (HPLC system). The fractions that showed the highest absorbance of reverse phase chromatography were sequenced, analyzed in a NCBI-Blast2 primary structure database (www.ebi.ac.uk/blastall) and aligned.

Statistical data processing

Data for Kaplan-Meyer survival curves, analyzed by Mantel-Haenszel from the in vivo experiments were performed were determined using

GraphPadPrism version 3.03 for Windows (GraphPad, San

Diego, CA; www.graphpad.com).

Data from cell adhesion, cell proliferation, cell migration and cell metabolism assays were analyzed using Excell (for Windows XP), to

44/108 determination of SD and statistical parameters (paired t test).

RESULTS

Characterization of the inhibitory activity of BbCI, rBbCI,

BbKI, rBbKI and EcTI

The inhibitory activities of BbCI, BbKI, rBbCI and rBbKI on the proteases were confirmed using the methodology described above, as described by

Batista et al., 1996; Oliva et al. , 1999; Oliveira et al.,

2001; Araújo et al., 2005 (Table II). EcTI inhibited HNE and the apparent inhibition constant was determined to be 55.00 nM. Through the inhibition curve in which increasing inhibitor concentration was used, it was possible to define the reaction stoichiometry as 1: 1 by extrapolation

linear to 100% of inhibition enzymatic (Figure 1) - Table II: Determination of the constants of inhibition of EcTI, BbCI, rBbCI, BbKI and rBbKI. Enzyme EcTI BbCI rBbCI BbKI rBbKI (K _iapp nM) (Ki _to pp nM) (Kiapp nM) (Κ _1βρ ρ nM) (Kiapp nM) Trypsin 0.88 Φ Φ 20.00 28.00 Chymotrypsin 1.11 Φ Φ 26.00 ND Plasmin 9.36 Φ Φ 330.00 ND HNE 55.00 5.30 1.70 Φ Φ

45/108

Enzyme EcTI BbCI rBbCI BbKI rBbKI (K _lapp nM) (K _iapp nM) (K _iapp nM) (Ki _app nü) (K _iapp nM) Factor Xa Φ Φ ND Φ ND Thrombin ND Φ ND Φ ND HuPK 6.15 Φ Φ 2.40 2.00 PoPK Φ Φ Φ 200.00 900.00 Cathepsin G ND 160.00 ND Φ Φ Cathepsin L ND 0.22 ND Φ ND Cruzaína ND 0.30 0.30 Φ Φ Cruzipaína ND 1.30 1.20 Φ Φ Φ = does not inhibit; ND = no determined

Zymography

Studies conducted by Baramova and collaborators (1997) have shown that the addition of plasminogen and or the combination with PMA induces the activation of pro-metalloprotease-2 (pro-MMP2) in HT1080 cells and that, in the presence of plasminogen, weak activation occurs of the active form of MMP-2 (Mr ~ 62 kDa). The authors propose that activation of pro-MMP-2 occurs in two phases. In the first phase, matrix metalloprotease type 1 (MT1-MMP) attached to the hydrolyzed cell surface proMMP-2, converting it to an intermediate form of 64 kDa.

Then, in the second phase, plasmin mediates processing into an active form of metalloprotease-2 (MMP46 / 108

2) 62 kDa.

EcTI inhibits the generation of the two active forms of proMMP-2, completely blocking the generation of MMP-2.

While the serine protease inhibitor aprotinin, which inhibits plasmin, is able to interfere only in the proteolytic action of this enzyme in the form of 64 kDa, blocking the generation of the mature form of MMP-2 (62-kDa), while the intermediate active form ( 64 kDa) is still produced (Figure 2).

On the other hand, the addition of plasminogen in cells

HT1080 results in the activation of pro-metalloprotease-9 (proMMP-9) in its 72 kDa form (Ramos-DeSimone, 1999). EcTI decreases plasmin-induced pro-MMP-9 activation, indicating that EcTI exhibits a direct influence on pro-MMP-9 activation by inhibiting plasmin activity, as was characterized and described earlier in the table

II, Aprotinin used as a control clearly inhibits the activation of pro-MMP-9. The results demonstrate that EcTI regulates the activity of MMP-2 and MMP-9 in the HT1080 strain and its effect is not only due to plasmin inhibitory activity, but also to the blocking of the first phase of MT1-mediated pro-MMP-2 activation -MMP through a process independent of the plasmin action (Figure 2).

Action of BbCI, rBbCI, BbKI, rBbKI and EcTI inhibitors on

47/108 Tm5 and melan-a strains

Action of inhibitors on cell adhesion

The results of the action of inhibitors on cell adhesion of the melan-a and TM5 lines to fibronectin, vitronectin and collagen IV showed that BbCI and its recombinant form (1.00-25.00 μΜ) showed similar activity and did not significantly interfere in the adhesion of melan-a and TM5 on fibronectin, vitronectin and collagen IV (Figure 3 and

4, respectively). BbKI and rBbKI (1.00-25.00 μΜ) also did not significantly inhibit the adhesion of these strains to the glycoproteins used (Figure 5 and 6, respectively).

On the other hand, EcTI inhibited the adhesion of melan-a and Tm5 to collagen IV, vitronectin and fibronectin. The results showed dose-dependent action caused by EcTI, and this inhibitor was more effective in inhibiting the adhesion of

Tm5 on the MEC proteins used (Figure 7).

Action of inhibitors on cell proliferation

Cell proliferation is one of the main factors for tumor development. In this way, the action of

BbCI, rBbCI, BbKI, rBbKI and EcTI (1.00-12.50 μΜ) were analyzed in the cell proliferation of melan-a and Tm5, at different incubation times (2h, 24h, 48h and 72h),

48/108 as previously described.

SQ

The results demonstrate that BbCI and rBbCI when incubated for 24 h-72 h interfere in the proliferation of melan-a (maximum inhibition = 30%). After 24 hours, inhibition was more effective for the transformed strain, Tm5 (maximum activity = 50%) (Figure 8 and 9, respectively).

Analyzing the effect of BbKI and rBbKI on the proliferation of melan-a and TM5, there was an inhibitory effect dependent on the incubation time and concentration, being more effective on TM5, especially after 24 and 48 hours of incubation (Figures 10 and 11, respectively).

EcTI inhibited the proliferation of the two cell lines in a dose and time dependent manner, reaching 8015 900% inhibition when used in the concentrations of

6.25 and 12.50 μΜ, after 48 hours of incubation (Figure 12).

Action of inhibitors on Tm5 metabolism and comparison with 5-FU

The cellular metabolism of Tm5 was analyzed after treatment with the inhibitors rBbCI, rBbKI and EcTI (1.0012.50 μΜ), and compared with the chemotherapeutic 5-FU, widely administered clinically for treatments of different types of tumors.

49/108

RBbCI inhibited dose-dependent being more effective than rBbKI, and both more effective than 5-FU (Figure

13).

rBbKI (12.50 μΜ, 24 hours) inhibited Tm5 metabolism by approximately 30%. And after 48 hours, rBbKI caused a decrease in inhibitory activity (Figure 13).

EcTI inhibited dose-dependent in 24 and 48 hours, yet EcTI proved to be more effective than 5FU in concentrations above 6.25 μΜ (Figure 13).

The chemotherapic 5-FU inhibits cell proliferation regardless of concentration and time.

Action of inhibitors in the phases of the cell cycle

The cell cycle phases of melan-a and Tm5 were analyzed after treatment with the inhibitors rBbCI, rBbKI and

EcTI.

rBbCI caused a significant increase in the number of melanin cells in the DNA fragmentation phase (Figure

14b), compared to cells that did not receive treatment (Figures 14a). And rBbCI does not significantly interfere in the phases of the Tm5 cycle (Figure 15b), compared to Tm5 cells that have not received treatment (Figure 15a).

rBbKI caused a significant increase in the percentage of

50/108 cells in the GO—> G1 phase of both strains studied (Figure 14c and 15c).

Treatment with EcTI caused an increase in the number of cells in Ml phase, corresponding to DNA fragmentation, when compared to the control (Figures 14d and 15d).

Action of inhibitors on cell migration

As shown in Figure 16, Tm5 migrated faster than melan-a on fibronectin, at all incubation times. Between 1 and 2 hours of incubation, the numbers of migrated Tm5 cells doubled, and between 2 and 3 hours there were no significant differences. Meanwhile, for melan-a there was a gradual increase in the number of cells migrated. In this way, the interference of inhibitors on the migration of the melan-a and TM5 strains using fibronectin, as a chemoattract, were evaluated after two hours of incubation.

The migration of melan-a and Tm5, treated with rBbCI was inhibited in a dose-dependent manner. And in concentrations of

6.25 and 12.50 μΜ was twice as effective for Tm5 (Figure

17).

The treatment of rBbKI (1.00 and 2.5 0 μΜ) in Tm5 caused an increase in the number of cells migrated in relation to the cells that did not receive any inhibitor, and only in the

51/108 concentrations of 6.25 and 12.50 μΜ showed inhibition of migration. In addition, the rBbKI effect in melan-a was similar at all concentrations (Figure 18).

EcTI (1.00 μΜ) did not significantly interfere with cell migration (Figure 19). In all assays, BSA was used as a control, to analyze the migration of the Tm5 and melanin strains in a non-specific way.

Tumorigenesis in vivo The effect of inhibitors on tumor growth in vivo was evaluated as previously described. The tumor volume was measured daily with the aid of a caliper, and the results showed that BbCI and rBbCI delayed tumor growth (Figure 20). EcTI delayed tumor growth in a dose-dependent manner (Figure 22). On the other hand, BbKI and rBbKI (Figure 21) stimulated the development of the tumor.

Analysis of proteins from tumors and linked to Sepharose inhibitor resin

Sepharose-inhibitor affinity chromatography and reverse phase chromatography

The tumor homogenate was subjected to rBbCI-Sepharose or rBbKISepharose or EcTI-Sepharose affinity chromatography, as described

52/108 previously. The materials retained on the support and eluted with 0.5 M KC1 / HCl solution, pH 2.0 were subjected to reverse phase chromatographic analysis (Figures 23-25).

Determination of the amino-terminal region

The sequences of the amino-terminal regions of the materials obtained with reverse phase chromatography were determined by the automated process of the degradation reaction of Edman (1956).

The sequence of the amino-terminal region of the material obtained from the affinity rBbCI-Sepharose and eluted at 38 minutes demonstrated similarity with proteins isolated from mice regulating G protein signaling. And the material eluted at 40 minutes demonstrated similarity with proteins associated with metastases (Figure 26 and 27, respectively).

The sequence of the material obtained from the rBbKI-Sepharose affinity chromatography, and subsequently eluted at 35 minutes, demonstrated similarity with P-selectin and with the factor 3 α-subunit induced by hypoxia (Figure 28).

The amino-terminal region of the eluted material at 37 minutes showed similarity with a receptor containing an immunoglobulin-like domain (Figure 29).

The amino-termini region of the eluted material with 31 minutes, referring to the fraction bound in the chromatography of

53/108 EcTI-Sepharose affinity, demonstrated similarity with inositol 1,4,5-triphosphate kinase (Figure 30). And the material eluted at 35 minutes showed similarity with natural killer cell-type lectin receptors and with protein associated with neoplastic progression (Figure 31). Metabolism studies in human tumorigenic cells treated with rBbCI, rBbKI and EcTI

The effects of the rBbCI, rBbKI and EcTI inhibitors on cell metabolism were compared with the chemotherapic

5-FU. The cell lines derived from different types of tumor were: MKN28, Hs746T, HT-29, HCT 116, MCF-7, SKBR3, K562 and THP-1. The results demonstrated that the effect of

5-FU does not change with increasing concentration.

The effect of rBbCI (24, 48 hours) in the MKN28 line (gastric cancer - less invasive line) inhibited metabolism in a dose and time dependent manner, being more effective than 5-FU, in concentrations greater than 6.25 μ. The rBbKI inhibitor in 24 hours inhibited dosedependently. After 48 hours and at a concentration of 12.50 μΜ it showed an effect 20% higher than the inhibition caused by 5FU. EcTI (6.25 μΜ and 12.5 μΜ) was approximately 50% more effective than 5-FU in the two incubation times (Figure

32).

54/108

In the line Hs746T (gastric cancer - most invasive line) rBbCI and rBbKI, in concentrations higher than 6.25 μΜ, presented an effect similar to that obtained in the line of gastric cancer - less invasive line. However, they were more effective at 24 hours of incubation and at 12.5 μΜ,

EcTI (6.25-12.50 μΜ, 24 hours) inhibited above 60%, while 5 - FU inhibited 15%. After 48 hours EcTI caused metabolism inhibition above 80%, while 5-FU inhibited only 25% (Figure 33).

In the HT-29 strain (colon cancer - less invasive strain) we observed that the inhibitors rBbCI and rBbKI (6.25 and 12.50 μΜ, 24 hours) were more effective than 5-FU. After 48 hours at 12.50 μΜ, they were approximately 20% more effective than chemotherapy. EcTI (6.25 and 12.50 μΜ) was more effective than 5-FU with dose-dependent action (Figure

34).

In contrast, in the HCT 116 less invasive colon cancer strain, the rBbCI and rBbKI inhibitors (1.00-6.25 μΜ, 24 and 48 hours) are less effective than

5-FU. However, EcTI (6.25 and 12.50 μΜ, 24 hours) has a greater effect than 5-FU (Figure 35).

In the MCF-7 (less invasive line) and SKBR-3 (more invasive line) breast cancer lines, the inhibitors

55/108 rBbCI and rBbKI (6.25 and 12.50 μΜ, 24 hours) were more effective than 5-FU and, after 48 hours with 12.50 μΜ, it was observed that both were more effective than 5-FU. EcTI (24 hours) was more effective than 5-FU in all concentrations used in breast cancer strains. Still, after 48 hours EcTI was approximately twice as effective as

5-FU when used with 12.50 μΜ (Figures 36 and 37).

In the K562 strain, rBbCI (12.50 μΜ, 24 hours) inhibited approximately 50% while 5-FU only 30%. On the other hand, rBbKI was not effective. The result demonstrated that EcTI (2.5-12.50 μΜ, 24 hours) is more effective than 5-FU (Figure

38).

The rBbCI (6.25 and 12.50 μΜ, 24 hours) caused inhibition of THP-1 metabolism and was more effective than 5-FU approximately 2 times. On the other hand, rBbKI was not effective for this strain. EcTI was more effective than 5-FU in all conditions analyzed at concentrations of 6.25 and 12.50 μΜ, (Figure 39).

Cell metabolism studies using combination therapy: EcTI with 5-FU and EcTI with carboplatin

Combination therapy is used to increase the activity of anti-neoplastic agents, as well as to minimize its toxicological effects. In this way, we analyze the

56/108 effect of the combined therapy of EcTI with chemotherapeutic agents that act on DNA.

The metabolism of gastric cancer strains MKN28 and

HS746T previously used in individual therapy, was analyzed with EcTI (1.00-6.25 μΜ) combined with 5-FU or with carboplatin, both in the same concentrations of EcTI.

Combination therapy (5-FU and EcTI) was more effective for inhibiting MKN28 metabolism, as there was a significant decrease in the concentration necessary to cause the same effect in relation to individual therapy. This effect is best seen in 48 hours at 1.00 μΜ (Figure

45).

At strain Hs746T (cancer gastric - lineage more invasive ) the positive effect of therapy combine was 15 observed after 48 hours and at concentration of 2.50 μΜ, doubling the effect caused by individual therapy (Figure

41).

Combined carboplatin therapy with EcTI showed efficacy over individual therapy in MKN28, when low concentrations (1.00 μΜ and 2.50 μΜ) were used in 24 hours (Figure 42). A similar effect was obtained with the invasive strain, Hs746T, of gastric cancer (Figure 43).

EFFECT OF BbCI ON TUMOR MODELS

57/108 (3

In the first stage, in models of tumor dissemination, we used the inhibitors in the melanocyte lines of mice, called melan-a, and melanoma Tm5, derived from the melan-a line through the forced impediment of cell-substrate adhesion (Corrêa et al ., 2005).

Studies carried out on melanoma have shown a direct correlation between changes in cell adhesion molecules, such as inaνβ ₃ integrin and tumor progression. Furthermore, the presence of this integrin, which was not detected in normal melanocytes, was related to the lower survival of patients with tumor (Edward, 1995; Natali et al., 1997; Ebrahimnejad et al., 2004). In this way, we use the inhibitors to verify their action in the adhesion process of the melan-a and Tm5 strains.

BbCI and rBbCI inhibitors with specificity for HNE, did not significantly interfere in the adhesion of the melanocyte strains of mice (melan-a) and melanoma (Tm5) to collagen IV, fibronectin and vitronectin (Figures 3 and 4, respectively).

Additionally, Champagne et al. (1998) demonstrated that HNE cleaves the intercellular binding of ICAM1, promoting the migration of neutrophils to the adhesion sites. The action of BbCI and rBbCI on elastase may have

58/108 interfered with the hydrolytic action of this enzyme on ICAM-1 interfering with the migration process.

In addition to the inhibitory activity BbCI and rBbCI could be linked to the family of adhesion molecules expressed in the melan-a and Tm5 lines, not contributing to Tm5 mobility. However, no structural similarity was observed with adhesion molecules that mediate this process, among proteins extracted from tumors retained in the affinity resin (rBbCI-Sepharose) (Figures 26 and 27).

The results of the proliferation of the strains analyzed demonstrated that both BbCI and rBbCI caused a more effective inhibition after 24 hours in the proliferation of the melanoma line (Tm5) than in the non-tumorigenic line of melanocytes (melan-a). The presence of a protease can induce an error in the DNA repair system through the hydrolysis of repressor proteins, resulting in cell transformation (Borek et al., 1977). Studies with UVB-induced mice deficient in elastase demonstrate that these animals are more resistant to the development of skin tumors (Starcher et al., 1996).

Therefore, the inhibition of Tm5 proliferation caused by

BbCI, may be related to its activity of inactivating HNE and cathepsin G, proteases related to tumor progression.

59/108

As with proliferation, rBbCI inhibits Tm5 cell metabolism, a fact observed through the activity of mitochondrial enzymes called dehydrogenases (Figure 13). Although the two assays use mitochondrial enzyme markers, in the proliferation assay the inhibitors were added simultaneously to the cells on the plate, while in the metabolism assay the cells were adhered to the plate and after 24 hours the added inhibitors. The results obtained 10 demonstrate that the inhibitor did not interfere in the adhesion of the cells to the plastic.

The rBbCI causes an increase in the number of melan-a cells in the DNA fragmentation phase, a fact that was not observed in Tm5 cells under the conditions analyzed. Sun and Yang (2004) suggest that the imbalance between elastase and allantitrippsin may promote carcinogenesis. The high concentration of HNE can result in the inhibition of apoptosis of tumor cells through its contribution to the degradation of ECM. Despite the low interference observed in melan-a, the results obtained on Tm5 can be explained due to the insufficient amount of inhibitor (6.25 μΜ), used to cause the same effect caused in melan-a. Since, in tumor cells occurs

60/108

the protease-inhibitor imbalance and since Tm5 is a melanoma strain, it may need a higher concentration of the inhibitor to produce the effect caused in the non-tumorigenic melanocyte strain.

On the other hand, Porteu et al. (1991) demonstrated that elastase is capable of removing a fragment of TNF receptor 2 (tumor necrosis factor) from the cell surface, reducing its affinity for the receptor, therefore, elastase may be interfering with the TNF response, preventing it from performing its function. Still, van Kessel et al. (1991) showed that cathepsin G inhibits TNF-ot activity. In addition, proteases secreted by neutrophils that are recruited in the inflammatory process, participate in the development of the tumor (Iwatsuki et al.,

2000). BbCI inhibiting cathepsin c elastase activity

G, can prevent these proteases from interfering with the apoptotic process through TNF, which is directly involved with the apoptosis signaling pathway.

The migration of the Tm5 strain of mouse melanoma (derived from mouse melan-a melanocytes) proved to be much faster on fibronectin, compared to the untransformed melan-a strain. Inhibition of Tm5 migration caused by rBbCI was twice as effective when compared to inhibition of melan-a migration. At the

61/108

The membrane-bound elastase cell migration process is preferably located in the direction of migration and can facilitate the leukocyte transendothelial passage by facilitating tumor development (Cepinskas et al.,

1999).

In addition to elastase, studies carried out with the

B16 (murine melanoma) in an in vitro invasion assay, showed that synthetic inhibitors of cathepsin B and L caused a decrease in invasion (Sever et al., 2002). The inhibitory activity of BbCI on cathepsins L, G and elastase, indicates that this may be the pathway to inhibit Tm5 migration.

In contrast to the results obtained with BbKI and rBbKI, studies with a tumorigenesis model in mice treated with BbCI and its recombinant form demonstrated a delay in tumor development when compared to the group that received PBS (Figure 20). Yamashita and collaborators (1996, 1997) demonstrated that the concentration of elastase is related to the progression of the tumor, with high concentrations of elastase being present in patients with larger tumors. These data probably suggest that localized HNE production is related to tumor invasion.

Overexpression of pro-cathepsin L in

62/108

melanoma increased its invasion potential and transformed the less invasive lineage phenotype into more invasive lineage {Frade et al. , 1998). Therefore, BbCI may be delaying tumor progression by inactivating proteases involved in the acquisition of an invasive phenotype.

Still, a possible role of BbCI in the development of the tumor, is through the indirect inactivation of other proteases, since elastase can activate other proteases, as components of the urokinase type plasminogen activator (uPA) system (Schmitt et al., 1991) , MMP-2 through MT1-MMP and metalloprotease-3 (MMP-3) (Okada et al., 1989; Shamamian et al., 2001), as well as cathepsin L can activate the plasminogen pro-activator single chain urokinase (pro-uPA) (Goretzki et al.,

1992).

In tumors, chemoattractive cytokines are responsible for the recruitment of leukocytes, which respond to the organization and suppression of cellular response to tissue reorganization. With the accumulation of mutations in the tumorigenic process, this organization's imbalance occurs, inducing aberrant cell proliferation, adhesion and migration, which can promote malignant behavior (Di Cario, 2001; Cerwenka and Lanier, 2001; Coussens and Werb,

63/108

2002; Lin and Pollard, 2004).

Data obtained with work developed on a model of pleurisy in rats induced by carrageenan, show that there is a 50% reduction in CINC-1 (IL-8 in humans) in the serum and exudate of animals treated with BbCI, when compared to the control group (de Oliveira, 2004).

Varney et al. (2003) demonstrated that the melanoma strain, human melanoma (A375SM), highly metastatic, expresses high levels of mRNA and the 10 proteins, CXCR1 and CXCR2 (chemokine receptors, such as

IL-8) when compared to the non-metastatic human melanoma strain SBC-2. In addition, treatment with recombinant IL-8 increased the potential for proliferation and invasion of these cells. Specific antibodies to these 15 receptors inhibited the potential for proliferation and invasion of human A375P melanoma cells, stimulated or not with IL-8. In addition, studies show that IL-8 can act as an autocrine growth factor for tumor cells (Schadendorf et al., 1993). In this way, BbCI can decrease the invasive potential and the proliferation of Tm5 by decreasing IL-8.

Another explanation of the observed BbCI effects may be related to its action on transmembrane PARs receptors, activated directly by divage

64/108

Vo of the amino-terminal region by serinoproteases (Loew et al., C

2000; Sambrano et al. , 2000; Cumashi et al. , 2001;

Macfarlane et al., 2001). Four members of this family are known (PAR-1 to PAR-4), who participate in platelet aggregation, inflammatory cell stimulation, cell proliferation and mobility (reviewed by Dery et al., 1998).

The protein eluted with 38 minutes of reverse phase chromatography showed similarity with proteins isolated from mice regulating G protein signaling.

(Figure 27) (Sierra et al., 2002). Thus, in addition to the inhibitory effect on proteases, rBbCI may be interfering with signaling through interaction with this protein.

Still, the amino-terminal region of the material eluted with minutes, shows similarity with proteins associated with metastasis, called (Mta), the family of genes of these proteins are called (MTA) (Kumar et al., 2003). The expression of these genes is being associated with the invasive potential in human cell lines and in tissues with tumor (Toh et al., 1997). Consequently, the inhibitor, when interacting with this protein, may be inhibiting the potential for invasion in vivo.

Effect of BbKI in tumor models

65/108

Components of the kallikrein system (plasmatic and / or / tissue) / kinins - KKS can be directly involved with tumors by increasing the expression and deregulation of proteolytic activity detected in a tumor in the pancreas (Yousef et al., 2004), prostate (Rittenhouse et al., 1998), breast (Luo et al., 2002), ovary (Obiezu et al.,

2001), among others, or indirectly by the generation of kinins and / or activation of other proteases (reviewed by

Bhoola, 1992; Shariat-Madar et al, 2002).

However, the treatment of cells with BbKI and rBbKI did not alter the process of melan-a and Tm5 adhesion to glycoproteins: collagen IV, fibronectin and vitronectin (Figures 4 and 5).

BbKI and its recombinant form were very effective in inhibiting the proliferation of the Tm5 cell line in relation to the melanocyte line, which can be explained by the effect of BbKI on plasma and tissue kallikreins. Results not shown showed a higher concentration of bradykinin in the Tm5 culture medium in relation to melan-a, indicating an important role of cinin-releasing enzymes in this strain. Overexpression of kallikreins in tumorigenic lines is already reported by different research groups (Denmeade et al., 2001; Yousef et al., 2004). Tissue kallikrein, hK3

66/108 (PSA), for example, is one of several human kallikreins used as a tumor marker for the diagnosis, prognosis and monitoring of prostate cancer (Rittenhouse et al., 1998; Diamandis and Yousef, 2002; Gray,

2005; Ryan and Small, 2005).

The results of the proliferation inhibitors were confirmed using the cell metabolism model (Figure 13) and, although there was interference in the cell metabolism, the effect presented was not better than that obtained with the 5-FU chemotherapy. The differences presented by rBbKI when used in low concentrations (Figure 13b, 48 hours) can be explained by the methodologies used in the two experiments. The previous addition of the cells to the plate modified the inhibition efficacy, which can be explained by the interference of the inhibitors in the adhesion of the cells to the plastic. Thus, a higher concentration would be necessary to interfere with the metabolism process, indicating that the inhibitor's functionality is better when it is previously incubated with the cells before they are added to the plate.

Analysis of the effect on the cell cycle shows that rBbKI causes an increase in the number of melan-a and Tm5 cells in the phases

G0—> G1 (Figures 14c and 15c, respectively), in relation to the

67/108 control in the absence of the inhibitor under the conditions used.

The GO phase is the period in which the cell maintains its metabolic rate, but does not grow in size, unless it receives extracellular signals and the G1 phase corresponds to the phase of metabolically active cells with continuous growth (Cooper, 1996).

The results of rBbKI in cell migration were surprising, since due to its antiproteolytic activity, a decrease in the invasive process was expected. Since, as mentioned, proteases have a prominent role, an active process of migration of neoplastic cells through the physical barrier formed by

MEC. Secreted proteases facilitate the degradation of this barrier (Liotta and Clair, 2000; Nguyen, 2004). However, in low concentrations, we observed a significant increase in the migration of melanoma cells, a fact not observed in non-transformed cells. On the other hand, migration inhibition occurs when it is used in high concentrations, probably due to its effect on proteases. Wolf et al. (2001) demonstrated the inhibition of adenocarcinoma invasion on matrigel (mimics ECM in vitro) by synthetic inhibitors of tissue kallikreins, indicating the participation of kallikreins in the invasion of the

tumor.

68/108

Tissue kallikreins participate in the invasion through the activation of metalloproteases such as MMP-2 and metalloprotease-9 (MMP-9) (Tschesche et al., 1989;

Desrivieres et al. , 1993). We emphasize that, in 1997,

Frenette and collaborators demonstrated that hK2 kallikrein activates uPA (urokinase-type plasminogen activator), triggering plasmin activation. Therefore, as rBbKI inhibits the activity of kallikreins and plasmin, it consequently inactivates the participation of these enzymes in this process.

In contrast, the increased migration of Tm5 by the action of rBbKI in lower concentrations may have occurred due to an insufficient amount of inhibitor to inactivate the kallikreins expressed in this strain, resulting in a proteolytic imbalance or the possible activation of other proteases involved with cell migration induced by kallikreins.

In an in vivo experimental model, BbKI caused a significant increase in tumor volume in the BbKI group, with some animals dying before the control group (data not shown). Shin et al. (1996) demonstrated the SbTI (soybean trypsin inhibitor) that has a low half-life due to the action of proteases on its degradation. However, only the processing of the inhibitor

69/108 does not allow us to explain the effect of tumor growth.

As mentioned earlier, the reactive BbKI site is similar to bradykinin (Cagliari et al.,

2003). Works developed by the present inventors (Santomauro-Vaz, 2005; Andrade, personal communication) demonstrated that synthetic peptides with the reactive site sequence act in a similar way to bradykinin (BK) in models of muscle contraction, release of calcium and

AT THE.

BK is involved in tumorigenesis processes associated with angiogenesis (Bhoola et al., 2001). The growth of the tumor is dependent on angiogenesis, and in its absence the growth of the tumor is restricted to a microscopic size and tumor cells do not leave the circulation (Folkman, 2003). BK has angiogenic activity by binding to B _x and B ₂ receptors in endothelial cells. The use of antagonists of these receptors has shown that blocking this link decreases vascular permeability, nitric oxide activation, cancer growth and progression (Hu and Fan, 1993; Maeda et al., 1999; Hayashi et al., 2001).

Results obtained by Santomauro-Vaz (2005), in an ischemia and reperfusion model in rats treated with BbKI, clearly demonstrate an increase in ONOO, indicating that the

70/108 protein also binds to the receptor. Free radicals such as NO can be induced by BK binding to the receptor, by cytokines and growth factors (Knowles and Moncada,

1994; Witte and Barbul, 2002). NO reacts quickly with superoxides forming 0N00 ‘(reviewed by Bongdan, 2001).

Okamoto et al. (1997) demonstrated that ONOO activates pro-MMPs, involved in the degradation of MEC (Maeda et al., 1999). Thus, BbKI may be showing pro-angiogenic activity due to its binding

BK and stimulate NO production.

The B ₂ receptor has been detected in different tissues and in tumor lines of mice (Wu et al., 2002), and there is no migration of neutrophils to inflamed tissue sites in receptor-deficient mice

Βχ, which results in the inhibition of the development of tumors after coinoculation of apoptotic cells and Tm5 cells (Corrêa et al., 2005). The results obtained with BbKI open perspectives to investigate the action of BbKI and the effects of peptides derived from BbKI, with a similar sequence to BK on Bi and B ₂ receptors, in the models of melanoma progression in vivo.

Still, the works developed by de Oliveira (2004), showed that the serum of animals treated with BbKI show increased expression of IL-ΐβ. Vidal-Vanaclocha

71/108 and collaborators (2000), demonstrated that IL-ΐβ deficient mice are more resistant to the development of metastases. In addition, IL-ΐβ increases the binding of lymphocytes and monocytes to endothelial cells, induces neovascularization and stimulates TNF-α production (reviewed by Adam et al., 2003). The participation of TNF-α can occur by inducing the production of

proteases important for cell invasion tumor and macrophages, such as HNE, and can cause a increase in expression of adhesion molecules in cells endothelial (Jonj ic et al. , 1992; Balkwill and Mantovani, 2001). Therefore, BbKI may be causing an increase IL-ΐβ and TNF-α in model used according to the present

invention, inducing tumor progression.

In the model studied by de Oliveira (2004) it was also found that BbKI stimulates the production of CINC-1 in rats (homologated to IL-8 in humans). Studies demonstrate that IL-8 promotes the growth of melanomas (Haghnegahdar et al.,

2000), is a potent angiogenic factor regulated by TNF-α and secreted by macrophages or monocytes infiltrated in the stroma (Torisu et al., 2000). Thus, BbKI can increase tumor growth by activating this cytokine.

eluted material with 35 minutes that presented

72/108 affinity to rBbKI-Sepharose demonstrated similarity to platelet selectin (P-selectin). P-selectin is a Ca ²⁺ dependent cell adhesion molecule that mediates the interaction of neutrophils with platelets as well as with endothelial cells (Buck, 1995). Mayer et al. (1998) demonstrated that vessel samples from patients with gastric cancer express high levels of P-selectins in the presence of leukocyte infiltrate. Still, some studies demonstrate the high expression of P-selectins in the plasma of cancer patients (Blann et al., 2001).

However, Caine et al. (2005), when administering anti-angiogenic drugs such as marismatati and captopril, demonstrated that these drugs did not modify the levels of P-selectin in the blood. Additionally, it has been shown that in the absence of P-selectin, there are other mechanisms involved in the leukocyte rolling during the inflammatory process (Ridger et al., 2005). Thus, rBbKI may be recognizing P-selectins, blocking the neutrophil bearing and, consequently, interfering with the inflammatory process.

This same sequence of the amino-terminal region is similar to the α subunit of factor 3 induced by hypoxia (Figure 28), which can contribute to the angiogenic process (reviewed by Browder et al., 2000;

73/108

Raghunand et al. , 2003). In this way, BbKI when recognizing this factor can amplify the local action of proangiogenic factors.

Still, we can emphasize that the material eluted at 37 minutes showed similarity with the receptor containing an immunoglobulin-like domain, a family of cell adhesion molecules that have several functions (reviewed by Buck,

1995). BbKI could be inhibiting the interaction of any member of this family with its ligand, resulting in dysfunction of anti-tumor processes.

Effect of EcTI on tumor models

The cellular adhesion of melan-a and Tm5 to collagen IV, fibronectin and vitronectin was inhibited by EcTI in a dose and time dependent manner. A possible action of EcTI would be to recognize cell membrane receptors _; and thereby prevent adherence. Integrins recognize several structural proteins, but also act as protease receptors, such as metalloproteases (MMPs), the activity of

MMP-2 has been shown to be dependent on interaction with Ovp3 (Hofmann et al., 2000). The interference in molecules involved in cell adhesion may be responsible for inhibiting the activation of MMP-2 by EcTI, since in melanoma cells, MMP-2 expression can be regulated

74/108 through the interaction of Ονββ with vitronectin (Albelda et al., 1990) and through the homotypic interaction mediated by the MCAM immunoglobulin (melanoma cell adhesion molecule) MMP-2 (Xie et al., 1997).

The action of EcTI in decreasing cell growth was confirmed by assays of cell proliferation and cell metabolism, showing to be more effective than chemotherapy 5-FU in high concentrations. The balance between cell proliferation and cell death controls tumor growth, and increased proliferation can be a determinant for malignancy (Reed, 1999). EcTI interferes in this process causing a decrease in cell proliferation.

The results showed that EcTI induces a high percentage increase in the DNA fragmentation of melan-a and Tm5.

Several studies demonstrate that MEC anchoring is crucial for the progression of the cell cycle between the G1- »S phases, the phase of metabolically active cells in continuous growth, and the DNA synthesis phase, respectively

0 (Cooper, 1996, - Lukashev and Web, 19 98). A variety of events can direct cell death, including cell adhesion (Vaux and Korsmeyer, 1999). Integrins play an important role in the anoikis process, that is, cell death

75/108 caused by the loss of anchorage (Parise et al.,

2000). Hoyt et al. (1996), when using cytotoxic agents, demonstrated that cell adhesion to ECM and activation of integrins suppress cell death induced by cytotoxicity. In addition, long-term deactivation results in the activation of caspases 8 and 9 and consequently activates effector caspase 3, inducing apoptosis (Bonfoco et al., 2000). In this way, EcTI can induce apoptosis by the anoikis process, preventing cell adhesion and consequently causing cell death, since it causes a decrease in the number of cells in the Gl—> S phases and an increase in the fragmentation phase.

In addition, the induction of cell death caused by

EcTI may also be due to its effect on plasmin and metalloproteases. since, recent studies have shown that cells resistant to cell death regulated by p53, a transcription factor that regulates the cell cycle, DNA repair and PKC, express high levels of

MMP-9, MMP-10 and metalloprotease-12 (MMP-12) (Meyer et al.,

2005). The importance of metalloproteases in melanoma is confirmed by the results of Toschi and collaborators (2000) overexpressed the wild gene of p53 in melanoma cells, showed a decrease in invasion. The authors relate this effect to the levels of MMP-2 secreted by

76/108 cells.

In the in vivo EcTI experiments caused a decrease in tumor volume, being more effective than BbCI, in addition, survival was superior to animals that received PBS (data 5 not shown). This decrease probably occurred in response to the inhibition of Tm5 proliferation.

In melanoma cell lines, the production of MMP2 and MMP-9 has been correlated with invasive potential (Vaisanen et al., 1996). In addition, type-2 metalloprotease inhibitor (TIMP-2) and synthetic inhibitors reduced melanoma growth and metastasis (Montgomery et al., 1994). Consequently, the decrease in tumor volume caused by EcTI may be due to its anti-proteolytic activity,

Gelatinase 2 can affect tumor growth by cleaving ICAM-1 by MMP-9, increasing the resistance of the tumor cell to cytotoxic agents (Fiore et al., 2002). Therefore, EcTI, by inhibiting the activation of proMMP-9, can prevent the hydrolysis of ICAM-1, decreasing the resistance of tumor cells.

Another explanation is based on the action of EcTI on cell death. Many tumors resist apoptosis by increasing the rate of anti-apoptotic effectors in relation to the pro-apoptotic ones (Evan and Vousden, 2001). Plasmin can activate

77/108

NF-κΒ in monocytes (Syrovets et al., 2001), NF-κΒ is a pro-apoptotic effector, that is, it induces the expression of apoptosis-inhibiting proteins, which bind and inhibit certain caspases, known as proteins that promote cell survival (Ghosh and Karin, 2002). Through inactivation of plasmin, EcTI can indirectly inhibit the expression of NF-κΒ and caspases, consequently promoting apoptosis.

Cell proliferation is maintained in an environment rich in inflammatory cells, growth factors, active stroma, agents that promote DNA damage, potentiating and / or promoting neoplastic risks (reviewed by

Coussens and Werb, 2002). Plasmin and gelatinases can activate and / or degrade inflammatory mediators that stimulate tumor development (reviewed by Syrovets and Simmet, 2004; Bjõrkund and Koivunen, 2005). Thus, considering that EcTI inhibits these proteases, a role on these mediators is possible.

The results obtained with affinity chromatography

EcTI-Sepharose and database analysis showed that the material eluted at 31 minutes, has similarity with inositol 1,4,5-triphosphate kinase (IP ₃ ), is released in the cytosol and signals the release of Ca ²⁺ essential for the

78/108

function of several physiological functions (Cooper, 1996). Therefore, EcTI may be involved in the release of

Ca ²⁺ .

And the material eluted in 35 minutes showed similarity with natural killer cell-type lectin receptors (Smyth et al., 2 002) and with proteins involved in neoplastic progression 1 (Sun et al., 1994) (Figure 31). Therefore, EcTI may be inducing a decrease in tumor growth through the recognition of proteins that contribute to tumor growth.

Effect of rBbCI on the metabolism of human tumor lines

The activity shown by rBbCI in the analyzed strains demonstrates efficient inhibition in relation to the chemotherapeutic 5-FU (Figures 32-39). In gastric cancer strains, acidic pH and the presence of pepsin in the stomach can activate precursors of cysteine proteases to the active form (Pagano et al., 1989). Several studies relate the presence of cathepsin L, in addition to the potential for invasion, as well as cystatine regulation (Saleh et al., 2003; Kielan et al., 2004).

In colon cancer, Herszènyi et al. (1999) observed in tissues of cancer patients that cathepsin L was one of the relevant short-term factors

79/108 survival and that high levels of this enzyme were correlated with high risk of death. Zajc et al. (2002) compared the expression of cathepsin L in breast cancer strains with different invasive potentials, MCF-7 was the least invasive strain with the lowest concentration of active cathepsin L and the highest concentration of inhibitor. Thus, we can emphasize the role of rBbCI on the inhibitory activity of cathepsin L in these types of tumors.

Additionally, cathepsin G and elastase induced the formation of multicellular spheroids in the breast cancer lineage (MCF-7), which may be contributing to the dissemination of cell aggregates (Yui et al., 2005), since in vivo experiments demonstrated that the intravenous injection of these aggregates results in the formation of tumors more efficiently than dispersed cells (Albini et al., 1987). These results demonstrate another possible rBbCI mechanism.

On the other hand, as oti-anti-trypsin was able to inhibit the release of growth factor-ot in MCF-7, while the soy trypsin inhibitor (BBI) did not affect this activity (Yavelow et al., 1997), rBbCI can cause inhibition of growth factors, since it inhibits

80/108

elastase and does not interfere with trypsin and chymotrypsin activity.

effect on the decrease in the metabolism of the rBbCI inhibitor in the THP-1 and K562 strains may have occurred as a result of blocking proteases secreted by neutrophils, such as HNE and cathepsin G.

Effect of rBbKI on the metabolism of human tumor strains

In the Hs746T invasive strain, rBbKI at higher concentrations after 24-hour incubation was more effective in inhibiting cell metabolism when compared to the non-metastatic strain MKN-28. Nagahara et al. (2005), compared the expression of the kallikrein gene, KLK6, and observed that its expression is proportional to the poor diagnosis of gastric cancer patients and, by silencing the gene in the MKN-28 lineage, there was a reduction in cell proliferation .

In contrast, kallikreins can negatively regulate tumor growth, as hK3 was able 20 to inhibit the growth of the MCF-7 breast cancer lineage through hormone regulation (Lai et al., 1996), however the same kallikrein promoted angiogenesis (Derynck et al., 2001). According to the present invention, results in breast cancer strains show the

81/108

rBbKI's ability to inhibit metabolism, through other mechanisms and / or over different proteases.

The high expression of trypsin and matrilisin (metalloprotease-7 [MMP-7]) activated by trypsin has been identified in colon cancer. These factors are related to the short life span of patients with this type of tumor (Yamamoto et al., 2003). Thus, rBbKI can be used as a study tool in colon cancer strains due to trypsin inhibition.

Therefore, the action of rBbKI on different tumor strains, demonstrates a better efficacy for inhibiting invasive strains. The effect is probably due to the inhibitory activity on proteases, according to studies that demonstrate overexpression of proteases in invasive strains. In addition, the inhibitor had a dose and time dependent effect, this action differentiates it from chemotherapy

5FU.

Effect of EcTI on the metabolism of human tumor strains

EcTI showed an important inhibitory action on cell metabolism (Figures 32-39). Several studies correlate the invasive potential with the presence of different proteases in gastric tumors. Park et al. (1997) demonstrated that Hs746T expresses uPA,

82/108 u-PAR and MMP-2 and their invasive potential was reduced with the presence of anti-uPA. High levels of MMP-2 and MMP-9 are associated with low survival in patients with gastric cancer (Sier et al., 1996; Bjõrrklund and Koivunen, 2005).

Ahmed et al. (2003) demonstrated that the decrease in Erk / MAP kinase activity in HCT 116 (colon cancer - more invasive lineage) with antibody to uPAR pro-MMP-9 activation was abolished, consequently a decrease in adherence , and when disrupting the uPAR / βι complex, the activation of pro-MMP-9 and pro-MMP-2 was blocked. Therefore, EcTI can inhibit the pathway of

Erk / MAP kinase, inhibiting the activation of pro-MMP-9, can still interact with the βι integrin chain by interfering with the activation of pro-gelatinases and or by inactivating plasmin.

In colon cancer, increased concentration of the components of the plasminogen / plasmin system and gelatinases are associated with a worse prognosis and invasive potential (Zeng et al., 1996; Herszènyi et al.,

2000; Yang et al., 2000; Berger, 2002).

The action of EcTI on the metabolism of breast cancer strains was similar to that obtained with colon cancer. The SKBR-3 metastatic strain expresses MMP-9,

83/108 while the non-metastatic MCF-7 strain does not express gelatinases. Different groups demonstrated the importance of the expression of uPA gelatinases and type-1 plasminogen activator inhibitor (PAI-1) in breast cancer, confirming the action on invasion and metastasis (Baramova et al., 1997;

Janicke et al. , 2001; Bjòrklund and Koivunen, 2005). In such a way, that the efficacy of EcTI on the inhibition of invasive colon and breast cancer lines, may have occurred through its action on plasmin and gelatinases.

In leukemic cells, EcTI was more effective than 5-FU especially with 24 hours of incubation. Researchers suggest that gelatinases are directly involved in leukemia, relating their role in angiogenesis, one of the diagnoses of leukemia (reviewed by Klein et al., 2004).

In 2000, Gazzanelli and collaborators demonstrated that the expression and / or release of metalloproteases can be regulated with the induction of apoptosis, therefore EcTI, by inducing cell death, can inhibit the activation of metalloproteases.

Although the activity of 5-FU in tumors is well recognized, resistance to this chemotherapeutic is frequently observed. Strategies such as co-treatment with other chemical agents or mutations have been developed to increase the activity of 5-FU (Bajetta,

84/108

1997; Klampfer et al. , 2005). Several studies are under development to identify new biomarkers, therapeutic targets and rational drug development to be used together. The effect of the combined therapy of EcTI and 5-FU or carboplatin decreases the dose necessary to inhibit the metabolism and the action in whole was more effective (Figures 40-43).

Although knowledge of the pharmacokinetic analysis of the combined therapy study is necessary, therapy with inhibitors may contribute to lessen the toxic effects caused by 5-FU or carboplatin.

The mechanism by which the 5-FU chemotherapy acts is different from the inhibitors according to the present invention,

BIBLIOGRAPHIC REFERENCES

Adam JK, Odhav B, Bhoola KD. Immune responses in cancer. Pharmacol Ther, 2003; 99: 113-32.

Ahmed N, Oliva K, Wang Y, Quinn M, Rice G.

Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR-betal integrin complex in colon cancer cells. Br J Cancer. 2003

Jul 21; 89 (2): 374-84.

Albelda SM, Mette SA, Elder DE, Stewart R, Damjanovich

L, Herlyn M, Buck CA. Integrin distribution in malignant

85/108 melanoma: association of the beta 3 subunit with tumor progression. Cancer Res. 1990 Oct 15; 50 (20): 6757-64.

Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson

SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for guantitating the invasive potential of tumor cells. Cancer

1987 Jun 15; 47 (12): 3239-45.

Andrade SS. Action of BbKI, human plasma kallikrtein inhibitor isolated from Bauhinia bauhinioides on calcium signaling. Beginning of Doctoral thesis 2 0 04,

Ongoing doctoral thesis, Federal University of São

Paulo.

Araújo AP, Hansen D, Vieira DF, Oliveira C, Santana

LA, Beltramini LM, Sampaio CA, Sampaio MU, Oliva ML.

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies. Biol Chem. 2005 Jun;

386 (6): 561-8.

Bajetta E, Di Bartolomeo M, Somma L, Del Vecchio M, Artale S, Zunino F, Bignami P, Magnani And, Buzzoni R, 20 Doxifluridine in colorectal cancer patients resistant to 5- fluorouracil (5-FU) containing regimens. Eur J Cancer. 1997

Apr; 33 (4): 687-90.

Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet. 2001 Feb 17; 357 (9255): 539-45.

86/108

Baramova ΕΝ, Bajou K, Remacle A, L’Hoir C, Krell HW,

Weidle UH, Noel A, Foidart JM. Involvement of PA / plasmin System in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation. FEBS Lett. 1997 Mar 24;

405 ¢ 2): 157-62.

Batista IFC, Oliva MLV, Araújo MS, Sampaio MU,

Richardson M, Fritz H, Sampaio CA. Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds. Phytochemistry. 1996; 41 (4): 101710 1022.

Berger DH. Plasmin / plasminogen system in colorectal cancer. World J Surg. 2002 Jul; 26 (7): 767-71.

Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol

Rev. 1992 Mar; 44 (1): 1-80.

Bhoola KD, Ramsaroop R, Plendl J, Cassin B, Dlamini Z,

Naicker S. Kallikrein and kinin receptor expression in inflammation and cancer. Biol Chem. 2001 Jan; 382 (1): 7789.

Bjõrkund M, Koivunen E. Gelatinase-mediated migration and invasion of cancer cells. Bioch Biophys Acta. 2005;

1755: 37-69.

Blann AD, Gurney D, Wadley M, Bareford D, Stonelake P,

Lip GY. Increased soluble P-selectin in patients with

87/108 haematological and breast cancer: a comparison with fibrinogen, plasminogen activator inhibitor and von

Willebrand factor. Blood Coagul Fibrinolysis. 2001 Jan;

(1): 43-50.

Bode W, Huber R. Structural basis of the endoproteaseprotein inhibitor interaction. Biochim Biophys Acta. 2000;

1477 (1-2): 241-52.

Bonfoco E, Chen W, Paul R, Cheresh DA, Cooper NR.

betai integrin antagonism on adherent, differentiated human neuroblastoma cells triggers an apoptotic signaling pathway. Neuroscience. 2000; 101 (4): 1145-52.

Bongdan C. Nitric oxide and the regulation of gene expression. TRENDS Cell Biol. 2001; 11 (2): 66-75.

Borek E, Baliga BS, Gehrke CW, Kuo CW, Belman S, Troll

W, Waalkes TP. High turnover rate of transfer RNA in tumor tissue. Cancer Res. 1977 Sep; 37 (9): 3362-6.

Browder T, Folkman J, Pirie-Shepherd S. The hemostatic system as a regulator of angiogenesis. J Biol Chem. 2000;

275 (3): 1521-1524.

Buck CA. Adhesion mechanisms controlling cell-cell matrix interactions during the metastatic process. In: The

Molecular Basis of Cancer, ed. Mendelson, J., Howley, P.,

Israel, M. and Liotta, W.B. Saundres Co., Philadelphia 1995,

172-205.

88/108

Cagliari Cl, De Caroli FP, Nakahata AM, Araújo MS,

Nakaie CR, Sampaio MU, Sampaio CA, Oliva ML. Action of

Bauhinia bauhinioides synthetic peptides on serine proteinases. Biochem Biophys Res Commun. 2003 Nov 7;

311 ¢ 1): 241-5.

Caine GJ, Harris AL, Christodoulos K, Lip GY, Blann

AD. Analysis of combination anti-angiogenesis therapy on markers of coagulation, platelet activation and angiogenesis in patients with advanced cancer. Cancer Lett.

2005 Mar 10; 219 (2): 163-7.

Cepinskas G, Sandig M, Kvietys PR. PAF-induced elastase-dependent neutrophil transendothelial migration is assoeiated with the mobilization of elastase to the neutrophil surface and localization to the migrating front.

J Cell I know. 1999 Jun; 112 (Pt 12): 1937-45.

Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immunol. 2001 Oct; 1 (1): 41-9.

Chammas R. Pathophysiological aspects of the spread of sarcomas. Acta Oncol Bras. 1998; 18 (1): 25-32.

Champagne B, Tremblay P, Cantin A, St. Pierre Y.

Proteolytic clevage of ICAM-1 by human neutrophil elastase,

J Immunol. 1998; 161: 6398-6405.

Cooper GM. In: The cell, ASM Press, Washington, 1996.

Corrêa M, Machado J Jr, Carneiro CR, Pesquero JB,

89/108

Α'Ί

Bader Μ, Travassos LR, Chammas R, Jasiulionis MG. Transient inflammatory response induced by apoptotic cells is an important mediator of melanoma cell engraftment and growth.

Int J Cancer. 2005 Apr 10; 114 (3): 356-63.

Coussens LM, Werb Z. Inflammation and cancer. Nature.

2002 Dec 19-26; 420 (6917): 860-7.

Cumashi A, Ansuini H, Celli N, De Blasi A, O'Brien PJ,

Brass LF, Molino M. Neutrophil proteases can inactivate human PAR3 and abolish the co-receptor function of PAR3 on murine platelets. Thromb Haemost. 2001 Mar; 85 (3): 533-8.

Curran S, Murray GI. Matrix metalloproteinases: molecular aspects of their roles in tumor invasion and metastasis. Eur J Cancer. 2000; 36: 1621-1630.

Denmeade SR, Sokoll LJ, Chan DW, Khan SR, Isaacs JT.

Concentration of enzymatically active prostate-epecific antigen (PSA) in the extracellular fluid of primary human prostate cancers and human prostate cancer xenograft models. Prostate. 2001 Jun 15; 48 (1): 1-6.

Dery O, Corvera CU, Steinhoff M, Bunnett NW.

Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol. 1998 Jun; 274 (6 Pt 1): C1429-52.

Desrivieres S, Lu H, Peyri N, Soria C, Legrand Y,

Menashi S. Activation of the 92 kDa type IV collagenase by

90/108

Μ tissue kallikrein. J Cell Physiol. 1993 Dec; 157 (3): 58793.

Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 2001

Nov; 29 (3): 351.

Di Cario E, Forni G, Lollini P, Colombo MP, Modesti A,

Musiani P. The intriguing role of polymorphonuclear neutrophils in antitumor reactions. Blood. 2001 Jan 15;

97 (2): 339-45.

Diamandis EP, Yousef GM. Human tissue kallikreins: a family of new cancer biomarkers. Clin Chem. 2002 Aug;

(8): 1198-205.

Ebrahimnejad A, Streichert T, Nollau P, Horst AK,

Wagener C, Bamberger AM, Brummer J. CEACAM1 enhances invasion and migration of melanocytic and melanoma cells.

Am J Pathol. 2004 Nov; 165 (5): 1781-7.

Edman P. Preparation of phenyl thiohydaltoins from some amino acids. Acta Chem Scand. 1956; 10: 761-5.

Edward M. Integrins and other adhesion molecules involved in melanocytic tumor progression. Curr Opin Oncol.

1995 Mar; 7 (2): 185-91.

Erlanger BF, Kokowsky N, Cohen N. Preparation and properties of two new chromogenic substrates of trypsin.

Arch Biochem Biophys. 1961; 95: 271-8.

91/108

Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001 May 17; 411 (6835): 342-8.

Fiore E, Fusco C, Romero P, Stamenkovic I. Matrix metal loproteinase 9 (ΜΜΡ-9 / gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity. Oncogene. 2002

Aug 8; 21 (34): 5213-23.

Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med. 2003 Nov; 3 (7): 643-51.

Frade R, Rodr igue s - Lima F, Huang S, Xie K, Guill aume

N, Bar-Eli M. Procathepsin-L, a proteinase that cleaves human C3 (the third component of complement), confers high tumorigenic and metastatic properties to human melanoma cells. Cancer Res. 1998 Jul 1; 58 (13): 2733-6.

Frenette G, Tremblay RR, Lazure C, Dube JY. Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), single-chain urokinase-type plasminogen activator activates. Int J Cancer. 1997 May 29; 71 (5): 897-9.

Fuchs EJ, Matzinger P. Is cancer dangerous to the immune system? Semin Immunol. 1996 Oct; 8 (5): 271-80.

Gazzanelli G, Luchetti F, Burattini S, Mannello F,

Falcieri E, Papa S. Matrix metalloproteinases expression in HL-6 0 promyelocytic leukemia cells during apoptosis.

Apoptosis. 2000 Apr; 5 (2): 165-72.

92/108

Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell. 2002 Apr; 109 Suppl: S81-96.

Goretzki L, Schmitt M, Mann K, Calvete J, Chucholowski

N, Kramer M, Gunzler WA, Janicke F, Graeff H. Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L. FEBS Lett. 1992 Feb 3; 297 (1-2): 112-8.

Gray MA. Clinicai use of prostate-specific antigen serum: a review. Clin Lab. 2005; 51 (3-4): 127-33.

Hansen D. BbCI, Bauhinia bauhinoid cruzipain inhibitor cDNA isolation and heterologous expression.

0 04. São Paulo, PhD thesis. Federal University of

Sao Paulo.

Haghnegahdar H, Du J, Wang D, Strieter RM, Burdick MD, 15 Nanney LB, Cardwell N, Luan J, Shattuck-Brandt R, Richmond A. The tumorigenic and angiogenic effects of MGSA / GRO proteins in melanoma. J Leukoc Biol. 2000 Jan; 67 (1): 5362.

Hayashi I, Ishihara K, Kumagai Y, Maj ima M.

Proinflammatory characteristics of a nonpeptide bradykinin mimic, FR190997, in vivo. Br J Pharmacol. 2001 Aug; 133 (8):

1296-306.

Herszènyi L, Plebani M, Carraro P, De Paoli M,

Roveroni G, Cardin R, Tulassay Z, Naccarato R, Farinati F.

93/108

The role of cysteine and serine proteases in colorectal carcinoma. Cancer 1999; 86: 1135-42.

Herszènyi L, Plebani M, Carraro P, De Paoli M,

Roveroni G, Cardín R, Foschia F, Tulassay Z, Naccarato R,

Farinati F. Proteases in gastrointestinal neoplastic diseases. Clin Chim Acta. 2000; 291: 171-87.

Hofmann UB, Westphal JR, Waas ET, Becker JC, Ruiter

DJ, van Muijen GN. Coexpression of integrin alpha (v) beta3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation: correlation with melanoma progression. J Invest

Dermatol. 2000 Oct; 115 (4): 625-32.

Hoyt DG, Rusnak JM, Mannix RJ, Modzelewski RA, Johnson

CS, Lazo JS. Integrin activation suppresses etoposideinduced DNA strand breakage in cultured murine tumor15 derived endothelial cells. Cancer Res. 1996 Sep 15; 56 (18):

4146-9.

Hu DE, Fan TP. [Leu8] des-Arg9-bradykinin inhibits the angiogenic effect of bradykinin and interleukin-1 in rats.

Br J Pharmacol. 1993 May; 109 (1): 14-7.

Iwatsuki K, Kumara E, Yoshimine T, Nakagawa H, Sato M,

Hayakawa T. Elastase expression by infiltrating neutrophils in gliomas. Neurol Res. 2000 Jul; 22 (5): 465-8.

Jonjic N, Peri G, Bernasconi S, Sciacca FL, Colotta F,

Pelícci P, Lanfrancone L, Mantovani A. Expression of

94/108 adhesion molecules and chemotactic cytokines in cultured human mesothelial cells. J Exp Med. 1992 Oct 1; 176 (4):

1165-74.

Káhâri V, Saarialho-Kere U. Matrix metalloproteinases and their inhibitors in tumor growth and invasion. Ann Med.

1999; 31: 34-45.

Kielan W, Suzanowicz J, Siewinski M, Saleh Y, Janocha

A, Swalski A, Tarnawa R. Evaluation of changes in the activity of proteolytic enzymes and their inhibitors in the processes that accompany the growth of gastric cancer.

Gastric cancer. 2004; 7: 17-23.

Klampfer L, Swaby LA, Huang J, Sasazuki T, Shirasawa

S, Augenlicht L. Oncogenic Ras increases sensitivity of colon cancer cells to 5-FU-induced apoptosis. Oncogene.

2005 Jun 2; 24 (24): 3932-41.

Klein G, Vellenga E, Fraaije MW, Kamps WA, by Bont ES. The possible role of matrix metalloproteinase (MMP) -2 and

MMP-9 in cancer, e.g. acute leukemia. Crit Rev Oncol

Hematol. 2004 May; 50 (2): 87-100.

Knight CG. The characterization of enzymes inhibition.

In: Barrett, AJ & Salvensen, G. eds. Proteinase inhibitors.

Amsterdam, Elsevier, 1986, p. 23-51.

Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994 Mar 1; 298 (Pt 2): 249-58.

95/108

Koblinski JE, Ahram M, Sloane BF. Unraveling the role of proteases in cancer. Clin Chim Acta. 2000 Feb 15;

291 (2): 113-35.

Kumar R, Wang RA, Bagheri-Yarmand R. Emerging roles of

MTA family members in human cancers. Semin Oncol. 2003 Oct;

30 (5 Suppl 16): 30-7.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;

227 (259): 680-5.

Lai LC, Erbas H, Lennard TW, Peaston RT. Prostatespecific antigen in breast cyst fluid: possible role of prostate-specific antigen in hormone-dependent breast cancer. Int J Cancer. 1996 Jun 11; 66 (6): 743-6.

Lin EY, Pollard JW. Role of infiltrated leucocytes in tumor growth and spread. Br J Cancer. 2004 Jun 1; 90 (11):

2053-8.

Liotta LA, Clair T. Cancer. Checkpoint for invasion.Nature. 2000 May 18; 405 (6784): 287-8.

Loew D, Perrault C, Morales M, Moog S, Ravanat C,

Schuhler S, Arcone R, Pietropaolo C, Cazenave JP, van

Dorsselaer A, Lanza F. Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry. Biochemistry. 2000 Sep 5; 39 (35): 10812-22.

96/108

Lukashev ME, Werb Z. ECM signalling: orchestrating cell behavior and misbehavior. Trends Cell Biol. 1998

Nov; 8 (11): 437-41.

Luo LY, Diamandis EP, Look MP, Soosaipillai AP, 5 Foekens JA. Higher expression of human kallíkrein 10 in breast cancer tissue predicts tamoxifen resistance. Br J

Cancer. 2002 Jun 5; 86 (11): 1790-6.

Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin

R. Proteinase-activated receptors. Pharmacol Rev. 2001 Jun;

53 (2): 245-82.

Maeda H, Wu J, Okamoto T, Maruo K, Akaike T.

Kallikrein-kinin in infection and cancer.

Immunopharmacology. 1999 Sep; 43 (2-3): 115-28.

Mayer B, Spatz H, Funke I, Johnson JP, Schildberg FW.

Again expression of the cell adhesion molecule E-selectin on gastric cancer endothelium. Langenbecks Arch Surg. 1998

Sea; 383 (1): 81-6.

Meyer E, Vollmer JY, Bovey R, Stamenkovic I. Matrix metalloproteinases 9 and 10 inhibit protein kinase C20 potentiated, p53-mediated apoptosis. Cancer Res. 2005 May

15; 65 (10): 4261-72.

Morrison JF. The slow-binding and slow-tight-binding inhibition of enzyme catalysed reactions. TIBS 1989; 3:

102-5.

97/108

Montgomery AM, Mueller BM, Reisfeld RA, Taylor SM,

DeClerck YA. Effect of tissue inhibitor of the matrix metalloproteinases-2 expression on the growth and spontaneous metastasis of a human melanoma cell line.

Cancer Res. 1994 Oct 15; 54 (20): 5467-73.

Nagahara H, Mimori K, Utsunomiya T, Barnard GF, Ohira

M, Hirakawa K, Mori M. Clinicopathologic and biological significance of kallikrein 6 overexpression in human gastric cancer. Clin Cancer Res. 2005 Oct 1; 11 (19 Pt 1):

6800-6.

Natal i PG, Hamby CV, Felding-Habermann B, Liang B,

Nicotra MR, Di Filippo F, Giannarelli D, Temponi M, Ferrone

S. Clinicai significance of alpha (v) beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions. Cancer Res. 1997 Apr 15; 57 (8):

1554-60.

Nguyen TH. Mechanisms of metastasis. Clin Dermatol.

2004 May-Jun; 22 (3): 209-16.

Obiezu CV, Scorilas A, Katsaros D, Massobrio M, Yousef

GM, Fracchioli S, Rigault de la Longrais IA, Arisio R,

Diamandis EP. Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis of ovarian cancer patients. Clin Cancer Res. 2001 Aug; 7 (8): 2380-6.

Okada Y, Nakanishi I. Activation of matrix

98/108 metalloproteinase-3 (stromelysin) and matrix metalloproteinase-2 (gelatinase) by human neutrophil elastase and Cathepsin G. FEBS Letters 1989; 249: 353-56.

Okamoto T, Akaike T, Nagano T, Miya j ima S, Suga M,

Ando M, Ichimori K, Maeda H. Activation of human neutrophil procollagenase by nitrogen dioxide and peroxynitrite: a novel mechanism for procollagenase activation involving nitric oxide. Arch Biochem Biophys. 1997 Jun 15; 342 (2):

261-74.

Oliva MLV, Sampaio MU, Sampaio CA. Purification and partial characterization of a thiol proteinase inhibitor from Enterolobium contortisiliquum beans. Biol Chem Hoppe

Seyler. 1988; 369: 229-32.

Oliva MLV, Mendes CR, et al. Characterization of a tissue kallikrein inhibitor isolated from Bauhinia bauhinoides seeds: inhibitor of the hydrolysis of kininogen related substrates. Immunopharmacology 1999; 45 (1-3): 1639.

Oliva ML, Mendes CR, Santomauro-Vaz EM, Juliano MA,

Mentele R, Auerswald EA, Sampaio MU, Sampaio CA. Bauhinia bauhinioides plasma kallikrein inhibitor: interaction with synthetic peptides and fluorogenic peptide substrates related to the reactive site sequence. Curr Med Chem. 2001a

Jul; 8 (8): 977-84.

99/108

Oliva ML, Santomauro-Vaz EM, Andrade SA, Juliano MA,

Pott VJ, Sampaio MU, Sampaio CA. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia:

interaction with human plasma kallikrein. Biol Chem. 2001b;

382 (1): 109-13.

de Oliveira C, Santana LA, Carmona AK, Cezari MH,

Sampaio MU, Sampaio CA, Oliva ML. Structure of cruzipain / cruzain inhibitors isolated from Bauhinia bauhinioides seeds. Biol Chem. 2001 May; 382 (5): 847-52.

de Oliveira C. Investigation of the anti-inflammatory action of inhibitors isolated from B. bauhinioides seeds.

2004. São Paulo, PhD thesis-Federal University of

Sao Paulo.

Pagano M, Dalet-Fumeron V, Engler R. The glycosylation

State of the precursors of the cathepsin B-like proteinase from human malignant ascitic fluid: possible implication in the secretory pathway of these proenzymes. Cancer Lett.

1989 Apr; 45 (1): 13-9.

Parise LV, Lee JW, Juliano RL. New aspects of integrin signaling in cancer. Semin Cancer Biol. 2000; 10: 407-414.

Park IK, Kim BJ, Goh YJ, Lyu MA, Park CG, Hwang ES,

Kook YH. Co-expression of urokinase-type plasminogen activator and its receptor in human gastric-cancer cell

100/108 lines correlates with their mvasiveness and

tumorigenicity. Int J Cancer. 1997 May 29; 71 (5): 867-73.

Polette M, Birembaut P. Membrane-type metalloproteinases in tumor invasion. Int J Biochem Cell

Biol. 1998; 30: 1195-1202.

Porteu F, Brockhaus M, Wallach D, Engelmann H, Nathan CF. Human neutrophil elastase releases a ligand-binding fragment from the 75-kDa tumor necrosis factor (TNF) receptor. Comparison with the proteolytic activity responsible for shedding of TNF receptors from stimulated neutrophils. J Biol Chem. 1991 Oct 5; 266 (28): 18846-53.

Raghunand N, Gat enby RA, Gillies RJ.

Microenvironmental and cellular consequences of altered blood flow in tumors. Br J Radiol. 2003; 76 Spec No 1:

Sll-22.

Ramos-DeSimone N, Hahn-Dantona E, Sipley J, Nagase H, French DL, Quigley JP. Activation of matrix metalloproteinase-9 (MMP-9) via converging plasmin / stromelysin-1 cascade enhances tumor cell invasion.

J Biol Chem. 1999 May 7; 274 (19): 13066-76.

Reed JC. Dysregulation of apoptosis in cancer. J Clin

Oncol. 1999 Sep; 17 (9): 2941-53.

Ridger VC, Hellewell PG, Norman KE. L- and P-selectins collaborate to support leukocyte rolling in vivo when high101 / 108 <Λ affinity P-selectin-P-selectin glycoprotein ligand-1 V interaction is inhibited. Am J Pathol. 2005 Mar; 166 (3):

945-52.

Rittenhouse HG, Finlay JA, Mikolajczyk SD, Partin AW.

Human Kallikrein 2 (hK2) and prostate-specific antigen (PSA): two closely related, but distinct, kallikreins in the prostate. Crit Rev Clin Lab Sci. 1998 Aug; 35 (4): 275368.

Ryan CJ, Small EJ. Progress in detection and treatment of prostate cancer. Curr Opin Oncol. 2005 May; 17 (3): 25760.

Saleh Y, Siewinski M, Kielan W, Ziólkowski P, Grybos

M, Rybka J. Regulat ion of catheps in B and L expression in vitro in gastric cancer tissue by egg cystatin. J. Exp.

The R. Oncol. 2003 Jul 3: 319-324.

Sambrano GR, Huang W, Faruqi T, Mahrus S, Craik C,

Coughlin SR. Cathepsin G activates protease-activated receptor-4 in human platelets. J Biol Chem. 2000 Mar 10;

275 (10): 6819-23.

Santomauro-Vaz, EM. Action of inhibitors isolated from Brazilian plants on ischemia and reperfusion syndrome.

São Paulo, 2005, Doctoral Thesis, Escola Paulista de

Medicine.

Schadendorf D, Moller A, Algermissen B, Worm M,

102/108

Sticherling M, Czarnetzki BM. IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor. J Immunol. 1993 Sep 1; 151 (5): 2667-75.

Schmitt M, Kanayama N, Janicke F, Hafter R, Graeff H.

Human tumor cell urokinase-type plasminogen activator (uPA): degradation of the proenzyme form (pro-uPA) by granulocyte elastase prevents subsequent activation by plasmin.Adv Exp Med Biol. 1991; 297: 111-28.

Sever N, Filipic M, Brzin J, Lah TT. Ef fect of cysteine proteinase inhibitors on murine B16 melanoma cell invasion in vitro. Biol Chem. 2002 May; 383 (5): 839-42.

Shamamian P, Schwartz JD, Pocock BJ, Monea S, Whiting

D, Marcus SG, Mignatti P. Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis.J Cell Physiol. 2001 Nov; 189 (2):

197-206.

Shariat-Madar Z, Mahdi F, Schmaier AH. Assembly and activation of the plasma kallikrein / kinin system: a new interpretation. Int Immunopharmacol. 2002 Dec; 2 (13-14):

1841-9.

Shin YH, Akaike T, Khan MM, Sakata Y, Maeda H. Further evidence of bradykinin involvement in septic shock:

reduction of kinin production in vivo and improved survival

103/108 in rats by use of polymer tailored SBTI with longer tl / 2.

Immunopharmacology. 1996 Jun; 33 (1-3): 369-73.

Sier CF, Kubben FJ, Ganesh S, Heerding MM, Griffioen

G, Hanemaaijer R, van Krieken JH, Lamers CB, Verspaget HW.

Tissue leveis of matrix metalloproteinases MMP-2 and MMP-9 are related to the overall survival of patients with gastric carcinoma. Br J Cancer. 1996 Aug; 74 (3): 413-7.

Sierra DA, Gilbert DJ, Householder D, Grishin NV, Yu

K, Ukidwe P, Barker SA, He W, Wensel TG, Otero G, Brown G,

Copeland NG, Jenkins NA, Wilkie TM. Evolution of the regulators of G-protein signaling multigene family in mouse and human. Genomics. 2002 Feb; 79 (2): 177-85.

Smyth MJ, Hayakawa Y, Takeda K, Yagita H. New aspects of natural-killer-cell surveillance and therapy of cancer.

Nat Rev Cancer. 2002 Nov; 2 (11): 850-61.

Solimene AC, Carneiro CR, Melati I, Lopes JD.

Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line. Braz J Med Biol Res. 2001 May; 34 (5): 653-61.

Starcher B, O'Neal P, Granstein RD, Beissert S.

Inhibition of neutrophil elastase suppresses the development of skin tumors in hairless mice. J Invest

Dermatol. 1996 Aug; 107 (2): 159-63.

Stetler-Stevenson WG, Yu AE. Proteases in invasion:

104/108 matrix metalloproteinases. Without Cancer Biol. 2001; 11: 143152.

Sun Y, Hegamyer G, Colburn NH. Molecular cloning of five messenger RNAs differentially expressed in preneoplastic or neoplastic JB6 mouse epidermal cells: one is homologous to human tissue inhibitor of metalloproteinases-3. Cancer Res. 1994 Mar 1; 54 (5): 113944.

Sun Z, Yang P. Role of imbalance between neutrophil elastase and αΐ-antitrypsin in cancer development and progression. Lancet Oncol. 2004 Marc; 5: 182-190.

Syrovets T, Jendrach M, Rohwedder A, Schule A, Simmet

T. Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKbeta15 mediated NF-kappaB activation. Blood. 2001 Junl5; 97 (12):

3941-50.

Syrovets T, Simmet T. Novel aspects and new roles for the serine protease plasmin. Cell Mol Life I know. 2 0 04 Apr;

61 (7-8): 873-85.

0 Toh Y, Oki E, Oda S, Tokunaga E, Ohno S, Maehara Y,

Nicolson GL, Sugimachi K. Overexpression of the MTA1 gene in gastrointestinal carcinomas: correlation with invasion and metastasis.Int J Cancer. 1997 Aug 22; 74 (4): 459-63.

Torisu H, Ono M, Kiryu H, Furue M, Ohmoto Y, Nakayama

105/108

J, Nishioka Y, Sone S, Kuwano M. Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: possible involvement of TNFalpha and

IL-lalpha. Int J Cancer. 2000 Jan 15; 85 (2): 182-8,

Toschi E, Route R, Antonini A, Melillo G, Capogrossi

MC. Wild-type p53 gene transfer inhibits invasion and reduces matrix mild metalloproteinase-2 in p53-mutated human melanoma cells. J Invest Dermatol. 2000 Jun; 114 (6):

1188-94.

Tschesche H, Michaelis J, Kohnert U, Fedrowitz J,

Oberhoff R. Tissue kallikrein effectively activates latent matrix degrading metalloenzymes. Adv Exp Med Biol. 1989;

247A: 545-8.

Vaisanen A, Tuominen H, Kallioinen M, Turpeenniemi15 Hujanen T. Matrix metalloproteinase-2 (72 kD type tv collagenase) expression occurs in the early stage of human melanocytic tumor progression and may have prognostic value. J Pathol. 1996 Nov; 180 (3): 283-9.

van Kessel KP, van Strijp JA, Verhoef J. Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils. J Immunol. 1991 Dec 1; 147 (11): 3862-8.

Varney ML, Li A, Dave BJ, Bucana CD, Johansson SL, Singh RK. Expression of CXCR1 and CXCR2 receptors in

106/108 malignant melanoma with different metastatic potential and their role in interleukin-8 (CXCL-8) -mediated modulation of metastatic phenotype. Clin Exp Metastasis. 2003; 20 (8):

723-31.

Vaux DL, Korsmeyer SJ. Cell death in development.

Cell. 1999 Jan 22; 96 (2): 245-54.

Vidal-Vanaclocha F, Fantuzzi G, Mendoza L, Fuentes AM,

Anasagasti MJ, Martin J, Carrascal T, Walsh P, Reznikov LL,

Kim SH, Novick D, Rubinstein M, Dinarello CA. IL-18 regulates IL-lbeta-dependent hepatic melanoma metastasis via vascular cell adhesion molecule-1. Proc Natl Acad Sei U

S. 2000 Jan 18; 97 (2): 734-9.

Witte MB, Barbul A. Role of nitric oxide in wound repair. Am J Surg. 2002; 183: 406-412.

Wolf WC, Evans DM, Chao L, Chao J. A synthetic tissue kallikrein inhibitor suppresses cancer cell invasiveness.

Am J Pathol. 2001 Nov; 159 (5): 1797-805

Wu J, Akaike T, Hayashida K, Miyamoto Y, Nakagawa T,

Miyakawa K, Müller-Esterl, Maeda H. Identification of bradykinin receptors in clinincal cancer specimens and murine tumor tissues. Int J Cancer. 2002; 98: 29-35.

Xie S, Luca M, Huang S, Gutman M, Reich R, Johnson JP, Bar-Eli M. Expression of MCAM / MUC18 by human melanoma cells leads to increased tumor growth and metastasis. Cancer Res.

107/108

1997 Jun 1; 57 (11): 2295-303.

Yamamoto H, Iku S, Adachi Y, Imsumran A, Taniguchi H,

Nosho K, Min Y, Horiuchi S, Yoshida M, Itoh F, Imai K.

Association of trypsin expression with tumor progression 5 and matrilysin expression in human colorectal cancer. J

Pathol. 2003 Feb; 199 (2): 176-84.

Yamashita J, Tashiro K, Yoneda S, Kawahara K,

Shirakusa T. Local increase in polymorphonuclear leukocyte elastase is associated with tumor invasiveness in non-small cell lung cancer. Chest. 1996 May; 109 (5): 1328-34.

Yamashita J, Ogawa M, Abe M, Hayashi N, Kurusu Y,

Kawahara K, Shirakusa T. Tumor neutrophil elastase is closely associated with the direct extension of non-small cell lung cancer into the aorta. Chest. 1997 Apr; 111 (4):

885-90.

Yang TS, Hsu KC, Tang R, Chang TC. Colon carcinoma with synchronous ovarian metastasis - report and discussion of fíve cases. Anticancer Drugs. 2000 Apr; 11 (4): 279-83.

Yavelow J, Tuccillo A, Kadner SS, Katz J, Finlay TH. 20 Alpha 1-antitrypsin blocks the release of transforming growth factor-alpha from MCF-7 human breast cancer cells. J

Clin Endocrinol Metab. 1997 Mar; 82 (3): 745-52.

Yousef GM, Borgono CA, Popalis C, Yacoub GM, Polymeris

ME, Soosaipillai A, Diamandis EP. In-silico analysis of

108/108 kallikrein gene expression in pancreatic and colon cancers.

Anticancer Res. 2004 Jan-Feb; 24 (1): 43-51.

Yui S, Tomita K, Kudo T, Ando S, Yamazaki M. Induction of multicellular 3-D spheroids of MCF-7 breast carcinoma 5 cells by neutrophil-derived cathepsin G and elastase.

Cancer I know. 2005 Sep; 96 (9): 560-70.

Za j c I, Sever N, Bervar A, Lah TT. Expression of cysteine protease cathepsin L and its inhibitors stefins A and B in relation to tumorigenicity of breast cancer cell lines. Cancer Letters. 2002 Jul; 187: 185-90.

Zeng ZS, Guillem JG. Placement of matrix metalloproteinase-9-mRNA and protein in human colorectal cancer stromal cells. Br J Cancer. 1996 Oct; 74 (8): 1161-7.

All publications mentioned in the specification described above are hereby incorporated by reference. Various modifications and variations of the present invention will be evident to those skilled in the art, without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with the specific preferred embodiment, it is to be understood that the invention, as claimed, should not be unduly limited to that specific embodiment.

1/1

Claims (8)

1. Use of protease inhibitors isolated from Bauhinia bauhinioides and / or Enterolobium contortisiliquum, as indicated in SEQ. IDs. we. 1, 2 and 3, characterized in that it is in the preparation of a pharmaceutical composition comprising at least one of said inhibitors for the treatment of cancer. 2. Use according to claim 1, characterized by the fact that said pharmaceutical composition additionally comprises a chemotherapeutic agent. 3. Use, according to claim 2, characterized by the fact that the said chemotherapeutic agent is 5 fluorouracil. 4. Use, according to claim 2, characterized by the fact that the referred chemotherapeutic agent is carboplatin. 5. Pharmaceutical composition comprising protease inhibitors isolated from Bauhinia bauhinioides and / or Enterolobium contortisiliquum, as indicated in SEQ. IDs. we. 1, 2 and 3, characterized in that it comprises an effective amount of at least one of said inhibitors and at least one pharmaceutically acceptable carrier. 6. Composition according to claim 5, characterized by the fact that it additionally comprises at least one chemotherapeutic agent. 7. Composition, according to the fact that claim 6, said agent characterized by chemotherapy is 5-fluorouracil. 8. Composition, according to characterized by the fact that chemotherapy is carboplatin. claim 6, said agent Petition 870170051966, of 7/24/2017, p. 6/12 1/40 Ι ι I I I _{t t} I I ι O 20 40 60 80 100 Io (nM)