TWI598105B - Medicinal composition with inhibition of interaction between MZF-1 and Elk-1 - Google Patents

Description

Medicinal composition with inhibition of interaction between MZF-1 and Elk-1

The present invention is a peptide, in particular, a peptide which inhibits the interaction between Elk-1 and MZF-1 and inhibits cancer.

The transcription factor Elk-1 (Ets-like protein-1) is a member of the Ets domain protein under the ternary complex factor (TCF). TCF regulates specificity through intermolecular and intramolecular interactions. Gene expression, TCF and SRF bind to the SRE (serum response element) to form a ternary complex. The Ets family contains Elk-1, Sap1 and Sap2, all of which have an Ets region and a DNA-bonded region of a winged helix-loop-helix (HLH), and Ets DNA binding at the N-terminus of Elk-1 The site is important for DNA identification. TCF has a B-box containing 20 amino acids (B box) and protein-protein interaction. The C-terminal transactivation region of Elk-1 can be phosphorylated by MAPK, Elk-1 The acceptance position of the D and FxFD regions when interacting with the MAPK. Elk-1 is involved in the regulation of cell proliferation, differentiation and development via mitogen-activated protein kinase (MAPK).

MZF (myeloid zinc finger, including MZF-1, MZF-1B, MZF-1C) belongs to the zinc finger protein in the Kruppel family. MZF-1 is usually located in hematopoietic progenitor cells, MZF. -1 overexpression can cause cancer cells Migration and erosion (Invasion). MZF-1 plays an important role in regulating homeostasis (hemopoiesis). In the in vitro experiment, it was found that MZF-1 can regulate the in vivo balance, that is, the cell transcription of non-in vivo balance, but the biological function of MZF-1 is still unclear.

Breast cancer is a cancer that occurs in women, and the incidence is increasing year by year. Therefore, breast cancer has become a major cause of rising female mortality. Most of the breast cancer cells carry three cell surface receptors, estrogen receptor (ER), progesterone receptors (PR) or HER2. However, 15% of breast cancer cells lack the expression of ER, PR and HER2, called triple-negative breast cancers (TNBC). For TNBC, because neither ER nor HER2 is expressed, the therapeutic effect of inhibiting ER or inhibiting HER2 target drugs is not good. Clinically, only traditional chemical drugs can be used for treatment, but high metastasis after cancer recurrence. There is currently no effective treatment. Many studies suggest that tumor-initiating cells (TIC) and cancer stem cells (CTS) are possible causes of cancer initiation and recurrence, which are related to cancer occurrence, metastasis, and drug resistance, and mammary tumor-initiating cells ( The breast tumor-initiating cell (BTIC) is thought to be involved in recurrence after treatment. The current TNBC/BTIC diagnosis is highly correct, but there is still a lack of clinically effective data.

Protein kinase C alpha (PKC α) plays a regulatory role in breast cancer stem cells (BCSC). Studies in cell lines and tumor samples have found that triple-negative breast cancer (TNBC)/breast The expression of protein kinase Cα on tumor-initiating cells (TICs) is associated with poor patient survival. Therefore, PKC α is considered to be a post-mortem indicator of TNBC and can even be used as a target for cancer treatment. In addition, when non-stem cells evolve into Cancer stem cells (CSCs), their signal transmission function is caused by epithelial cell growth. Epidermal growth factor receptor (EGFR) signal transduction is regulated by platelet-derived growth factor receptor during progression (PDGFR) signaling, which is responsible for protein kinase Cα expression, epithelial cell embryo transformation (epithelial) The -mesenchymal transition (EMT) mechanism is also involved in the expression of protein kinase Cα.

Existing PKC alpha inhibitors include or not (Riluzole), anti-translation oligonucleotides (Aprinocarsen) and peptide inhibitors (αV5-3). These protein kinase Cα inhibitors are not only directed against cancer cells, so how The design of a peptide, which only targets the specific mechanism of cancer cells, further affects the protein kinase Cα to inhibit cancer, and has become an important subject to be solved by the present invention.

In view of the inhibition of the growth and research of cancer cells in the past, the emphasis is on the regulation of protein kinase Cα, but protein kinase Cα is present in both cancer cells and normal cells, so inhibition of protein kinase Cα inhibits cancer cell growth and also inhibits normal cell growth. How to affect only the protein kinase Cα in cancer cells without affecting normal cells is a difficult challenge. After long-term conception and research, the inventors found a synergistic interaction between MZF-1 and Elk-1 in triple-negative breast cancer. It will regulate the expression of protein kinase Cα, and we also found that MZF-1 and Elk-1 each utilize a specific region to combine two genes to form a heterodimer. The purpose of the present invention is to provide a novel The peptide can inhibit the expression of endogenous Elk-1 or MZF-1, and also interrupt the interaction between Elk-1 and MZF-1, reduce the expression of protein kinase Cα, and inhibit the growth of cancer cells.

Object of the present invention is to provide a peptide for the preparation of a pharmaceutical composition the treatment of breast cancer, the peptide comprising the at least one of the following, or any combination of: (A) peptide MZF-1 _60-72, the sequence of SEQ ID NO (65) the peptide Elk-1 _145-157 , the sequence is identical to SEQ ID NO: 66; the peptide has a breast cancer therapeutic effect by inhibiting the interaction of MZF-1 with Elk-1.

Wherein, the pharmaceutical composition may further comprise the pharmaceutically acceptable carrier, wherein the carrier comprises an excipient, a diluent, a thickener, a filler, a binder, a disintegrant, a lubricant, a grease or a non- A base for oils, a surfactant, a suspending agent, a gelling agent, an adjuvant, a preservative, an antioxidant, a stabilizer, a colorant or a fragrance.

Wherein, the pharmaceutical composition is administered by oral, soaking, injecting, smearing or patching.

Among them, the breast cancer is a triple-negative breast cancer.

Figure 1 shows the expression of MZF-1, Elk-1, and protein kinase Cα by immunohistochemical staining with human breast cancer tissue sections.

Fig. 2 shows the expression of MZF-1, Elk-1, and protein kinase Cα by immunohistochemical staining with human liver cancer tissue sections.

Figure 3 shows the expression of MZF-1, Elk-1, and protein kinase Cα by immunohistochemical staining with human lung cancer tissue sections.

Figure 4 shows the expression of MZF-1, Elk-1, and protein kinase Cα by immunohistochemical staining with human bladder cancer tissue sections.

Figure 5 shows the expression of MZF-1, Elk-1, and protein kinase Cα by immunohistochemical staining with human triple negative breast cancer tissue sections. And the normal activity of MZF-1 and Elk-1 was transferred by fluorescence activity analysis. The effect of protein kinase Cα expression.

Figure 6 is a diagram showing the effect of normal and transgenic MZF-1 and Elk-1 on the expression of protein kinase Cα by fluorescence activity in HCC cells.

Figure 7 is a schematic representation of the sequence of the wild type and mutant MZF-1/Elk-1 at the position of the PRKCA promoter.

Figure 8 shows the analysis of anti-Elk-1 and anti-MZF-1 antibodies in human triple negative breast cancer DMA-MB-231 cell nuclear extract by electrophoretic mobility analysis (EMSA). The wild type and mutant MZF- were observed. 1/Elk-1 DNA-protein binding ability.

Figure 9 shows the electrophoretic mobility analysis (EMSA) analysis of human triple negative breast cancer DMA-MB-231 cell nuclear extract, identified by anti-Elk-1, anti-MZF-1 antibody, observed 20-100 times without labeling The probe affects the binding of DNA to protein in a competitive manner.

Figure 10 shows the MZF-1/Elk-1 binding ability of human triple-negative breast cancer DMA-MB-231 cells by co-immunoprecipitation, first immunoprecipitation with anti-MZF-1 or anti-Elk-1 antibody (IP) After electrophoresis, it was identified by anti-MZF-1 or anti-Elk-1 antibody.

Figure 11 shows the MZF-1/Elk-1 binding ability of human triple-negative breast cancer DMA-MB-231 cells by co-immunoprecipitation, cytoplast construction (Elk-1-c-Myc-ΔDBD, FLAG-MZF-1- ΔDBD), immunoprecipitation (IP) with anti-c-Myc or anti-FLAG antibody, and identified by anti-MZF-1 or anti-Elk-1 antibody after electrophoresis.

Figure 12 shows the DNA binding activity of SK-Hep-1 cells MZF-1/Elk-1 by chromatin immunoprecipitation (ChIP) and secondary chromatin immunoprecipitation (Re-ChIP). The first ChIP was anti- MZF-1 or anti-Elk-1 antibody was adsorbed, and the second ChIP was first adsorbed with anti-Elk-1 antibody and then with anti-MZF-1 antibody, and amplified by PCR, and confirmed by electrophoresis.

Figure 13 shows the binding activity of MZF-1/Elk-1 in SK-Hep-1 cells Elk-1 or MZF-1 by antisense oligonucleotides to chromatin immunoprecipitation (ChIP) And secondary chromatin immunoprecipitation (Re-ChIP) analysis.

Figure 14 shows the schematic diagram of MZF-1 fragments of different lengths. The following figure shows HEK-293 cells transfected with different length fragments of MZF-1. The binding activity of MZF-1/Elk-1 was analyzed by immunoprecipitation. The -c-Myc antibody was subjected to immunoprecipitation (IP), and after electrophoresis, it was identified by an anti-FLAG-1 antibody.

15 is a schematic view showing the wild-type and mutant _{MZF-1 60-72, MZF-1} 1-72 amino acid sequence.

Figure 16 is a train bound wild-type and mutant MZF-1 _60-72 and the MZF-1 / of Elk-1 activity, electrophoretic mobility shift assay (EMSA) analysis.

Figure 17, above is a schematic diagram of Elk-1 fragments of different lengths; the following figure shows HEK-293 cells transfected with different length fragments of MZF-1, and the binding activity of MZF-1/Elk-1 was analyzed by immunoprecipitation. The -c-Myc antibody was subjected to immunoprecipitation (IP), and after electrophoresis, it was identified by an anti-FLAG-1 antibody.

Figure 18 is a schematic representation of the wild type and mutant Elk-1 _145-157 and MZF-1 _145-428 amino acid sequences.

Figure 19 shows the binding activity of wild-type and mutant Elk-1 _145-157 to MZF-1/Elk-1, and analyzed by electrophoretic mobility analysis (EMSA).

FIG. 20, the left viewing FIG microscope observation of MDA-MB-231 cells transfected with expression patterns _60-72 of the MZF-1 changes right picture cells MZF-1, Elk-1, expression of protein kinase Cα, beta] -actin is the control group and c-myc is the marker of the transgenic cells.

FIG 21, to analyze the confocal microscope (Confocal microscopy), MDA-MB -231 cells were transfected after the performance MZF-1 _60-72, cells, Elk-1, showed MZF-1, antibodies to -Elk-1 The expression of Elk-1 protein was identified by FITC secondary antibody, and the expression of MZF-1 protein was identified by anti-MZF-1 antibody and rhodamine secondary antibody. The thickness of Z-axis slice was 0.5-0.6 μm.

FIG 22 is a line stably expressing MZF-1 _60-72 Hs578T and MDA-MB-231 cells, which MZF-1 and Elk-1 protein expression changes.

FIG 23 is a line stably expressing MZF-1 _60-72 Hs578T and MDA-MB-231 cells, which MZF-1 degradation.

FIG line 24 after the MZF-1 _60-72, to chromatin immunoprecipitation (ChIP) analysis of endogenous MZF-1 is transfected MDA-MB-231 cells expressing colonization, Elk-1, expression of protein kinase Cα; on The figure was immunoprecipitated (IP) with an anti-MZF-1 antibody, and the following figure was subjected to immunoprecipitation (IP) with an anti-Elk-1 antibody, and the PRKCA promoter was identified by PCR after electrophoresis.

FIG 25 is a first anti-cancer drugs based impact flavin piperidine (acriflavine) and cis-platinum (cisplatin) showed MZF-1 _60-72 of cells MDA-MB-231 transfected the upper graph of glycoprotein (P-gp) The effect of performance, the following figure is the impact on cell proliferation.

FIG 26 is a line MDA-MB-231 cells transfected with MZF-1 _60-72, the picture shows the change patterns for the cells, under the influence of the picture shows the migration and proliferation of cancer cells.

FIG 27 is a line of breast cancer xenograft mouse model, MDA-MB-231 cells transfected with and HS578T MZF-1 _60-72 implanted mice (left), upper right panel of tumor size lines so as to form, following figure Tumor tissue section map.

FIG 28 is a line transfected _{MZF-1 60-72 MDA-MB-} 231 cells and HS578T cells of mesodermal epithelial transition (EMT) associated gene expression.

Figure 29 is a breast cancer cell line MDA-MB-468 and MCF-7 (low malignancy), to immunoblot blot analysis transfected MZF-1 _60-72 the Ca kinase protein, skin cell embryos transition (EMT) etc. The effect of related protein expression.

Figure 30 shows the cell-transformed full-length protein kinase Cα, the upper panel shows cell type changes, and the lower panel shows the effect on cancer cell migration.

Figure 31 is a diagram showing the effect of cell transfection of full-length protein kinase Cα on protein kinase Cα, dermal cell embryo transfer (EMT) and other related proteins.

Figure 32 is a schematic representation of TAT fusion wild type and mutant Elk-1, MZF-1 peptides.

Figure 33 shows the cell TAT fusion Elk-1, MZF-1 peptide, the left picture shows cell type changes, and the right picture shows the effect on cancer cell migration.

Figure 34 is the effect of cellular TAT fusion Elk-1, MZF-1 peptide on protein kinase Cα, dermal cell embryo transfer (EMT) and other related proteins.

Figure 35 is the effect of TEK fusion Elk-1 and MZF-1 peptide on the interaction between MZF-1 and Elk-1 in HEK-293 cells. Immunoprecipitation analysis with anti-c-Myc antibody (IP), after electrophoresis, identified by anti-c-Myc or anti-FLAG antibody (IB).

Figure 36 is a schematic representation of the interaction of MZF-1 with Elk-1 to regulate protein kinase Cα.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

The present invention discloses the correlation between the interaction of MZF-1 and Elk-1 and cancer. MZF-1 peptide or Elk-1 peptide is provided to inhibit the interaction of MZF-1 with Elk-1, which can inhibit cancer.

Plasmid Construction

In this experiment, pcDNA3.0 (Invitrogen) was used as the expression vector, and the gene expression was controlled by the Cytomegalovirus (CMV) promoter. Human MZF-1 (GenBank Accession No. AF161886 10781-12235bp) and Elk-1 (GenBank Accession) No. AB016193 101-1384bp) The open reading frame (ORF) of the gene was obtained from SK-Hep-1 cells by RT-PCR, and pcDNA-MZF-1 and pcDNA-Elk-1 were synthesized. Primers were used. The sequence and restriction enzyme (EZ) are listed in Table 1. The PCR product was isolated and transfected into pcDNATM 3.1/myc-His vector (Invitrogen).

Cell culture and growth conditions

Hepatocellular carcinoma (HCC) HepG2 (BCRC no. 60434), human breast cancer cells Hs578T (BCRC no. 60120), MDA-MB-231 (MB-231, BCRC no.60425), MCF-7 (BCRC no) .60436), human embryonic kidney cell HEK-293 (BCRC no.60019), the above five cells were purchased from the Center for Bioassess Conservation and Research of the Food Industry Development Research Institute (Hsinchu, Taiwan); human liver cancer cell SK-Hep-1 ( ATCC no.HTB-52), Huh-7 (ATCC no.JCRB-0403), human breast cancer cell line MDA-MB-468 (ATCC no.HTB-132), the above three cells were purchased from the American model culture collection library. (American type culture collection, ATCC); cell line in DMEM medium (Gibco-BRL) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin at 5% CO ₂ , 37 ° C to cultivate.

Immunohistochemical analysis

Cancer tissue sections, stained with antibodies, identified by anti-PKCα antibody (BD Biosciences), anti-Elk-1 antibody (Santa Cruz), anti-MZF-1 antibody (Santa Cruz), and correlated according to color intensity It is weak, medium and strong.

Chromatin immunoprecipitation Assay (ChIP)

The cells were fixed in 1% formaldehyde solution (formadehyde), stopped by formaldehyde reaction with glycine (Glycine), and dissolved in lysate containing protease inhibitor (0.1% SDS, 1% sodium deoxycholate, 150 mM NaCl, 10 mM NaPO ₄ (pH 7.2), 2mM EDTA, 0.2 mM NaVO ₃ , 1% NP-40), immunofluorescent (IP) with anti-MZF-1 antibody or anti-Elk-1 antibody, and chromatin with protein A agarose Chromatin ), the -760 to -550 region of the PRKCA promoter, to the 5'-GGTACAGGCAGCTAAAACAC-3', as in the sequence SEQ ID NO: 47, and the anti-translation 5'-GTCTTCCTTCTCCCACTCC-3', Sequence SEQ ID NO: 48, amplified, product size 210 bp, confirmed by electrophoresis on 2% agarose film.

Secondary Chromatin Immunoprecipitation (Re-ChIP) is a precipitation complex that immunoprecipitates the first anti-Elk-1 antibody or anti-MZF-1 antibody to anti-MZF-1 antibody or anti-Elk-1 antibody Immunoprecipitation (IP), chromatin was obtained with protein A agarose, eluted with 30 μl of ChIP elution buffer (50 mM NaHCO ₃ , 1% SDS), multiple eluates were collected, and the supernatant was collected by centrifugation and eluted. The solution was diluted with a protease inhibitor buffer (1% Triton X-100, 5 mM EDTA, 150 mM NaCl, and 25 mM Tris, pH 8), and the subsequent PCR and electrophoresis analysis methods were the same as the ChIP analysis method described above.

Electrophoretic Mobility Shift Assay (EMSA)

Reagent used LightShift ^TM chemiluminescent EMSA (Pierce), taking 15μg nuclear extract, biotin-labeled (Biotin-labeled) of the double stranded oligonucleotide (double-stranded oligonucleotides) used as primers, the sequence is as follows: normal wild type MZF-1/normal Elk-1 (positively translated 5'-CCTGAGGATGGGGAAGGGGCTTCCTGCTGCGGTG-3', as in sequence SEQ ID NO: 49, anti-translation 5'-CACCGCAGCAGGAAGCCCCTTCCCCATCCTCAGG-3', as in sequence SEQ ID NO: 50), mutation MZF -1/Normal Elk-1 (normal translation 5'-CCTGCGTATTTTTAAGGGGCTTCCTGCTGCGGTG-3', as in sequence SEQ ID NO: 51, anti-translation 5'-CACCGCAGCAGGAAGCCCCTTAAAAATACGCAGG-3', as in sequence SEQ ID NO: 52), normal MZF- 1/mutant Elk-1 (transformed 5'-CCTGCGGATGGGGAAGGGGATTAATGATGAGGTG-3', as in sequence SEQ ID NO: 53, reverse-translation 5'-CACCTCATCATTAATCCCCTTCCCCATCCGCAGG-3', as in SEQ ID NO: 54), mutation MZF-1 /mutant Elk-1 (transformed 5'-CCTGCGTATTTTTAAGGGGATTAATGATGAGGTG-3', as in sequence SEQ ID NO: 55, reverse translation strand 5'-CACCTCATCATTAATCCCCTTAAAAATACGCAGG-3', as in SEQ ID NO: 56). Add 20-100-fold concentration without biotin-labeled sequences (such as SEQ ID NO: 49-56) to compete for specific binding, and the combined DNA-protein complex was electrophoresed with 6% native polyacrylamide gel and run for 3 hours at 100V. The buffer was transferred to a positively-charged nylon membrane using a buffer of 0.5 times Tris borate/EDTA buffer and analyzed by chemiluminescence.

Plasamic transfection

The cells were cultured in 6 cm medium. The medium was replaced with DMEM containing no fetal bovine serum 18 hours before the transfer. After removing the medium, 1 ml of 15 μg Lipofectamine 2000 transfection reagent (Invitrogen) and different concentrations of plastids were cultured for 6 hours. The medium was DMEM containing 20% fetal calf serum. After 18 hours of culture, the medium was replaced with fetal bovine serum, and culture was continued for 48 hours, and collected for subsequent experiments.

Stable expression of cell lines (Stable Clone Establishment)

Algebraically earlier (low-passage) the cells (3 × 10 ⁵ cells) in 6 cm culture medium to Lipofectamine 2000 rpm colonization 5μg MZF-1 _60-72 plasmid to geneticin (G418; 600μg / ml) cultured A stable expression cell line was selected for 5 weeks.

TAT-Fused Peptide treatment

Design access TAT HIV TAT 47-57 amino acid residues in a protein sequence (Residue), the human MZF-1 fusion protein the amino acid sequence _60-72 (MZF-1 60-72) or human protein Elk-1-amine The base acid sequence 145-157 (Elk-1 _145-157 ), the sequence is as follows: TAT fusion normal MZF-1 (60-72; YGRKKRRQRRRGGGSDLRSEQDPTDED, as SEQ ID NO: 57), TAT fusion mutation MZF-1 (60-72; YGRKKRRQRRRGGGAEATPAQESRLAS, as SEQ ID NO: 58), TAT fusion normal Elk-1 (145-157; YGRKKRRQRRRGGGLARSSRNEYMRSG, SEQ ID NO: 59), TAT fusion mutation Elk-1 (145-157; YGRKKRRQRRRGGGLAASSANEYMASG, SEQ ID NO: 60 The cells were grown to a density of 50-60%, replaced with serum-free medium, and the specified concentration of TAT fusion peptide was added. After 3 days of expression, the cells were used for subsequent experiments.

In vitro tumorgenesis assay

In this experiment, 4-6 weeks old female nude mice (BALB/c) were purchased from the National Institutes of Health (Taipei, Taiwan), and kept in a sterile environment. The nude mice were fed with sterile water and feed, and collected by trypsin. For cancer cells, 1×10 ⁷ cells (in 100 μl DMEM) were injected into the right posterior flank, and after 2-3 months of culture, the nude mice were sacrificed and the tumor was taken out. Tumor volume calculation formula: 0.5236 × L1 (L2) ² , L1 represents length, L2 represents width, tumor volume in control group-tumor volume in test group / (tumor volume in control group) × 100%.

Cell proliferation test

The cell proliferation assay was performed by tetrazolium chloride analysis (MTT assay), and 1×10 ⁴ cells were cultured in a 24-well culture dish and cultured overnight in DMEM containing 10% serum. After culturing for 24 or 48 hours with varying plasmids, fresh medium was replaced, MTT (1 mg/ml) was added for 3 hours, and 1 ml of DMSO was added for 30 minutes. The absorbance at a wavelength of 570 nm was analyzed by a spectrophotometer.

Cell migration test

In the cell migration experiment, cells in serum-free DMEM (2 × 10 ⁵ /well) were placed on top of a 48-well Boyden chamber (Neuro Probe), and the lower layer was placed in a serum containing 20%. In DMEM, cells were cultured at 37 ° C, 5% CO _{2 for} 12 hours, and the cells were fixed with methanol, stained with 0.05% Giemsa for 1 hour, and observed under a microscope.

Cell invasion test

The upper layer of the 48-well Bodeng trough was first treated with 10 μg/ml Matrigel (BD Biosciences), and the cells in serum-free DMEM (2×105/well) were placed on top of the 48-well Boden trough, and the lower layer was placed in 20% serum. In DMEM, cells were cultured at 37 ° C, 5% CO _{2 for} 24 hours, and the cells were fixed with methanol, stained with 0.05% Giemsa for 1 hour, and observed under a microscope.

Antisense Knockout Assays

The anti-translation stocks and the translated stocks (control group) used in the anti-translation stock inhibition performance test were as follows: Elk-1 (anti-translation stock 5 ' -CAGCGTCACAGATGGGTCCAT-3 ' , as the sequence SEQ ID NO: 61, the translation stock 5 ' -ATGGACCCATCTGTGACGCTG-3 ' , such as the sequence SEQ ID NO: 62), MZF-1 (anti-translation 5 ' -TACACAAGGGGACCATTCATTC-3 ' , as the sequence SEQ ID NO: 63, the translation of the stock 5 ' -GAATGAATGGTCCCCTTGTGTA-3 ' , As in the sequence SEQ ID NO: 64).

Example 1

Correlation between protein kinase Cα expression and MZF-1/Elk-1

Human breast cancer (Fig. 1), liver cancer (Fig. 2), lung cancer (Fig. 3), bladder cancer (Fig. 4), and analysis of protein kinase Cα and MZF-1 by immunohistochemical staining (irmmunohistochemical staining) /Elk-1 correlation, protein expression of protein kinase Cα in human breast cancer and liver cancer tissues has a positive correlation with MZF-1 or Elk-1 expression, and most of the medium-strength positive correlation results from phase 2 The sample with the third stage cancer; however, in the lung cancer and bladder cancer samples, the amount of protein kinase Cα expression was not correlated with the amount of MZF-1 or Elk-1 expression. Further analysis of cancer cell lines confirmed the correlation between protein kinase Cα expression and MZF-1/Elk-1. In TNBC breast cancer (MDA-MB-231) and liver cancer (SK-Hep-1) cell lines, siRNA interfered with Elk-1. Protein expression also reduced the expression of protein kinase Cα protein, but in lung cancer (A549) and bladder cancer (bladder 5637) cell lines, interference with Elk-1 protein expression by siRNA did not change the protein kinase Cα protein expression. The TNBC breast cancer specimen was analyzed by immunohistochemical staining. As shown in Fig. 5, the protein kinase Cα expression amount was also positively correlated with the MZF-1 or Elk-1 expression. Further, in the TNBC breast cancer (MDA-MB-231 and Hs578T) cell lines, gene transfer increased MZF-1 or / and Elk-1, and the protein kinase Cα transcription activity was found to increase several times. From the above results, it can be inferred that in liver cancer, breast cancer and even TNBC, MZF-1/Elk1 can regulate protein kinase Cα expression.

Example 2

Combination of MZF-1/Elk-1 complex with PRKCA promoter region

Two hepatocellular carcinoma cells (HCC, Huh-7 and HepG2) cells were transfected into full-length MZF-1 or Elk-1, and the transtriptional activity of protein kinase Cα was found to increase, as shown in Fig. 6. Simultaneous transduction of full-length MZF-1 and Elk-1 by HCC cells resulted in higher translational activity of PKC α, but if DNA plastids were constructed, the DNA binding domain of Elk-1 (Elk-1ΔDBD, Elk-1) _87-428 ) or the DNA binding region of MZF-1 (MZF-1ΔDBD, MZF-1 _1-72 ) deleted did not affect the translational activity of protein kinase Cα, and the results were shown in the MZF-1/Elk-1 direct and PRKCA promoter region. Binding to modulate the translational activity of protein kinase Cα.

The nucleotide guanine (G) of the PRKCA promoter and the MZF-1 binding region were all replaced with thymine (T), and the nucleotide cytosine (C) of the Elk-1 binding region was all Replace with adenine (A), as shown in Fig. 7, analyze the binding of DNA to protein by electrophoresis mobility shift assay (EMSA). The results are shown in Fig. 8, where P: Indicates that only probes, V: indicates only nuclear extract, N: indicates nuclear extract plus probe, FP: free probe, triple negative breast cancer cells (MDA-MB-231) nuclear extract for EMSA analysis The wild-type PRKCA promoter probe can be bound to MZF-1 or Elk-1 by a slower moving speed. Conversely, the PRKCA promoter MZF-1 binding region or the Elk-1 binding region nucleotide is replaced. The binding ability to MZF-1 or Elk-1 was decreased, and the results are shown in Fig. 9, wherein P: indicates only a probe, N: indicates a nuclear extract plus a probe, and FP: a free probe. From these results, it was confirmed that MZF-1 or/and Elk-1 bind to the PRKCA promoter to affect its translation activity.

MZF-1 can be found in the complex by anti-Elk-1 antibody by co-immunoprecipitation. The results are shown in Fig. 10, and conversely, anti-MZF-1 antibody can also be used from the complex. Elk-1 was found. In addition, DMA-MB-231 cells were transfected into Elk-1 plastid construct (Elk-1-c-Myc-ΔDBD lacked the N-terminal region via deletion mutation), with anti-c-Myc The MZF-1 protein was found in the antibody immunoprecipitated complex. As shown in Fig. 11, the cells were transfected with Flag-MZF-1 ΔDBD plastid, and Elk-1 protein was found in the complex immunoprecipitated with anti-FLAG antibody. The results showed that Elk-1 was found to bind to the N-terminal region of MZF-1 in all cell lines, and MZF-1 binds to the N-terminal region of Elk-1. It is speculated that MZF-1/Elk-1 will form a heterologous complex. (heterodimeric complex).

The chromatin immunoprecipitation assay (ChIP) analysis, immunoprecipitation with anti-Elk-1 or anti-MZF-1 antibody can amplify the PRKCA promoter fragment in the complex, analyzed by Re-ChIP assay, the results show Elk-1 forms a complex with MZF-1 and binds to the PRKCA promoter. The results are shown in Fig. 12; inhibition of Elk-1 or MZF-1 expression by antisense oligonucleotides, inhibition of complexation The body is combined with the PRKCA promoter, and the results are shown in Fig. 13, wherein S: indicates a positive translation (sense), AS: indicates an anti-sense, and the result indicates that Elk-1 and MZF need to be formed first. A heterologous complex of 1 binds to the PRKCA promoter region.

From the results, it was found that Elk-1 forms a complex with MZF-1 and binds to the PRKCA promoter, thereby inhibiting the translational activity of protein kinase Cα.

Example 3

Interaction between the MZF-1 Acidic Domain and the Heparin-Binding Domain of Elk-1

The MZF-1 acidic region was designed with different MZF-1 peptide fragments at the 60-72 amino acid sites. The results were analyzed by co-immunoprecipitation assay, including MZF, as shown in Figure 14. -1 full length acidic area _{MZF-1, MZF-1 1-72} , MZF-1 1-141, MZF-1 60-72 are combined and Elk-1, but _{MZF-1 1-60, MZF-1} 73- ₄₈₅ did not bind to Elk-1, and it was confirmed that the 60-72 amino acid peptide fragment of the acidic region of MZF-1 was in a binding position to Elk-1. Design of mutant _{MZF-1 60-72, MZF-1} 1-72, as shown in FIG. 15, wherein the amino acid sequence of aspartate 61,67,70,72 position of positively charged (Aspartic acid, Asp, D) is substituted with uncharged alanine (Alanine, Ala, A), which has reduced binding capacity to Elk-1.

In the saturated protein - protein binding domains (saturating the protein-protein binding domains ) corresponding to MZF-1 _60-72 peptide fragment Gan scrambling endogenous Elk-1 and MZF-1, show the results from the EMSA, MZF- 1 _60-72 can reduce the DNA binding activity of Elk-1 and MZF-1, and has a dose-dependent effect. As shown in Fig. 16, the mutant pattern of the fragment does not affect their binding activity, and the results of these studies are further verified. MZF-1 interacts with Elk-1 through its acidic region.

Several Elk-1 fragments were constructed. As shown in Figure 17, the peptide fragment of 145-157 amino acids of Elk-1 is the heparin-binding region, and Elk-1 _145-157 interacts with MZF-1. In addition, Elk -1 _145-157 reduced the DNA binding activity of Elk-1 and MZF-1 in a dose-dependent manner, and the mutant type did not have this property. The result showed that the heparin-binding region of Elk-1 amino acid 145-157 was associated with MZF. -1 binding position in which the region interacts with MZF-1. Design mutations Elk-1 _145-157 , MZF-1 _145-428 , as shown in Figure 18, where arginine (Arg, R, Arg, R) in the ₁₄₇ , ₁₅₀ , and 155 positions of the amino acid sequence is alanine (Alanine, Ala, A) replaced, its ability to bind to MZF-1 decreased. The addition of Elk-1 _145-157 reduced the binding activity of MZE-1 and Elk-1 to DNA, and the results are shown in Fig. 19.

From the above results, it was found that MZF-1 interacts with Elk-1 through its acidic region, and Elk-1 interacts with MZF-1 through its heparin-binding region.

Example 4

Effect of MZF-1/ELK-1 heterodimer on the expression of protein kinase Cα

Forming MZF-1 heterodimer, and ELK-1 protein kinase Cα via reduction performance decreases resistance of breast cancer cells and the malignant phenotype, as MZF-1 _60-72 endogenous MZF-1 competition Elk- 1 binding sites and reduces the activity of the endogenous DNA binding and protein kinase Cα performance, in triple negative breast cancer cells stably expressing MZF-1 _60-72 compared to non-performance MZF-1 _60-72 a control group of cells morphology More round, the result is shown in Figure 20. In addition, to reduce the MDA-MB-231 cells stably expressing MZF-1 _60-72 and protein kinase Cα cells of the MZF-1 expression level and Elk-1 concentrations remain relatively constant, to staining by immunofluorescence microscopy cross conjugate ( confocal microscopy) analyzes, Elk-1 / MZF-1 distributed mainly in the cytoplasm, and the control group were Elk-1 / MZF-1 evenly distributed in the cytoplasm and nucleus, consistent performance MZF-1 _60-72 of Elk-1 cells /MZF-1 is located in the cytoplasm, and the results are shown in Fig. 21, wherein N: represents a nucleus and C: represents a cytoplasm.

Immune blot (Immunoblotiing) analysis, breast cancer cells (Hs578T and MDA-MB-231) stably expressing MZF-1 _60-72, Elk-1 phosphorylation in the cell has not changed, but the nucleus (nuclear translocation) is inhibited, The result is shown in Figure 22. After treatment with cycloheximide and proteasome inhibitor MG132, MZF-1 is reduced due to increased protein degradation. As shown in Figure 23, these phenomena indicate two endogenous transcription factors, MZF- The interaction between 1 and Elk-1 promotes the stability of the MZF-1 protein and its entry into the nucleus.

In ChIP experiments analysis, stably expressing MZF-1 _60-72 triple negative breast cancer cells (MDA-MB-231) reduces -Elk-1 or anti-anti-amplification of -MZF-1 immunoprecipitated PRKCA promoter, as a result of Figure 24 shows. P- glycoprotein (P-glycoprotein, P-gp ) is the multidrug resistance associated proteins, stable expression MZF-1 _60-72 triple negative breast cancer cells (MDA-MB-231) of P-gp significantly reduced, as a result of Figure 25 shows. Can be known from the above results, MZF-1 _60-72 as 1 Elk-binding endogenous Elk 1-interrupted and the interaction between endogenous MZF-1, and inhibit their binding PRKCA promoter.

Stably expressing MDA-MB-231 cells MZF-1 _60-72 cisplatin (cisplatin) was treated with piperidine flavin general breast cancer drug (acriflavine), cell viability was measured and the results are shown in FIG. 25, flavin piperidine the half inhibitory concentration (IC ₅₀₎ of 1.49 m 2.43μM reduced, cisplatin by the IC ₅₀ fell 27.97μM 21.14μM, can be presumed from the above results, the inhibition of endogenous MZF1 _60-72 MZF-1 and Elk-1 post It acts to reduce binding to the PRKCA promoter, thereby reducing the expression of protein kinases Cα and P-gp and increasing sensitivity to chemotherapeutic drugs.

In MZF-1 _60-72 stably expressing breast cancer cells (MDA-MB-231 and HS578T) Cell migration distance compared with the control group decreased 80-90% (with significant), the results as shown in FIG. 26, but the cell growth No increase. Analysis MZF-1 _60-72 oncogenic efficacy in a breast cancer xenograft mouse model, the mice were injected stably expressing MZF-1 _60-72 transplanted breast cancer cells (MDA-MB-231-M (v4) and HS578T- M(s3)) formed tumors at a relatively slow rate, and the results are shown in Fig. 27 (top left panel). Stable performance MZF-1 MDA-MB-231 cells, the maximum tumor growth inhibition was 91.0% ± 5.2% (n = 5) 60-72 , and stable performance MZF-1 HS578T cells _60-72, tumor growth The maximum inhibition rate was 90.7% ± 4.6% (n = 5) of the control group HS578T cells. Stable performance MZF-1 Hs578T cells _60-72, the average tumor growth inhibition was 85.6% ± 4.1%, stable performance MZF-1 MDA-MB-231 cells _60-72, the average tumor growth inhibition was 84.4% ± 6.7 %, is injected into the MZF-1 _60-72 stably expressing cells also generate significant reduction of tumor weight, as the results shown in FIG. 27 (top right), the separation of tumor cells exhibit a number of interstitial tissue (interstitial tissue) a begins to form a tubular structure, as shown in the results (lower panel) Figure 27, outer layer of white blood cells found tumor invasion, tumor growth indicates restricted, from the results of inference, MZF-1 _60-72 Effect MZF-1 / Elk-1 Interaction and PRKCA translation activity, but have inhibitory effects on tumors.

A large number of peptides MZF-1 or Elk-1 are provided to inhibit endogenous MZF-1 or Elk-1 production, affect MZF-1/Elk-1 interaction and inhibit PRKCA translation activity, thereby inhibiting tumors.

Example 5

Inhibition of MZF-1/Elk-1 heterodimer formation to transform epithelial-mesenchymal transition (EMT)

Stability Analysis of expression in triple negative breast cancer cells MZF-1 _60-72 a HS578T-M (S3), and MDA-MB-231-M ( v4), for 22,203 genes, in HS578T-M (S3) cells, 1209 The gene expression showed a 2-fold increase, and 1557 genes showed a 2-fold decrease. As shown in Figure 28, in the expression of MDA-MB-231-M (v4) cells, there was a 2-fold increase in the expression of the 1272 gene. 1494 gene expression 2-fold decrease, two cells have both 821 gene showed increased 931 gene expression is reduced, the affected gene comprising EMT, such as 24 EMT-core-upregulated genes ( CDH11, CTGF, EMP3, FBN1, FN1, FSTL1, HAS2, LOX, MAP1B, MYL9, PLAT, PMP22, PRKCA, PTX3, RGS4, SERPINE1, - SERPINE2, SNAI2, SRGN, TFPI, TGM2, VIM, ZEB1, and ZEB2) decreased, 24 EMT-core-downregulated Genes ( AGR2 , ANK3 , CA2 , CD24 , CDH1 , CDS1 , CXCL16 , ELF3 , EPCAM , FGFR2 , FXYD3 , JUP , MAP7 , MPZL2 , MTUS1 , OCLN , PRRG4 , S100P , SLC27A2 , ST6GALNAC2 , TMEM30B , TPD52L1 , TSPAN1 , and ZHX2 Increase, PKCα, Slug ( SNAI2 ), Vimentin ( VIM ) protein significantly decreased, cadherin ( CDH1 ) As the protein mass increased, the amount of PKCδ protein did not change, and the results are shown in Fig. 29.

To verify the function of protein kinase Cα in EMT, the results are shown in Figure 30, MDA-MB-231-M (V4) and HS578T-M (S3) cells are transfected to express full-length protein kinase Cα, which exhibits protein kinase Cα. Cell migration rate increased significantly from 5% to 41% (MDA-MB-231-M(V4)), from 9% to 62% (Hs578T-M(s3) cells), EMT-related genes (Slug, Vimentin) , E-cadherin) The amount of performance increased, the results are shown in Figure 31.

These results suggest that the MZF-1/Elk-1 interaction is interrupted in cancer cells, and the negative regulation of protein kinase Cα is reduced, thus affecting EMT-related genes.

Example 6

Effect of TAT (HIV trans- activating regulatory protein) fusion peptide on the formation of MZF-1/Elk-1 heterodimer

HS578T, and MDA-MB-231 cells TAT- fusion peptide MZF-1 _60-72, or the fusion TAT- Elk-1 _145-157, sequence design as shown in FIG. 32 peptides, the cell migration is reduced, as a result As shown in Fig. 33, the EMT-related protein (PKCα, Slug and Vimentin) increased, and the mesenchymal-epithelial transition (MET)-related protein (E-cadherin) decreased, and the results are shown in Fig. 34. In terms of TAT- mutant fusion peptides (e.g., FIG. 16), does not affect protein expression by co-immunoprecipitation analysis revealed MZF-1 _60-72 and Elk-1 _145-157 reduction MZF-1 and Elk-1 binding There is concentration dependence, and the use of TAT-fusion mutant peptide will not affect, the results are shown in Figure 35.

From the results, it can be proved that TAT-fusion peptide inhibits the formation of MZF-1/Elk-1 heterodimer, and MZF-1 and Elk-1 interaction play an important role in tumor progression, and the interaction between MZF-1 and Elk-1 is suspended. Treat cancer effects.

In the present invention, cancer cells are found there MZF-1 and Elk-1 interact in the mechanism as shown in FIG. 36, and will bind to the promoter PRKCA; In terms of peptide MZF-1 or Elk-1 inhibition The endogenous peptide MZF-1 or Elk-1 can inhibit the interaction between MZF-1 and Elk-1, inhibit the translational activity of protein kinase Cα, and inhibit cancer; therefore, the peptide in the present invention inhibits the MZF unique to cancer cells. The interaction between -1 and Elk-1 further affects the expression of protein kinase Cα and has no effect on non-cancerous cells, thus having a better potential to solve cancer treatment problems than existing protein kinase Cα inhibitors.

The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention, and the equivalent implementations or modifications of the present invention should be included in the present invention. In the scope of patents.

The above-mentioned multiple functions are the statutory invention patents that fully meet the novelty and progressiveness. If you apply in accordance with the law, you are requested to approve the invention patent application.

<110> China Medical University

<120> Peptide inhibits the interaction between MZF-1 and Elk-1

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Claims (4)

One kind of peptide for preparing a pharmaceutical composition the treatment of breast cancer, the peptide which comprises at least one of the following, or any combination of: (A) peptide MZF-1 _60-72, the sequence of Ser Asp Leu Arg Ser Glu Gln Asp Pro Thr Asp Glu Asp (SEQ ID NO: 65); (B) peptide Elk-1 _145-157 , sequence of Leu Ala Arg Ser Ser Arg Asn Glu Tyr Met Arg Ser Gly (SEQ ID NO: 66); It has a breast cancer treatment effect by inhibiting the interaction of MZF-1 and Elk-1. The pharmaceutical composition may further comprise the pharmaceutically acceptable carrier, wherein the carrier comprises an excipient, a diluent, a thickener, a filler, a binder, and disintegration, as claimed in claim 1. Agents, lubricants, grease or non-greasy bases, surfactants, suspending agents, gelling agents, adjuvants, preservatives, antioxidants, stabilizers, colorants or perfumes. The use of the first aspect of the patent application, wherein the pharmaceutical composition is administered orally, by soaking, injecting, smearing or patching. As for the use of the first item of the patent application, the breast cancer is a triple-negative breast cancer.