CN116870167B

CN116870167B - A combined drug and drug composition for treating solid tumors

Info

Publication number: CN116870167B
Application number: CN202311074572.XA
Authority: CN
Inventors: 肖智雄; 易勇; 秦可为; 喻姝菡
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-08-24
Filing date: 2023-08-24
Publication date: 2024-11-08
Anticipated expiration: 2043-08-24
Also published as: CN116870167A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a combined medicine and a pharmaceutical composition for treating solid tumors. The present invention studies have found that activating the JNK-USP36-Snail1 signaling pathway is critical for the resistance of solid tumors to ribosomal inhibitors including homoharringtonine. Targeted inhibition of the JNK-USP36-Snail signaling pathway will enhance the sensitivity of solid tumor cells to ribosomal inhibitors. Based on the above, the invention provides a brand-new strategy for combining a ribosome inhibitor and a JNK-USP36-Snail pathway inhibitor to treat solid tumors, and can provide more choices for clinical treatment of cancer patients.

Description

Combination drug and pharmaceutical composition for treating solid tumors

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a combined medicine and a pharmaceutical composition for treating solid tumors.

Background

Ribosomes are intracellular protein synthesis centers. Tumor growth requires an increase in ribosome function to meet the protein synthesis required for its rapid growth. Inhibition of ribosomal function is therefore considered an important strategy for tumor treatment. Homoharringtonine (HHT) is a plant alkaloid separated from harringtonine, and is an efficient antitumor drug independently developed in China. HHT can bind to the ribosomal peptide transferase center and thus block protein synthesis. HHT is currently the only ribosome inhibitor for clinical cancer therapy and is widely used in the treatment of acute non-lymphoblastic leukemia. However, clinical studies have shown that solid tumors, including head and neck cancer, breast cancer, colorectal cancer, renal cancer, etc., do not respond to HHT treatment for reasons that are not yet clear. Therefore, the research on the molecular mechanism of the solid tumor cells against HHT has important significance for expanding the application of the HHT in tumor treatment.

Snail1 is an important transcription factor mainly localized in the cytoplasm, playing an important role in Epithelial Mesenchymal Transition (EMT), tumor metastasis and survival, metabolic reprogramming, tumor stem cell characteristics, and the like. However, whether Snial1 can be localized to nucleoli and participate in regulating ribosomal production has not been reported. The Snail1 protein is mainly degraded by ubiquitin-dependent proteasome pathways, so ubiquitination and de-ubiquitination regulation are key factors affecting the stability of the Snail1 protein. USP36 is a deubiquitinase specifically targeted to nucleoli, which promotes ribosomal rRNA transcription and processing by stabilizing B23, fibrin (FBL), and c-Myc proteins, etc.

At present, no other report is seen on the relation between the USP36-Snail1 related pathway and anti-tumor.

Disclosure of Invention

In view of the problems of the prior art, the present invention provides a combination and pharmaceutical composition for the treatment of solid tumors, aiming at the combined use of ribosomal inhibitors including homoharringtonine and JNK-USP36-Snail pathway inhibitors for the treatment of solid tumors.

Use of a JNK-USP36-Snail pathway inhibitor for the manufacture of a medicament for the elimination of a solid tumor resistant ribosomal inhibitor.

The invention also provides the use of a ribosome inhibitor in combination with a JNK-USP36-Snail pathway inhibitor for the manufacture of a medicament for the treatment of solid tumors.

Preferably, the ribosome inhibitor is selected from at least one of homoharringtonine, anisomycin, puromycin, G418 or blasticidin.

Preferably, the JNK-USP36-Snail pathway inhibitor is selected from at least one of a JNK inhibitor, a USP36 inhibitor or a Snail1 inhibitor.

Preferably, the JNK inhibitor is selected from at least one of SP600125, JNK-IN-8, JNK-930 or Bentamapimod; the USP36 inhibitor is selected from specific siRNA against USP 36; the Snail1 inhibitor is selected from specific sirnas for Snail 1.

Preferably, the solid tumor is lung cancer, head and neck cancer, breast cancer, colorectal cancer or renal cancer.

The present invention also provides a combination for the treatment of solid tumors, which is a ribosome inhibitor and JNK-USP36-Snail pathway inhibitor administered separately or simultaneously.

Preferably, the combination is for the treatment of lung cancer, head and neck cancer, breast cancer, colorectal cancer or renal cancer.

The invention also provides a pharmaceutical composition which is prepared by taking a ribosome inhibitor and a JNK-USP36-Snail pathway inhibitor as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Preferably, the ribosome inhibitor and JNK-USP36-Snail pathway inhibitor are used in a weight ratio of 0.5:7.5-3:25.

The invention discovers that activating the JNK-USP36-Snail1 signaling pathway is key to resisting ribosome inhibitors including homoharringtonine of solid tumors through research. Further using cell and animal model studies found that: 1) The JNK inhibitor SP600125 and the ribosome inhibitor can be combined to obviously inhibit the survival of in vitro tumor cells and the growth of in vivo solid tumors; 2) Silencing USP36 or Snail1 using RNAi interference techniques can significantly promote ribosome inhibitors mediated in vitro tumor cell death and in vivo tumor growth inhibition. The experimental results show that: targeted inhibition of JNK-USP36-Snail signaling will enhance the sensitivity of solid tumor cells to ribosomal inhibitors; therefore, the invention provides a brand-new strategy for combining a ribosome inhibitor and a JNK-USP36-Snail pathway inhibitor to treat solid tumors, and can provide more choices for clinical treatment of cancer patients.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 shows that HHT promotes nucleolus aggregation of Snail1 protein. (A) The indicated cells were treated with 20ng/mL HHT for 24 hours and then subcellular localization of the Snail1 protein was detected by immunofluorescent staining, B23 as nucleolar marker, DAPI as nuclear dye, scale bar 25 μm. (B) Co-localization of the Snail1 and B23 proteins in (A) was calculated using Pearson's correlation coefficient correlation coefficients, and 40 cells from three independent experiments were used for this calculation. (C) HCC1806 cells were treated with 20ng/mL HHT for 24 hours, then the cell fractions (CP, cytosol; NP, nucleoplasm; no, nucleolus) were isolated and the expression of Snail1 protein in each fraction was examined by immunoblotting. Tubulin is a marker of cytoplasms and SP1 is a marker of nucleoplasms.

FIG. 2 shows that HHT promotes nucleolus aggregation of Snail1 protein by increasing USP36 expression. (A) Cells were treated with 20ng/mL HHT for 24 hours, then 50 μg/mL protein synthesis inhibitor Cycloheximide (CHX) for the indicated time, and then immunoblotted for detection of Snail1 protein expression. Quantitative analysis is carried out on the Snail1 protein, and then a half-life map of the Snail1 protein is drawn. (B) HCC1806 cells were treated with 20ng/mL HHT, 20nM mTOR inhibitor RAPAMYCIN (RAP), 200ng/mL ribosomal inhibitor puromycin (Puro), and 2 μg/mL ribosomal inhibitor blasticidin (Blasti) for 24 hours, and then immunoblotted for USP36 and Snail1 protein expression. (C) Wild-type USP36 (USP 36-WT) or USP36 deubiquitinase-inactivated mutant USP36-C131A was stably expressed in HCC1806 and SUM159 cells and then immunoblotted for detection of expression of Snail1 protein. (D) The expression of the Snail1 protein was examined by immunoblotting after the replacement of USP36-WT or USP36-C131A in HCC1806 cells knocked down with USP36 (shUSP). (E) Stable expression of USP36-WT or USP36-C131A in HCC1806 cells followed by immunofluorescent staining to detect Snail1 protein subcellular localization. (F) HCC1806 cells knocked down (shUSP) or not knocked down (shGFP) USP36 were treated with 20ng/mL HHT for 24 hours and then immunofluorescent staining was performed to detect Snail1 protein subcellular localization. The scale bar is 25 μm.

FIG. 3 shows that USP36 is the deubiquitinase of nucleolus Snail 1. (A) The immunoprecipitation assay detects endogenous USP36 and Snail1 protein complex formation in nucleoli. (B) Wild-type USP36 (USP 36-WT) or deubiquitinated enzyme-inactivated mutant USP36-C131A was stably expressed in HEK-293 cells, and then the cells were treated with 50. Mu.g/mL of the protein synthesis inhibitor Cycloheximide (CHX) for the indicated time, and then immunoblots were performed to detect the expression of the Snail1 protein, quantitatively analyze the Snail1 protein, and then map the half-life of the Snail1 protein. (C) USP36 (shUSP) was knocked down in HCC1806 cells, then the cells were treated with 50. Mu.g/mL protein synthesis inhibitor Cycloheximide (CHX) for the indicated time, then immunoblotted to detect the expression of the Snail1 protein, quantitative analysis of the Snail1 protein was performed, and then a Snail1 protein half-life profile was drawn. (D) The effect of USP36-WT or USP36-C131A on ubiquitination of Snail1 protein was examined by co-immunoprecipitation experiments. (E) The effect of knockdown USP36 (shUSP) on ubiquitination of Snail1 protein was examined using a co-immunoprecipitation assay. (F) The effect of USP36 on ubiquitination of the indicated Snail1 protein was examined by co-immunoprecipitation experiments. (G-H) expression of wild type Snail1 (Snail 1-WT) or Snail1-K146R/K206R mutant (Snail 1-2 KR) stably in HEK-293 cells, treatment of the cells with 50. Mu.g/mL protein synthesis inhibitor Cycloheximide (CHX) for a period of time indicated, followed by immunoblotting to detect Snail1 protein expression (G); the Snail1 protein was quantitatively analyzed, and then a Snail1 protein half-life profile (H) was drawn.

FIG. 4 shows that lysine 157 (K157) on the Snail1 protein is a key site for the formation of the complex of the Snail1-USP36 protein and the localization of the nucleolus of Snail 1. (A) ZDOCK predict the key amino acid residues that Snail1 binds to USP 36. (B) Co-immunoprecipitation detects binding of wild type Snail1 (Snail 1-WT) or Snail1-K157R to USP36 in nucleoli. (C-D) cell fraction isolation experiments the effect of USP36 or HHT on the expression of Snail1 protein in nucleoli (CP, cytosol; NP, nucleoplasm; no, nucleoli). (E-F) immunofluorescent staining the effect of USP36 or HHT on the subcellular localization of the Snail1 protein. The scale bar is 25 μm.

FIG. 5 is a graph showing that HHT upregulates USP36 expression by activating JNK. (A) HCC1806 cells were treated with 20ng/mL HHT for 24 hours, and then USP36 mRNA expression was detected using fluorescent quantitative PCR. (B-C) HCC1806 cells were treated with 20ng/mL HHT alone or in combination with 20. Mu.M JNK inhibitor SP600125 for 24 hours, and then examined for USP36 protein and mRNA expression by immunoblotting or fluorescent quantitative PCR. (D) HCC1806 cells were treated with 20ng/mL HHT alone or in combination with 20 μm SP600125 for 24 hours, and then the cell fractions (CP, cytosol; NP, nucleoplasm; no, nucleolus) were isolated and the expression of Snail1 protein in each fraction was examined by immunoblotting. Tubulin is a marker of cytoplasms and SP1 is a marker of nucleoplasms.

Fig. 6 shows that gene silencing Snail1 or USP36 significantly promotes HHT mediated in vitro tumor cell death and in vivo tumor growth inhibition. (A-B) the wild-type Snail1 (Snial-WT) or the Snail1 mutant Snail1-K157R with loss of nucleolar localisation was back-filled in the HCC1806 cells knocked down with Snail1, HCC1806 cells were treated with 20ng/mL HHT for 24 hours, and then (1) the protein expression profile shown was examined by immunoblotting (A); (2) detecting apoptosis (B) by flow cytometry. (C) 5×10 ⁵ of the indicated HCC1806 cells were inoculated subcutaneously (n=5/group) on the right neck of female BALB/c nude mice; on day 3 post inoculation, HHT (1 mg/kg, daily) was injected intraperitoneally; mice were sacrificed on day 14 and tumor photographs were taken (C) and each tumor weight was detected (D). (E-F) supplementing the cells with either Snail1-WT or Snail1-K157R in HCC1806 cells knocked down with USP36, treating the cells with 20ng/mL HHT for 24 hours, and then (1) detecting the expression of the indicated proteins by immunoblotting; (2) detecting apoptosis by using a flow cytometer.

Fig. 7 shows that the combination of JNK inhibitor SP600125 and HHT significantly inhibited tumor cell survival in vitro and tumor growth in vivo. (a-B) treating the indicated cells with 20ng/mL HHT alone or in combination with 20 μm JNK inhibitor SP600125 for 24 hours, followed by (1) detecting the indicated protein expression by immunoblotting (a); (2) detecting apoptosis (B) by flow cytometry. (C-D) 5×10 ⁵ HCC1806 cells were inoculated subcutaneously (n=5/group) in the right neck of female BALB/C nude mice; on day 3 post inoculation, the abdominal cavity was injected with HHT (1 mg/kg) and SP600125 (15 mg/kg) either alone or in combination per day; mice were sacrificed on day 17 and tumors were removed and each tumor weight was examined (C); immunoblots were used to detect the expression of the proteins shown in each tumor tissue.

Fig. 8 is a graph showing that the combination JNK inhibitor SP600125 and the ribosome inhibitor anisomycin or blasticidin significantly inhibited tumor cell survival in vitro. (A) HCC1806 cells were treated with 50ng/mL anisomycin (Aniso), 200ng/mL puromycin (Puro), 1 μg/mL G418, or 2 μg/mL blasticidin (Blasti) for 24 hours, then subcellular localization of the Snail1 protein was detected using immunofluorescent staining, B23 as nucleolar marker, DAPI as nuclear dye, and scale bar 25 μm. (B) Co-localization of the Snail1 and B23 proteins in (A) was calculated using Pearson's correlation coefficient correlation coefficients, and 40 cells from three independent experiments were used for this calculation. (C) The indicated HCC1806 cells were treated with ribosomal inhibitor HHT (20 ng/mL), anisomycin (50 ng/mL), puromycin (200 ng/mL), G418 (1. Mu.g/mL), or blasticidin (2. Mu.g/mL) for 24 hours, then the nuclear localization of the Snail1 protein was detected using immunofluorescent staining, about 200 cells in each group were randomly selected, and then the percentage of nucleolar Snail1 positive cells was counted. (D-E) treating the indicated cells with 20ng/mL HHT, 50ng/mL anisomycin (Aniso), 2 μg/mL blasticidin (Blasti) alone or in combination with 20 μM JNK inhibitor SP600125 for 24 hours, and then (1) detecting the indicated protein expression by immunoblotting (D); (2) detecting apoptosis (E) by flow cytometry.

Detailed Description

In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available.

EXAMPLE 1 combination of homoharringtonine and SP600125 for the treatment of cancer

The embodiment provides a combination drug of homoharringtonine and SP600125, which can be used simultaneously or separately, wherein the dosage ratio of homoharringtonine to SP600125 is as follows: 1:15.

As a preferred embodiment, the combination is 1mg/kg of aqueous homoharringtonine and 15mg/kg of aqueous SP600125, each independently used.

In other embodiments, the homoharringtonine of this embodiment can be replaced with other ribosome inhibitors, such as: anisomycin, puromycin, G418 or blasticidin.

The technical scheme of the invention is further described through experiments.

Experimental example 1 molecular mechanism of solid tumor cells against HHT

1. Experimental method

1. Cell culture

A549 (human non-small cell lung cancer), hs 578T (human breast cancer), HCC1806 (human breast squamous carcinoma) and SUM159 (human breast cancer) cells were cultured in DMEM medium (Gibco, rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS, hyClone, logan, UT, USA), 100 units/mL penicillin (Gibco, rockville, MD, USA) and 100 μg/mL streptomycin (Gibco, rockville, MD, USA). Cells were grown in a humidified incubator with 5% CO2 at 37 ℃.

2. Apoptosis and cell viability assays

Cells were cultured in 6-well plates, and when the cell rate was about 70%, cells were treated with 20ng/mL of 20. Mu.M JNK inhibitor SP600125 (S1460, houston, USA) alone or in combination for 24 hours, then incubated with Annexin V-FITC and PI (Beyotime, C1052, china), and analyzed for apoptosis by flow cytometry (FACScan, BD Biosciences, USA); for cell viability, MTS assay was performed using CellTiter 96 kit (Promega, USA).

3. Cell fraction isolation and in vivo Snail1 ubiquitination assay

Separation of cellular components of cytoplasm, nucleoplasm and nucleolus: cells were collected, washed twice with cold PBS, resuspended in 1mL buffer A (10 mM Tris-HCl pH 7.8;10mM KCl;1.5mM MgCl2;0.5mM DTT) and placed on ice for 10 minutes; then, at 4 ℃,228g was centrifuged for 5 minutes, and the supernatant was a cytoplasmic fraction. The cell pellet was washed with buffer A and then layered with 1mL of buffer S1 (0.25M sucrose; 10mM MgCl2) and centrifuged at 1430g for 10min at 4℃on 1mL of buffer S2 (0.35M sucrose, 0.5mM MgCl2). Resuspending the clean cell pellet in 0.5mL buffer S2; the samples were sonicated in an ice bath at 20% full power for 12X 10 seconds (10 seconds rest between each sonication) to prevent overheating, then 0.5mL buffer S3 (0.88M sucrose; 0.5mM MgCl2) was added. Centrifuge at 3,000g for 20 min at 4 ℃. The supernatant (nucleolus fraction) and pellet (nucleolus fraction) were collected. Nucleolus was resuspended in 0.5mL buffer S1 and 0.5mL 0.35m buffer S2; centrifugation was carried out at 2500rpm (1430 g) for 10 minutes at 4 ℃. For Western blot analysis, insoluble fractions containing nucleoli were lysed in 1 XSDS sample buffer (# 7722, CST), if necessary sonicated. In a co-immunoprecipitation experiment, nucleoli was lysed in high salt RIPA buffer (50mM Tris pH 7.5,500mM NaCl,1%Nonidet P-40,0.5% deoxycholate and proteasome inhibitor).

For the Snail1 protein ubiquitination experiments, the collected cells were lysed in pre-boiled denaturing cell lysis buffer (50 mM pH7.4 Tris-HCl,70mM beta-ME, freshly added beta-ME). Cell lysates were boiled for 10 min, and 4 volumes of dilution buffer (20 mM pH7.4 Tris-HCl,300mM NaCl,1mM EDTA,1mM EGTA,1% Triton X-100,2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na3VO4, 1. Mu.g/mL pancreatic peptide) were added. Cell lysates were centrifuged at 12000g for 30min, and then immunoprecipitated and western blot analyzed.

4. Western blot, co-immunoprecipitation and immunofluorescent staining analysis

For western blot analysis, cells were collected, washed twice with cold PBS, and then lysed in 1x SDS sample buffer (# 7722,Cell Signaling Technology,USA). Equal amounts of proteins were loaded into SDS-PAGE gels and electrophoretically separated, and then proteins were transferred onto PVDF membranes (Millipore, darmstadt, germany). Membranes were blocked in 4% skim milk, then hybridized to primary antibody and horseradish peroxidase (HRP) conjugated secondary antibody, followed by detection by chemiluminescence (Bio-Rad). Analysis of gel and blot images using Image Lab Software 5.0.0 antibodies to .Snail1(#3895,1:1000),GAPDH(#5174,1:2000),p38(#9212,1:1000),p-p38(#9216,1:1000),JNK(#9252,1:1000),p-JNK(#9251,1:1000),Tubulin(#3873,1:1000),SP1(#5931,1:1000),HA-Tag(#5017,1:1000),Cleaved-caspase 3(#9654,1:1000) and Flag-Tag (# 8146, 1:1000) were purchased from CELL SIGNALING Technology (Danvers, MA, USA). USP36 antibody (14783-1-AP, 1:1000) was purchased from Proteintech (Chicago, IL, USA). B23 antibody (MA 5-12508, 1:1000) was purchased from ThermoFisher Scientific (Wilmington, DE, USA).

For endogenous Co-immunoprecipitation (Co-IP), nucleoli is lysed in high salt RIPA buffer (50mM Tris pH 7.5,500mM NaCl,1%Nonidet P-40,0.5% deoxycholate and proteasome inhibitor). Cell lysates were centrifuged at 12000G for 30min, and an equivalent amount of total protein was incubated with primary or normal IgG overnight at 4℃and then 30. Mu.l of protein A/G beads were added and incubated for a further 2h. For exogenous Co-IP, anti-HA (or anti-Flag) beads were added to an equal amount of protein solution for overnight incubation, then the beads were centrifuged (500 g,30 s) and washed three times with wash buffer (20 mM Tris-HCl,250mM NaCl,0.2mM EGTA,0.1%Nonidet P-40). Before western blot analysis, heat was applied at 100℃for 10 minutes. Anti-FLAG M2 affinity gel (A2220) was purchased from Sigma-Aldrich (St. Louis, USA). PIERCE ANTI-HA magnetic beads (# 88836) were purchased from Thermo FISHER SCIENTIFIC (Waltham, mass., USA).

Immunofluorescent staining: cells grown on coverslips were fixed with 4% paraformaldehyde, cells were perforated with 0.1% Triton X-100, blocked with 4% bovine serum albumin, and then the cells were hybridized (snail1:50,sc-271977,Santa Cruz Biotechnology(CA,USA);USP36:1:4000,14783-1-AP,Proteintech;B23:1:50,MA5-12508,ThermoFisher;HA-Tag:1:1000,#5017,CST),Flag-Tag:1:1000,#8146,CST), with the appropriate primary antibody followed by incubation with secondary antibodies (goat anti-mouse Alexa Fluor 488, a-11029 or goat anti-rabbit Alexa Fluor 514, a-31558, thermoFisher) with DAPI (# 82961, CST)Gold ANTIFADE REAGENT counterstained cells and photographed using a Leica TCS SP5II confocal laser scanning microscope. To determine that Snail1 is co-localized with nucleolus B23 or USP36, the dual fluorescence correlation coefficients are calculated using free software Image J.Fiji in combination with Coloc plug-ins and Pearson correlation coefficients, and the co-localized fluorescence is given by a scatter plot.

2. Experimental results

1. HHT promotes nucleolus aggregation of Snail1 protein

Snail1 is a key transcription factor localized in the nuclear cytoplasm and plays an important role in regulating biological processes such as epithelial-mesenchymal transition (epithelial-to-MESENCHYMAL TRANSITION, EMT), cell survival, metabolic reprogramming, and tumor cell stem properties. To investigate the role of Snail1 in solid tumor cell resistance to HHT, the present experimental example first examined the effect of HHT on Snail1 protein subcellular localization using immunofluorescent staining. As shown in fig. 1A-B, HHT significantly promotes co-localization of the Snail1 protein with nucleolar marker B23 in breast cancer cells Hs578T, SUM159 and lung cancer cells a 549. In addition, cell fraction isolation experiments also showed that HHT significantly promoted Snail1 protein expression in HCC1806 nucleoli in breast cancer cells (fig. 1C). The above results indicate that HHT can significantly promote the aggregation of Snail1 protein in nucleolus.

2. HHT promotes nucleolus aggregation of Snail1 protein by increasing USP36 expression

Next, this experimental example explores the molecular mechanism by which HHT promotes nucleolus aggregation of Snail1 protein. Through protein half-life analysis experiments, HHT was found to significantly increase the stability of the Snail1 protein (FIG. 2A). Thus, it is speculated that HHT might promote its aggregation in nucleoli by increasing Snail1 protein stability. USP36 is a deubiquitinase specifically localized in the nucleolus. This experimental example found that HHT significantly increased the expression of USP36 and Snail1 proteins (fig. 2B). Heterologous expression of wild-type USP36 (USP 36-WT) significantly increased expression of Snail1, but USP36 de-ubiquitination enzyme activation inactivating mutant USP36-C131A had no significant effect on Snail1 protein expression (figure 2C). In addition, knocking down USP36 also significantly reduced Snail1 protein expression (fig. 2D). The recruitment USP36-WT was able to fully restore knockdown USP 36-mediated reduction of Snail1 protein, but the recruitment USP36-C131A was unable to achieve this (FIG. 2D). The results indicate that USP36 positively regulates Sanil protein expression and that this process is dependent on the deubiquitinase activity of USP 36. Further, the effect of USP36 on nucleolus localization of Snail1 protein was examined using immunofluorescent staining. As shown in FIG. 2E, the heterologous expression of USP36-WT significantly promoted the aggregation of Snail1 protein in nucleolus, but USP36-C131A had no significant effect on the location of Snail1 protein. Importantly, knocking down USP36 significantly inhibited HHT mediated Snail1 protein nucleolus aggregation. Taken together, the results indicate that HHT promotes nucleolar aggregation of Snail1 protein by up-regulating USP36 expression.

3. USP36 is deubiquitinase of nucleolus Snail1

The above studies indicate that HHT promotes Snail1 protein nucleolus aggregation by increasing USP36 expression. It was further investigated whether USP36 is a deubiquitinase for nucleolus Snail1 protein, as shown in fig. 3A, USP36 forms a stable protein complex with Snail1 in nucleolus. Heterologous expression of USP36-WT significantly increased Snail1 protein stability, but the deubiquitinated enzyme-activated inactivating mutant USP36-C131A had no significant effect on Snail1 protein stability (fig. 3B). Conversely, knocking down USP36 significantly reduced Snal1 protein stability (fig. 3C). Next, the effect of USP36 on ubiquitination of Snail1 protein was examined. As shown in FIG. 3D, the heterologous expression of USP36-WT significantly inhibited ubiquitination of the Snail1 protein, while USP36-C131A had no significant effect on ubiquitination of the Snail1 protein. Knocking down USP36 significantly increased ubiquitination of Snail1 protein (fig. 3E). Further, it was found by analysis of the point mutation technique that lysine 146 (K146) and lysine 206 (K206) on the Snail1 protein are deubiquitination sites of USP 36. As shown in FIG. 3F, the heterologous expression USP36 significantly reduced ubiquitination of wild-type Snail1 (Snail 1-WT) proteins, but had no significant effect on Snail1-K146R/K206R (Snail 1-2 KR) ubiquitination. The protein half-life experimental analysis shows that the heterologous expression USP36 significantly increases the stability of the Snail1-WT protein, but has no obvious effect on the stability of the Snail1-2KR protein (FIG. 3G-H). Taken together, the study of this experimental example shows that (1) USP36 is a deubiquitinase of nucleolus Snail1 protein; (2) USP36 stabilizes the Snail1 protein by removing ubiquitin chains on Snail1 proteins K146 and K206.

4. Lysine 157 (K157) on the Snail1 protein is a key site for the formation of the complex of the Snail1-USP36 protein and the localization of the nucleolus of Snail1

Bioinformatic predictions suggest that lysine 157 (K157) on the Snail1 protein may be a critical amino acid residue for the formation of the Snail1-USP36 protein complex (FIG. 4A). Indeed, analysis by point mutation and co-immunoprecipitation experiments revealed that wild type Snail1 protein forms a stable protein complex with USP36 in nucleolus, but Snail1-K157R does not form a protein complex with USP36 (fig. 4B). Further analysis by cell fraction isolation and immunofluorescence staining experiments revealed that neither treatment with either over-expressed USP36 nor HHT promoted nucleolus aggregation of the Snail1-K157R proteins (FIGS. 4C-F). The results indicate that K157 on the Snail1 protein is a key amino acid residue for regulating the formation of the Snail1-USP36 protein complex and affecting the location of the nucleolus of Snail 1.

5. HHT upregulates USP36 expression by activating JNK

Next, the molecular mechanism of HHT up-regulation of USP36 expression was resolved: fluorescent quantitative PCR analysis showed that HHT significantly increased mRNA expression levels of USP36 gene (fig. 5A). HHT was suggested to promote USP36 transcription. Studies have shown that HHT can significantly activate JNK signaling pathways, thereby regulating transcription of genes. We speculate that HHT may promote USP36 expression through JNK signaling pathways. As shown in fig. five B-C, HHT significantly activated JNK, while JNK-specific inhibitor SP600125 significantly inhibited HHT-mediated increase in USP36 protein and mRNA expression levels. In addition, SP600125 also significantly inhibited HHT-mediated nucleolus aggregation of Snail1 protein (fig. 5D). The above results indicate that HHT upregulates USP36 expression and Snail1 nucleolus aggregation by activating JNK.

In conclusion, the research of the experimental example shows that HHT can significantly activate JNK so as to up-regulate nucleolus deubiquitinase USP36 expression; increased USP36 promotes nucleolar aggregation and tumor cell survival by increasing the stability of nucleolar Snail1 protein. Thus, activation of the JNK-USP36-Snail1 signaling pathway is critical for the resistance of solid tumors against HHT.

Experimental example 2 Gene silencing Snail1 or USP36 significantly promotes HHT-mediated ex vivo tumor cell death and in vivo tumor growth inhibition

1. Experimental method

1. Lentiviral infection and RNA interference

Lentivirus-based shRNA targeting Snail1(#1:5′-CCACTCAGATGTCAAGAAGTA-3'(SEQ ID NO.1);#2:5′-CCAGGCTCGAAAGGCCTTCAA-3′(SEQ ID NO.2))、USP36(#1:5′-GCGGTCAGTCAGGATGCTATT-3′(SEQ ID NO.3);#2:5′-ACAGAACATCCAACGTCTTAA-3′(SEQ ID NO.4)) or green fluorescent protein (GFP, 5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID No. 5)) was constructed into plko.1 plasmid. pMD2G and psPAX packaging plasmid and pLKO.1 plasmid were transfected into HEK-293FT cells with 70% confluence using Lipofectamine 2000 (Invitrogen, carlsbad, calif., USA) and lentiviruses were collected 60 hours after transfection. And (3) infecting target cells with the confluence of 60% by using recombinant lentivirus under the condition of 10 mug/mL polystyrene, and finally obtaining the stable cell strain with the knockdown target gene.

2. Apoptosis and cell viability assays

HCC1806 (human breast squamous carcinoma) cells were cultured in 6-well plates, and when the cell synthesis rate was about 70%, cells were treated with 20ng/mL of JNK inhibitor SP600125 (S1460, houston, USA) alone or in combination for 24 hours, then incubated with Annexin V-FITC and PI (Beyotime, C1052, china), and analyzed for apoptosis by flow cytometry (FACScan, BD Biosciences, USA); for cell viability, MTS assay was performed using CellTiter 96 kit (Promega, USA).

3. Animal model

Animal experiments of the study are all carried out according to national legislation and institutional ethical guidelines of China and are approved by the institutional review board of Sichuan university. Female BALB/c nude mice (BALB/cNj-Foxn 1 nu/Gpt) were purchased from GEMPHARMATECH (Chengdu China). 5X 10 ⁵ of the indicated HCC1806 cells were inoculated subcutaneously in the right neck of 6 week old female BALB/c nude mice (n=5/group). Mice were intraperitoneally injected with HHT (1 mg/kg) daily on day 3 post-inoculation. Tumor growth in mice was dynamically observed and mice were sacrificed on day 14 post-dose, tumors removed and tumor volume and weight were determined.

2. Experimental results

The experimental example explores the role of the JNK-USP36-Snail1 pathway in the resistance of solid tumor cells to HHT. As shown in fig. 6A-B, knocking down Snail1 significantly promoted HHT-mediated clear-caspase 3 (CC 3, apoptosis marker) expression and apoptosis, a process that could be recovered by wild-type Snail1 expressed in nucleolus (Snail 1-WT), but not by Snail1-K157R that lost nucleolus localization. Importantly, knocking down Snail1 also significantly promoted the inhibition of tumor growth in vivo by HHT, a process that was also recovered by Snail1-WT but not by Snail1-K157R (fig. 6C-D). Furthermore, consistent with knockdown of Snail1, knockdown of USP36 also significantly promoted HHT-mediated CC3 expression and apoptosis, which was also restored by Snail1-WT, but not by Snail1-K157R (fig. six E-F). The above results indicate that (1) gene silencing Snail1 or USP36 significantly promotes HHT mediated tumor cell death in vitro and tumor growth inhibition in vivo; (2) nucleolus Snail1 is critical for the resistance of tumor cells to HHT.

Experimental example 3 significant inhibition of in vitro tumor cell survival and in vivo tumor growth in combination with JNK inhibitor SP600125 and HHT

1. Experimental method

1. Apoptosis and cell viability assays

Hs 578T (human breast cancer), HCC1806 (human breast squamous carcinoma) and SUM159 (human breast cancer) cells were cultured in 6-well plates, and when the cell rate was about 70%, cells were treated with 20ng/mL HHT alone or in combination with 20. Mu.M JNK inhibitor SP600125 (S1460, houston, USA) for 24 hours, then incubated with Annexin V-FITC and PI (Beyotime, C1052, china), and the flow cytometer analyzed for apoptosis (FACScan, BD Biosciences, USA); for cell viability, MTS assay was performed using CellTiter 96 kit (Promega, USA).

2. Animal model

Animal experiments of the study are all carried out according to national legislation and institutional ethical guidelines of China and are approved by the institutional review board of Sichuan university. Female BALB/c nude mice (BALB/cNj-Foxn 1 nu/Gpt) were purchased from GEMPHARMATECH (Chengdu China). 5×10 ⁵ HCC1806 cells were inoculated subcutaneously in the right neck of 6 week old female BALB/c nude mice (n=5/group). On day 3 post inoculation, mice were intraperitoneally injected daily with DMSO (5% V/V) or SP600125 (15 mg/kg) and/or HHT (1 mg/kg). Mice were monitored daily for tumor size, sacrificed on day 17 after intraperitoneal administration, tumors removed and tumor weights were determined.

2. Experimental results

The experimental example explores the role of JNK in the resistance of solid tumor cells against HHT. As shown in fig. 7A-B, the combination of HHT and SP600125 significantly promoted clear-caspase 3 (CC 3) expression and apoptosis compared to HHT or JNK inhibitor SP600125 alone. Importantly, the combination of HHT and SP600125 significantly promoted expression of CC3 in tumor tissue in vivo and significantly inhibited tumor growth in vivo (fig. 7C-D). The results indicate that the combined JNK inhibitors SP600125 and HHT are potential new strategies for tumor treatment.

Experimental example 4 significant inhibition of tumor cell survival in vitro by combination of JNK inhibitor SP600125 and ribosomal inhibitor anisomycin or blasticidin

1. Experimental method

1. Apoptosis assay

HCC1806 (human breast squamous carcinoma) cells were cultured in 6-well plates, and when the cell synthesis rate was about 70%, the cells were treated with 20ng/mL HHT, 50ng/mL anisomycin, 200ng/mL puromycin, 1 μg/mL G418, 2 μg/mL blasticidin alone or in combination with 20 μm JNK inhibitor SP600125 (S1460, houston, USA) for 24 hours, then incubated with Annexin V-FITC and PI (Beyotime, C1052, china), and the flow cytometer analyzed for apoptosis (FACScan, BD Biosciences, USA).

2. Experimental results

The experimental example explores the role of JNK in the resistance of solid tumor cells to ribosomal inhibitors anisomycin or blasticidin. As shown in fig. 8A-C, consistent with HHT, the ribosome inhibitors anisomycin (Aniso), puromycin (Puro), G418, or blasticidin (Blasti) all significantly promote nucleolar localization of Snail1 protein. Importantly, JNK inhibitors SP600125 and anisomycin or blasticidin significantly promoted intracellular CC3 expression and significantly promoted apoptosis (fig. 8D-E).

The results of this experimental example show that the mechanism of the solid tumor against various ribosome inhibitors is the same, namely, the solid tumor is realized through a JNK-USP36-Snail signaling pathway. Thus, combining JNK-USP36-Snail pathway inhibitors with ribosomal inhibitors is a potential new strategy for tumor treatment.

From the above experimental examples, it can be seen that targeted inhibition of JNK-USP36-Snail signaling pathway will enhance the sensitivity of solid tumor cells to ribosomal inhibitors; the combination of ribosome inhibitors and inhibitors of the JNK-USP36-Snail pathway is a novel strategy for the treatment of solid tumors. The invention provides a new choice for clinical treatment of solid tumors and has good application prospect.

Claims

1. Use of a ribosome inhibitor in combination with a JNK-USP36-Snail pathway inhibitor for the manufacture of a medicament for the treatment of breast cancer, characterized in that: the ribosome inhibitor is selected from homoharringtonine;

The JNK-USP36-Snail pathway inhibitor is at least one of shRNA-Snail1, shRNA-USP36 and SP 600125;

The shRNA-Snail1 nucleotide sequence is at least one of SEQ ID NO. 1-SEQ ID NO. 2;

The shRNA-USP36 nucleotide sequence is selected from at least one of SEQ ID NO. 3-SEQ ID NO. 4.

2. Use of a ribosome inhibitor in combination with a JNK-USP36-Snail pathway inhibitor for the manufacture of a medicament for the treatment of breast cancer, characterized in that: at least one ribosome inhibitor selected from anisomycin, blasticidin;

the JNK-USP36-Snail pathway inhibitor is selected from SP600125.