CN111420059B - Medicine composition for overcoming drug resistance of liver cancer and kidney cancer tumors and application thereof - Google Patents

Abstract

The invention discloses a pharmaceutical composition for overcoming drug resistance of liver cancer and kidney cancer tumors and application thereof. The research of the invention shows that the acetylase inhibitor Anacardic Acid (AA) and the EZH2 inhibitor GSK-126 are combined for use, the drug combination jointly enhances the tumor sensitivity of the EZH2 inhibitor from two aspects of reducing the self stability of the EZH2 and inhibiting the activity of the EZH2, the effect is obvious, and a new method and a new thought are provided for improving the drug effect and the application range of the EZH2 inhibitor. Experiments show that the combined use of the acetylase inhibitor and the EZH2 inhibitor has stronger killing effect on hepatocellular carcinoma and renal clear cell carcinoma cells, the effect is obviously better than that of the single use of the EZH2 inhibitor, and the combined use of the acetylase inhibitor and the EZH2 inhibitor can play a role in synergy, so that a new method is provided for improving the drug effect and the application range of the EZH2 inhibitor.

Description

Medicine composition for overcoming drug resistance of liver cancer and kidney cancer tumors and application thereof

Technical Field

The present invention belongs to the field of biomedicine technology. More particularly, relates to a drug combination for overcoming drug resistance of liver cancer and kidney cancer tumors and application thereof.

Background

Among the globally prevalent human malignancies, both the morbidity and mortality of hepatocellular carcinoma (HCC) and renal clear cell carcinoma (ccRCC) are at the forefront. However, the current drug treatment for the two tumors has the problem of drug resistance, which is the main reason for the frequent recurrence of the tumors.

Targeted therapy is an important conventional therapeutic approach in cancer treatment. The research, development and application of targeted drugs are the main content and purpose of targeted therapy. EZH2(enhancer of zeste homolog 2) and SUZ12, EED jointly form PRC2(Polycomb regenerative Complex 2) protein Complex, and the Complex carries out trimethylation (H3K27me3) modification on the 27 th lysine of histone 3 mainly through the methyltransferase activity of EZH2, thereby mediating gene silencing. Research finds that EZH2 is highly expressed in various solid tumors such as hepatocellular carcinoma, renal clear cell carcinoma and the like, is closely related to the occurrence and development of tumors, and plays an important role in promoting the proliferation, the diffusion and the metastasis of tumor cells. In clinical tissue samples of liver and kidney, the expression of EZH2 in cancer tissues is also found to be abnormally increased, and the total survival time of hepatocellular carcinoma and renal clear cell carcinoma can be obviously reduced, namely the prognosis of patients with high EZH2 expression is poor, so the expression level of EZH2 has been clinically used as one of important indexes for judging the prognosis of malignant tumor patients. In cytological experiments, the knocking-down expression level of EZH2 can obviously inhibit the growth of a plurality of liver and kidney tumor cells, and the tumorigenic capacity of the nude mice is also obviously reduced. There are many inhibitors developed by targeting histone methyltransferase EZH2 currently entering clinical trials (e.g., GSK-126), which have some effect in treating hematological tumors, whereas EZH2 inhibitors have poor therapeutic effect on solid tumors in clinical trials.

Therefore, improving the response rate of solid tumors to EZH2 inhibitors and expanding the clinical applications of EZH2 inhibitors in solid tumors are important research directions.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the drug resistance of hepatocellular carcinoma and renal clear cell carcinoma to an EZH2 active inhibitor GSK-126 and provides a scheme which can break through the drug resistance problem of an EZH2 inhibitor; particularly, two small molecule drugs are used in a combined mode to overcome the problem of tumor drug resistance, and the drug combination can provide a new solution for breaking through the problem of drug resistance of the EZH2 inhibitor.

The invention aims to provide application of an acetylation modified inhibitor and an EZH2 activity inhibitor in preparation of a preparation for improving drug sensitivity of liver cancer and kidney cancer tumors.

The invention also aims to provide application of the acetylation modified inhibitor and an EZH2 activity inhibitor in preparation of medicines for preventing and treating liver cancer and kidney cancer.

The invention further aims to provide application of Anacardic Acid (AA) and GSK126 in preparation of a preparation for improving drug sensitivity of liver cancer and kidney cancer tumors.

The invention also aims to provide the application of anacardic acid combined with GSK126 in preparing medicines for preventing and treating liver cancer and kidney cancer.

The above purpose of the invention is realized by the following technical scheme:

the invention researches the drug resistance of two tumors, namely hepatocellular carcinoma and renal clear cell carcinoma, to the EZH2 activity inhibitor GSK-126 by combining an acetylation modification inhibitor and an EZH2 activity inhibitor, and experiments and results show that: hepatocytes treated with 0.2 μ M acetylase inhibitor (TSA) for HL 770224 hours, an increased level of acetylation of EZH2 was found by an acetylation co-immunoprecipitation experiment, suggesting that acetylation of EZH2 may occur in hepatocytes (fig. 1). Also, TSA-treated hepatocyte HL7702 cell line 24 hours, TSA-significantly increased the level of EZH2 in response (fig. 2). Next, the EZH 2K 384 site was also observed as the major acetylation site in the hepatocellular carcinoma Hep3B cell line (fig. 3). Acetylation levels decreased significantly after mutation of EZH2 lysine 384 to arginine, which was unable to undergo acetylation, whereas there was no decrease in the acetylation level of EZH2 after mutation of lysine 234 (as a control) to arginine, EZH2 (fig. 3). Further exploration found that acetylation of EZH 2K 348 increased the stability of EZH 2. Hepatocellular carcinoma Hep3B 0 was treated with protein synthesis inhibitor CHX for 0-6 hours, and EZH 2K 348Q (mimicking K348 acetylation) had significantly increased stability relative to EZH2 wild-type (WT); whereas EZH 2K 348R (mimicking K348 deacetylation) protein stability was significantly reduced (figure 4). The known EZH2 inhibitor has poor treatment effect on solid tumors in clinical tests, and has the problems of high use concentration, strong toxic and side effects and the like which need to be solved urgently. The invention provides that an acetylase inhibitor Anacardic Acid (AA) can inhibit the activity of acetylase PCAF of EZH2, so that the acetylation level of EZH2 is weakened, and the degradation of EZH2 is induced, so that a medicine combination can be formed with an EZH2 activity inhibitor to play a synergistic effect.

To further validate the above conclusions, classical hepatocellular carcinoma (SNU449 and SNU475) and renal clear cell carcinoma cell lines (a498 and RCC10), respectively, were first selected. GSK-126, an inhibitor of EZH2 activity, was found to inhibit Cell survival (Cell viability) of hepatoma cells SNU449 and SNU475 and renal carcinoma cells A498 and RCC10 in a dose-responsive manner 6 days after treatment with GSK-126 (FIG. 5). In addition, the half inhibitory concentrations (IC50) of GSK-126 on SNU449 and SNU475 of hepatoma cells and A498 and RCC10 of renal carcinoma cells were 8.941. mu.M, 7.369. mu.M, 7.210. mu.M and 5.886. mu.M, respectively (FIG. 5). Meanwhile, it was also found that the inflection point treatment concentrations of the activity curves of the acetylase inhibitors Anacardic Acid (AA) for 6 days were 60 μ M, 40 μ M and 60 μ M (no significant effect, but critical treatment concentrations that would have significant inhibitory effects) for the liver cancer cells SNU449 and SNU475 and the renal cell carcinoma a498 and RCC10 (fig. 6). The doses around IC50 of EZH2 GSK-126 for 6 days, i.e., GSK-1264. mu.M and 6. mu.M, were selected respectively (FIG. 5), and liver cancer cells (SNU449 and SNU475) and kidney cancer cells (A498 and RCC10) were treated for 6 days using the selected doses, respectively, and it was found that Anacardic Acid 60 or 40. mu.M was substantially non-toxic to the above cancer cells, and that the inhibition rates of GSK-1264. mu.M and 6. mu.M were both lower than 50% (FIG. 7). However, GSK-1264 μ M and 6 μ M treated liver cancer cells (SNU449 and SNU475) and kidney cancer cells (a498 and RCC10) for 6 days in combination with Anacardic Acid 60 μ M, respectively, and the effect of the combination was found to be significantly increased (fig. 7), indicating that the combination with the acetylation inhibitor Anacardic Acid can increase the sensitivity of EZH2 inhibitor GSK-126 to liver and kidney cancer cells.

Therefore, the following applications should be within the scope of the present invention:

the acetylation modification inhibitor and the EZH2 activity inhibitor are applied to the preparation of the preparation for improving the sensitivity of tumor drugs.

The acetylation modified inhibitor is combined with an EZH2 activity inhibitor to prepare the tumor prevention and treatment medicine.

Application of anacardic acid combined with GSK126 in preparing a preparation for improving tumor drug sensitivity.

Application of anacardic acid combined with GSK126 in preparing medicine for preventing and treating tumor is provided.

Application of anacardic acid in preparing antitumor synergist of GSK126 is provided.

An antitumor agent contains acetylation modification inhibitor and EZH2 activity inhibitor.

An antitumor drug contains anacardic acid and GSK 126.

Particularly preferably, the tumor is liver cancer and/or kidney cancer. More particularly hepatocellular carcinoma and renal clear cell carcinoma cells.

The invention has the following beneficial effects:

the application effect of the EZH2 inhibitor GSK-126 in solid tumor hepatocellular carcinoma and renal clear cell carcinoma is detected, and the EZH2 inhibitor GSK-126 and an acetylase inhibitor Anacardic Acid (AA) are combined for use, so that the drug combination is found to effectively improve the sensitivity of the hepatocellular carcinoma and the renal clear cell carcinoma to the GSK-126 inhibitor, and a new method and a new thought are provided for improving the drug effect and the application range of the EZH2 inhibitor.

EZH2 is highly expressed in various solid tumors, and inhibition of EZH2 activity can inhibit tumor cell growth and tumor growth in vivo. However, in the actual use process of the inhibitor, the insensitivity of tumors is easy to occur, and the inhibitor usually needs higher use concentration and is accompanied with high toxic and side effects. The present invention thus jointly enhances the tumor sensitivity of EZH2 inhibitors, both in terms of reducing EZH2 stability itself and inhibiting EZH2 activity, by combining the acetylase inhibitor Anacardic Acid with the regimen of EZH2 inhibitor GSK 126. Experiments show that the combined use of the acetylase inhibitor and the EZH2 inhibitor has stronger killing effect on hepatocellular carcinoma and renal clear cell carcinoma cells, the effect is obviously better than that of the single use of the EZH2 inhibitor, and the combined use of the acetylase inhibitor and the EZH2 inhibitor can play a role in synergy, so that a new method is provided for improving the drug effect and the application range of the EZH2 inhibitor.

Drawings

FIG. 1 is a graph showing the acetylation modification level of EZH2 protein after TSA treatment of hepatic normal cells HL 7702.

FIG. 2 is a graph showing the protein expression level of EZH2 after TSA treatment of hepatic normal cells HL 7702.

FIG. 3 is a diagram of proteins modified by acetylation of lysine 384 in EZH 2.

FIG. 4 is a graph of protein stability of EZH2 with respect to the absence or acetylation of amino acid 384.

FIG. 5 is a graph of cell survival curves for hepatocellular carcinoma and renal clear cell carcinoma cell lines at different concentrations of GSK 126.

FIG. 6 is a graph of cell survival curves for hepatocellular carcinoma and renal clear cell carcinoma cell lines at different concentrations of Anacardic Acid.

FIG. 7 is a bar graph of the activity of hepatocellular carcinoma and renal clear cell carcinoma cell line cells following the combination of GSK126 with Anacardic Acid.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Various materials and reagents used in the present invention: HL7702, SNU449, SNU475, A498 and RCC10 cells were cultured with Dulbecco's modified eagle medium or Roswell Park mental Institute-1640, plus 10% fetal bovine serum (Hyclone) and 1% penicilin-Streptomycin Solution (Hyclone) at 5% carbon dioxide, 37 ℃. TSA (MCE, HY-15144), GSK-126(MCE, HY-13470) and Anacardic Acid (MCE, HY-N2020) are dissolved in DMSO.

The various experimental procedures of the following examples of the invention are as follows:

(1) plasmid construction:

extracting mRNA of HEK-293T cells, carrying out reverse transcription to obtain cDNA, amplifying an EZH2 gene by using the cDNA as a template and a PCR technology through designing a primer, carrying out enzyme digestion and connection on a carrier and an amplified fragment, transferring the carrier and the amplified fragment into an escherichia coli competent cell Stabl3, selecting positive clones on an agar plate containing ampicillin, and carrying out first-generation sequencing verification on the clones by a biological engineering (Shanghai) corporation. And for the EZH2 point mutation plasmid, adopting the EZH2 plasmid as a template, designing a point mutation primer, carrying out PCR amplification to obtain the mutation plasmid, and carrying out subsequent steps consistent with plasmid construction.

(2) And (3) packaging the virus:

after seeding approximately 50% HEK-293T cells on a 10cm cell culture dish overnight, the medium was changed to serum-free medium before transfection, and then the lentiviral expression plasmid (10. mu.g), the viral packaging plasmid psPAX2 (5. mu.g) and the viral envelope plasmid pMD2.G (2. mu.g) were co-transfected into HEK-293T cells with the transfection reagent PEI (Polyethylenimine, Linear, MW 25000, Polysciences Inc.) and changed to 10% FBS medium after 3-4 hours. After 48 hours, the culture supernatant was collected, the virus solution was filtered through a 0.45 μm filter, and the filtrate was dispensed into an EP tube and frozen in a freezer at-80 ℃ for use.

(3) Viral infection:

cells were seeded in 6-well plates at a density of about 30% the first day of virus infection; adding a proper amount of virus liquid into the culture medium on the next day, and simultaneously adding polybrene with the final concentration of 8 mg/mL; removing virus liquid on the third day, and replacing with new culture medium; on the fourth day, cells were digested from 6-well plates and transferred to 10cm petri dishes for culture; and adding puromycin for screening on the fifth day to obtain a cell strain infected by the virus for subsequent experiments.

(4) Protein immunoblotting:

after lysis of cells by adding a lysis solution of protease inhibitor to RIPA buffer (1% Triton X-100or NP-40), BCA protein was quantified, SDS loading buffer was added, and the cells were heated at 95 ℃ for 10 minutes, followed by SDS-PAGE of 10 to 50. mu.g of protein sample. And transferring the protein on the PAGE gel to a PVDF membrane through a transfer membrane system after electrophoresis, and respectively carrying out primary antibody incubation and secondary antibody incubation. Antibodies used include: Anti-Histone H3(Cell Signaling Technology,4499), Anti-Histone H3K27me3(Cell Signaling Technology,9733), Anti-AcK antibody (Cell Signaling Technology,9441), EZH2(Cell Signaling Technology,5246), and V5-tag (Cell Signaling Technology, 13202).

(5) Co-immunoprecipitation:

the treated cells were lysed with 1% NP-40 in IP lysate, quantified by BCA protein, and the cell lysate over-expressing the biotin-tagged protein was added to streptavidin-conjugated magnetic beads (Promega, Z5482) in the same mass, and incubated at 4 ℃ for 4 hours with slow rotation in a refrigerator. Then, washing the magnetic beads for 4 times by using lysis solution, and finally eluting the protein combined on the magnetic beads by using 2X SDS loading buffer for western blot analysis; the acetylation co-immunoprecipitation method comprises the following steps: adding an anti-AcK antibody into cell lysate for incubation overnight, then incubating with Protein A/G magnetic beads for 4 hours, washing the agarose beads with the lysate for 4 times, and finally eluting proteins bound on the magnetic beads by using a 2X SDS loading buffer for western blot analysis.

(6) Cell proliferation assay:

cells were seeded into 96-well plates at a density of 1000-. Corresponding drug treatment, changing the drug every 2 days, and detecting the Cell proliferation by using Cell Counting Kit-8 (Biyunyan, C0039).

(7) The statistical analysis method of the invention comprises the following steps:

SPSS is adopted in software analysis, the experimental result is represented by Mean value plus or minus standard error (Mean plus or minus SD), Student's-t test is adopted for comparing the difference between groups divided into two groups in the experiment, and the difference is statistically significant when P is less than 0.05, wherein the mark is P less than 0.05; p < 0.01 is marked x; p < 0.001 marked x; n.s. indicates that the difference is not statistically significant.

Example 1 acetylation modification of EZH2 can enhance its stability

1. Experiment one:

cells treated with the acetylation inhibitor TSA were lysed with an IP lysate, and protein was quantified by BCA protein quantification. Lysine-acetylated antibodies were added to the lysates of the lysis control (DMSO) and TSA-treated groups, respectively, and incubated overnight at 4 ℃ with slow shaking. Then, an appropriate amount of protein A/G magnetic beads were taken, and overnight incubated cell lysate and protein A/G magnetic beads were added. Subsequently, the cells were centrifuged at 500g for 1 minute at 4 ℃ to remove the supernatant lysate, washed 3 times with 500. mu.L of the lysate, and finally 30. mu.L of 2 XSDS loading buffer was added, heated at 95 ℃ for 10 minutes, centrifuged, and the supernatant sample was collected. Samples were subjected to western blot analysis.

The results demonstrate that when hepatocytes treated with 0.2 μ M acetylase inhibitor (TSA) for HL 770224 hours, the acetylation level of EZH2 was found to increase by an acetylation co-immunoprecipitation experiment, indicating that EZH2 has a significant level of acetylation modification in hepatocytes HL7702 (fig. 1).

2. Experiment two:

treatment of HL7702 with the deacetylase inhibitor TSA inhibited the activity of the deacetylase, thereby increasing the level of acetylation modification of the EZH2 protein (fig. 1). Cells were treated with TSA (0.01, 0.05, 0.1, 0.2 and 0.3. mu.M) at various concentrations for 24 hours and subjected to Western blot analysis.

The results demonstrate that the protein level of EZH2 shows a trend towards an increase in the dose response relationship after inhibition of deacetylase activity (figure 2).

3. Experiment three:

to further search for specific sites of acetylation modification of EZH2, we mutated lysine 234 and lysine 384 of EZH2 to arginine (EZH 2K 234R and K384R), respectively. After HL7702 birA cells were infected with lentivirus with biotin-labeled wild-type EZH2 and mutant EZH2, the cells were lysed with an IP lysate, and a biotin co-immunoprecipitation experiment was performed.

The results showed that the acetylation modification level of EZH2 protein decreased after mutation of lysine 384 of EZH2 (K384R), demonstrating that lysine 384 of EZH2 is the main site for acetylation modification of EZH2 (fig. 3).

4. Experiment four:

post-translational modification of proteins plays an important role in the activity, stability, interaction, etc. of proteins. Next, we further explored the effect of acetylation modification of EZH2 on the stability of EZH2 protein. Lysine 384 of EZH2 was mutated to arginine (K384R) and glutamine (K384Q), respectively, mimicking lysine non-acetylation and acetylation modifications, respectively. Then, wild-type EZH2 and mutant EZH2 (both wild-type and mutant are introduced into V5 label) are transferred into hepatocyte HL7702 cell by slow virus infection method. After introduction of different EZH2 mutants into the cell lines, the cells were seeded in 6-well plates separately and samples were collected at different time points (0, 2, 4 and 6 hours) after addition of the protein synthesis inhibitor CHX for western blot analysis.

The experimental results show that compared with the wild type EZH2, the protein degradation rate is accelerated when EZH2 is not acetylated and modified (K348R). In contrast, EZH2 protein was acetylated (K348Q), EZH2 protein was stable and not prone to proteolytic degradation (fig. 4).

The experimental results show that the expression level of the EZH2 protein is increased in the process of tumorigenesis and development, and more importantly, the EZH2 is subjected to acetylation modification in the process. After acetylation modification, the stability of the EZH2 protein is obviously enhanced, so that the EZH2 can be gradually accumulated in tumors, and further plays the role of cancer-promoting genes.

Example 2 enhancing the killing sensitivity of the EZH2 activity inhibitor GSK-126 to liver and kidney cancer cells using the acetylase inhibitor Anacardic Acid in combination

1. Experiment one:

the inhibition effect of the EZH2 inhibitor GSK126 on liver cancer and kidney cancer cells is detected. Logarithmic growth of cell lines from liver cancers, SNU449 and SNU475 and kidney cancer, RCC10 and A498, were seeded into 96-well plates at 1000 cells per well and the seeded plates were placed in a cell incubator overnight. After 24 hours, the cells were treated with GSK-126 after the cells were adherent, with drug concentrations in concentration gradients of 0, 1, 2, 4, 6, 8 and 10 μ M, 3 replicate wells per group. After 6 days of GSK-126 treatment, the CCK-8 solution was incubated with medium for 1: 10 dilution, then the 96 well plate medium is removed, 100 u L CCK-8 dilution is added, 37 ℃ incubator incubation for 2 hours, using the plate reader to detect the OD 450nm absorbance of each well. Taking the average value of each multiple hole, and calculating the survival rate of the cells after the drug treatment, wherein the calculation formula is as follows: experiment group absorbance/no-drug group absorbance x 100%, the IC50 concentration of GSK-126 and the survival rate curve were finally obtained, and each experiment was repeated 3 times (fig. 5).

2. Experiment two:

detecting the inhibiting effect of an acetylase inhibitor Anacardic Acid on liver cancer and kidney cancer cells. The experimental procedure was similar to that of example 2. The drug concentration gradient of acetylase inhibitor Anacardic Acid (AA) is 0, 2, 5, 10, 20, 40, 60, 80 and 100 μ M, the average value of each multiple hole is taken to calculate the survival rate of the cells after drug treatment, and the calculation formula is as follows: the experiment group absorbance/unadditized group absorbance was multiplied by 100%, and the survival rate curve of Anacardic Acid and the inflection point treatment concentration thereof were finally obtained, and each group of experiments was repeated 3 times (fig. 6).

3. Experiment three:

to enhance the effect of EZH2 inhibitor GSK-126 in inhibiting solid tumor cells, we observed the effect of using GSK-126 in combination with the acetylase inhibitor Anacardic Acid on hepatocellular carcinoma and renal clear cell carcinoma cell proliferation. The cells were divided into four groups, i.e., DMSO group, GSK-126 group, Anacardic Acid group, GSK126+ Anacardic Acid combination treatment group for 6 days, and cell survival was measured by CCK-8. The treatment concentrations of GSK-126 and Anacardic Acid were selected based on the results of fig. 5 and 6. IC50 (half inhibition concentration) of liver cancer cells SNU449 and SNU475 is 8.941 and 7.369. mu.M, respectively, and IC50 of kidney cancer cells A498 and RCC10 is 7.21 and 5.886. mu.M, respectively, so 4. mu.M and 6. mu.M are selected as doses for subsequent combination treatment in the range of about IC 50. For Anacardic Acid, inflection point concentrations were selected based on the cancer cell survival curves after Anacardic Acid treatment, i.e., the Anacardic Acid treatment concentrations for SNU449, SNU475, A498 and RCC10 were 60. mu.M, 40. mu.M and 60. mu.M, respectively. Treatment with Anacardic Acid alone at the above concentrations did not significantly affect cell growth (fig. 6 and 7). Importantly, however, the inhibitory effect of the EZH2 activity inhibitor GSK-126 on hepatocellular carcinoma and renal clear cell carcinoma was significantly increased in combination with the treatment with the acetylase inhibitor, Anacardic Acid (fig. 7).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (5)

1. Application of Anacardic Acid (Anacardic Acid) and GSK126 in preparation of a preparation for improving sensitivity of a liver cancer and/or kidney cancer treatment drug.

2. Application of Anacardic Acid (Anacardic Acid) and GSK126 in preparing medicine for treating hepatocarcinoma and/or renal carcinoma is provided.

3. Application of Anacardic Acid (Anacardic Acid) in preparing synergist for resisting liver cancer and/or kidney cancer of GSK126 is provided.

4. The use according to any one of claims 1 to 3, wherein the liver cancer is hepatocellular carcinoma and the kidney cancer is renal clear cell carcinoma.

5. A medicine for resisting liver cancer and/or kidney cancer, which is characterized by comprising anacardic acid and GSK 126.

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