Application of disulfiram in reversing regorafenib resistance in hepatocellular carcinoma

Abstract

The present invention relates to the field of tumor treatment, and specifically to the use of small molecule compounds in the preparation of drugs for treating hepatocellular carcinoma. For the first time, we used disulfiram to reverse the regorafenib resistance of MHCC-97H / REGO cells. The experimental results showed that after using disulfiram, the same concentration of regorafenib inhibited the proliferation, migration and invasion of MHCC-97H / REGO cells more significantly, and could significantly inhibit the tolerance of hepatocellular carcinoma to regorafenib. Therefore, the disulfiram provided by the present invention can be used to prepare drugs for prolonging the life of patients after hepatocellular carcinoma becomes regorafenib-resistant. The present invention provides disulfiram for the preparation of drugs for treating regorafenib-resistant hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and specifically refers to the application of disulfiram in reversing regorafenib resistance in hepatocellular carcinoma. Background Art

[0002] Hepatocellular carcinoma (HCC) is a major disease that threatens human life. HCC has the characteristic of insidious onset, so many patients have lost the opportunity for surgery by the time of diagnosis. Drug therapy is the main treatment for these patients. Regorafenib is a second-line treatment for advanced HCC approved by the US Food and Drug Administration (FDA) and can prolong the lives of HCC patients. However, its therapeutic effect is limited due to acquired drug resistance. Therefore, finding methods to reverse regorafenib resistance is a key research direction in the field of HCC treatment. Drug repurposing is an important strategy for reversing acquired drug resistance. Disulfiram (DSF) is an early FDA-approved drug for alcohol withdrawal. So far, there are no reports on whether it can be used to treat regorafenib resistance in hepatocellular carcinoma.

[0003] This study will explore the effects of disulfiram on the proliferation and migration of hepatocellular carcinoma through in vitro and in vivo models, and explore its role in reversing regorafenib resistance in hepatocellular carcinoma, providing ideas for the development of new applications and new treatment strategies for the clinical treatment of hepatocellular carcinoma after drug resistance. Summary of the Invention

[0004] The present invention confirmed the effects of ezetimibe on breast cancer cell migration, invasion, and EMT through wound healing, invasion, and Western blotting experiments. After ezetimibe treatment of breast cancer cells, differentially expressed genes were analyzed by transcriptome sequencing and verified by Western blot and qRT-PCR. Breast cancer cells overexpressing TGFβ2 were then constructed, and the effects of TGFβ2 on the migration and invasion of ezetimibe-treated breast cancer cells were confirmed through wound healing and invasion experiments.

[0005] Therefore, the main contents of the present invention are summarized as follows: activation of p-ERK1 / 2 is the mechanism leading to regorafenib resistance in hepatocellular carcinoma; disulfiram can inhibit the proliferation, migration and invasion of hepatocellular carcinoma through p-ERK1 / 2; and disulfiram has the effect of reversing regorafenib resistance in hepatocellular carcinoma both in vivo and in vitro.

[0006] The details of the present invention are as follows:

[0007] 1. We successfully constructed the regorafenib-resistant cell line MHCC-97H / REGO from MHCC-97H for the first time and found that the proliferation, migration and invasion abilities of MHCC-97H / REGO were significantly improved compared with MHCC-97H. We also found that the expression of downstream cyclin B1, N-cadherin, vimentin, snail and slug in regorafenib-resistant HCC cells was affected by p-ERK1 / 2 regulation.

[0008] 2. We revealed for the first time that a p-ERK1 / 2 inhibitor (SCH772984) can suppress the resistance of hepatocellular carcinoma cells (MHCC-97H / REGO) to regorafenib, and can inhibit the proliferation, migration and invasion ability of MHCC-97H / REGO.

[0009] 3. We first revealed that the p-ERK1 / 2 agonist (TBHQ) can reduce the sensitivity of MHCC-97H to regorafenib and promote the proliferation, migration and invasion of MHCC-97H.

[0010] 4. We revealed for the first time that disulfiram copper chelate can reverse regorafenib resistance in hepatocellular carcinoma in vitro, and found that disulfiram reverses regorafenib resistance by regulating downstream cyclin B1, N-cadherin, vimentin, snail, and slug of p-ERK1 / 2.

[0011] 5. We show for the first time that oral disulfiram can reverse regorafenib resistance in HCC in vivo. Our results may provide some help for the clinical treatment of regorafenib-resistant patients with advanced HCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Establishment and morphological characteristics of regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H and MHCC-97H / REGO cells were treated with different concentrations of regorafenib (0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM) for 72 hours, and CCK8 assay was used to assess cell viability. (B) MHCC-97H and MHCC-97H / REGO cells were photographed under a microscope 24 hours after seeding. Scale bar = 100 μm.

[0013] Figure 2Changes in the proliferation of regorafenib-resistant hepatocellular carcinoma cells: (A) 1000 cells / well were seeded in MHCC-97H and MHCC-97H / REGO cells and cultured for 7 days. The number of colonies formed was determined by a colony formation assay. (B) The results of the colony formation assay were stained, counted, and statistically analyzed. (C) Cell cycle distribution was determined in MHCC-97H and MHCC-97H / REGO cells after 24 hours of culture. (D) Statistical analysis of cell cycle distribution in MHCC-97H and MHCC-97H / REGO cells.

[0014] Figure 3 Changes in migration and invasion abilities of regorafenib-resistant hepatocellular carcinoma cells: (A) Wound-healing assay to examine the healing rates of MHCC-97H and MHCC-97H / REGO cells cultured in serum-free medium at 0 h, 24 h, and 48 h. Microscopic images were taken. Scale bar = 100 μm. (B) Statistical results of the wound healing rate. (C) Transwell migration assay to examine the number of cells that passed through the chamber after 24 h of culture in serum-free medium for MHCC-97H and MHCC-97H / REGO cells. Microscopic images were taken. Scale bar = 100 μm. (D) Statistical results of the Transwell migration assay to examine the number of cells that passed through the chamber after 24 h of culture in serum-free medium for MHCC-97H and MHCC-97H / REGO cells. Transwell invasion assay to examine the number of cells that passed through the chamber after 24 h of culture in serum-free medium for MHCC-97H and MHCC-97H / REGO cells. Microscopic images were taken. Scale bar = 100 μm. (F) Statistical results of the Transwell invasion assay to examine the number of cells that passed through the chamber after 24 h of culture in serum-free medium for MHCC-97H and MHCC-97H / REGO cells.

[0015] Figure 4 Changes in p-ERK1 / 2 and cellular phenotypic protein expression in regorafenib-resistant hepatocellular carcinoma cells: (A) 48 hours after seeding MHCC-97H and MHCC-97H / REGO cells, Western blot analysis was performed to examine changes in E-cadherin, N-cadherin, vimentin, snail, slug, cyclin B1, p-ERK1 / 2, and ERK1 / 2 protein expression. (B) Grayscale analysis of relative protein expression.

[0016] Figure 5Effect of SCH772984 on Regorafenib-resistant HCC Cells: (A) MHCC-97H / REGO cells treated with SCH772984 and the control were analyzed by Western blot analysis of p-ERK1 / 2 expression, and the results were statistically analyzed. (B) MHCC-97H / REGO cells treated with SCH772984 and the control were treated with different concentrations of Regorafenib (0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM) for 72 hours, and cell viability was assessed by CCK8 assay.

[0017] Figure 6 Effect of SCH772984 on the proliferation of regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells were seeded at 1000 cells / well in 6-well plates. 24 hours after SCH772984 treatment and control treatment, cells were cultured in DMEM for 7 days, stained, photographed, and counted, and the results were statistically analyzed. (B) 24 hours after SCH772984 treatment and control treatment, cell cycle distribution in MHCC-97H / REGO cells was assessed by flow cytometry, and the results were statistically analyzed.

[0018] Figure 7 Effect of SCH772984 on the migration and invasion of regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells were treated with SCH772984 and control treatments for 0 and 24 hours, and cell healing rates were recorded using a wound healing assay. Scale bar = 100 μm. The results were statistically analyzed. (B) MHCC-97H / REGO cells were treated with SCH772984 and control treatments for 24 hours, and cell migration was assessed using a Transwell migration assay. The number of cells entering the lower chamber was recorded using a microscope. Scale bar = 100 μm. The results were statistically analyzed. (C) MHCC-97H / REGO cells were treated with SCH772984 and control treatments for 24 hours, and cell invasion was assessed using a Transwell invasion assay. The number of cells entering the lower chamber was recorded using a microscope. Scale bar = 100 μm.

[0019] Figure 8Effects of SCH772984 on p-ERK1 / 2 and cellular phenotypic proteins in regorafenib-resistant hepatocellular carcinoma cells: (A) Proteins were extracted from MHCC-97H / REGO cells 24 hours after SCH772984 treatment and control treatment, and Western blot analysis was performed to examine changes in E-cadherin, N-cadherin, vimentin, snail, slug, and cyclin B1 protein expression. (B) Relative expression levels of each protein were recorded, and the results were statistically analyzed.

[0020] Figure 9 Effect of TBHQ on Regorafenib Resistance in Hepatocellular Carcinoma Cells: (A) MHCC-97H cells treated with TBHQ and the control were analyzed by Western blot analysis for changes in p-ERK1 / 2 expression, and the results were statistically analyzed. (B) MHCC-97H cells treated with regorafenib at different concentrations (0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM) for 72 hours were analyzed by CCK8 assay for cell viability. The results were statistically analyzed.

[0021] Figure 10 Effect of TBHQ on the proliferation of hepatocellular carcinoma cells: (A) MHCC-97H cells were seeded at 1000 cells / well in 6-well plates and cultured in DMEM for 7 days after TBHQ treatment or control treatment for 24 hours. The cells were then stained, photographed, and counted. (B) Cell cycle distribution in MHCC-97H cells was assessed by flow cytometry after TBHQ treatment or control treatment for 24 hours, and the results were statistically analyzed.

[0022] Figure 11 Effect of TBHQ on the migration and invasion of hepatocellular carcinoma cells: (A) MHCC-97H cells, seeded at 40,000 cells / well in scratch chambers, were treated with TBHQ and control at 0 and 24 hours. Cell healing rates were recorded under a microscope. Scale bar = 100 μm. The results were statistically analyzed. (B) MHCC-97H cells were treated with TBHQ and control for 24 hours and then subjected to a Transwell migration assay to assess cell migration. The number of cells entering the lower chamber was recorded under a microscope. Scale bar = 100 μm. The results were statistically analyzed. (C) MHCC-97H cells were treated with TBHQ and control for 24 hours and then subjected to a Transwell invasion assay to assess cell invasion. The number of cells entering the lower chamber was recorded under a microscope. Scale bar = 100 μm. The results were statistically analyzed.

[0023] Figure 12Effects of TBHQ on p-ERK1 / 2 and cellular phenotypic proteins in hepatocellular carcinoma cells: (A) Proteins were extracted from MHCC-97H cells 24 hours after TBHQ treatment and control treatment, and Western blot analysis was performed to examine changes in E-cadherin, N-cadherin, vimentin, snail, slug, cyclin B1, and p-ERK1 / 2 protein expression. (B) The relative expression levels of each protein were recorded, and the results were statistically analyzed.

[0024] Figure 13 Effect of copper disulfiram chelate on the proliferation of hepatocellular carcinoma cells: (A) MHCC-97H / REGO, MHCC-97H, SMCC-7721, and MHCC-LM3 HCC cell lines were exposed to the indicated concentrations of CuET (0 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, and 0.5 μM) for 24 hours, and cell viability was measured using the CCK8 assay. (B) MHCC-97H / REGO, MHCC-97H, SMCC-7721, and MHCC-LM3 HCC cell lines were seeded at 1000 cells / well in 6-well plates and treated with the indicated concentrations of CuET for 24 hours. After culture in DMEM for 7 days, the cells were stained and photographed.

[0025] Figure 14Effects of copper disulfiram chelate on the cell cycle and cyclins in hepatocellular carcinoma cells: (A) After MHCC-97H / REGO cells were treated with CuET (0.1 μM) for 24 hours, changes in cell cycle distribution were detected by flow cytometry, and the proportions of cells in different phases were statistically analyzed. (B) After MHCC-97H cells were treated with CuET (0.1 μM) for 24 hours, changes in cell cycle distribution were detected by flow cytometry, and the proportions of cells in different phases were statistically analyzed. (C) After SMCC-7721 cells were treated with CuET (0.1 μM) for 24 hours, changes in cell cycle distribution were detected by flow cytometry, and the proportions of cells in different phases were statistically analyzed. (D) After MHCC-LM3 cells were treated with CuET (0.1 μM) for 24 hours, changes in cell cycle distribution were detected by flow cytometry, and the proportions of cells in different phases were statistically analyzed. (E) After MHCC-97H / REGO cells were treated with CuET (0.1 μM) for 24 hours, the expression of the proliferation-related protein cyclin B1 was detected by Western blot, and the relative protein expression levels were statistically analyzed. (F) After MHCC-97H cells were treated with CuET (0.1 μM) for 24 hours, the expression of the proliferation-related protein cyclin B1 was detected by Western blot, and the relative protein expression levels were statistically analyzed. (G) After SMCC-7721 cells were treated with CuET (0.1 μM) for 24 hours, the expression of the proliferation-related protein cyclin B1 was detected by Western blot, and the relative protein expression levels were statistically analyzed. (H) After MHCC-LM3 cells were treated with CuET (0.1 μM) for 24 hours, the expression of the proliferation-related protein cyclin B1 was detected by Western blot, and the relative protein expression levels were statistically analyzed.

[0026] Figure 15Effects of copper disulfiram chelate on the migration and invasion of hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells, 40,000 cells / scratch chamber, were treated with CuET (0 μM, 0.1 μM) for 24 and 48 hours. Microscopic images were taken and the healing rate was calculated. Scale bar = 100 μm. The results were statistically analyzed. (B) MHCC-97H cells, 40,000 cells / scratch chamber, were treated with CuET (0 μM, 0.1 μM) for 24 and 48 hours. Microscopic images were taken and the healing rate was calculated. Scale bar = 100 μm. The results were statistically analyzed. (C) SMCC-7721 cells, 40,000 cells / scratch chamber, were treated with CuET (0 μM, 0.1 μM) for 24 and 48 hours. Microscopic images were taken and the healing rate was calculated. Scale bar = 100 μm. The results were statistically analyzed. (D) MHCC-LM3 cells, 40,000 cells / well, were treated with CuET (0 μM, 0.1 μM) for 24 and 48 hours. Microscopic images were taken and the healing rate was calculated. Scale bar = 100 μm, and the results were statistically analyzed. (E) MHCC-97H / REGO cells, 80,000 cells / well, were seeded into the upper chamber of a Transwell (upper: uncoated with Matrigel, lower: coated with Matrigel). After treatment with CuET (0 μM, 0.1 μM) for 24 hours, the wound was stained and photographed. Scale bar = 100 μm, and the number of cells entering the lower chamber was counted. The results were statistically analyzed. (F) MHCC-97H cells, seeded at 80,000 cells / well in the upper chamber of a Transwell plate (upper: uncoated with Matrigel, lower: coated with Matrigel), were treated with CuET (0 μM, 0.1 μM) for 24 hours, stained, and photographed under a microscope. Scale bar = 100 μm. The number of cells that entered the lower chamber was counted, and the results were statistically analyzed. (G) SMCC-7721 cells, seeded at 80,000 cells / well in the upper chamber of a Transwell plate (upper: uncoated with Matrigel, lower: coated with Matrigel), were treated with CuET (0 μM, 0.1 μM) for 24 hours, stained, photographed under a microscope. Scale bar = 100 μm. The number of cells that entered the lower chamber was counted, and the results were statistically analyzed. (H) MHCC-LM3 cells, 80,000 cells / well, were implanted into the upper chamber of Transwell (upper: uncoated with Matrigel, lower: coated with Matrigel). After treatment with CuET (0 μM, 0.1 μM) for 24 h, the cells were stained and photographed under a microscope (scale bar = 100 μm). The number of cells entering the lower chamber was counted, and the results were statistically analyzed.

[0027] Figure 16Effects of copper disulfiram chelate on migration and invasion phenotype proteins in hepatocellular carcinoma cells: (A) After 24 hours of treatment with CuET (0 μM, 0.1 μM), MHCC-97H / REGO cells were analyzed by Western blot for changes in intracellular expression of E-cadherin, N-cadherin, vimentin, snail, and slug. Relative protein expression levels were calculated as grayscale values, and the results were statistically analyzed. (B) After 24 hours of treatment with CuET (0 μM, 0.1 μM), MHCC-97H cells were analyzed by Western blot for changes in intracellular expression of E-cadherin, N-cadherin, vimentin, snail, and slug. Relative protein expression levels were calculated as grayscale values, and the results were statistically analyzed. (C) After treatment with CuET (0 μM, 0.1 μM) for 24 hours, SMCC-7721 cells were analyzed by Western blot for changes in the expression of E-cadherin, N-cadherin, vimentin, snail, and slug. Relative protein expression levels were calculated as grayscale values, and the results were statistically analyzed. (D) After treatment with CuET (0 μM, 0.1 μM) for 24 hours, MHCC-LM3 cells were analyzed by Western blot for changes in the expression of E-cadherin, N-cadherin, vimentin, snail, and slug. Relative protein expression levels were calculated as grayscale values, and the results were statistically analyzed.

[0028] Figure 17 Effects of copper disulfiram chelate on p-ERK1 / 2 in hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells treated with CuET (0 μM, 0.1 μM) were analyzed by Western blot for changes in ERK1 / 2 and p-ERK1 / 2 expression, as well as statistical graphs. (B) MHCC-97H cells treated with CuET (0 μM, 0.1 μM) were analyzed by Western blot for changes in ERK1 / 2 and p-ERK1 / 2 expression, as well as statistical graphs. (C) SMCC-7721 cells treated with CuET (0 μM, 0.1 μM) were analyzed by Western blot for changes in ERK1 / 2 and p-ERK1 / 2 expression, as well as statistical graphs. (D) MHCC-LM3 cells treated with CuET (0 μM, 0.1 μM) were analyzed by Western blot for changes in ERK1 / 2 and p-ERK1 / 2 expression, as well as statistical graphs.

[0029] Figure 18Effect of copper disulfiram chelate on regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells treated with CuET at different concentrations of regorafenib, and cell viability measured by CCK8 assay. (B) MHCC-97H / REGO cells treated with different concentrations of CuET, and IC50 values ​​calculated based on cell viability inhibition curves.

[0030] Figure 19 Effect of disulfiram copper chelate on the proliferation of regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells were seeded at 500 cells / well in 12-well plates and treated for 24 hours with the control, disulfiram, regorafenib, and combination treatment groups. The cells were then cultured in DMEM for 7 days and observed with crystal violet staining. (B) Statistical analysis of the number of colonies formed in MHCC-97H / REGO cells after treatment with different drugs. (C) Flow cytometry analysis of cell cycle distribution in MHCC-97H / REGO cells after 24 hours of treatment with the control, disulfiram, regorafenib, and combination treatment groups. (D) Statistical analysis of flow cytometry results of drug-treated MHCC-97H / REGO cells.

[0031] Figure 20 Effects of copper disulfiram chelate on the migration and invasion of regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells. From left to right, the control group, CuET-treated group, regorafenib-treated group, and combination-treated group are shown. Cell healing rates were observed under a microscope at 24 and 48 hours after treatment. Scale bar = 100 μm. (B) Statistical analysis of cell healing rates in MHCC-97H / REGO cells at 24 and 48 hours. (C) MHCC-97H / REGO cells. From left to right, the control group, CuET-treated group, regorafenib-treated group, and combination-treated group are shown. Cell numbers crossing the Transwell chamber were recorded under a microscope at 24 hours after drug treatment. Scale bar = 100 μm. (D) MHCC-97H / REGO cells. Statistical analysis of cell numbers crossing the chamber in a Transwell migration assay. (E) MHCC-97H / REGO cells. From left to right, the control group, CuET-treated group, regorafenib-treated group, and combination-treated group are shown. 24 hours after drug treatment, the number of cells that crossed the Matrigel-coated Transwell chamber was recorded under a microscope. Scale bar = 100 μm. (F) MHCC-97H / REGO cells. Statistical analysis of the number of cells that crossed the chamber in a Transwell invasion assay.

[0032] Figure 21Effects of copper disulfiram chelate on p-ERK1 / 2 and cellular phenotypic proteins in regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells were treated with the control, CuET, regorafenib, and combination treatment groups for 24 hours. Western blot analysis was performed to examine the expression of E-cadherin, N-cadherin, vimentin, snail, slug, cyclin B1, and p-ERK1 / 2 proteins. (B) MHCC-97H / REGO cells were treated with the control, CuET, regorafenib, and combination treatment groups. Statistical analysis was performed using three Western blots.

[0033] Figure 22 Effect of disulfiram on the growth curve of subcutaneous tumors of regorafenib-resistant hepatocellular carcinoma cells: (A) Tumor growth curves of subcutaneous tumors in MHCC-97H / REGO nude mice after treatment with the control group, disulfiram treatment group, regorafenib treatment group, and combination treatment group.

[0034] Figure 23 Effect of disulfiram on subcutaneous tumor size in regorafenib-resistant hepatocellular carcinoma cells: (A) Tumor size of MHCC-97H / REGO nude mice after treatment with the control, disulfiram, regorafenib, and combination treatment groups. Figure 2 shows tumor size when the average diameter of the control mice reached 20 mm. (B) Statistical analysis of tumor weight of MHCC-97H / REGO nude mice after treatment with the control, disulfiram, regorafenib, and combination treatment groups. DETAILED DESCRIPTION

[0035] All experimental materials and methods mentioned in the Examples section

[0036] 1. Cell Recovery

[0037] (1) Preparation: Preheat a water bath, pipette 10 mL of DMEM medium into a culture dish, and place it in a 37°C constant temperature incubator to preheat.

[0038] (2) Thawing cells: When the water bath temperature reaches 37°C, remove the MHCC-97H cells from liquid nitrogen and quickly place them in a water bath. After 2 minutes, remove the MHCC-97H cells.

[0039] (3) Cell collection: Prepare a 15 mL centrifuge tube, transfer 1 mL of thawed MHCC-97H cells into the centrifuge tube, add 3 mL of preheated DMEM medium to the centrifuge tube, balance with another 15 mL centrifuge tube with 4 mL of water, and centrifuge at 200 g for 5 min.

[0040] (4) Cell seeding: After centrifugation, remove the centrifuge tube, aspirate the supernatant with a pipette, discard it into the waste tank, aspirate 1 mL of preheated DMEM medium and slowly and gently resuspend the cells. Transfer the cells into a culture dish and culture them in a 37°C 5% CO2 incubator. After 6-8 hours, observe the cell adhesion. If the cell survival rate is high, culture them overnight. If a large number of cells float, replace the culture medium with new one and continue the culture.

[0041] (5) Cell Culture: MHCC-97H cells were revived and cultured in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured in a 37°C, 5% CO2 incubator. The culture medium was observed and replaced daily. The culture dish was washed with 1 mL-2 mL PBS each time the culture medium was changed. Cells were passaged when the confluence reached a level that covered the entire bottom surface.

[0042] (6) Cell passaging: Remove the confluent cells from the incubator, remove the culture medium, and add 1 mL of PBS for washing. After removing the PBS, add 1 mL of trypsin to digest the cells for 3 minutes. Collect the cells and centrifuge them at 200g for 5 minutes. Discard the supernatant and resuspend the cells in 2 mL of culture medium. Divide the cells evenly and culture them in 2-3 culture dishes. Add 7-8 mL of complete culture medium to each dish.

[0043] 2. Construction of Regorafenib-resistant HCC cell lines

[0044] (1) Selection of initial induction concentration: The initial induction concentration is slightly lower than the IC of regorafenib for MHCC-97H. 50 The preliminary experimental results showed that the IC of regorafenib for MHCC-97H 50 It is about 7 μM, so this study used 5 μM regorafenib as the initial concentration to screen the cells. The old culture medium was discarded every two days, and the cells were washed with PBS and replaced with fresh culture medium.

[0045] (2) Concentration gradient induction: Slowly increase the concentration of regorafenib by 0.5 μM each week as the next induction concentration. After the concentration is increased, half of the cells are cultured at the original concentration and the other half are cultured at a concentration increased by 0.5 μM. If excessive cell death occurs after the concentration is increased, continue culturing at the original concentration for a period of time. If the cells can continue to grow and passage after the concentration is increased, culture them at the increased concentration. Repeat this cycle and continuously stimulate the culture concentration to about 14 μM to obtain the regorafenib-resistant cell line MHCC-97H / REGO. The obtained resistant cell line is cultured in a medium containing 4 μM regorafenib to maintain its resistance.

[0046] 3. Cytotoxicity Assay (CCK8 Assay)

[0047] (1) Cell seeding: Select MHCC-97H cells and MHCC-97H / REGO cells in the logarithmic growth phase, discard the culture medium and add 1 mL of PBS for washing. Add 1 mL of trypsin to digest the cells for 3 minutes. Then, use 1 mL of serum-containing culture medium to terminate the digestion. Centrifuge at 200g for 5 minutes, remove the supernatant, and add culture medium to resuspend the cells. The cells are configured to a concentration of 50,000 cells / mL. Seed into a 96-well plate, add 100 μL of cell suspension to each well, and the final concentration per well is 5,000 cells / well. Add 100 μL of PBS to the wells around the 96-well plate to avoid edge effects.

[0048] (2) Drug treatment: After overnight cell culture, the culture medium was discarded and the cells were treated with different concentrations of regorafenib for 72 h (the concentrations of regorafenib selected in this study were 0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM).

[0049] (3) Cell viability assay: CCK8 and culture medium were mixed at a ratio of 1:10 as CCK8 assay solution. After removing the cell supernatant from the treated cells, 100 μL of CCK8 assay solution was added to each well. Six blank controls were set up. Only 100 μL of CCK8 assay solution was added to the blank controls. After addition, the cells were transferred to a 37°C 5% CO2 constant temperature incubator. After reacting for 0.5 h, 1 h, 1.5 h, and 2 h, the absorbance at 450 nm was measured using a microplate reader. The control group with an absorbance between 1.2 and 2 was selected.

[0050] (4) Data processing: The blank control was subtracted from the obtained data to obtain the cell viability of cells treated with different concentrations of regorafenib. The IC values ​​of regorafenib for MHCC-97H and MHCC-97H / REGO were calculated. 50 .

[0051] 4. Cell proliferation ability detection (clone formation assay, flow cytometry)

[0052] Clone formation assay

[0053] (1) Cell plating: Take MHCC-97H / REGO and MHCC-97H cells in the logarithmic growth phase, add 1 mL of trypsin, digest for 3 minutes, and add 1 mL of complete culture medium to terminate the digestion. Collect the cells, place them in a 15 mL centrifuge tube, and centrifuge them at 200g for 5 minutes. Discard the supernatant, add culture medium to resuspend the cells, and seed them into 6-well plates. The cell number is set to 1000 cells / well.

[0054] (2) Cell clone formation: After the cells adhere to the wall, replace the culture medium and observe the cell growth and the number of cell clusters under a microscope every day. Replace the culture medium every 2 days. Culture for about 7-8 days. When the cells grow into clusters and the number of cells is large, staining and observation can be performed.

[0055] (3) Cell staining observation: remove the culture medium, add 1-2 mL PBS to wash the cells, add 4% paraformaldehyde to fix the cells for 20 min, add PBS to wash the cells again, add 0.5% crystal violet solution to stain the cells for 15 min, recover the crystal violet, add PBS to wash the cells again, dry them, take pictures, and calculate the number of colonies formed in each well to obtain the cell cloning experiment results.

[0056] Each experiment was repeated 3 times independently.

[0057] Flow cytometry

[0058] (1) Cell plating: Take MHCC-97H / REGO and MHCC-97H cells in the logarithmic growth phase, add 1 mL of trypsin, digest for 3 minutes, and add 1 mL of complete culture medium to terminate the digestion. Collect the cells, place them in a 15 mL centrifuge tube, and centrifuge them at 200g for 5 minutes. Discard the supernatant, add culture medium to resuspend the cells, and seed them into a 12-well plate. The cell number is set to 400,000 cells / well.

[0059] (2) Cell collection: After trypsin digestion, 100,000 cells were collected and fixed with 1 mL of 70% alcohol for 30 min. The cells were centrifuged at 200 g for 5 min and the supernatant was discarded.

[0060] (3) Cell staining: Add 1 mL of PBS solution to resuspend the cells, add 5 μL of PI dye, and incubate at room temperature in the dark for 30 min.

[0061] (4) Flow cytometry: The stained cell suspension was detected by BD FACSCalibur flow cytometer, and the results were analyzed by Flow Jo software.

[0062] 5. Cell migration and invasion ability detection (scratch assay, Transwell assay)

[0063] Scratch test

[0064] (1) Prepare the chamber: Place the scratch chamber in a clean culture dish, disinfect with alcohol, irradiate under UV light for 30 minutes, and dry. Prepare a 24-well plate, draw a line on the bottom, and stick the chamber on the lined area. Continue to prepare under UV light.

[0065] (2) Cell plate: Take logarithmically growing MHCC-97H and MHCC-97H / REGO cells, add 1 mL of trypsin, and digest for 3 minutes. After digestion is complete, add 1 mL of complete culture medium to terminate digestion. Collect the cells and centrifuge at 200g for 5 minutes. Discard the supernatant and resuspend the cells in culture medium. The cell suspension is prepared to a concentration of 100,000 cells / mL. Add 350 μL-400 μL into the chamber and culture overnight.

[0066] (3) Scratch test record: On the second day, the chamber was removed, washed with PBS, and serum-free medium was added. At 0 h, 24 h, and 48 h, the chamber was photographed under a microscope at 100x magnification. The black line drawn in advance was used as a reference to ensure that each photograph was taken at the same location. This allowed us to understand changes in cell migration ability.

[0067] Transwell migration assay

[0068] (1) Prepare the Transwell device: The Transwell device should be used immediately. After opening, place it directly into a 24-well plate and irradiate with ultraviolet light for 30 minutes.

[0069] (2) Cell plate: MHCC-97H and MHCC-97H / REGO cells in the logarithmic growth phase were selected. After removing the culture medium, 1 mL of trypsin was added and digested for 3 min. Complete culture medium was added to terminate the digestion. The cells were collected and centrifuged at 200 g for 5 min. The supernatant was discarded and the cells were resuspended in serum-free culture medium. The cells were configured to 200,000 cells / mL and 400 μL of the culture medium was added to the upper chamber of the Transwell chamber. 1 mL of serum-containing culture medium was added to the lower chamber.

[0070] (3) Cell staining observation: After culturing in a 37°C 5% CO2 constant temperature incubator for 24 hours, the Transwell device was removed from the 24-well plate, the culture medium was aspirated, the cells were washed with PBS, and the cells were fixed in 4% paraformaldehyde for 15 minutes, washed with PBS, and stained in 0.5% crystal violet for 15 minutes. After staining, the cells were washed again with PBS, and the cells remaining in the upper chamber were slowly wiped with a hairless cotton swab until the cells in the upper chamber were completely wiped off. After treatment, the cells were photographed with a microscope at a magnification of 200 times, and 5 photos were taken at random positions. The obtained photos were counted to detect the cell migration ability.

[0071] Transwell invasion assay

[0072] (1) Prepare the Transwell device: The Transwell device should be used immediately. After opening, place it directly into a 24-well plate and irradiate with ultraviolet light for 30 minutes.

[0073] (2) Transwell coating: Matrigel and serum-free culture medium were prepared in a ratio of 1:8 and coated on the upper chamber of the Transwell chamber. The Transwell device was placed in a constant temperature incubator at 37°C and 5% CO2 for 30 minutes to allow the Matrigel to form a gel.

[0074] (3) Cell plating: MHCC-97H and MHCC-97H / REGO cells in the logarithmic growth phase were selected. After removing the culture medium, 1 mL of trypsin was added and digested for 3 min. Complete culture medium was added to terminate the digestion. The cells were collected and centrifuged at 200 g for 5 min. The supernatant was discarded and the cells were resuspended in serum-free culture medium. The cells were configured to 200,000 cells / mL and 400 μL of the culture medium was added to the upper chamber of the Transwell chamber. 1 mL of serum-containing culture medium was added to the lower chamber.

[0075] (4) Cell staining observation: After culturing in a 37°C 5% CO2 constant temperature incubator for 24 hours, the Transwell device was removed from the 24-well plate, the culture medium was aspirated, the cells were washed with PBS, and the cells were fixed in 4% paraformaldehyde for 15 minutes, washed with PBS, and stained in 0.5% crystal violet for 15 minutes. After staining, the cells were washed again with PBS, and the cells remaining in the upper chamber were slowly wiped with a hairless cotton swab until the cells in the upper chamber were completely wiped off. After treatment, the cells were photographed under a microscope at a magnification of 200 times, and 5 photos were taken at random positions. The obtained photos were counted to detect the cell migration ability.

[0076] 6. Total Cell Protein Extraction

[0077] (1) Cell lysis buffer preparation: NP40 lysis buffer was prepared in advance. Each mL of lysis buffer contained 10 μL of phosphatase inhibitors, 1 μL of protease inhibitors, and 5 μL of 100 nM PMSF.

[0078] (2) Cell collection: Select MHCC-97H and MHCC-97H / REGO cells in the logarithmic growth phase, remove the supernatant from the culture dish, and wash with PBS.

[0079] (3) Cell lysis: Prepare an ice pack and place the culture dish on the ice pack. Add 200 μL NP40 to each well and treat on ice for 15 min. Then use a cell brush to scrape the cells from the bottom of the culture dish and transfer them into a 1.5 mL EP tube. Continue lysis for 10 min and shake the EP tube several times to ensure that the cells are fully digested.

[0080] (4) Collect protein: Take another EP tube and balance it, place it in a low-temperature centrifuge, adjust it to 12000g, 15min, 4℃. After centrifugation, carefully remove the EP tube and collect the supernatant to obtain the protein.

[0081] 7. Protein Concentration Determination (BCA Method)

[0082] (1) Sample preparation: Total protein from MHCC-97H and MHCC-97H / REGO cells was obtained as the sample to be tested. 16 μL of standard diluent and 4 μL of the sample to be tested were added to each well of a 96-well plate.

[0083] (2) Preparation of standard concentration curve: The standard protein concentration is 0.5 mg / mL. In a 96-well plate, the standard protein is added at the following levels: 0 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL. The standard diluent is then used to fill each well to 20 μL.

[0084] (3) Preparation of BCA working solution: BCA is divided into liquid A and liquid B. When using, pre-mix them in a ratio of 50:1 and use them after they are fully mixed without precipitation.

[0085] (4) Protein concentration measurement: Add 100 μL of BCA working solution to each well. Incubate at 60°C for 30 min. Measure the absorbance at 540 nm using a microplate reader. Create a standard curve and calculate the protein concentration of the sample to be tested.

[0086] 8. Protein expression detection (Western blot experiment)

[0087] (1) Sample preparation: The extracted total proteins of MHCC-97H and MHCC-97H / REGO cells were boiled and used as test samples to detect proteins related to malignant phenotype and drug resistance in MHCC-97H and MHCC-97H / REGO cells.

[0088] (2) Gel preparation: Separating gel and stacking gel for Western blot experiments were prepared according to the manufacturer's instructions. The separating gel composition was: 3.1 mL H2O, 2.0 mL 1.5 M Tris-HCL pH 8.8, 2.7 mL Aceylamide / Bis, 80 μL 10% SDS, 80 μL 10% AP, 10 μL TEMED; the stacking gel composition was: 2.4 mL H2O, 1.0 mL 1.5 M Tris-HCL pH 6.8, 0.5 mL Aceylamide / Bis, 40 μL 10% SDS, 40 μL 10% AP, 10 μL TEMED.

[0089] (3) Sample loading: The total amount of sampled protein is 20 μg. After 30 minutes of gel preparation, sample loading begins. The same total amount of protein is added to each well. The experiment can be repeated on the same plate gel. Markers need to be added between different groups to indicate protein quality. 5 μL of 1× loading buffer needs to be added to the leftmost and rightmost wells of the gel to reduce edge drift during protein electrophoresis.

[0090] (4) Electrophoresis: Use constant voltage electrophoresis, 80V, 30min, 120V, 60min. Observe the process at all times to prevent the protein from escaping from the gel.

[0091] (5) Transfer: Prepare filter paper and PVDF membrane, with the membrane larger than the filter paper, then the gel. Prepare three containers and add methanol, transbuffer, and ddH2O to each container. Activate the PVDF membrane in methanol for 15 seconds, transfer it to ddH2O, and then transfer it to transbuffer. Wet the filter paper with transbuffer. Sandwich the gel and membrane between the filter paper and begin transfer. Constant current: 120 mA, 60 min.

[0092] (6) Blocking: The PVDF membrane was placed in pre-prepared 5% skim milk powder and blocked at room temperature for 1 hour. After blocking, it was washed with TBST three times for 5 minutes each time.

[0093] (7) Incubation with primary antibody: Separate the protein to be measured from the PVDF membrane according to the marker instructions. Dilute the primary antibody according to the instructions in the instructions. Soak the protein bands to be detected in the corresponding primary antibody overnight. Remove the bands and wash them three times with TBST for 15 minutes each time.

[0094] (8) Incubation with secondary antibody: After washing, the strips were immersed in secondary antibody for 2 h. After the secondary antibody incubation, they were washed three times with TBST for 15 min each time.

[0095] (9) Exposure: The obtained strips were placed in TBST for exposure. The exposure instrument was cooled in advance. After the temperature reached -40°C, the camera was started. The ECL developer solution A and solution B were mixed in a 1:1 ratio to form a working solution. After removing any water marks from the strips, they were placed on the exposure instrument with the protein side facing up. The working solution was then added dropwise. The strips were developed and the images were saved. The grayscale analysis of the images was performed using ImageJ, and the analysis results were statistically analyzed.

[0096] 9. In vivo drug sensitivity test (nude mouse tumor-bearing oral drug treatment experiment)

[0097] (1) Animal preparation: The animals used in the experiment were 6-week-old male BALB / c nude mice weighing approximately 20 g. They were raised in the laboratory for 3-4 days to reduce stress response. The experimental mice were housed in an SPF environment with an ambient temperature of 18℃-26℃, a relative humidity of 40%-70%, and a 12:12 hour day and night light cycle to maintain the circadian rhythm of the nude mice. The cages were sterile plastic boxes and sterile cage covers. The drinking water was sterilized ddH2O. The bedding was corn cob bedding, which was changed once a week and cleaned each time. The feed was Co60 irradiated. The mouse chow and drinking water were changed every 3-4 days.

[0098] (2) Construction of subcutaneous tumors in nude mice: The loaded tumor cells were MHCC-97H / REGO cell line, which was established in the laboratory. MHCC-97H / REGO cells were injected subcutaneously into nude mice at a rate of 1×10 7 Each mouse was injected with 100 μL of cell suspension in the axilla. The skin was disinfected with alcohol before injection. The needle was inserted 1.5 cm from the axilla and moved 1 cm to 1.5 cm subcutaneously. When the needle reached the axilla, the cell suspension in the syringe was ejected and the needle was gently pressed and quickly withdrawn.

[0099] (3) Nude mouse grouping: After tumor loading, nude mice were uniformly placed in cages to ensure that there were no more than 7 mice per cage. Every two days, the long and short diameters of the subcutaneous tumors of nude mice were calculated with a vernier caliper. The formula for calculating the tumor volume of mice was: long diameter × short diameter × short diameter / 2. The tumor volume of all nude mice was calculated, and the average subcutaneous tumor volume was calculated every day. When the average volume of the subcutaneous tumor of nude mice reached 100 mm, the tumor volume was calculated. 3 At about 3 d, the mice were randomly divided into control group, regorafenib treatment group, disulfiram treatment group, and combination treatment group.

[0100] (4) In vivo tumor treatment experiment: The administration method is gavage, and the administration time is three times in the morning, noon and evening. Regorafenib is administered by gavage at noon, once a day for three weeks, and then stopped for one week. The dosage is 20 mg / kg. Disulfiram is administered by gavage in the morning, and the dosage is 50 mg / kg of disulfiram, which is continuously administered until the end of the experiment. Copper gluconate is administered by gavage at night, and the dosage is 0.2 mg / kg of copper gluconate, which is continuously administered until the end of the experiment. The non-treated group is gavaged with the corresponding solvent in the morning, noon and evening. The specific administration forms are:

[0101] 1) Control group: solvent;

[0102] 2) Regorafenib treatment group: Regorafenib was administered;

[0103] 3) Disulfiram treatment group: disulfiram and copper gluconate administration;

[0104] 4) Combination treatment group: regorafenib, disulfiram, and copper gluconate were administered.

[0105] (5) Nude mouse tumor measurement: At the beginning of the experiment, the long and short diameters of the subcutaneous tumors of nude mice were measured every two days with a vernier caliper. The mouse tumor volume was calculated using the formula: long diameter × short diameter × short diameter / 2. The mouse body weight and tumor volume were recorded.

[0106] According to animal ethics requirements, nude mice were euthanized when the longest diameter of the tumor reached 20 mm to minimize animal suffering. For this study, the experiment was terminated when the average diameter of the control group tumor reached 20 mm. Upon completion of the experiment, all nude mice were euthanized, and the tumors were removed and photographed.

[0107] 10. Tumor Tissue Protein Extraction

[0108] After the mouse tumor was isolated, it was cut into 5 mm pieces and placed in 1 mL of NP40. The tissue was broken up using an ultrasonic grinder at 100 Hz for 1 min. After digestion for 30 min, 5× loading buffer was added for dilution and the mixture was boiled at 95°C for 5 min.

[0109] 11. Statistical processing

[0110] The experimental data were analyzed using SPSS 22.0 statistical software and graphed using GraphPad. Multiple groups of data were statistically analyzed using the LSD method in ANOVA, and a p value less than 0.05 indicated statistical significance.

[0111] Example 1 Construction of Regorafenib-resistant hepatocellular carcinoma strain MHCC-97H / REGO

[0112] A regorafenib-resistant cell line was constructed by the gradient concentration increase method. After 6 months of regorafenib stimulation of MHCC-97H cells, the obtained cell line was named MHCC-97H / REGO. The results of the CCK8 experiment showed that the cell survival rate of MHCC-97H / REGO was significantly higher than that of MHCC-97H cells under the action of 1μM, 5μM, 10μM, and 20μM regorafenib. Statistical analysis of the CCK8 experimental results showed that the IC of regorafenib in MHCC-97H / REGO was 2.34, 2.73, and 2.37, respectively. 50 (13.78μM±1.02μM) significantly higher than the IC of regorafenib in MHCC-97H 50 (7.16μM±0.46μM)( Figure 1 A). This result indicates that MHCC-97H / REGO has reduced sensitivity to regorafenib, indicating that the MHCC-97H / REGO cell line constructed in this study is regorafenib-resistant. Morphological observation results showed that the parental cell MHCC-97H was flat in shape, while the resistant cell line MHCC-97H / REGO was elongated in shape ( Figure 1 B) The experimental results show that during the process of regorafenib resistance in HCC cells, the cell morphology tends to transform from epithelial cells to mesenchymal cells, and also demonstrate the successful establishment of regorafenib-resistant HCC cells.

[0113] Example 2 Changes in proliferation, migration and invasion abilities of regorafenib-resistant hepatocellular carcinoma cell line MHCC-97H / REGO

[0114] The results of the clone formation experiment showed that the number of clones formed by MHCC-97H / REGO was significantly higher than that of MHCC-97H, which was 3.94±0.73 times that of MHCC-97H ( Figure 2 A- Figure 2 B). The experimental results showed that MHCC-97H / REGO had improved proliferation ability compared to MHCC-97H. The results of the cell cycle experiment showed that the proportion of cells in the G2 / M phase of MHCC-97H / REGO was significantly lower than that of MHCC-97H, which was 64.6% ± 11.8% of that of MHCC-97H ( Figure 2 C- Figure 2 D). The experimental results showed that the G2 / M phase cell cycle progression in MHCC-97H / REGO may be accelerated compared with MHCC-97H. The scratch test results showed that the cell healing rate of MHCC-97H / REGO was significantly higher than that of MHCC-97H. At 24 hours, it was 2.16±0.96 times that of MHCC-97H ( Figure 3 A- Figure 3 B), at 48h, it was 1.75±0.52 times that of MHCC-97H ( Figure 3 A- Figure 3 B). The experimental results show that compared with MHCC-97H, MHCC-97H / REGO has enhanced migration ability under serum-free conditions. Tranwell migration assay results show that the number of cells that passed through the chamber of MHCC-97H / REGO was significantly increased compared with MHCC-97H, which was 3.49±1.37 times that of MHCC-97H ( Figure 3 C- Figure 3 D). The experimental results showed that compared with MHCC-97H, MHCC-97H / REGO had enhanced migration ability under serum conditions. Transwell invasion assay results showed that the number of cells that passed through the Matrigel chamber in MHCC-97H / REGO was significantly increased compared with MHCC-97H, which was 2.65±0.61 times that of MHCC-97H ( Figure 3 E- Figure 3 F) The experimental results show that MHCC-97H / REGO has enhanced invasive ability compared with MHCC-97H.

[0115] Example 3 Detection of protein expression between regorafenib-resistant hepatocellular carcinoma cell line MHCC-97H / REGO and MHCC-97H

[0116] The results of cell cycle-related protein detection showed that the expression of cyclin B1 in MHCC-97H / REGO cells was significantly increased, which was 4.94±1.38 times that of MHCC-97H ( Figure 4 A- Figure 4B). The experimental results indicate that cyclin B1 expression may be positively correlated with regorafenib resistance. EMT-related protein detection results showed that the expression of epithelial marker E-cadherin in MHCC-97H / REGO was significantly lower than that in MHCC-97H, accounting for 51.1% ± 10.1% of that in MHCC-97H ( Figure 4 A- Figure 4 B). The expression levels of mesenchymal cell markers N-cadherin and vimentin, and EMT-related transcription factors snail and slug in MHCC-97H / REGO were significantly increased compared with MHCC-97H, which were 7.73±2.28 times (N-cadherin), 3.23±0.52 times (vimentin), 6.68±2.18 times (snail), and 2.82±0.67 times (slug), respectively ( Figure 4 A- Figure 4 B) The experimental results suggest that the expression of mesenchymal markers N-cadherin and vimentin, as well as EMT-related transcription factors snail and slug, may be positively correlated with regorafenib resistance, while the expression of epithelial marker E-cadherin may be negatively correlated with regorafenib resistance.

[0117] Example 4 Effect of p-ERK1 / 2 Inhibition on Regorafenib Resistance in MHCC-97H / REGO

[0118] Western blot results showed that the expression level of p-ERK1 / 2 in the SHC772984-treated group was significantly lower than that in the control group ( Figure 5 A). The experimental results show that SCH772984 can inhibit the expression of p-ERK1 / 2 in MHCC-97H / REGO. The CCK8 assay results show that under the treatment conditions of different concentrations of regorafenib (0μM, 1μM, 5μM, 10μM, 20μM, 40μM), the cell viability of the SHC772984-treated group was significantly reduced compared with the control group. The IC value of regorafenib in MHCC-97H / REGO cells treated with SHC772984 was significantly lower than that in the control group. 50 Compared with the control group, the IC 50 :13.78μM±1.02μM, IC of SCH772984-treated group 50 :7.501μM±0.77μM( Figure 5 B) Experimental results show that reducing the expression of p-ERK1 / 2 can reduce the resistance of MHCC-97H / REGO to regorafenib.

[0119] Example 5 Effects of p-ERK1 / 2 Inhibition on Proliferation, Migration and Invasion of MHCC-97H / REGO Cells

[0120] The results of the clone formation experiment showed that the number of MHCC-97H / REGO clones in the SCH772984 treatment group decreased significantly, which was 50%±4% of that in the control group ( Figure 6 A). The experimental results show that reducing the expression of p-ERK1 / 2 can inhibit the proliferation of MHCC-97H / REGO. The cell cycle results showed that the number of cells arrested in the G2 / M phase in the SCH772984 treatment group increased significantly, which was 1.67±0.11 times that of the control group ( Figure 6 B). The experimental results show that reducing the expression of p-ERK1 / 2 can inhibit the cell cycle progression of MHCC-97H / REGO cells. The results of the scratch test showed that the healing rate of MHCC-97H / REGO cells in the SCH772984 treatment group was significantly reduced, which was 1% ± 0.6% of that in the control group ( Figure 7 A). The experimental results show that reducing the expression of p-ERK1 / 2 can inhibit the migration ability of MHCC-97H / REGO cells in the absence of serum. The results of the Transwell migration assay showed that the number of MHCC-97H / REGO cells that passed through the chamber in the SCH772984 treatment group was significantly reduced, reaching 9.6% ± 1.8% of the control group ( Figure 7 B). Experimental results indicate that reducing the expression of p-ERK1 / 2 can inhibit the migration ability of MHCC-97H / REGO cells in the presence of serum. Transwell invasion assay results showed that the number of MHCC-97H / REGO cells that passed through the chamber in the SCH772984-treated group was significantly reduced, reaching 3.5% ± 2.2% of that in the control group ( Figure 7 C) The experimental results show that reducing the expression of p-ERK1 / 2 can inhibit the invasion ability of MHCC-97H / REGO.

[0121] Example 6 Effects of Inhibiting p-ERK1 / 2 on Cell Cycle and EMT-Related Proteins in MHCC-97H / REGO Cells

[0122] The results of EMT-related protein detection showed that the expression of epithelial marker E-cadherin in the SCH772984 treatment group was significantly increased, which was 2.7±0.64 times that of the control group. The expression of mesenchymal markers N-cadherin and vimentin, and EMT-related transcription factors snail and slug was significantly decreased, which was 32.3%±13.3% (N-cadherin), 39.2%±12.5% ​​(vimentin), 26.3%±15% (snail) and 44.7%±14% (slug) of the control group ( Figure 8 A- Figure 8 B). Experimental results indicate that reducing the expression of p-ERK1 / 2 can inhibit the expression of mesenchymal markers N-cadherin and vimentin, as well as EMT-related transcription factors snail and slug, and promote the expression of E-cadherin. Cell cycle protein detection results showed that the expression of cyclin B1 in the SCH772984 treatment group was significantly reduced, reaching 37.4% ± 12.1% of the control group ( Figure 8 A- Figure 8 B). The experimental results show that reducing the expression of p-ERK1 / 2 can inhibit the expression of cyclin B1.

[0123] Example 7 Effect of Activating p-ERK1 / 2 on Regorafenib Sensitivity in MHCC-97H

[0124] Western blot results showed that the expression level of p-ERK1 / 2 in the TBHQ-treated group was significantly increased compared with the control group ( Figure 9 A). The experimental results show that TBHQ can stimulate the expression of p-ERK1 / 2 in MHCC-97H cells. The CCK8 assay results show that under the treatment conditions of different concentrations of regorafenib (0μM, 1μM, 5μM, 10μM, 20μM, 40μM), the cell viability of the TBHQ-treated group was significantly increased compared with the control group. The IC of regorafenib in the TBHQ-treated group was 1. 50 Compared with the control group, the IC 50 :9.61μM±0.59μM, control group IC 50 :7.31μM±0.44μM( Figure 9 B) Experimental results show that increasing the expression of p-ERK1 / 2 can reduce the sensitivity of MHCC-97H to regorafenib.

[0125] Example 8 Effects of Activating p-ERK1 / 2 on the Proliferation, Migration and Invasion Abilities of MHCC-97H

[0126] The results of the clone formation experiment showed that the number of MHCC-97H cell clones in the TBHQ treatment group increased significantly, which was 2.29±0.25 times that of the control group ( Figure 10 A). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the proliferation of MHCC-97H. The cell cycle results showed that the proportion of G2 / M phase cells in the TBHQ treatment group decreased significantly, which was 76.4% ± 5.3% of the control group ( Figure 10B). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the progression of MHCC-97H cell cycle. The results of the scratch test showed that the healing rate of MHCC-97H cells in the TBHQ treatment group was significantly increased, which was 5.24±1.22 times that of the control group ( Figure 11 A). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the migration ability of MHCC-97H in serum-free conditions. The results of the Transwell migration assay showed that the number of MHCC-97H cells that passed through the chamber in the TBHQ-treated group was significantly increased, which was 7.95±0.41 times that of the control group ( Figure 11 B). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the migration ability of MHCC-97H cells in the presence of serum. The results of the Transwell invasion assay showed that the number of MHCC-97H cells that passed through the chamber in the TBHQ-treated group was significantly increased, which was 15.5±1.54 times that of the control group ( Figure 11 C) The experimental results show that increasing the expression of p-ERK1 / 2 can promote the invasion ability of MHCC-97H.

[0127] Example 9 Effects of Activating p-ERK1 / 2 on MHCC-97H Cell Cycle and EMT-Related Proteins

[0128] The results of EMT-related protein detection showed that the expression of E-cadheirn in the TBHQ-treated group was significantly decreased, reaching 53.3%±10.7% of the expression level in the control group. The expression of mesenchymal markers N-cadherin and vimentin, and EMT-related transcription factors snail and slug were significantly increased, reaching 2.92±0.57 times (N-cadherin), 1.97±0.38 times (vimentin), 4.67±1.78 times (snail), and 2.42±1.04 times (slug) of the expression levels in the control group ( Figure 12 A- Figure 12 B). The experimental results showed that increasing the expression of p-ERK1 / 2 could inhibit the expression of epithelial marker E-cadheirn and promote the expression of mesenchymal markers N-cadherin and vimentin as well as EMT-related transcription factors snail and slug. The results of cell cycle-related protein detection showed that the expression of cyclin B1 in the TBHQ-treated group was significantly increased, which was 2.24±0.34 times the expression level of the control group ( Figure 12 A- Figure 12 B). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the expression of cyclin B1.

[0129] Example 10 Effects of disulfiram copper chelate on proliferation, cell cycle and related proteins of hepatocellular carcinoma cells

[0130] The results of CCK8 experiments showed that different concentrations of CuET (0.1μM-0.5μM) could significantly inhibit the cell viability of MHCC-97H / REGO, MHCC-97H, MHCC-LM3 and SMCC-7721 cells ( Figure 13 A). The experimental results show that CuET can inhibit the survival of HCC cells. The results of the clone formation experiment found that different concentrations of CuET can significantly reduce the number of clones formed by MHCC-97H / REGO, MHCC-97H, MHCC-LM3 and SMCC-7721 ( Figure 13 B). The experimental results show that CuET can inhibit the proliferation of HCC cells. Through cell cycle experiments, it was found that CuET treatment can significantly increase the proportion of cells arrested in the G2 / M phase of MHCC-97H / REGO, MHCC-97H, MHCC-LM3 and SMCC-7721 cells ( Figure 14 A- Figure 14 D). The experimental results indicate that CuET can block the cell cycle progression of HCC cells. Western blot results showed that the expression of cyclin B1 in MHCC-97H / REGO, MHCC-97H, SMCC-7721, and MHCC-LM3 cells treated with CuET was significantly decreased, reaching 44.1% ± 4.3% of the control group (MHCC-97H / REGO) ( Figure 14 E), 46.3%±2.4%(MHCC-97H)( Figure 14 F), 31.2%±1.1%(SMCC-7721)( Figure 14 G) and 32.1% ± 9.7% (MHCC-LM3) ( Figure 14 H). The experimental results show that CuET can inhibit the expression of cyclin B1.

[0131] Example 11 Effects of disulfiram copper chelate on migration, invasion and EMT-related proteins in hepatocellular carcinoma cells

[0132] The results of the scratch test showed that in MHCC-97H / REGO cells, the 24h cell healing rate of CuET-treated MHCC-97H / REGO was 18.2%±12.1% of that of the control group; and the 48h cell healing rate was 32.5%±14.9% of that of the control group. Figure 15 A). In MHCC-97H cells, the 24h cell healing rate of CuET-treated MHCC-97H was 18.2%±12.1% of that of the control group; and the 48h cell healing rate was 32.5%±14.9% of that of the control group ( Figure 15B). In SMCC-7721 cells, the 24h cell healing rate of CuET-treated SMCC-7721 was 35.2%±3.8% of that of the control group; and the 48h cell healing rate was 39.9%±4.5% of that of the control group ( Figure 15 C). In MHCC-LM3 cells, the 24h cell healing rate of CuET-treated MHCC-LM3 cells was 69.9%±4.9% of that of the control group; and the 48h cell healing rate was 55.2%±3.9% of that of the control group ( Figure 15 D). The experimental results show that CuET can inhibit the migration ability of HCC cells in the absence of serum. The results of the Transwell migration assay showed that the number of MHCC-97H / REGO cells that passed through the chamber after 24 hours in the CuET-treated group was significantly reduced, which was 35.6% ± 8.6% of that in the control group ( Figure 15 E). The number of MHCC-97H cells that passed through the chamber in the CuET-treated group decreased significantly after 24 hours, which was 35.6%±8.6% of that in the control group ( Figure 15 F). The number of SMCC-7721 cells that passed through the chamber in the CuET-treated group after 24 hours showed no significant difference compared with the control group ( Figure 15 G). The number of MHCC-LM3 cells that passed through the chamber in the CuET-treated group decreased significantly after 24 hours, which was 60.3%±10.2% of that in the control group ( Figure 15 H). The experimental results show that CuET can inhibit the migration ability of HCC cells under serum conditions. The results of the Transwell invasion assay showed that the number of MHCC-97H / REGO cells that passed through the chamber in the CuET-treated group was significantly reduced after 24 hours, which was 52% ± 20.5% of that in the control group ( Figure 15 E). The number of MHCC-97H cells that passed through the chamber in the CuET-treated group decreased significantly after 24 hours, which was 14.9%±6.5% of that in the control group ( Figure 15 F). The number of SMCC-7721 cells that passed through the chamber in the CuET-treated group decreased significantly after 24 hours, which was 6.7%±5.5% of that in the control group ( Figure 15 G). The number of MHCC-LM3 cells that passed through the chamber in the CuET-treated group was significantly decreased, which was 4.5%±2.2% of that in the control group ( Figure 15H). The experimental results showed that CuET can inhibit the invasive ability of HCC cells. The results of Western blot experiments on EMT-related proteins showed that in MHCC-97H / REGO, the expression of epithelial marker E-cadherin in the CuET-treated group was significantly increased, which was 3.36±2.41 times that of the control group. The expression of mesenchymal markers N-cadherin and vimentin as well as EMT-related transcription factors snail and slug in the CuET-treated group was significantly decreased, which was 49.2%±13.4% (N-cadherin), 45.5%±12.8% (vimentin), 22.2%±10.1% (snail) and 44.4%±11.8% (slug) of the control group ( Figure 16 A). In MHCC-97H cells, the expression of the epithelial marker E-cadherin was significantly increased in the CuET-treated group, reaching 2±0.43 times that of the control group. The expression of the mesenchymal markers N-cadherin and vimentin, as well as the EMT-related transcription factors snail and slug, was significantly decreased in the CuET-treated group, reaching 49.2%±13.4% (N-cadherin), 45.5%±12.8% (vimentin), 33.5%±10.8% (snail), and 47.2%±20.9% (slug) of the control group ( Figure 16 B). In SMCC-7721 cells, the expression of the epithelial marker E-cadherin was significantly increased in the CuET-treated group, reaching 1.71±0.68 times that of the control group. The expression of the mesenchymal markers N-cadherin and vimentin, as well as the EMT-related transcription factors snail and slug, was significantly decreased in the CuET-treated group, reaching 26.2%±5.5% (N-cadherin), 38.5%±11.8% (vimentin), 42.1%±18.8% (snail), and 46.1%±24.5% (slug) of the control group ( Figure 16 C). In MHCC-LM3 cells, the expression of E-cadherin in the CuET-treated group was significantly increased, reaching 2.32±0.85 times that of the control group. The expression of mesenchymal markers N-cadherin and vimentin, as well as EMT-related transcription factors snail and slug, was significantly decreased in the CuET-treated group, reaching 35.9%±13.2% (N-cadherin), 66.5%±7.2% (vimentin), 49.9%±25.2% (snail), and 75.9%±9.8% (slug) of the control group ( Figure 16D) Experimental results show that CuET can promote the expression of the epithelial marker E-cadherin in HCC cells, and inhibit the expression of mesenchymal markers N-cadherin and vimentin, as well as the EMT-related transcription factors snail and slug.

[0133] Example 12: Disulfiram copper chelate inhibits regorafenib resistance-associated protein p-ERK1 / 2

[0134] Western blot results showed that the expression of p-ERK1 / 2 in MHCC-97H / REGO cells in the CuET-treated group was 40.9%±1.7% of that in the control group ( Figure 17 A). The expression of p-ERK1 / 2 in MHCC-97H cells in the CuET-treated group was 48.2%±1.5% of that in the control group ( Figure 17 B). The expression of p-ERK1 / 2 in SMCC-7721 cells in the CuET-treated group was 49%±10.2% of that in the control group ( Figure 17 C). The expression of p-ERK1 / 2 in MHCC-LM3 cells in the CuET-treated group was 51.8%±5.8% of that in the control group ( Figure 17 D) The experimental results show that CuET can inhibit the expression of p-ERK1 / 2 in HCC cells.

[0135] Example 13 Effect of Disulfiram Copper Chelate on Regorafenib Resistance in MHCC-97H / REGO

[0136] The CCK8 assay was used to detect the IC50 of regorafenib in MHCC-97H / REGO cells treated with different concentrations of CuET. 50 The experimental results showed that the IC value of regorafenib on MHCC-97H / REGO in the control group was 50 The IC values ​​of regorafenib against MHCC-97H / REGO in the 0.1μM, 0.15μM, and 0.2μM CuET treatment groups were 13.77μM±1.03μM. 50 7.38 μM ± 0.94 μM (0.1 μM CuET), 0.42 μM ± 0.27 μM (0.15 μM CuET) and 0.15 μM ± 0.09 μM (0.2 μM CuET), respectively. Figure 18 A- Figure 18 B) The experimental results show that compared with the control group, the inhibitory effect of regorafenib on MHCC-97H / REGO was significantly increased in the CuET-treated group, and the inhibitory effect became more pronounced with increasing CuET concentration. Specifically, 0.1 μM CuET restored regorafenib resistance in MHCC-97H / REGO cells to the level of parental MHCC-97H cells.

[0137] Example 14 Effects of disulfiram copper chelate combined with regorafenib on the proliferation, migration and invasion of MHCC-97H / REGO

[0138] The results of the clone formation experiment showed that the number of MHCC-97H / REGO clones in the combined treatment group was significantly reduced, which was 12.2%±3.5% of the regorafenib treatment group ( Figure 19 A- Figure 19 B), and 18.7%±7.6% of the CuET-treated group ( Figure 19 A- Figure 19 B). The experimental results show that compared with the single-use of regorafenib or CuET alone, the combination of CuET and regorafenib can significantly inhibit the proliferation of MHCC-97H / REGO cells. Flow cytometry results showed that the proportion of MHCC-97H / REGO cells arrested in the G2 / M phase in the combination treatment group was significantly increased, which was 1.41±0.17 times that of the regorafenib treatment group ( Figure 19 C- Figure 19 D), which was 1.63±0.16 times that of the CuET-treated group ( Figure 19 C- Figure 19 D). The experimental results showed that compared with regorafenib alone or CuET alone, the combination of CuET and regorafenib significantly arrested the MHCC-97H / REGO cell cycle at the G2 / M phase. The scratch assay results showed that the 48-hour cell healing rate of MHCC-97H / REGO cells in the combination treatment group was significantly reduced, reaching 56.2% ± 8.5% of that in the regorafenib treatment group ( Figure 20 A- Figure 20 B), and 56.5%±20.9% of the CuET-treated group ( Figure 20 A- Figure 20 B). The experimental results show that compared with regorafenib alone or CuET alone, the combination of CuET and regorafenib significantly inhibited the migration ability of MHCC-97H / REGO cells in the serum-free condition. Transwell migration assay results showed that the number of MHCC-97H / REGO cells that crossed the chamber in the combination treatment group was significantly reduced, accounting for 21.6% ± 3.7% of that in the regorafenib treatment group ( Figure 20 C- Figure 20 D), which was 10%±4% of the CuET-treated group ( Figure 20 C- Figure 20 D). The experimental results showed that compared with regorafenib alone or CuET alone, the combination of CuET and regorafenib significantly inhibited the migration ability of MHCC-97H / REGO cells in the presence of serum. Transwell invasion assay results showed that the number of MHCC-97H / REGO cells that passed through the chamber in the combination treatment group was significantly reduced, reaching 8.2% ± 3.8% of that in the regorafenib treatment group ( Figure 20 E- Figure 20 F), which was 10.1%±5% of the CuET-treated group ( Figure 20 E- Figure 20 F) The experimental results showed that compared with regorafenib alone or CuET alone, the combination of CuET and regorafenib significantly inhibited the invasion ability of MHCC-97H / REGO cells.

[0139] Example 15 Effects of disulfiram copper chelate combined with regorafenib on p-ERK1 / 2, cell cycle, and EMT-related proteins in MHCC-97H / REGO

[0140] The experiment set up four groups: control group (no drug treatment), regorafenib alone treatment group (10μM regorafenib), disulfiram alone treatment group (0.1μM CuET treatment), and combination treatment group (10μM regorafenib + 0.1μM CuET combined treatment). After 72 hours of treatment, total protein was extracted from MHCC-97H / REGO cells in each group. Western blot analysis of p-ERK1 / 2 showed that the expression of p-ERK1 / 2 in MHCC-97H / REGO cells in the combination treatment group was significantly reduced, reaching 58.6% ± 20.5% of the regorafenib treatment group and 48.1% ± 25.5% of the CuET treatment group ( Figure 21 A- Figure 21 B). The experimental results showed that the combination of CuET and regorafenib significantly inhibited the expression of p-ERK1 / 2 compared with regorafenib or CuET alone. Western blot analysis of cell cycle proteins showed that the expression of cyclin B1 in MHCC-97H / REGO cells was significantly reduced in the combination treatment group, reaching 37.5% ± 22.6% of that in the regorafenib group and 27.9% ± 15.5% of that in the CuET group ( Figure 21 A- Figure 21B) The experimental results show that compared with regorafenib or CuET alone, the combination of CuET and regorafenib can significantly inhibit the expression of cyclin B1. Western blot analysis of EMT-related proteins Blot results showed that the expression of epithelial marker E-cadherin in MHCC-97H / REGO cells in the combined treatment group was significantly increased, while the expression of mesenchymal markers N-cadherin and vimentin, and EMT-related transcription factors snail and slug were significantly decreased, which were 1.75±0.58 times (E-cadherin), 45.7%±13.9% (N-cadherin), 23.8%±8.4% (vimentin), 45.5%±5.8% (snail), and 46.1%±19.4% (slug) in the regorafenib treatment group, and 2.39±0.44 times (E-cadherin), 48.2%±23.2% (N-cadherin), 21.1%±15.1% (vimentin), 33.5%±19.2% (snail), and 74.7%±12.5% ​​(slug) in the CuET treatment group ( Figure 21 A- Figure 21 B) The experimental results showed that compared with regorafenib or CuET alone, the combination of CuET and regorafenib significantly activated the expression of the epithelial marker E-cadherin and inhibited the expression of the mesenchymal markers N-cadherin and vimentin, as well as the EMT-related transcription factors snail and slug.

[0141] Example 16 Effect of Disulfiram on Regorafenib Resistance in MHCC-97H / REGO Subcutaneous Tumors

[0142] The experimental nude mice were randomly divided into 4 groups, and the nude mice in each group were treated according to the following: control group (normal saline gavage), disulfiram treatment group (disulfiram plus copper gluconate gavage), regorafenib treatment group (regorafenib gavage), and combination treatment group (disulfiram plus copper gluconate plus regorafenib gavage). The main purpose was to compare the differences between the regorafenib treatment group and the combination treatment group, and to analyze the effect of disulfiram in reversing regorafenib resistance in vivo. The results of the nude mouse treatment experiment showed that during the treatment process, the subcutaneous tumor volumes of the nude mice in the combination treatment group, regorafenib treatment group, and disulfiram treatment group were basically the same for the first 13 days; subsequently, the growth trends of the subcutaneous tumor volumes of the nude mice in the three treatment groups showed significant differences. The subcutaneous tumor volumes of the nude mice in the disulfiram treatment group and regorafenib treatment group showed an increasing trend, while the subcutaneous tumor volumes of the nude mice in the combination treatment group showed a decreasing trend ( Figure 22 A). After 31 days of treatment, the tumors of the nude mice in the control group, disulfiram treatment group, regorafenib treatment group, and combination treatment group were isolated. Photographs and records showed that the tumor volume of the nude mice in the combination treatment group was significantly reduced compared with the regorafenib treatment group or the disulfiram treatment group ( Figure 23 A). The size of the isolated MHCC-97H / REGO tumor tissue was weighed and statistically analyzed. The experimental results showed that the tumor weight of the nude mice in the combination treatment group was significantly lower than that in the other treatment groups, which was 27.4% ± 6.1% of the regorafenib treatment group and 11.1% ± 5% of the CuET treatment group ( Figure 23 B) The experiment showed that the combination of disulfiram and regorafenib significantly inhibited the growth of MHCC-97H / REGO subcutaneous tumors compared with disulfiram alone or regorafenib alone.

[0143] Summary of the invention

[0144] Acquired resistance to regorafenib has been a major factor limiting its use and widespread adoption in the clinical application and research of HCC. This study designed multiple experimental protocols to investigate the mechanisms of regorafenib resistance in HCC, explore the therapeutic effects of disulfiram, and analyze the feasibility of reversing regorafenib resistance in HCC with disulfiram both in vivo and in vitro.

[0145] We have revealed for the first time that p-ERK1 / 2 plays a regulatory role in regorafenib resistance in MHCC-97H cells, primarily through its regulation of cell proliferation, migration, and invasion. More importantly, we have used disulfiram for the first time to reverse regorafenib resistance in MHCC-97H / REGO cells. The experimental results showed that disulfiram significantly inhibited the proliferation, migration, and invasion of MHCC-97H / REGO cells at the same concentration of regorafenib. In summary, the disulfiram provided by the present invention can be used as a drug to prolong the lives of patients with regorafenib-resistant hepatocellular carcinoma.

Claims

1. Application of disulfiram in the preparation of drugs for enhancing the sensitivity of hepatocellular carcinoma to regorafenib.

2. Application of disulfiram in the preparation of drugs to reverse regorafenib resistance in hepatocellular carcinoma.

3. Use of a combination of disulfiram and regorafenib in the preparation of a drug for treating hepatocellular carcinoma.

4. The use of disulfiram in the preparation of drugs for the treatment of patients with advanced hepatocellular carcinoma who are resistant to regorafenib.

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