Application of disulfiram in reversing regorafenib drug tolerance of hepatocellular carcinoma

Abstract

The invention relates to the field of tumor treatment, in particular to application of a small molecule compound in preparation of a medicine for treating hepatocellular carcinoma. Disulfiram is used for reversing the regorafenib drug resistance of MHCC-97H / REGO cells for the first time, and experimental results show that after disulfiram is used, regorafenib with the same concentration has more obvious effects of inhibiting proliferation, migration and invasion of MHCC-97H / REGO, and can significantly inhibit the tolerance of hepatocellular carcinoma to regorafenib. Therefore, the disulfiram provided by the invention can be used for preparing a medicine for prolonging the life of a patient after the hepatocellular carcinoma regorafenib resistance. The invention provides application of disulfiram in preparation of a regorafenib-resistant drug for treating 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 when diagnosed, and drug therapy is the main treatment for these patients. Regorafenib is a second-line treatment drug for advanced HCC approved by the US Food and Drug Administration (FDA), which can prolong the life of HCC patients, but its therapeutic effect is limited due to acquired drug resistance. Therefore, finding a way to reverse regorafenib resistance is a key research direction in the field of HCC treatment. Drug repurposing is an important strategy to reverse acquired drug resistance. Disulfiram (DSF) is an early FDA-approved drug for alcohol withdrawal. So far, there has been no report on whether it can be used for regorafenib resistance in hepatocellular carcinoma.

[0003] This study will explore the effects of disulfiram on the proliferation and migration of hepatocellular carcinoma through in vivo and in vitro 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 resistance. Summary of the invention

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

[0005] Therefore, the main contents of the present invention are summarized as follows: the 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. For the first time, we successfully constructed a regorafenib-resistant cell line MHCC-97H / REGO derived from MHCC-97H, 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 of hepatocellular carcinoma cells was regulated by p-ERK1 / 2 to affect the cell phenotype.

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

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

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

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

[0013] Figure 2.Changes in proliferation ability of regorafenib-resistant hepatocellular carcinoma cells: (A) 1000 cells / well were planted in MHCC-97H and MHCC-97H / REGO and cultured for 7 days. The number of clones formed was detected by clone formation experiment. (B) After staining and counting of clone formation experiment results, the experimental results were statistically analyzed. (C) After 24h of culture of MHCC-97H and MHCC-97H / REGO, the cell cycle distribution was detected by cell cycle experiment. (D) Statistical analysis of cell cycle distribution of MHCC-97H and MHCC-97H / REGO.

[0014] Figure 3 Changes in migration and invasion ability of regorafenib-resistant hepatocellular carcinoma cells: (A) Scratch assay to detect changes in cell healing rate at 0h, 24h and 48h in MHCC-97H and MHCC-97H / REGO cells cultured in serum-free medium, photographed and recorded under a microscope, scale = 100μm. (B) Statistical results of cell healing rate in the scratch assay. (C) Transwell migration assay to detect changes in the number of cells that passed through the chamber in 24h in MHCC-97H and MHCC-97H / REGO cells cultured in serum-free medium, photographed and recorded under a microscope, scale = 100μm. (D) Statistical results of the number of cells that passed through the chamber in the Transwell migration assay. (E) Transwell invasion assay to detect changes in the number of cells that passed through the chamber in 24h in MHCC-97H and MHCC-97H / REGO cells cultured in serum-free medium, photographed and recorded under a microscope, scale = 100μm. (F) Statistical results of the number of cells that passed through the chamber in the Transwell invasion assay.

[0015] Figure 4 Changes of p-ERK1 / 2 and cell phenotype proteins in regorafenib-resistant hepatocellular carcinoma cells: (A) 48 hours after MHCC-97H and MHCC-97H / REGO cells were plated, the expression of E-cadheirn, N-cadherin, vimentin, snail, slug, cyclin B1, p-ERK1 / 2, and ERK1 / 2 proteins were detected by Western blot. (B) The relative amount of protein expression was analyzed by grayscale statistical results.

[0016] Figure 5Effect of SCH772984 on the drug resistance of hepatocellular carcinoma regorafenib-resistant cells: (A) MHCC-97H / REGO cells, under SCH772984 treatment and control treatment conditions, Western blot assay was used to detect the changes in p-ERK1 / 2 expression, and the experimental results were statistically analyzed. (B) MHCC-97H / REGO cells, under SCH772984 treatment and control treatment conditions, after 72 hours of treatment with different concentrations of regorafenib (0μM, 1μM, 5μM, 10μM, 20μM, 40μM), CCK8 assay was used to detect cell viability.

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

[0018] Figure 7 Effect of SCH772984 on the migration and invasion of regorafenib-resistant hepatocellular carcinoma cells: (A) Scratch assay was used to detect the healing rate of MHCC-97H / REGO cells under SCH772984 treatment and control treatment conditions. At 0h and 24h, the healing rate was recorded under a microscope. The scale bar = 100μm, and the experimental results were statistically analyzed. (B) After 24h of SCH772984 treatment and control treatment, the Transwell migration assay was used to detect the changes in cell migration ability of MHCC-97H / REGO cells. The number of cells entering the lower chamber was recorded under a microscope. The scale bar = 100μm, and the experimental results were statistically analyzed. (C) After 24h of SCH772984 treatment and control treatment, the Transwell invasion assay was used to detect the changes in cell invasion ability of MHCC-97H / REGO cells. The number of cells entering the lower chamber was recorded under a microscope. The scale bar = 100μm.

[0019] Figure 8Effects of SCH772984 on p-ERK1 / 2 and cell phenotype proteins in regorafenib-resistant hepatocellular carcinoma cells: (A) After 24 hours of SCH772984 treatment and control treatment, proteins were extracted from MHCC-97H / REGO cells, and Western blot was used to detect the expression changes of E-cadherin, N-cadherin, vimentin, snail, slug, and cyclin B1 proteins. (B) The relative expression of each protein was recorded, and the experimental results were statistically analyzed.

[0020] Fig. 9 Effect of TBHQ on regorafenib resistance in hepatocellular carcinoma cells: (A) MHCC-97H cells, under TBHQ treatment and control treatment conditions, Western blot assay was used to detect changes in p-ERK1 / 2 expression, and the experimental results were statistically analyzed. (B) MHCC-97H cells, under TBHQ treatment and control treatment conditions, were treated with different concentrations (0μM, 1μM, 5μM, 10μM, 20μM, 40μM) of regorafenib for 72h, and CCK8 assay was used to detect cell viability. The experimental results were statistically analyzed.

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

[0022] Fig.11 Effect of TBHQ on the migration and invasion of hepatocellular carcinoma cells: (A) MHCC-97H cells, 40,000 cells / well, were seeded in the scratch chamber. Under TBHQ treatment and control treatment conditions, at 0h and 24h, the cell healing rate was recorded under a microscope. The scale bar = 100μm, and the experimental results were statistically analyzed. (B) After 24h of TBHQ treatment and control treatment of MHCC-97H cells, the Transwell migration assay was used to detect the changes in cell migration ability. The number of cells entering the lower chamber was recorded under a microscope. The scale bar = 100μm, and the experimental results were statistically analyzed. (C) After 24h of TBHQ treatment and control treatment of MHCC-97H cells, the Transwell invasion assay was used to detect the changes in cell invasion ability. The number of cells entering the lower chamber was recorded under a microscope. The scale bar = 100μm, and the experimental results were statistically analyzed.

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

[0024] Fig.13 Effect of copper disulfiram chelate on the proliferation of hepatocellular carcinoma cells: (A) Four HCC cell lines, MHCC-97H / REGO, MHCC-97H, SMCC-7721 and MHCC-LM3, were exposed to the specified concentrations of CuET (0μM, 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM) for 24h, and the cell survival rate was determined by CCK8 method. (B) Four HCC cell lines, MHCC-97H / REGO, MHCC-97H, SMCC-7721 and MHCC-LM3, were seeded in 6-well plates at a density of 1000 cells / well, and then treated with the specified concentrations of CuET for 24h, cultured in DMEM medium for 7 days, stained and photographed.

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

[0026] Fig.15Effects of copper disulfiram chelate on migration and invasion of hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells, 40,000 cells / scratch chamber, after being treated with CuET (0 μM, 0.1 μM) for 24 h and 48 h, photographs were taken under a microscope to record and calculate the healing rate, scale = 100 μm, and the experimental results were statistically analyzed. (B) MHCC-97H cells, 40,000 cells / scratch chamber, after being treated with CuET (0 μM, 0.1 μM) for 24 h and 48 h, photographs were taken under a microscope to record and calculate the healing rate, scale = 100 μm, and the experimental results were statistically analyzed. (C) SMCC-7721 cells, 40,000 cells / scratch chamber, after being treated with CuET (0 μM, 0.1 μM) for 24 h and 48 h, photographs were taken under a microscope to record and calculate the healing rate, scale = 100 μm, and the experimental results were statistically analyzed. (D) MHCC-LM3 cells, 40,000 cells / scratch chamber, after being treated with CuET (0 μM, 0.1 μM) for 24 h and 48 h, photos were taken under a microscope to record and calculate the healing rate, scale bar = 100 μm, and the experimental results were statistically analyzed. (E) MHCC-97H / REGO cells, 80,000 cells / well were implanted in the upper chamber of Transwell (upper: uncoated with matrix gel, lower: coated with matrix gel), after being treated with CuET (0 μM, 0.1 μM) for 24 h, stained, and photos were taken under a microscope, scale bar = 100 μm, and the number of cells entering the lower chamber was calculated, and the experimental results were statistically analyzed. (F) MHCC-97H cells, 80,000 cells / well were implanted in the upper chamber of Transwell (upper: uncoated with Matrigel, lower: coated with Matrigel), treated with CuET (0 μM, 0.1 μM) for 24 h, stained, photographed under a microscope, scale bar = 100 μm, the number of cells entering the lower chamber was calculated, and the experimental results were statistically analyzed. (G) SMCC-7721 cells, 80,000 cells / well were implanted in the upper chamber of Transwell (upper: uncoated with Matrigel, lower: coated with Matrigel), treated with CuET (0 μM, 0.1 μM) for 24 h, stained, photographed under a microscope, scale bar = 100 μm, the number of cells entering the lower chamber was calculated, and the experimental 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), and treated with CuET (0 μM, 0.1 μM) for 24 h, stained, and photographed under a microscope, scale bar = 100 μm. The number of cells entering the lower chamber was calculated, and the experimental results were statistically analyzed.

[0027] Fig.16Effects of copper disulfiram chelate on migration and invasion phenotype proteins in hepatocellular carcinoma cells: (A) After MHCC-97H / REGO cells were treated with CuET (0μM, 0.1μM) for 24h, Western blot was used to detect the expression changes of E-cadherin, N-cadherin, vimentin, snail and slug in the cells, and the grayscale values ​​were calculated based on the relative expression of proteins. The experimental results were statistically analyzed. (B) After MHCC-97H cells were treated with CuET (0μM, 0.1μM) for 24h, Western blot was used to detect the expression changes of E-cadherin, N-cadherin, vimentin, snail and slug in the cells, and the grayscale values ​​were calculated based on the relative expression of proteins. The experimental results were statistically analyzed. (C) After SMCC-7721 cells were treated with CuET (0μM, 0.1μM) for 24h, the expression changes of E-cadherin, N-cadherin, vimentin, snail and slug in the cells were detected by Western blot, the gray value was calculated based on the relative expression of proteins, and the experimental results were statistically analyzed. (D) After MHCC-LM3 cells were treated with CuET (0μM, 0.1μM) for 24h, the expression changes of E-cadherin, N-cadherin, vimentin, snail and slug in the cells were detected by Western blot, the gray value was calculated based on the relative expression of proteins, and the experimental results were statistically analyzed.

[0028] Fig.17 Effects of copper disulfiram chelate on p-ERK1 / 2 in hepatocellular carcinoma cells: (A) MHCC-97H / REGO, after treatment with CuET (0μM, 0.1μM), Western blot was used to detect the expression changes of ERK1 / 2 and p-ERK1 / 2, as well as the statistical graph. (B) MHCC-97H, after treatment with CuET (0μM, 0.1μM), Western blot was used to detect the expression changes of ERK1 / 2 and p-ERK1 / 2, as well as the statistical graph. (C) SMCC-7721, after treatment with CuET (0μM, 0.1μM), Western blot was used to detect the expression changes of ERK1 / 2 and p-ERK1 / 2, as well as the statistical graph. (D) MHCC-LM3, after treatment with CuET (0μM, 0.1μM), Western blot was used to detect the expression changes of ERK1 / 2 and p-ERK1 / 2, as well as the statistical graph.

[0029] Fig.18Effect of copper disulfiram chelate on the drug resistance of hepatocellular carcinoma cells resistant to regorafenib: (A) After adding CuET, MHCC-97H / REGO cells were treated with different concentrations of regorafenib, and the cell viability was measured by CCK8 assay. (B) After adding different concentrations of CuET to MHCC-97H / REGO cells, IC50 was calculated based on the cell viability inhibition curve.

[0030] Fig.19 Effect of disulfiram copper chelate on the proliferation of hepatocellular carcinoma regorafenib-resistant cells: (A) MHCC-97H / REGO cells were seeded in 12-well plates at 500 cells / well. After 24 hours of treatment in the control group, disulfiram treatment group, regorafenib treatment group and combination treatment group, they were cultured in DMEM medium for 7 days and observed by crystal violet staining. (B) After MHCC-97H / REGO was treated with different drugs, the number of clones formed was statistically analyzed. (C) After 24 hours of treatment in the control group, disulfiram treatment group, regorafenib treatment group and combination treatment group, the cell cycle distribution of MHCC-97H / REGO cells was detected by flow cytometry. (D) The results of flow cytometry cycle of MHCC-97H / REGO drug treatment were statistically analyzed.

[0031] Fig. 20 Effect of copper disulfiram chelate on the migration and invasion ability of regorafenib-resistant hepatocellular carcinoma cells: (A) MHCC-97H / REGO cells, from left to right, control group, CuET treatment group, regorafenib treatment group, and combination treatment group. After 24h and 48h of treatment, the cell healing rate was observed under a microscope, and the scale bar = 100μm. (B) Statistical analysis of the cell healing rate of MHCC-97H / REGO cells at 24h and 48h. (C) MHCC-97H / REGO cells, from left to right, control group, CuET treatment group, regorafenib treatment group, and combination treatment group. After 24h of drug treatment, the number of cells that passed through the Transwell chamber was recorded under a microscope, and the scale bar = 100μm. (D) MHCC-97H / REGO cells, the number of cells that passed through the chamber in the Transwell migration experiment was statistically analyzed. (E) MHCC-97H / REGO cells, from left to right: control group, CuET treatment group, regorafenib treatment group, and combination treatment group. After 24 hours of drug treatment, the number of cells that passed through the Transwell chamber (coated with matrix gel) was recorded under a microscope, and the scale bar = 100 μm. (F) MHCC-97H / REGO cells, the number of cells that passed through the chamber in the Transwell invasion assay was statistically analyzed.

[0032] Fig.21Effects of copper disulfiram chelate on p-ERK1 / 2 and cell phenotype proteins in hepatocellular carcinoma cells resistant to regorafenib: (A) After 24 hours of treatment with the control group, CuET treatment group, regorafenib treatment group and combination treatment group, Western blot experiments were used to detect the changes in the expression of E-cadherin, N-cadherin, vimentin, snail, slug, cyclin B1 and p-ERK1 / 2 proteins in MHCC-97H / REGO cells. (B) After 3 Western blot experiments were performed on MHCC-97H / REGO cells treated with the control group, CuET treatment group, regorafenib treatment group and combination treatment group, statistical analysis was performed.

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

[0034] Fig.23 Effect of disulfiram on the size of subcutaneous tumors of hepatocellular carcinoma regorafenib-resistant cells: (A) MHCC-97H / REGO nude mouse subcutaneous tumors, after treatment with the control group, disulfiram treatment group, regorafenib treatment group, and combination treatment group, the tumor size chart was taken when the average long diameter of the mice in the control group reached 20 mm. (B) MHCC-97H / REGO nude mouse subcutaneous tumors, after treatment with the control group, disulfiram treatment group, regorafenib treatment group, and combination treatment group, the tumor weight was taken and statistically analyzed. 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 the dish in a 37°C constant temperature incubator to preheat.

[0038] (2) Thawing cells: When the temperature of the water bath reaches 37°C, take out the MHCC-97H cells from the liquid nitrogen and quickly put them into the water bath. After 2 minutes, take out the MHCC-97H cells.

[0039] (3) Collecting cells: 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 plating: After centrifugation, remove the centrifuge tube, aspirate the supernatant with a pipette and discard it into the waste container. Aspirate 1 mL of preheated DMEM medium to 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 culturing.

[0041] (5) Cell culture: MHCC-97H cells were cultured after recovery in DMEM medium containing 10% FBS, 100U / mL penicillin, and 100ug / mL streptomycin. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The culture medium was observed and replaced every day. Each time the culture medium was replaced, the culture dish was washed with 1mL-2mL PBS. When the cell confluence increased to cover the entire bottom surface, it was necessary to pass the cells.

[0042] (6) Cell passaging: Take out the overgrown cells from the incubator, remove the culture medium, add 1 mL of PBS to wash, remove the PBS, add 1 mL of trypsin to digest the cells for 3 min; collect the cells and centrifuge them at 200 g for 5 min; discard the supernatant, add 2 mL of culture medium to resuspend the cells, and divide the cells evenly into 2-3 culture dishes for culture. 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 of 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 every 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 of the cells are cultured at a concentration increased by 0.5 μM. If too many cells die after the concentration is increased, continue to culture at the original concentration for a period of time. If the cells can continue to grow and pass after the concentration is increased, culture them at the increased concentration. This cycle is repeated, and the culture concentration is continuously stimulated to increase 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 method)

[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 PBS for washing, add 1 mL trypsin to digest the cells for 3 min, then terminate the digestion with 1 mL serum-containing culture medium, centrifuge at 200g for 5 min, remove the supernatant, add culture medium to resuspend the cells, and configure the cells 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 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 detection: CCK8 and culture medium were mixed at a ratio of 1:10 as CCK8 detection solution. After removing the cell supernatant from the treated cells, 100 μL of CCK8 detection solution was added to each well. Six blank controls were set up, and only 100 μL of CCK8 detection solution was added to the blank controls. After adding, the cells were transferred to a 37°C 5% CO2 constant temperature incubator. After reacting for 0.5h, 1h, 1.5h, and 2h, the absorbance at 450nm was measured using an ELISA reader. The results with an absorbance between 1.2 and 2 in the control group were 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 experiment, flow cytometry)

[0052] Colony formation assay

[0053] (1) Cell seeding: Take MHCC-97H / REGO and MHCC-97H cells in the logarithmic growth phase, add 1 mL of trypsin, digest for 3 min, and add 1 mL of complete medium to terminate the digestion. Collect the cells, put them into a 15 mL centrifuge tube, centrifuge at 200g for 5 min, discard the cell supernatant, add medium to resuspend the cells, and seed them into a 6-well plate. 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 cells in clusters under a microscope every day. Replace the culture medium every 2 days and 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, take pictures, and calculate the number of colonies formed in each well to obtain the results of the cell cloning experiment.

[0056] Each experiment was repeated 3 times independently.

[0057] Flow cytometry

[0058] (1) Cell seeding: Take MHCC-97H / REGO and MHCC-97H cells in the logarithmic growth phase, add 1 mL of trypsin, digest for 3 min, and add 1 mL of complete medium to terminate the digestion. Collect the cells, put them into a 15 mL centrifuge tube, centrifuge at 200g for 5 min, discard the cell supernatant, add 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 cell 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, sterilize with alcohol, irradiate under ultraviolet light for 30 min, and dry. Prepare a 24-well plate, draw a line on the bottom, stick the chamber on the drawn area, and continue to prepare under ultraviolet light.

[0065] (2) Cell plate: Take logarithmically growing MHCC-97H and MHCC-97H / REGO cells, add 1 mL of trypsin, digest for 3 min, and after digestion is complete, add 1 mL of complete medium to terminate digestion. Collect cells and centrifuge at 200g for 5 min, discard the supernatant, and resuspend the cells in 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 experiment record: The next day, the chamber was removed, washed with PBS, and serum-free culture medium was added. At 0h, 24h, and 48h, the microscope was magnified 100 times, and photos were taken. The black line drawn in advance was used as the positioning to ensure that each photo was taken at the same position. The changes in cell migration ability were understood.

[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 min.

[0069] (2) Cell plate: MHCC-97H and MHCC-97H / REGO cells in the logarithmic growth phase were selected. After the culture medium was removed, 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 cells were 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, fixed in 4% paraformaldehyde for 15 min, washed with PBS, and stained in 0.5% crystal violet for 15 min. 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.

[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 min.

[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 min to allow the Matrigel to form a gel.

[0074] (3) Cell plate: MHCC-97H and MHCC-97H / REGO cells in the logarithmic growth phase were selected. After the culture medium was removed, 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 cells were 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, fixed in 4% paraformaldehyde for 15 min, washed with PBS, and stained in 0.5% crystal violet for 15 min. 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) Preparation of cell lysis buffer: NP40 lysis buffer was prepared in advance. Each mL of lysis buffer contained 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor, 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 lysing for 10 min and shake the EP tube several times to ensure that the cells are fully digested.

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

[0081] 7. Protein concentration determination (BCA method)

[0082] (1) Preparation of test samples: Total protein of MHCC-97H and MHCC-97H / REGO cells was obtained as test samples. 16 μL of standard diluent and 4 μL of test sample 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 0 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL. Then, each well is filled to 20 μL with standard diluent.

[0084] (3) Preparation of BCA working solution: BCA is divided into liquid A and liquid B. Before use, 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. After addition, treat at 60°C for 30 min, then use an ELISA reader to measure the absorbance at a wavelength of 540 nm, make 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: Separation gel and stacking gel for Western blot experiments were prepared according to the manufacturer's instructions. The components of the separation gel are: 3.1 mL H2O, 2.0 mL 1.5 M Tris-HCL pH8.8, 2.7 mL Aceylamide / Bis, 80 μL 10% SDS, 80 μL 10% AP, 10 μL TEMED; the components of the stacking gel are: 2.4 mL H2O, 1.0 mL 1.5 M Tris-HCL pH6.8, 0.5 mL Aceylamide / Bis, 40 μL 10% SDS, 40 μL 10% AP, 10 μL TEMED.

[0089] (3) Loading: The total amount of protein to be loaded is 20 μg. After 30 minutes of gel preparation, loading begins. The same total amount of protein is added to each well. The experiment can be repeated on the same 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, 80 V, 30 min, 120 V, 60 min. Observe at all times during the electrophoresis process to prevent the protein from escaping from the gel.

[0091] (5) Transfer: Prepare filter paper and PVDF membrane in the order of membrane larger than filter paper and larger than glue. 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 for later use. Wet the filter paper with transbuffer first. Sandwich the glue and membrane between the filter paper and start the transfer. Constant current, 120mA, 60min.

[0092] (6) Blocking: The PVDF membrane was blocked in pre-prepared 5% skimmed milk powder at room temperature for 1 h. After blocking, it was washed three times with TBST for 5 min 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. 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: The washed 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 are placed in TBST for exposure. The exposure instrument is cooled in advance, and the camera is started after the temperature reaches -40°C. The A solution and the B solution of the ECL colorimetric solution are prepared in a 1:1 ratio to form a working solution. After removing the water marks from the strips, they are placed on the exposure instrument with the protein side facing up, and the working solution is added dropwise. Develop and save the image. The image results are analyzed in grayscale using Imagej, and the analysis results are 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 about 20g, and raised in the laboratory for 3-4 days to reduce stress response. The experimental mice were raised in an SPF environment with an ambient temperature of 18℃-26℃, a relative humidity of 40%-70%, a 12:12 hour day and night light cycle to maintain the circadian rhythm of the nude mice, sterile plastic boxes and sterile cage covers, drinking water was sterilized ddH2O, bedding was corn cob bedding, changed once a week, cleaned each time, feed was Co60 irradiated, and mouse food and drinking water were changed every 3-4 days.

[0098] (2) Construction of subcutaneous tumors in nude mice: The tumor cells loaded were MHCC-97H / REGO cell lines, which were self-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 at the axilla of nude mice. The skin was disinfected with alcohol during injection. The needle was inserted 1.5 cm away from the axilla and moved 1 cm-1.5 cm along the subcutaneous tissue. When the needle reached the axilla, the cell suspension in the syringe was pushed out and the needle was gently pressed to quickly withdraw.

[0099] (3) Grouping of nude mice: After tumor loading, nude mice were uniformly placed in cages to ensure that there were no more than 7 mice in each cage. The long and short diameters of the subcutaneous tumors of nude mice were calculated with a vernier caliper every two days. 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 3 At about 12 h, the mice were randomly divided into a control group, a regorafenib treatment group, a disulfiram treatment group, and a combination treatment group.

[0100] (4) In vivo tumor treatment experiment: The administration method is oral gavage, and the administration time is three times in the morning, noon and evening. Regorafenib is set to be administered by oral 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 oral 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 oral gavage at night, and the copper gluconate is 0.2 mg / kg, 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 dosage forms are:

[0101] 1) Control group: solvent;

[0102] 2) Regorafenib treatment group: Regorafenib administration;

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

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

[0105] (5) Measurement of nude mouse tumors: 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 should be killed when the longest diameter of tumor load reaches 20mm to reduce the pain of experimental animals. This study selected the average long diameter of the tumor in the control group reaching 20mm as the end point of the animal experiment time. After the experiment was completed, 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 small pieces of 5 mm in size and placed in 1 mL of NP40. The tissue was broken 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 analysis

[0110] The experimental data were analyzed using SPSS22.0 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 a statistically significant difference.

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

[0112] The regorafenib-resistant cell line was constructed by the gradient concentration increase method. After MHCC-97H cells were stimulated with regorafenib for 6 months, 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 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%, 1.32%, and 1.71%, 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 constructed in this study is a regorafenib-resistant cell line. 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 the cell morphology of hepatocellular carcinoma cells tends to change from epithelial cell morphology to mesenchymal cell morphology during the process of regorafenib resistance, and also indicate the successful construction of regorafenib-resistant hepatocellular carcinoma cells.

[0113] Example 2 Changes in proliferation, migration and invasion abilities of regorafenib-resistant hepatocellular carcinoma strain 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 compared with MHCC-97H, MHCC-97H / REGO had improved proliferation ability. 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 24h, 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. The results of Tranwell migration assay showed that the number of cells that passed through the chamber of MHCC-97H / REGO increased significantly compared with MHCC-97H, which was 3.49±1.37 times that of MHCC-97H ( Figure 3 C- Figure 3 D). The experimental results show that compared with MHCC-97H, MHCC-97H / REGO has enhanced migration ability under serum conditions. The results of the Transwell invasion assay showed that the number of cells that passed through the matrix gel chamber of 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 the invasion ability of MHCC-97H / REGO is enhanced compared with MHCC-97H.

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

[0116] The results of cell cycle-related protein detection showed that the expression of cyclin B1 in MHCC-97H / REGO was significantly increased, which was 4.94±1.38 times that of MHCC-97H ( Figure 4 A- Figure 4B). The experimental results showed that the expression of cyclin B1 may be positively correlated with regorafenib resistance. The results of EMT-related protein detection showed that the expression of epithelial marker E-cadherin in MHCC-97H / REGO was significantly lower than that in MHCC-97H, which was 51.1%±10.1% of that in MHCC-97H ( Figure 4 A- Figure 4 B). The expression levels of N-cadherin and vimentin, markers of mesenchymal cells, and snail and slug, the EMT-related transcription factors, in MHCC-97H / REGO were significantly increased compared with those in 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 show that the expression of mesenchymal markers N-cadherin and vimentin and EMT-related transcription factors snail and slug may be positively correlated with regorafenib resistance, and 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 results of CCK8 experiments showed 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, and the IC of regorafenib in MHCC-97H / REGO in the SHC772984-treated group was significantly lower than that in the control group. 50 Compared with the control group, the IC 50 :13.78μM±1.02μM, SCH772984 treatment group IC 50 :7.501μM±0.77μM( Figure 5 B). The 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 inhibiting p-ERK1 / 2 on proliferation, migration and invasion of MHCC-97H / REGO

[0120] The results of the clone formation experiment showed that the number of clones formed by MHCC-97H / REGO in the SCH772984 treatment group was significantly reduced to 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 blocked 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. 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 under serum-free conditions. 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, which was 9.6%±1.8% of that in the control group ( Figure 7 B). The experimental results show that reducing the expression of p-ERK1 / 2 can inhibit the migration ability of MHCC-97H / REGO 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 SCH772984 treatment group was significantly reduced, which was 3.5%±2.2% of that in the control group ( Figure 7 C). The experimental results showed that reducing the expression of p-ERK1 / 2 could inhibit the invasion ability of MHCC-97H / REGO.

[0121] Example 6 Effects of inhibiting p-ERK1 / 2 on MHCC-97H / REGO cell cycle and EMT-related proteins

[0122] The results of EMT-related protein detection showed that the expression of epithelial marker E-cadherin in the SCH772984-treated 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). The experimental results show that reducing the expression of p-ERK1 / 2 can inhibit the expression of mesenchymal markers N-cadherin and vimentin and EMT-related transcription factors snail and slug, and promote the expression of E-cadherin. The results of cell cycle protein detection showed that the expression of cyclin B1 in the SCH772984 treatment group was significantly decreased, which was 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 that in the control group ( Fig. 9 A). The experimental results show that TBHQ can stimulate the expression of p-ERK1 / 2 in MHCC-97H. The results of CCK8 experiments showed 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, and the IC of regorafenib in the TBHQ-treated group MHCC-97H was 50 Compared with the control group, the IC 50 :9.61μM±0.59μM, control group IC 50 :7.31μM±0.44μM( Fig. 9 B). The 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 Ability 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 ( Fig.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 show 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 ( Fig.10B). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the progression of MHCC-97H cell cycle. The scratch test results 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 ( Fig.11 A). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the migration ability of MHCC-97H under serum-free conditions. The results of the Transwell migration experiment showed that the number of MHCC-97H cells that passed through the chamber in the TBHQ-treated group increased significantly, which was 7.95±0.41 times that of the control group ( Fig.11 B). The experimental results show that increasing the expression of p-ERK1 / 2 can promote the migration ability of MHCC-97H under serum conditions. The results of the Transwell invasion experiment showed that the number of MHCC-97H cells that passed through the chamber in the TBHQ treatment group increased significantly, which was 15.5±1.54 times that of the control group ( Fig.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, accounting for 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 was significantly increased, accounting for 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 ( Fig.12 A- Fig.12 B). The experimental results show that increasing the expression of p-ERK1 / 2 can 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 ( Fig.12 A- Fig.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 (0.1 μM-0.5 μM) could significantly inhibit the cell viability of MHCC-97H / REGO, MHCC-97H, MHCC-LM3 and SMCC-7721 cells ( Fig.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 ( Fig.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 blocked in the G2 / M phase of MHCC-97H / REGO, MHCC-97H, MHCC-LM3 and SMCC-7721 cells ( Fig.14 A- Fig.14 D). The experimental results show that CuET can block the cell cycle progression of HCC cells. Western blot experimental results showed that the expression of cyclin B1 in MHCC-97H / REGO, MHCC-97H, SMCC-7721 and MHCC-LM3 cells in the CuET treatment group was significantly decreased, which was 44.1%±4.3% of the control group (MHCC-97H / REGO)( Fig.14 E), 46.3%±2.4%(MHCC-97H)( Fig.14 F), 31.2%±1.1%(SMCC-7721)( Fig.14 G) and 32.1%±9.7% (MHCC-LM3) ( Fig.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. Fig.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 ( Fig.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 ( Fig.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 ( Fig.15 D). The experimental results show that CuET can inhibit the migration ability of HCC cells in serum-free conditions. The results of the Transwell migration experiment showed that the number of MHCC-97H / REGO cells that passed through the chamber in the CuET-treated group was significantly reduced in 24 hours, which was 35.6%±8.6% of that in the control group ( Fig.15 E). The number of MHCC-97H cells that passed through the chamber in the CuET-treated group was significantly decreased at 24 h, which was 35.6%±8.6% of that in the control group ( Fig.15 F). There was no significant difference in the number of SMCC-7721 cells that passed through the chamber in the CuET treatment group and the control group at 24 hours ( Fig.15 G). The number of MHCC-LM3 cells that passed through the chamber in the CuET-treated group was significantly reduced at 24 hours, which was 60.3%±10.2% of that in the control group ( Fig.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 experiment showed that the number of MHCC-97H / REGO cells that passed through the chamber in the CuET-treated group was significantly reduced at 24 hours, which was 52%±20.5% of that in the control group ( Fig.15 E). The number of MHCC-97H cells that passed through the chamber in the CuET-treated group was significantly reduced to 14.9%±6.5% of that in the control group ( Fig.15 F). The number of SMCC-7721 cells that passed through the chamber in the CuET-treated group was significantly reduced after 24 hours, which was 6.7%±5.5% of that in the control group ( Fig.15 G). The number of MHCC-LM3 cells that passed through the chamber in the CuET-treated group was significantly decreased to 4.5%±2.2% of that in the control group ( Fig.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 ( Fig.16 A). In MHCC-97H cells, the expression of epithelial marker E-cadherin in the CuET-treated group was significantly increased, which was 2±0.43 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), 33.5%±10.8% (snail) and 47.2%±20.9% (slug) of the control group ( Fig.16 B). In SMCC-7721 cells, the expression of epithelial marker E-cadherin in the CuET-treated group was significantly increased, which was 1.71±0.68 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 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 ( Fig.16 C). In MHCC-LM3 cells, the expression of E-cadherin in the CuET-treated group was significantly increased, which was 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 in the CuET-treated group was significantly decreased, which was 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 ( Fig.16D). The experimental results showed that CuET can promote the expression of epithelial marker E-cadherin in HCC cells, and inhibit the expression of mesenchymal markers N-cadherin and vimentin as well as 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 ( Fig.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 ( Fig.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 ( Fig.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 ( Fig.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] CCK8 assay was used to detect the IC 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 7.38μM±0.94μM (0.1μM CuET), 0.42μM±0.27μM (0.15μM CuET) and 0.15μM±0.09μM (0.2μM CuET) respectively. Fig.18 A- Fig.18 B). The experimental results show that compared with the control group, the inhibitory effect of regorafenib on MHCC-97H / REGO in the CuET treatment group was significantly increased, and the inhibitory effect became more significant with the increase of concentration. Among them, 0.1μM CuET can restore the resistance of MHCC-97H / REGO to regorafenib to the level of parental cell MHCC-97H.

[0137] Example 14 Effects of disulfiram copper chelate combined with regorafenib on 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 combination treatment group was significantly reduced, which was 12.2%±3.5% of the regorafenib treatment group ( Fig.19 A- Fig.19 B), and 18.7%±7.6% in the CuET-treated group ( Fig.19 A- Fig.19 B). The experimental results show that compared with the single-use of regorafenib or the single-use of CuET, the combination of CuET and regorafenib can significantly inhibit the proliferation of MHCC-97H / REGO. Flow cytometry results showed that the proportion of MHCC-97H / REGO cells blocked in the G2 / M phase in the combined treatment group was significantly increased, which was 1.41±0.17 times that of the regorafenib treatment group ( Fig.19 C- Fig.19 D), which was 1.63±0.16 times that of the CuET-treated group ( Fig.19 C- Fig.19 D). The experimental results show that compared with the single-use of regorafenib or the single-use of CuET, the combination of CuET and regorafenib can significantly block the MHCC-97H / REGO cell cycle in the G2 / M phase. The scratch test results showed that the 48h cell healing rate of MHCC-97H / REGO in the combination treatment group was significantly reduced, which was 56.2%±8.5% of that in the regorafenib treatment group ( Fig. 20 A- Fig. 20 B), and 56.5%±20.9% of the CuET-treated group ( Fig. 20 A- Fig. 20 B). The experimental results show that compared with the single-use of regorafenib or the single-use of CuET, the combination of CuET and regorafenib can significantly inhibit the migration ability of MHCC-97H / REGO under serum-free conditions. The results of the Transwell migration experiment showed that the number of MHCC-97H / REGO cells that passed through the chamber in the combined treatment group was significantly reduced, which was 21.6%±3.7% of that in the regorafenib treatment group ( Fig. 20 C- Fig. 20 D), which was 10%±4% of the CuET-treated group ( Fig. 20 C- Fig. 20 D). The experimental results show that compared with the single-use of regorafenib or the single-use of CuET, the combination of CuET and regorafenib can significantly inhibit the migration ability of MHCC-97H / REGO under serum conditions. The results of the Transwell invasion experiment showed that the number of MHCC-97H / REGO cells that passed through the chamber in the combination treatment group was significantly reduced, which was 8.2%±3.8% of that in the regorafenib treatment group ( Fig. 20 E- Fig. 20 F), which was 10.1%±5% of the CuET-treated group ( Fig. 20 E- Fig. 20 F). The experimental results showed that compared with the single use of regorafenib or CuET alone, the combination of CuET and regorafenib could significantly inhibit the invasion ability of MHCC-97H / REGO.

[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 combined treatment group (10μM regorafenib + 0.1μM CuET combined treatment). After MHCC-97H / REGO treatment for 72h, total protein was extracted. The results of Western blot experiment of p-ERK1 / 2 showed that the expression of p-ERK1 / 2 in MHCC-97H / REGO in the combined treatment group was significantly reduced, which was 58.6%±20.5% of that in the regorafenib treatment group and 48.1%±25.5% of that in the CuET treatment group ( Fig.21 A- Fig.21 B). The experimental results showed that compared with regorafenib alone or CuET alone, CuET combined with regorafenib could significantly inhibit the expression of p-ERK1 / 2. The Western blot results of cell cycle proteins showed that the expression of cyclin B1 in MHCC-97H / REGO in the combined treatment group was significantly reduced, which was 37.5%±22.6% of that in the regorafenib treatment group and 27.9%±15.5% of that in the CuET treatment group ( Fig.21 A- Fig.21B) The experimental results show that compared with regorafenib alone or CuET alone, CuET combined with regorafenib can significantly inhibit the expression of cyclin B1. Western blot of EMT-related proteins The results of blot experiments showed that the expression of epithelial marker E-cadherin in MHCC-97H / REGO in the combined treatment group was significantly increased, and the expression of mesenchymal markers N-cadherin and vimentin, and EMT-related transcription factors snail and slug was significantly decreased, which was 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 ( Fig.21 A- Fig.21 B). The experimental results showed that compared with regorafenib alone or CuET alone, CuET combined with regorafenib could significantly activate the expression of epithelial marker E-cadherin and inhibit the expression of mesenchymal markers N-cadherin and vimentin as well as 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 experimental nude mice in each group were treated according to the 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 volumes of subcutaneous tumors in the combination treatment group, regorafenib treatment group and disulfiram treatment group were basically the same in 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 volumes of subcutaneous tumors in the disulfiram treatment group and regorafenib treatment group showed an increasing trend, while the volumes of subcutaneous tumors in the combination treatment group showed a decreasing trend ( Fig. 22 A). After 31 days of treatment, the tumors of the experimental 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 ( Fig.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 combined 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 ( Fig.23 B). The experiment showed that compared with the treatment regimen of disulfiram alone and regorafenib alone, the treatment regimen of disulfiram combined with regorafenib could significantly inhibit the growth of MHCC-97H / REGO subcutaneous tumors.

[0143] Summary of the invention

[0144] In the clinical application and research of regorafenib in the treatment of HCC, acquired drug resistance has always been the main factor limiting its use and promotion. This paper designed multiple experimental schemes to study the mechanism of regorafenib resistance in HCC, explore the effect of disulfiram in the treatment of HCC, and analyze the feasibility of disulfiram in reversing regorafenib resistance in HCC in vivo and in vitro.

[0145] We revealed for the first time that p-ERK1 / 2 has a regulatory role in the process of regorafenib resistance in MHCC-97H cells, which is mainly manifested in the regulation of cell proliferation, migration and invasion. Of particular importance is that we used disulfiram for the first time 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 more significantly. In summary, the disulfiram provided by the present invention can be used as a drug to prolong the life of patients with hepatocellular carcinoma after regorafenib resistance.

Claims

1. Application of disulfiram in the preparation of drugs for the treatment of hepatocellular carcinoma.

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

3. Application of disulfiram in the preparation of drugs for reversing regorafenib resistance in hepatocellular carcinoma.

4. Use of a combination of disulfiram and regorafenib in the preparation of drugs for treating hepatocellular carcinoma.

5. Use of disulfiram in the preparation of preparations that inhibit cyclin B1, N-cadherin, vimentin, snail and slug protein levels.

6. Use of disulfiram in the preparation of drugs for the treatment of patients with advanced hepatocellular carcinoma who are resistant to regorafenib.

7. Application of disulfiram in the preparation of preparations for inhibiting proliferation, migration and EMT of hepatocellular carcinoma cells.

8. Use of disulfiram in the preparation of drugs for the treatment of metastasis and recurrence of hepatocellular carcinoma.

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