Application of Protein Kinase CK2 Inhibitor in Enhancing Therapeutic Efficacy of Liver Cancer Treatment

Abstract

The present invention discloses the application of a protein kinase CK2 inhibitor in enhancing the therapeutic efficacy of liver cancer treatment, which relates to the field of biomedicine. The present invention proves that the combined use of CX-4945 and lenvatinib has a synergistic effect on the proliferation inhibition of liver cancer cells resistant to lenvatinib, and has a good tumor inhibitory effect in the liver cancer xenograft mouse experiment, significantly inhibiting the growth of liver cancer both in vitro and in vivo. At the same time, it provides a potential combined drug target for the treatment of liver cancer patients resistant to lenvatinib. Moreover, CX-4945 plays a role in reversing lenvatinib resistance with a small dosage, and has very little toxic and side effects, showing broad application prospects in the treatment of liver cancer resistant to lenvatinib.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a protein kinase CK2 inhibitor in enhancing the therapeutic efficacy of liver cancer. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, accounting for approximately 90% of all primary liver cancer cases and is the fourth leading cause of cancer-related deaths worldwide.

[0003] Lenvatinib (trade name: LENVIMA), the active ingredient is lenvatinib mesylate, molecular formula: C 21 H 19 ClN4O4·CH4O3S, with a molecular weight of 522.96, is a multikinase inhibitor that inhibits liver cancer proliferation primarily by inhibiting angiogenesis. It can inhibit multiple tyrosine kinase receptors, including fibroblast growth factor receptors (FGFR) 1-4, vascular endothelial growth factor receptors (VEGFR) 1-4, platelet-derived growth factor receptor (PDGR) α, the proto-oncogene RET, and c-KIT. Lenvatinib inhibits tumor angiogenesis by blocking VEGF receptors, and inhibits tumor cell proliferation by blocking FGFR, PDFRα, RET, and c-KIT. Ultimately, lenvatinib exhibits potent antitumor effects, primarily demonstrating its ability to prolong overall survival and progression-free survival in patients with advanced liver cancer and convert some unresectable liver cancers to resectable ones. Currently, lenvatinib has been approved by the FDA as a first-line targeted therapy for patients with advanced liver cancer. However, in clinical practice, some patients with liver cancer have shown a poor response to lenvatinib or experience tumor progression after treatment. The reasons for this can be broadly divided into two categories: one is that the tumor is naturally resistant to lenvatinib; the other is that the tumor adapts to lenvatinib, allowing tumor cells to survive high concentrations of lenvatinib. However, the mechanisms of lenvatinib resistance in liver cancer and the remedial treatment options after resistance remain urgent scientific issues.

[0004] MYH9, also known as myosin IIa or non-muscle myosin heavy chain 9 (NMMHC-IIA), interacts with actin in the cytoplasm and participates in different cellular processes such as cell movement, cell migration, and cell adhesion. Based on the analysis of the TCGA database, MYH9 is elevated in hepatocellular carcinoma and leads to poor prognosis. In clinical samples, high expression levels of MYH9 predict poor prognosis in patients with hepatocellular carcinoma. Studies have confirmed that targeted inhibition of MYH9 significantly improves the survival rate of mice with liver cancer and enhances the sensitivity of liver cancer cells to sorafenib in vivo, indicating that MYH9 is a key therapeutic target for HCC. Sorafenib and lenvatinib are both preferred targeted drugs for patients with advanced liver cancer and have a high degree of overlap in the drug action target pathway. This study attempts to explore the application of inhibiting MYH9 phosphorylation in reversing and reducing the drug resistance of lenvatinib in the treatment of liver cancer.

[0005] Silmitasertib (CX-4945) is an inhibitor targeting the CK2 pathway, with the Chinese name 5-[(3-chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylic acid, and the molecular formula C 19 H 12 ClN3O2, with a molecular weight of 349.77. Preclinical studies have confirmed that by inhibiting the CK2 pathway, DNA repair can be blocked, thereby inducing cell apoptosis, and it also has the effect of enhancing the anti-tumor activity of chemotherapy drugs such as gemcitabine and cisplatin. Currently, the FDA has granted orphan drug status to Silmitasertib (CX-4945) for the treatment of patients with cholangiocarcinoma. Its clinical trials are underway, and it is reported that this drug can achieve relatively ideal therapeutic effects as a first-line treatment. The results of the mid-term analysis showed that the overall response rate (ORR) of Silmitasertib treatment was 32.1%, and the disease control rate (DCR) was 79.3%; the median progression-free survival (PFS) of patients was 11.2 months, and the median overall survival (OS) was 17.4 months. However, the role of Silmitasertib (CX-4945) in lenvatinib-resistant liver cancer has not been reported at home and abroad. Summary of the Invention

[0006] Therefore, the present invention provides the application of a protein kinase CK2 inhibitor in enhancing the therapeutic efficacy of liver cancer. Through the synergistic effect of Silmitasertib (CX-4945) and lenvatinib, the inhibitory effect on lenvatinib-resistant liver cancer cells is significantly improved. At the same time, it can significantly inhibit the proliferation, invasion, and colony formation of liver cancer drug-resistant cells, promote the apoptosis of liver cancer drug-resistant cells, and achieve good tumor suppression effects in the liver cancer xenograft nude mouse experiment.

[0007] The present invention provides the use of a protein kinase CK2 inhibitor in preparing a drug for treating liver cancer. The protein kinase CK2 inhibitor is a small molecule compound CX-4945, and the drug is used in combination with lenvatinib.

[0008] The protein kinase CK2 inhibitor is designed to reverse the resistance of liver cancer cells to lenvatinib. Specifically, when used in combination with lenvatinib, it can significantly inhibit the growth of liver cancer cells in vitro and in vivo. Its function is to inhibit the phosphorylation of MYH9, thereby reversing the resistance of liver cancer cells to lenvatinib.

[0009] The liver cancer is lenvatinib-resistant liver cancer and is primary hepatocellular carcinoma.

[0010] When used in combination with lenvatinib, the molar ratio of the protein kinase CK2 inhibitor to lenvatinib is 1:0.5-5.

[0011] The combination therapy includes in vitro applications, where CX-4945 combined with lenvatinib significantly inhibited the growth of liver cancer cells in lenvatinib-resistant cells, and in vivo applications, where CX-4945 combined with lenvatinib significantly inhibited the growth of liver cancer cells in xenografted nude mice.

[0012] The present invention also provides a pharmaceutical composition for treating liver cancer, comprising lenvatinib and a protein kinase CK2 inhibitor, wherein the protein kinase CK2 inhibitor is a small molecule compound CX-4945.

[0013] The liver cancer is lenvatinib-resistant liver cancer and is primary hepatocellular carcinoma.

[0014] Specifically, the molar ratio of the protein kinase CK2 inhibitor to lenvatinib is 1:0.5-5.

[0015] In liver cancer cells, the combined use of CX-4945 and lenvatinib can significantly inhibit the proliferation of lenvatinib-resistant cells and increase cell apoptosis.

[0016] In xenografted mice, the combined use of CX-4945 and lenvatinib by gavage significantly inhibited the growth of liver cancer xenografts.

[0017] In this application, the CK2 inhibitor CX-4945 is used to reduce the resistance of liver cancer cells to lenvatinib.

[0018] The present invention proves that the combined use of CX-4945 and lenvatinib has a better inhibitory effect on liver cancer than the use of CX-4945 or lenvatinib alone.

[0019] CX-4945 significantly enhances the efficacy of lenvatinib both in vitro and in vivo, and the two drugs have a synergistic effect to inhibit the growth of liver cancer.

[0020] In the above application, the concentrations of CX-4945 and lenvatinib used in the drug-resistant liver cancer cell lines Huh-7 and Hep-3B are 2 μM and 1 μM respectively.

[0021] In the above application, the concentrations of CX-4945 and lenvatinib used in nude mice with liver cancer xenografts are 20 mg / kg / day and 30 mg / kg / day respectively.

[0022] In the above product for anti-liver cancer or anti-liver cancer cells, the liver cancer is primary hepatocellular carcinoma.

[0023] In this application, anti-liver cancer can specifically be manifested as inhibiting the proliferation, invasion, colony formation, apoptosis of liver cancer cells and inhibiting the growth of liver cancer xenografts.

[0024] In the present invention, CX-4945 significantly enhances the efficacy of lenvatinib both in vitro and in vivo, and the two drugs have a synergistic effect to inhibit the growth of liver cancer. By using the pharmaceutical composition of the present invention, especially for tissues or individuals resistant to lenvatinib, the combined use of drugs can significantly inhibit the proliferation and formation of drug-resistant cells and promote the apoptosis of drug-resistant cells. Therefore, the pharmaceutical composition of the present invention can be used to prepare drugs for treating liver cancer.

[0025] Beneficial effects:

[0026] The present invention confirms that the combined use of CX-4945 and lenvatinib has a synergistic effect on the proliferation inhibition of lenvatinib-resistant liver cancer cells, and has a good tumor inhibitory effect in the nude mouse experiment of liver cancer xenografts, and significantly inhibits the growth of liver cancer both in vitro and in vivo. At the same time, it provides a potential combined drug target for the treatment of liver cancer patients resistant to lenvatinib. Moreover, CX-4945 plays a role in reversing lenvatinib resistance with a small amount of dosage, and its toxic and side effects are very small, and it has broad application prospects in the treatment of liver cancer resistant to lenvatinib. Description of the drawings

[0027] Figure 1 To establish lenvatinib-resistant cell lines of Huh-7 cells and Hep-3B cells in liver cancer cell lines; among them, A is the schematic diagram of the establishment process of establishing lenvatinib-resistant cell lines of Huh-7 cells and Hep-3B cells in liver cancer cell lines; B is the IC50 detection results of the proliferation experiments of Huh-7 cells, Hep-3B cells, Huh-7-LR cells and Hep-3B-LR cells; C is the determination result of cell colony formation.

[0028] Figure 2Identification diagram of drug resistance of Huh-7-LR cells and Hep-3B-LR cells; among them, Figure A shows the results of cell invasion assay, and the scale bars are all 100 μm; Figure B shows the results of flow cytometry detecting the effect of lenvatinib on cell line apoptosis; Figure C shows the results of the maximum tumor formation in nude mice, * indicates p < 0.05.

[0029] Figure 3 Expression levels of MYH9 protein in Huh-7 cells and Hep-3B cells under different treatments. Among them, Figure A shows the expression of MYH9 protein detected by Western Blot in Huh-7 cells, Hep-3B cells, Huh-7-LR cells and Hep-3B-LR cells; Figure B shows the protein expression level of MYH9 after overexpression of MYH9 gene in Huh-7 cells and Hep-3B cells; Figure C shows the protein expression level of MYH9 after knockdown of MYH9 gene in Huh-7-LR cells and Hep-3B-LR cells.

[0030] Figure 4 Effect of MYH9 gene on colony formation of lenvatinib-resistant cell lines. Among them, Figure A shows the results of cell colony formation assay after overexpression of MYH9 gene in Huh-7 cells and Hep-3B cells; Figure B shows the results of cell colony formation assay after knockdown of MYH9 gene in Huh-7-LR cells and Hep-3B-LR cells.

[0031] Figure 5 Effect of MYH9 gene on invasion of lenvatinib-resistant cell lines. Among them, Figure A shows the invasion results of Huh-7 cells and Hep-3B cells after overexpression of MYH9 gene; Figure B shows the transwell invasion results of Huh-7-LR cells and Hep-3B-LR cells after knockdown of MYH9 gene, * indicates p < 0.05, ** indicates p < 0.01, and the scale bars are all 100 μm.

[0032] Figure 6 Effect of MYH9 gene on apoptosis of lenvatinib-resistant cell lines. Among them, Figure A shows the results of flow cytometry analysis of cell apoptosis detection after treatment of Huh-7 cells, Hep-3B cells and Huh-7 cells and Hep-3B cells overexpressing MYH9 gene with 5 μM lenvatinib; Figure B shows the results of flow cytometry analysis of cell apoptosis detection after treatment of Huh-7-LR cells, Hep-3B-LR cells and Huh-7-LR cells and Hep-3B-LR cells with knocked-down MYH9 gene with 5 μM lenvatinib, *P < 0.05.

[0033] Figure 7The expression levels of MYH9 and p-MYH9 proteins in Huh-7 cells, Hep-3B cells, Huh-7-LR cells and Hep-3B-LR cells after the application of CX-4945.

[0034] Figure 8 The cell counting results of Huh-7-LR cells and Hep-3B-LR cells after being treated with 2 μM, 5 μM lenvatinib and 1 μM CX-4945 in combination.

[0035] Figure 9 The apoptosis detection results by flow cytometry analysis of Huh-7-LR cells and Hep-3B-LR cells after being treated with 5 μM lenvatinib alone and 5 μM lenvatinib combined with 0 μM, 1 μM, 2 μM CX-4945 for 48 h, * indicates p < 0.05.

[0036] Figure 10 The changes in tumor and body weight of nude mice in different treatment groups. Among them, Figure A shows the tumor size and morphology of each group after 3 weeks of treatment with the control group, lenvatinib group, CX-4945 group, and CX-4945 + lenvatinib group; Figure B shows the measurement of the tumor volume of each group at the time of drug administration; Figure C shows the changes in the body weight of nude mice in each group during the treatment process.

[0037] Figure 11 The expression of p-MYH9 in xenografts after treatment in different treatment groups detected by immunohistochemistry, * indicates p < 0.05. Detailed implementation mode

[0038] Example 1: Preparation of lenvatinib-resistant hepatocellular carcinoma cell lines Huh-7-LR cells and Hep-3B-LR cells

[0039] Induce and obtain lenvatinib-resistant hepatocellular carcinoma cell lines.

[0040] The human HCC cell lines Huh-7 and Hep-3B were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The authenticity of all cells was verified by short tandem repeat (STR) analysis. Hep-3B cells and Huh-7 cells were cultured in MEM and DMEM media containing 10% fetal bovine serum and 1% penicillin-streptomycin-gentamicin, and incubated in an incubator at 37 °C and 5% carbon dioxide. By continuously administering increasing concentrations of lenvatinib to Huh-7 and Hep-3B cells until it stabilized at 20 μmol / L, and then maintaining the culture at a concentration of 2 μmol / L, lenvatinib-resistant Huh-7-LR and Hep-3B-LR ( Figure 1 A).

[0041] Example 2: Detection of drug resistance of Huh-7-LR cells and Hep-3B-LR cells

[0042] 1. Cell viability was determined by the CCK-8 method:

[0043] 10% CCK-8 solution was added to the Huh-7-LR and Hep-3B-LR cells obtained in Example 1 and incubated at 37 °C for 4 hours. Then the culture medium was taken out, and the absorbance at 450 nm was measured with a microplate reader.

[0044] The results showed that the IC50 of Huh-7 cells increased from 3.864 to 15.55 μmol / L; the IC50 of Hep-3B cells increased from 1.852 to 17.03 μmol / L; the difference in IC50 between the two was significant (p < 0.05)( Figure 1 B).

[0045] 2. Colony formation assay: 3×10 3 ~5×10 3 The Huh-7-LR and Hep-3B-LR cells obtained in Example 1 were respectively implanted into 12-well plates, cultured at 37 °C and 5% CO2, and treated with 0, 1, and 2 μmol / L lenvatinib. After adding the drug, the culture medium of each group was changed every 2 days starting from the first day and cultured for 2 weeks. Then the cell colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet for 20 minutes, and photographed. The results of the colony formation experiment( Figure 1 C) showed that the proliferation rate of lenvatinib-resistant Huh-7-LR cells and Hep-3B-LR cells in the lenvatinib-containing culture medium was faster than that of Huh-7 cells and Hep-3B cells (p < 0.05);

[0046] 3. Cell invasion assay: 3×10 4 ~5×10 4 The Huh-7-LR and Hep-3B-LR cells obtained in Example 1 were respectively suspended in 200 μL of serum-free medium and loaded onto the upper compartment of a chamber containing a polycarbonate membrane (Corning). The serum concentration in the lower chamber was 15%. After culturing for 24 h, the cells that had passed through the membrane were stained with crystal violet. Photos of 4 randomly selected fixed cells were captured, and the cells were counted. The experiment was independently repeated three times. Staining and counting were performed.

[0047] Cell counting experiment: 3×10 4 ~5×10 4 Hepatoma cells were implanted into 12-well plates. After culturing at 37 °C and 5% CO2 for 48 hours, the cells were digested into single-cell suspensions, stained with trypan blue solution, and counted in a cell counting chamber under a microscope.

[0048] Results of the transwell invasion assay( Figure 2A) It was shown that the invasion ability of lenvatinib-resistant Huh-7-LR cells and Hep-3B-LR cells in lenvatinib-containing medium was enhanced compared with that of Huh-7 cells and Hep-3B cells (p < 0.05);

[0049] 4. Flow cytometry: 1.5×10 5 Huh-7-LR and Hep-3B-LR cells obtained in Example 1 were respectively placed in six-well plates and harvested after treatment. The cells were resuspended with binding buffer and stained with Annexin V-APC / 7-AAD double-staining cell apoptosis detection kit (KeyGen Biotech) according to the manufacturer's instructions. Analysis was performed using a FACSCantoii flow cytometer (BD Biosciences). To detect surface protein expression, HCC cells were collected and suspended in cell staining buffer (KeyGen Biotech). Coupled fluorescent reagents such as Annexin V-APC and 7-AAD were added to the cell suspension. The reaction was carried out at room temperature in the dark for 5 - 10 min, and the samples were analyzed using a flow cytometer within 1 hour.

[0050] Results of flow cytometry ( Figure 2 B) showed that compared with Huh-7 cells and Hep-3B cells, apoptosis of lenvatinib-resistant Huh-7-LR cells and Hep-3B-LR cells in lenvatinib-containing medium was significantly reduced (p < 0.05), demonstrating the formation of drug-resistant strains;

[0051] 5. Different numbers of Huh-7 cells and Huh-7-LR cells were resuspended in 100 μL of sterile PBS and basement membrane matrix (1:1) and subcutaneously injected into the right axilla of 5-week-old male BALB / c mice to observe the tumor formation results.

[0052] Results of tumor formation in BALB / c nude mice at the limit Figure 2 C) showed that when establishing a tumor formation model in BALB / c nude mice with 1×10 4 Huh-7 cells and Huh-7-LR cells, the tumor formation rate of Huh-7 cells was 0%, and that of Huh-7-LR cells was 20%; when 1×10 5 cells formed tumors, the tumor formation rate of Huh-7 cells was 0%, and that of Huh-7-LR cells was 80%; when 1×10 6 cells formed tumors, the tumor formation rate of Huh-7 cells was 40%, and that of Huh-7-LR cells was 80%, demonstrating that the tumorigenicity of Huh-7-LR cells was enhanced compared with that of Huh-7 cells.

[0053] Example 3: MYH9 gene affects apoptosis, invasion and colony formation of lenvatinib-resistant cell lines

[0054] 1. Seed 3×10 5 Huh-7 cells, Hep-3B cells, Huh-7-LR cells, and Hep-3B-LR cells into 6-cm culture dishes. After culturing for 48 h, collect the cells in each group into 1.5-mL EP tubes, add 100 μL of lysis buffer (RIPA:PMSF = 100:1, 1% phosphatase inhibitor), lyse by sonication twice, centrifuge at 4°C, 12,000 r / min for 10 min, collect the protein supernatant, determine the protein concentration by the BCA method, and perform SDS-PAGE gel electrophoresis with a loading amount of 30 ng. After electrophoresis, transfer the proteins onto a PVDF membrane by wet transfer, block with 5% skim milk in TBST for 2 h. After blocking, incubate with primary antibodies (MYH9, p-MYH9, β-actin) overnight. After rinsing three times with TBST, incubate with the corresponding secondary antibodies, incubate at room temperature for 2 h, and then analyze the bands by exposure with a gel imager.

[0055] The results of Western blotting ([ Figure 3 A) showed that compared with Hep-3B cells and Huh-7 cells, the expression of MYH9 protein was increased in lenvatinib-resistant Huh-7-LR cells and Hep-3B-LR cells.

[0056] Primary antibodies:

[0057] Non-muscle myosin 2A antibody (abcam; cat#ab138498);

[0058] Phosphorylated MYH9 (Ser1943) antibody (p-MYH9) (affinity, cat#AF4425);

[0059] β-actin (proteintech, cat#66009-1-Ig);

[0060] Secondary antibodies:

[0061] Horseradish peroxidase (HRP)-labeled affinity-purified goat anti-mouse IgG (H+L) (Proteintech, Cat.SA00001-1);

[0062] Horseradish peroxidase (HRP)-labeled affinity-purified goat anti-rabbit IgG (H+L) (Proteintech, Cat.SA00001-2).

[0063] 2. Lentiviral transfection of cells: Seed 3×10 5Huh-7-LR cells and Hep-3B-LR cells were seeded into 6-cm culture dishes and transfected with negative control lentivirus (NC), MYH9 lentivirus, or shMYH9 lentivirus. After 48 hours, the cells were treated with puromycin. After another 48 hours, the cells were harvested for gene expression assays.

[0064] Among them, MYH9 shRNA-1: 5‘-ACCGGGCAAGCTGCCGATAAGTATTCTCGAGAATACTTATCGGCAGCTTGCTTTTTTGAATTC-3’;

[0065] MYH9 shRNA-2: 5‘-ACCGGGCAAATTCATTCGCATCAATCTCGAGATTGATGCGAATGAATTTGCTTTTTTGAATTC-3’.

[0066] Compared with the control group (NC), the expression level of MYH9 protein in Huh-7 and Hep-3B cells overexpressing MYH9 increased ( Figure 3 B); the expression of MYH9 protein in Huh-7-LR cells and Hep-3B-LR cells with MYH9 knockdown decreased ( Figure 3 C);

[0067] 3. Colony formation assay (experimental procedure same as step 2 of Example 2) detected that: compared with the control group (NC), overexpression of MYH9 promoted colony formation in Huh-7 cells and Hep-3B cells ( Figure 4 A); knockdown of MYH9 inhibited colony formation in Huh-7-LR cells and Hep-3B-LR cells ( Figure 4 B).

[0068] 4. Cell invasion assay: The experimental procedure was the same as step 3 in Example 2.

[0069] Through the transwell assay ( Figure 5 B) it was detected that: compared with the control group (NC), overexpression of MYH9 increased the invasion ability of Huh-7 cells and Hep-3B cells, p < 0.05 ( Figure 5 A); knockdown of MYH9 decreased the invasion ability of Huh-7-LR cells and Hep-3B-LR cells, p < 0.05.

[0070] 5. Flow cytometry: The experimental procedure was the same as step 4 in Example 2.

[0071] Through the flow cytometry experiment ( Figure 6A) Detection: Compared with the control group (NC), overexpression of MYH9 inhibited apoptosis of Huh-7 cells and Hep-3B cells; knockdown of MYH9 promoted apoptosis of Huh-7-LR cells and Hep-3B-LR cells( Figure 6 B).

[0072] Example 4: CX-4945 inhibits phosphorylation of MYH9

[0073] 1. Silmitasertib (CX-4945) was purchased from SELLECK; Huh-7-LR cells and Hep-3B-LR cells were treated with 0 μM, 1 μM, and 2 μM CX-4945 for 48 h respectively. Detection by Western blotting (experimental steps were the same as step 1 in Example 3) showed that compared with Huh-7 cells and Hep-3B cells, the expression levels of MYH9 protein and p-MYH9 protein in Huh-7-LR cells and Hep-3B-LR cells were decreased( Figure 7 ).

[0074] Example 5: Combined application of CX-4945 and Lenvatinib inhibits the growth of drug-resistant liver cancer cells in vitro

[0075] 1. The Lenvatinib-resistant Huh-7-LR cells and Hep-3B-LR cells were treated with 0 μM, 2 μM, and 5 μM Lenvatinib alone for 48 h, and 1 μM CX-4945 combined with 0 μM, 2 μM, and 5 μM Lenvatinib for 48 h. Cell counting experiments showed that the combined use of CX-4945 and Lenvatinib could significantly inhibit the proliferation of Huh-7-LR cells and Hep-3B-LR cells, *p < 0.05( Figure 8 ).

[0076] 2. The Lenvatinib-resistant Huh-7-LR cells and Hep-3B-LR cells were treated with 5 μM Lenvatinib alone for 48 h, and 5 μM Lenvatinib combined with 0 μM, 1 μM, and 2 μM CX-4945 for 48 h. Flow cytometry analysis showed that the combined use of CX-4945 and Lenvatinib could significantly promote apoptosis of Huh-7-LR cells and Hep-3B-LR cells, *p < 0.05( Figure 9 ).

[0077] Example 6: Combined application of CX-4945 and Lenvatinib inhibits the growth of drug-resistant liver cancer cells in vivo

[0078] A xenograft tumor model was constructed using BALB / c nude mice:

[0079] The Huh-7-LR cells (2×10 6 ) were resuspended in 100 μL of sterile PBS and basement membrane matrix (1:1), and subcutaneously injected into the right axilla of 5-week-old male BALB / c nude mice. After observing for 4 weeks, when the tumor grew to 50 mm 3 , the mice were sacrificed, the tumors were isolated, cut into appropriate sizes, and implanted into the right axilla of 8-week-old male BALB / c nude mice to establish a subcutaneous tumor xenograft model. To confirm the anti-tumor effect of CX-4945, one week after subcutaneous tumor inoculation, the mice were randomly divided into 4 groups (n = 5): control group, lenvatinib group (30 mg / kg / d), CX-4945 group (20 mg / kg / d), CX-4945 (20 mg / kg / d) + lenvatinib (30 mg / kg / d) group. All groups were administered by gavage with saline containing 0.5% sodium carboxymethylcellulose for 3 weeks (6 times / week). The control group was given an equal volume of saline containing 0.5% sodium carboxymethylcellulose daily. During the whole experiment, each mouse was designated and tracked individually. At each injection, the length (a) and width (b) of the tumor and the body weight of the mice were measured until the end of the experiment. The tumor volume calculation formula: a×b 2 ×0.5 (mm 3 ). All mice were from the animal facilities of Zhejiang University.

[0080] The mice were divided into 4 treatment groups (n = 5), namely the control group (NS), Lenvatinib group (30 mg / kg / d), CX-4945 group (20 mg / kg / d), CX-4945 (20 mg / kg / d) + Lenvatinib (30 mg / kg / d) group. During the gavage administration, the appearance of the tumors in different drug treatment groups was observed and the tumor volume ( Figure 10 B) was measured and the body weight of the mice ( Figure 10 C) was recorded. As shown in Figure 10 A, compared with the control group (NS), the use of CX-4945 alone could not inhibit tumor growth; compared with the Lenvatinib group, the CX-4945 + Lenvatinib group had a more significant effect on inhibiting tumor growth. At the same time, during the medication process, the body weight fluctuations of mice in different groups were not significant, indicating that the combination medication did not produce obvious side effects.

[0081] The expression of p-MYH9 protein in the 4 treatment groups was detected by immunohistochemistry. The tumor specimens of nude mice were fixed with formalin and embedded in paraffin, and immunohistochemical staining was performed according to the protocol of the immunohistochemistry detection kit (Proteintech) manufacturer. The immunohistochemical staining was scanned using Pannoramic MIDI (3DHISTECH Ltd.).

[0082] The results showed that after the combination of CX-4945 and Lenvatinib, compared with the control group (NS), the expression of p-MYH9 in the CX-4945 (20 mg / kg / d) + Lenvatinib (30 mg / kg / d) group was significantly decreased ( Figure 11 ).

Claims

1. Use of a protein kinase CK2 inhibitor and lenvatinib in the preparation of a medicament for treating liver cancer, wherein the protein kinase CK2 inhibitor is a small molecule compound CX-4945; The type of liver cancer is liver cancer resistant to lenvatinib.

2. The application according to claim 1, characterized in that, The liver cancer is primary hepatocellular carcinoma.

3. The application according to claim 1, wherein The molar ratio of the protein kinase CK2 inhibitor to lenvatinib is 1:0.5 - 5.

4. A pharmaceutical composition for treating liver cancer, characterized in that, It includes lenvatinib and a protein kinase CK2 inhibitor, and the protein kinase CK2 inhibitor is a small molecule compound CX-4945.

5. The pharmaceutical composition for treating liver cancer according to claim 4, wherein The type of liver cancer is liver cancer resistant to lenvatinib.

6. The pharmaceutical composition for treating liver cancer according to claim 5, wherein The liver cancer is primary hepatocellular carcinoma.

7. The pharmaceutical composition for treating liver cancer according to claim 4, wherein The molar ratio of the protein kinase CK2 inhibitor to lenvatinib is 1:0.5 - 5.

Images