Composition for treating sorafenib-resistant hepatocellular carcinoma and its application

Abstract

The present invention belongs to the field of medicine and relates to a composition for treating sorafenib-resistant hepatocellular carcinoma. The composition for treating sorafenib-resistant hepatocellular carcinoma described in the present invention comprises: an effective therapeutic dose of licochalcone A and sorafenib. The present invention also provides the use of a composition containing licochalcone A and sorafenib in the preparation of a drug for treating sorafenib-resistant hepatocellular carcinoma, and the composition containing licochalcone A and sorafenib is used as an active ingredient to prepare an oral dosage form. The composition described in the present invention can inhibit the autophagy level of drug-resistant cells by blocking autophagosome-lysosome fusion, resulting in a significant increase in ROS in drug-resistant cells, inducing apoptosis of drug-resistant cells, and thereby increasing the sensitivity of drug-resistant cells to sorafenib.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a composition for treating sorafenib-resistant hepatocellular carcinoma, in particular to a composition for improving the drug sensitivity of sorafenib-resistant hepatocellular carcinoma cells and applications of the composition. Background Art

[0002] Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate. It is one of the most lethal malignancies worldwide. According to the World Health Organization's 2020 Global Cancer Data, liver cancer ranks third among the top 10 cancer deaths. In China, HBV carriers are numerous, and secondary diseases such as cirrhosis are a major risk factor for HCC, contributing to the high death toll from liver cancer in the country. Common treatments for liver cancer include surgery, chemotherapy, and radiotherapy, but these are associated with significant side effects and a high risk of metastasis and recurrence, often leading to advanced HCC.

[0003] Sorafenib (SORA) is a multikinase inhibitor used clinically to treat inoperable advanced renal cell carcinoma and metastatic primary hepatocellular carcinoma. It is a first-line treatment for advanced HCC. However, sorafenib's inherent resistance has hampered its clinical efficacy. The renowned Sorafenib in Hepatocellular Carcinoma Assessment Randomized Protocol (SHARP) trial demonstrated that sorafenib improved overall survival by an additional 2.8 months. However, resistance develops within 6 months, resulting in very limited clinical efficacy. Therefore, there is an urgent need to find ways to reverse sorafenib resistance in liver cancer and improve its clinical efficacy.

[0004] Licochalcone A (LCA) is a natural chalcone compound extracted from licorice root, which has a wide range of biological activities and pharmacological effects, including anti-inflammatory, antibacterial, antifungal, antiviral, antiparasitic and antitumor. In the field of anti-tumor, a variety of tumor cell lines have been used in the anti-tumor research of LCA, including bile duct cancer, lung cancer, nasopharyngeal cancer, gastric cancer, liver cancer, etc. However, in terms of drug resistance, there have been no reports on the effect of LCA on liver cancer drug resistance. Therefore, Summary of the Invention

[0005] The object of the present invention is to provide a composition for treating sorafenib-resistant hepatocellular carcinoma, which can improve the clinical efficacy of sorafenib on patients with resistant liver cancer.

[0006] The composition for treating sorafenib-resistant hepatocellular carcinoma of the present invention comprises: licochalcone A and sorafenib in an effective therapeutic dose.

[0007] According to a further feature of the composition of the present invention, the effective therapeutic dose ratio of the licochalcone A to sorafenib is 1:1 to 1:2.

[0008] Preferably, the effective therapeutic dose ratio of licochalcone A to sorafenib is 1:1.5.

[0009] The present invention also provides use of a composition containing licochalcone A and sorafenib in preparing a drug for treating sorafenib-resistant hepatocellular carcinoma.

[0010] According to a further feature of the use of the present invention, a composition containing licochalcone A and sorafenib is used as an active ingredient to prepare an oral dosage form.

[0011] Preferably, the oral dosage form is a solid tablet or a solution.

[0012] The inventors have discovered a novel adjuvant drug, licochalcone A (LCA), for the combined treatment of sorafenib-resistant hepatocellular carcinoma (HCC). The authors have validated the combined effect of LCA and sorafenib on HCC-resistant cells and identified the underlying molecular mechanisms. This drug combination inhibits autophagy in resistant cells by blocking autophagosome-lysosome fusion, significantly increasing ROS production within the cells and inducing apoptosis, thereby increasing their sensitivity to sorafenib.

[0013] The present invention shows through experiments that after 24 hours of combined treatment of liver cancer resistant cells with CA and sorafenib, cell viability was significantly decreased, autophagy level was reduced, and ROS was significantly increased compared with the no-drug group and the group given LCA or sorafenib alone. The combined use of the two drugs greatly improved the sensitivity of liver cancer resistant cells to sorafenib. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shows the IC50 of CCK-8 in detecting HepG2 and HepG2-SR.

[0015] Figure 2A The results show the effect of combined use of LCA (10 μM) and sorafenib (10 μM, 15 μM, 20 μM) on the viability of HepG2-SR cells detected by CCK-8.

[0016] Figure 2B The results show the effect of LCA (10 μM, 15 μM) combined with sorafenib on the viability of HepG2-SR cells detected by CCK-8.

[0017] Figures 3A to 3B Shown are the effects of LCA (10 μM) combined with sorafenib (15 μM) on the expression of Beclin-1 and P62 in HepG2-SR cells.

[0018] Figure 4A Shows the confocal microscopy observation of the effects of combined application of LCA (10 μM) and sorafenib (15 μM) on autophagosomes and lysosomes in HepG2-SR cells.

[0019] Figure 4B Shown are the effects of LCA (10 μM) combined with sorafenib (15 μM) on SANP29 expression in HepG2-SR cells.

[0020] Figure 5 Shown are the effects of LCA (10 μM) combined with sorafenib (15 μM) on ROS levels in HepG2-SR cells.

[0021] Figures 6A to 6B Shown are the effects of LCA (10 μM) combined with sorafenib (15 μM) on the expression of BCL-2 and BAX in HepG2-SR cells.

[0022] Figure 6C The flow cytometry analysis shows the effect of LCA (10 μM) combined with sorafenib (15 μM) on apoptosis in HepG2-SR cells. DETAILED DESCRIPTION

[0023] Example 1: Establishment of Sorafenib-resistant liver cancer cells

[0024] Cell source: HepG2 sensitive cells, purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

[0025] HepG2 sensitive cells were cultured in minimum essential medium (MEM) supplemented with 12% fetal bovine serum, 1% glutamine, 1% sodium pyruvate, and 1% glutathione dipeptide (all purchased from Wuhan Punosai Biotechnology Co., Ltd., China). All cells were cultured in a CO2 incubator at 37°C and 5% (v / v) humidity.

[0026] Drug source: Sorafenib, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0027] HepG2 cells were cultured with increasing concentrations of sorafenib until the maximum tolerated dose was reached. HepG2 sensitive and resistant hepatoma cells were cultured at 3×10 4 Cells were plated in 96-well plates and incubated at 37°C overnight. After the cells attached to the wall, they were stimulated with gradient concentrations of sorafenib for 24 h, and cell viability was detected by CCK-8.

[0028] The results are as follows Figure 1As shown, the IC50 of HepG2 sensitive cells was 7.025 (6.65-7.41) μM, while the IC50 of HepG2 resistant liver cancer cells (HepG2-SR) was 39.29 (36.28 - 42.29) μM, and the resistance index reached 5.6, indicating that sorafenib-resistant liver cancer cells (HepG2-SR) were successfully established.

[0029] Example 2: Experiment on the effect of combined use of licochalcone A (LCA) and sorafenib on the sensitivity of liver cancer resistant cells to sorafenib

[0030] Drug source: Licorice chalcone A (LCA), purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0031] HepG2-SR cells established in Example 1 were used, with 3×10 4 Cells were plated in 96-well plates and incubated at 37°C overnight. After the cells attached, they were stimulated with 10 μM LCA and 10 μM, 15 μM, or 20 μM sorafenib for 24 h, i.e., the ratio of LCA to sorafenib was 1:1, 1:1.5, or 1:2. Cell viability was detected by CCK-8.

[0032] The results are as follows Figure 2A As shown, after administration of 10 μM LCA, 10 μM sorafenib, and 15 μM sorafenib alone, HepG2-SR cell viability remained above 80%, demonstrating virtually no cytotoxicity. After administration of 20 μM sorafenib, HepG2-SR cell viability decreased slightly to approximately 60%. However, when 10 μM LCA was combined with 10 μM, 15 μM, and 20 μM sorafenib, respectively, all exerted potent cytotoxicity against HepG2-SR cells, significantly increasing the sensitivity of drug-resistant HCC cells to sorafenib. Therefore, the effective therapeutic dose ratio of licochalcone A to sorafenib was preliminarily determined to be 1:1 to 1:2.

[0033] When 10 μM LCA was used in combination with 15 μM sorafenib, this dosage ratio ensured that the single drugs had little killing effect on drug-resistant cells, while also ensuring sufficient killing effect when used in combination. Therefore, in subsequent experiments, 10 μM LCA and 15 μM sorafenib were selected for combination therapy, that is, the optimal LCA to sorafenib dosage ratio was 1:1.5.

[0034] HepG2-SR cells established in Example 1 were used, with 3×10 4 Cells were plated in 96-well plates and incubated at 37°C overnight. After the cells attached to the wall, they were stimulated with fixed concentrations of LCA (10 μM and 15 μM) and gradient concentrations of sorafenib for 24 h. Cell viability was detected by CCK-8.

[0035] The results are as follows Figure 2B As shown in the results, the IC50 of HepG2-SR cells was 48.38 (46.31-50.45) μM. When combined with 10 μM LCA, the IC50 decreased to 6.242 (5.85-6.63) μM. When combined with 15 μM LCA, the IC50 decreased to 2.305 (2.16 - 2.45) μM, indicating that the combination of LCA and sorafenib can significantly improve the sensitivity of drug-resistant liver cancer cells.

[0036] Example 3: Combination of LCA and Sorafenib to Inhibit Autophagy in Drug-Resistant Liver Cancer Cells

[0037] HepG2-SR cells prepared in Example 1 were plated in 6-well plates. When the cell density reached approximately 80%, cells were treated with 10 μM LCA and 15 μM sorafenib for 24 hours. Cells were then harvested with lysis buffer for total protein extraction. Protein was then quantified according to the instructions of the BCA protein quantification kit (purchased from KeyGEN, Jiangsu). Proteins were separated on a 12% polyacrylamide gel and transferred to a PVDF membrane. The membranes were incubated with antibodies against p62, Beclin-1 (purchased from Cell Signaling Technology), and β-actin (purchased from Fude Biotechnology), followed by further incubation with a goat anti-rabbit secondary antibody (purchased from Fude Biotechnology). Finally, ECL imaging was performed. Protein bands were analyzed by calculating the grayscale value of each band in the film using Image J software.

[0038] Western blot results are as follows Figure 3A As shown, the expression of P62 in the combined treatment group of the two drugs was significantly increased, the expression of Beclin-1 was reduced, and the autophagy level of liver cancer resistant cells was significantly inhibited.

[0039] HepG2-SR cells prepared in Example 1 were plated in 6-well plates. When the cell density reached approximately 80%, cells were treated with 10 μM LCA and 15 μM sorafenib for 0, 2, 4, 6, 12, and 24 hours. Cells were then harvested with lysis buffer for total protein extraction. Protein was then quantified according to the instructions of the BCA protein quantification kit (purchased from KeyGEN, Jiangsu). Proteins were separated on a 12% polyacrylamide gel and transferred to a PVDF membrane. Antibodies to p62, Beclin-1 (purchased from Cell Signaling Technology), and β-actin (purchased from Fude Biotechnology) were then incubated with a goat anti-rabbit secondary antibody (purchased from Fude Biotechnology). Finally, ECL imaging was performed. Protein bands were analyzed by calculating the grayscale value of each band in the film using Image J software.

[0040] Western blot results are as follows Figure 3B As shown in the figure, it shows that after combined treatment with the two drugs, the expression of P62 increased with the increase of administration time, while the expression of Beclin-1 decreased, and the autophagy level of liver cancer resistant cells was significantly inhibited.

[0041] Example 4: Combination of LCA and Sorafenib Inhibits Autophagosome-Lysosome Fusion in Drug-Resistant Liver Cancer Cells

[0042] HepG2-SR cells established in Example 1 were used, with 1×10 5 The cells were spread on confocal plates and incubated at 37°C overnight. After the cells adhered to the wall, 1×10 6 The cells were incubated with recombinant adenovirus constructed by expressing pfu mCherry-eGFP-labeled LC3B for 18 h, then replaced with normal culture medium for 24 h, and finally treated with 10 μM LCA and 15 μM sorafenib for 24 h. The fusion of lysosomes and autophagosomes was observed under confocal microscopy.

[0043] The results are as follows Figure 4A As shown in the results, it was found that combined treatment of LCA and sorafenib could block the fusion of lysosomes and autophagosomes and inhibit autophagy.

[0044] HepG2-SR cells prepared in Example 1 were plated in 6-well plates. When the cell density reached approximately 80%, cells were treated with 10 μM LCA and 15 μM sorafenib for 24 hours. Cells were then harvested with lysis buffer for total protein extraction. Protein was then quantified according to the instructions of the BCA protein quantification kit (purchased from KeyGEN, Jiangsu). Proteins were separated on a 12% polyacrylamide gel and transferred to a PVDF membrane. Antibodies against SANP29 (a functional protein that regulates autophagosome-lysosome fusion) (purchased from Huaan Biotechnology) and β-actin (purchased from Fude Biotechnology) were then incubated with a goat anti-rabbit secondary antibody (purchased from Fude Biotechnology). Finally, ECL imaging was performed. Protein bands were analyzed by calculating the grayscale value of each band in the film using Image J software.

[0045] Western blot results are as follows Figure 4B As shown, the expression of SANP29 was reduced in the combined treatment group, indicating that the fusion of lysosomes and autophagosomes in drug-resistant cells was blocked.

[0046] Example 5: Combination of LCA and sorafenib significantly increases ROS in drug-resistant liver cancer cells

[0047] The HepG2-SR cells established in Example 1 were plated at 1×10 5 Cells were plated into 12-well plates and incubated overnight at 37°C. After cell attachment, they were treated with 10 μM LCA and 15 μM sorafenib for 24 hours. ROS levels were detected using a ROS detection kit (purchased from Beyotime Biotechnology, Shanghai). Fluorescence intensity of each group was observed under a Nikon inverted microscope. Fluorescence images were calculated and analyzed using Image J software.

[0048] The results are as follows Figure 5 As shown in the data, ROS in the LCA and sorafenib combination treatment group was significantly increased compared with the no-drug group and the LCA or sorafenib single-drug group.

[0049] Example 6: Combination of LCA and Sorafenib Induces Apoptosis in Drug-Resistant Liver Cancer Cells

[0050] HepG2-SR cells prepared in Example 1 were plated in 6-well plates. When the cell density reached approximately 80%, cells were treated with 10 μM LCA and 15 μM sorafenib for 24 hours. Cells were then harvested with lysis buffer for total protein extraction. Protein was then quantified according to the instructions for the BCA protein quantification kit (purchased from KeyGEN, Jiangsu). Proteins were separated on a 12% polyacrylamide gel and transferred to a PVDF membrane. The membranes were incubated with antibodies against BCL-2 (purchased from Huaan Biotechnology), BAX (purchased from Proteintech), and β-actin (purchased from Fude Biotechnology), followed by incubation with a goat anti-rabbit secondary antibody (purchased from Fude Biotechnology). Finally, ECL imaging was performed. Protein bands were analyzed by calculating the grayscale value of each band in the film using Image J software.

[0051] The results are as follows Figure 6A As shown in the figure, the BAX / BCL-2 value of LCA combined with sorafenib treatment was significantly increased, indicating that the combination of the two drugs induced apoptosis of drug-resistant cells and increased the sensitivity of drug-resistant cells to sorafenib.

[0052] HepG2-SR cells prepared in Example 1 were plated in 6-well plates. When the cell density reached approximately 80%, cells were treated with 10 μM LCA and 15 μM sorafenib for 0, 2, 4, 6, 12, and 24 hours. Cells were then harvested with lysis buffer for total protein extraction. Protein was then quantified according to the instructions of the BCA protein quantification kit (purchased from KeyGEN, Jiangsu). Proteins were separated on a 12% polyacrylamide gel and transferred to a PVDF membrane. After incubation with antibodies against BCL-2 (purchased from Huaan Biotechnology), BAX (purchased from Proteintech), and β-actin (purchased from Fude Biotechnology), the membrane was incubated again with a goat anti-rabbit secondary antibody (purchased from Fude Biotechnology), and finally imaged using ECL technology. Protein bands were analyzed by calculating the grayscale value of each band in the film using Image J software.

[0053] The results are as follows Figure 6B As shown in the figure, the BAX / BCL-2 values ​​of LCA combined with sorafenib increased with the increase of administration time, indicating that the apoptosis of drug-resistant cells induced by the combination of the two drugs is time-dependent.

[0054] HepG2-SR cells prepared in Example 1 were plated in 6-well plates. When the cell density reached approximately 80%, 10 μM LCA and 15 μM sorafenib were administered for 24 h. The cells were then harvested using EDTA-free trypsin digestion, resuspended in binding buffer, and incubated with Annexin V-FITC and PI for 20 min at room temperature in the dark. For each sample, at least 1×10 cells were detected using a flow cytometer (purchased from DxFLEX, Beckman-Colter, Suzhou, Jiangsu, China). 4 cells. Figure 6C The apoptosis rate of LCA combined with sorafenib was the highest, and it was mainly concentrated in the early stage of apoptosis, indicating that the combination of the two drugs induced apoptosis of drug-resistant cells and increased the sensitivity of drug-resistant cells to sorafenib.

[0055] Example 7: Preparation of the pharmaceutical dosage form of the composition of the present invention

[0056] According to existing pharmaceutical processes, pharmaceutically acceptable excipients, adjuvants or carriers can be used to prepare oral dosage forms of sorafenib and licochalcone A respectively, and then the two can be used in combination.

[0057] According to the experimental data of the present invention, the effective therapeutic dose ratio of licochalcone A to sorafenib is 1:1 to 1:2, preferably 1:1.5.

[0058] Sorafenib is an oral drug that has been approved for marketing. The current dosage forms of the drug are oral dosage forms, including solid tablets and solutions.

[0059] Licorice chalcone A, as a raw material, is usually sold in powder form and can be prepared into oral dosage forms, including solid tablets and solutions.

[0060] A typical process is: mixing licochalcone A powder with intragranular excipients, fluidized bed granulation, grinding, and blending with extragranular excipients to obtain a final powder mixture of licochalcone A; tablet compression to produce tablet cores; film coating to produce film-coated tablets; and packaging.

[0061] Another typical process is: mixing licochalcone A powder with intragranular excipients, dry granulating, grinding, and blending with extragranular excipients to obtain a final powder mixture of licochalcone A; tablet compression to produce tablet cores; film coating to produce film-coated tablets; and packaging.

Claims

1. Use of a composition containing licochalcone A and sorafenib in the preparation of a medicament for treating sorafenib-resistant hepatocellular carcinoma, wherein the dosage ratio of licochalcone A to sorafenib is 1:1 to 1:

2.

2. The use according to claim 1, characterized in that: The composition containing licochalcone A and sorafenib is used as active ingredients to prepare an oral dosage form.

3. The use according to claim 2, characterized in that: The oral dosage form is a solid tablet or a solution.

4. The use according to claim 1, characterized in that: The dosage ratio of the licorice chalcone A to sorafenib is 1:1.5.

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