Application of temozolomide and metformin combination in treatment of liver cancer

Abstract

The invention discloses combined application of temozolomide and metformin to treatment of liver cancer, and belongs to the field of biological medicine. In-vivo and in-vitro experiments prove that combined administration of temozolomide and metformin can remarkably inhibit proliferation and growth of liver cancer cells, animal experiments find that combined use of temozolomide and metformin can remarkably inhibit growth of tumors, obvious liver and kidney toxicity cannot be generated, the liver cancer treatment effect can be improved, and the application of temozolomide and metformin in liver cancer treatment can be promoted. And a new drug combination scheme is provided for liver cancer treatment.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically relates to the application of temozolomide and metformin in combination in the treatment of liver cancer. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for 80-90% of primary liver cancer cases. Currently, treatment options for liver cancer include liver transplantation, hepatectomy, transarterial chemoembolization, stereotactic radiotherapy, radiofrequency ablation, and molecular targeted therapy. Most liver cancer patients are already in the advanced stage at the time of diagnosis, having lost the opportunity for surgery or liver transplantation. Therefore, systemic chemotherapy may be the only effective treatment. However, liver cancer has a low sensitivity to systemic chemotherapy, resulting in a poor prognosis for these patients. Therefore, there is an urgent need to find new systemic chemotherapy methods for the treatment of liver cancer.

[0003] Temozolomide (TMZ) is an oral alkylating agent that transfers alkyl groups to guanine bases, causing DNA damage that, if not repaired, can lead to cell apoptosis. Clinical trials have shown that concurrent radiotherapy and adjuvant TMZ significantly prolong patient survival. Currently, this drug is primarily used to treat neuroblastomas, glioblastomas, and astrocytomas. There are currently no clinical cases of temozolomide used to treat liver cancer.

[0004] Metformin (Met), a well-known small molecule derived from natural plants, has been used as a first-line treatment for type 2 diabetes mellitus (T2DM). Given that T2DM is a major risk factor for hepatocellular carcinoma (HCC), recent studies suggest that metformin may be a preventive measure for HCC. Retrospective observational studies and meta-analyses have shown that metformin reduces the risk of HCC in patients with diabetes. In addition to its preventive effects, several preclinical studies suggest that metformin may enhance the effects of cytotoxic drugs, radiofrequency ablation, or radiotherapy for HCC.

[0005] However, there is currently no combination of temozolomide and metformin for the treatment of liver cancer. Therefore, there is an urgent need to find new systemic chemotherapy methods for the treatment of liver cancer. Summary of the Invention

[0006] In view of this, the primary purpose of this application is to provide the use of temozolomide and metformin in combination for the treatment of liver cancer, wherein metformin increases the sensitivity of liver cancer cells to temozolomide, and at the same time, temozolomide promotes the effect of metformin on liver cancer cells. The combination of temozolomide and metformin for the treatment of liver cancer has a significant synergistic effect. The combination of the two significantly enhances the inhibition of liver cancer cells, improves the treatment effect of liver cancer, and provides a new combination drug regimen for the treatment of liver cancer.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions: One aspect of the present application discloses the use of an effective amount of temozolomide and metformin in combination in the preparation of a drug or pharmaceutical composition for treating liver cancer.

[0008] Another aspect of the present application discloses an in vitro method for inhibiting or delaying the proliferation or growth of liver cancer cells for non-therapeutic and non-diagnostic purposes, comprising the step of administering an effective amount of temozolomide and metformin to the liver cancer cells.

[0009] Beneficial effects of this application: This application proposes for the first time the combined use of temozolomide and metformin for the treatment of liver cancer, achieving significant results. Specifically, metformin enhances the sensitivity of liver cancer cells to temozolomide, while temozolomide promotes the effects of metformin on liver cancer cells. The combined use of the two drugs for the treatment of liver cancer has a significant synergistic effect, effectively inhibiting the growth of liver cancer cells and slowing the progression of liver cancer.

[0010] Furthermore, both drugs are FDA-approved, eliminating the need for the full R&D, testing, registration, and marketing process required for new drug development. Specifically, both drugs have undergone rigorous pharmacological studies, with clear conclusions regarding their activity, formulation, dosage form, and effective dose. Furthermore, both drugs have undergone rigorous pharmacokinetic and pharmacodynamic studies, with rigorous safety and toxicity assessments and guidance. Third, both drugs utilize readily available raw materials, employ mature manufacturing processes, and have received marketing approval from drug regulatory authorities. These advantages allow for faster entry into clinical trials, offering significant practical and development advantages.

[0011] In general, the combination of temozolomide and metformin for the treatment of liver cancer provided in this application has important practical significance, provides a new medication regimen for the clinical treatment of liver cancer, and provides an important basis for improving the treatment effect of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of temozolomide and metformin alone and in combination inhibiting the proliferation of liver cancer cells HepG2 are shown in Figure 2. Figure 1A in the figure represents the proliferation inhibition effect of different concentrations of temozolomide on liver cancer cell HepG2; Figure 1 B in the figure shows the inhibitory effect of different concentrations of metformin on the proliferation of liver cancer cells HepG2; Figure 1 C in the figure represents the inhibition of proliferation of HepG2 liver cancer cells by the combination of 0.05 mM temozolomide and different concentrations of metformin.

[0013] Figure 2 This is the test result of the combination index (CI) of temozolomide and metformin.

[0014] Figure 3 This is the result of the cell cloning experiment detecting the inhibition of liver cancer cell formation by the control group (Ctrl), temozolomide alone group (TMZ), metformin alone group (Met), and temozolomide and metformin combination group (TMZ&Met).

[0015] Figure 4 The results of Western blot detection of DNA damage in liver cancer cells in the control group (Ctrl), temozolomide alone group (TMZ), metformin alone group (Met), and temozolomide and metformin combined group (TMZ&Met) were shown. Figure 4 A in the figure is a Western blot image of γH2AX, a DNA damage indicator in liver cancer cells in different drug-treated groups. Figure 4 B in the sentence is for Figure 4 Statistical results of the band diagram of γH2AX in panel A.

[0016] Figure 5 The results of immunofluorescence detection of DNA damage in liver cancer cells in the control group (Ctrl), temozolomide alone group (TMZ), metformin alone group (Met), and temozolomide and metformin combined group (TMZ&Met) are shown. Figure 5 A in the figure is the immunofluorescence result of different drug-administered groups. Figure 5 B in Figure 5 Statistical results of γH2AX expression in immunofluorescence results in A.

[0017] Figure 6 The figure shows the effects of the control group (Ctrl), temozolomide alone group (TMZ), metformin alone group (Met), and temozolomide and metformin combined group (TMZ&Met) on the growth of mouse liver cancer cells in vivo. Figure 6 A in the figure shows the photographic results of livers taken from mice with liver cancer in different drug-treated groups; Figure 6 Figure B is a statistical chart of weight changes in liver cancer mice in different drug-dosing groups during the drug-dosing period; Figure 6 C in the figure is a statistical graph of the ratio of mouse liver weight to body weight; Figure 6 D and E are the ratios of spleen and kidney to body weight, respectively; Figure 6 F in the figure shows the HE staining results of spleen and kidney. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the embodiments of the present application. The technical solutions in the embodiments described below are exemplary and are only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can fully combine the embodiments of the present application to obtain other embodiments without creative work, and these embodiments are also within the scope of protection of the present application.

[0019] The first aspect of the present application discloses the use of temozolomide and metformin in combination in the preparation of a drug or pharmaceutical composition for treating liver cancer.

[0020] This application proposes for the first time the combined use of temozolomide and metformin for the treatment of liver cancer. In vitro and in vivo experiments confirm that the combined administration of temozolomide and metformin has a significant therapeutic effect on liver cancer. Metformin enhances the sensitivity of liver cancer cells to temozolomide, while temozolomide promotes the effects of metformin on liver cancer cells. The combination of the two has a significant synergistic effect in the treatment of liver cancer cells.

[0021] In the present application, the dosage ratio of temozolomide to metformin in the drug or pharmaceutical composition can be determined experimentally. In some specific examples, the dosage ratio of temozolomide to metformin is 0.05:(0.5-8), for example, it can be any ratio of 0.05:0.5, 0.05:1, 0.05:1.5, 0.05:2, 0.05:2.5, 0.05:3, 0.05:3.5, 0.05:4, 0.05:4.5, 0.05:5, 0.05:6, 0.05:7, 0.05:8, or a range between any two of them.

[0022] In the present application, the drug or pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. Specific examples of the excipient include, but are not limited to, at least one of a diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, colorant, pH regulator, antioxidant, antibacterial agent, buffer, and carrier. The specific type of excipient is not particularly limited and can be selected based on the route of administration of the drug, etc., in accordance with relevant standards or guidelines.

[0023] In this application, the dosage form or administration method of the drug or pharmaceutical composition is not particularly limited, and any pharmaceutical dosage form known in the art is suitable for use in the pharmaceutical composition of this application. Representative administration methods include gastrointestinal administration or subcutaneous injection, and representative dosage forms include oral administration or injection. As examples, the oral administration includes but is not limited to capsules, tablets, pills, granules, oral liquids, suspensions, emulsions, syrups, and the like.

[0024] In the present application, the drug or pharmaceutical composition for treating liver cancer includes at least one of the following manifestations: a: Inhibit the proliferation of liver cancer cells; b: Inhibit or delay the growth of liver cancer cells; c: Promote the damage of liver cancer cell DNA.

[0025] The second aspect of the present application discloses an in vitro method for inhibiting or delaying the proliferation or growth of liver cancer cells for non-therapeutic and non-diagnostic purposes, comprising the step of administering an effective amount of temozolomide and metformin to the liver cancer cells.

[0026] In the present application, there is no particular limitation on the type of liver cancer cells. As an example, the liver cancer cells are liver cancer cell line HepG2, but the invention is not limited thereto.

[0027] The effective amount of the specific active ingredient can be determined by experiment without special requirements. In some examples, the concentration of temozolomide is 0.05 mM, and the concentration of metformin is 0.5-8 mM.

[0028] In this application, the term "liver cancer" refers to a malignant tumor occurring in the liver, i.e., cancerous tissue formed by abnormal proliferation of liver cells due to gene mutation.

[0029] The term "effective amount" or "therapeutically effective amount" refers to a dose of a drug that produces the desired therapeutic effect when treating a disease or alleviating symptoms. When such a dose is administered to a patient, it produces the desired therapeutic effect. Such effects include, but are not limited to, alleviating the degree of disease or symptoms; preventing the onset of disease; inhibiting or slowing the progression of disease, thereby preventing further deterioration of the disease; promoting disease cure, thereby completely eliminating the disease; and providing a biological or medical response, i.e., inducing a specific pharmacological response, to achieve the therapeutic goal.

[0030] The present application will be further explained below in conjunction with specific embodiments of the present application. It should be noted that the following specific embodiments are only for illustrative purposes and do not limit the scope of the present application in any way.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0032] In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are all commercially available.

[0033] In the following examples, a control group (Ctrl), a temozolomide monotherapy group (TMZ), a metformin monotherapy group (Met), and a combination therapy group (TMZ&Met) were set up.

[0034] The drug configuration in the cell experiment is as follows: Metformin (TargetMol, #1115-70-4) powder was prepared with sterile ultrapure water to a 0.5 M stock solution. This solution was added to cell culture medium to achieve the experimental concentrations used for cell culture in subsequent experiments.

[0035] Temozolomide (shyuanye, #B34588) powder was prepared in DMSO to a 100 mM stock solution. This was added to cell culture medium to achieve the desired concentration for the experimental group.

[0036] Unless otherwise noted, in cell-based experiments, the concentrations of temozolomide and metformin in the combination group were 50 μM and 2 mM, respectively. The concentrations in the temozolomide monotherapy group were 50 μM, and the concentrations in the metformin monotherapy group were 2 mM. In mouse experiments, the doses of temozolomide and metformin were 5 mg / kg / 2 days and 250 mg / kg / day, respectively.

[0037] Example 1 In this example, the CCK8 assay was used to detect the effect of drugs on the proliferation of liver cancer cells. The specific steps were as follows: HepG2 liver cancer cells (from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) were plated into 96-well plates and allowed to adhere overnight. A series of drug concentrations, ranging from high to low, were then added to the wells, along with a control group, with triplicate wells for each concentration. After incubation for 24 hours in a 37°C, 5% CO2 cell culture incubator, CCK-8 (1 / 10 the volume of culture medium) was added. Three hours later, the cell OD values at a wavelength of 450 nm were measured using a multi-functional microplate reader to calculate the inhibitory effect of different drug concentrations on liver cancer cell proliferation.

[0038] The HepG2 liver cancer cell culture medium was Dulbecco's modified Eagle's medium (DMEM; Gibco, #C11995500BT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, #10091148) and 1% penicillin / streptomycin (Gibco, #15140122).

[0039] The treatments for the single-drug group and the combination-drug group were achieved by adding a certain amount of drugs to the above-mentioned culture medium and culturing cells in the culture medium containing the drugs.

[0040] The control group was achieved by adding the same volume of DMSO as that of the experimental group and the same volume of sterile ultrapure water as that of the highest concentration group into the above culture medium and culturing cells in the corresponding culture medium.

[0041] Results see Figure 1 It can be seen that when temozolomide and metformin were used in combination to treat liver cancer cells HepG2, cell proliferation was detected by the CCK8 method, and the statistical difference calculation results showed that metformin would increase the sensitivity of liver cancer cells to TMZ, and TMZ would promote the effect of metformin on liver cancer cells. The combination of drugs had a significant synergistic inhibitory effect on cell proliferation.

[0042] Example 2 In this example, the concentrations and corresponding inhibition rates of the single-drug group and the combination-drug group obtained from the experimental results of CCK8 in Example 1 were used to calculate the combination index (CI) using Compusyn software.

[0043] Among them, if CI < 1, it means that the drug combination has a synergistic effect, that is, the effect when used in combination is greater than the sum of the effects of the two drugs used alone; if CI = 1, it means that the drug combination has an additive effect, that is, the effect when used in combination is equal to the sum of the effects of the two drugs used alone; if CI > 1, it means that the drug combination has an antagonistic effect, that is, the effect when used in combination is less than the sum of the effects of the two drugs used alone.

[0044] Results see Figure 2 and as shown in Table 1.

[0045] Table 1 Combination index (CI) active ingredient concentration and calculation results

[0046] pass Figure 2 As can be seen from Table 1, the CI of 0.05 mM temozolomide and different concentrations of metformin combined for liver cancer cells is less than 1, indicating that temozolomide and metformin combined for liver cancer cells have a significant synergistic effect, and the effect of the two drugs combined is better than that of single drugs.

[0047] Example 3 In this embodiment, the effect of drugs on the formation of liver cancer cell clones is detected by cell cloning. The specific steps are as follows: HepG2 liver cancer cells were plated in 6-well plates and allowed to adhere overnight. The cells were then divided into a control group, a temozolomide monotherapy group (50 μM), a metformin monotherapy group (2 mM), and a temozolomide and metformin combination group (50 μM + 2 mM). Each group had triplicate wells filled with the corresponding culture medium or drug solution. The cells were incubated at 37°C, with the medium changed every 3 days, maintaining the same drug concentration in the fresh culture medium. After 2 weeks, the culture medium was aspirated, the cells were rinsed twice with 1× PBS buffer, and crystal violet was added for staining for 5 minutes. The crystal violet was aspirated, and the cells were rinsed twice with 1× PBS buffer. The cells were photographed, and the number of colonies formed in each well was counted. The number of colonies formed in each replicate well of each group was counted, and a bar graph of the counts was plotted.

[0048] Results see Figure 3 The results showed that compared with the control group and the single-drug group, the combination drug had a significant synergistic inhibitory effect on cell clone formation, and the combination drug group had the fewest cell clones and the smallest size.

[0049] Example 4 In this example, Western Blot was used to detect the expression of marker molecules that indicate drug-induced DNA damage in liver cancer cells. The specific steps were as follows: HepG2 liver cancer cells were plated in 6-well plates and adhered overnight. The cells were divided into a control group, a temozolomide monotherapy group, a metformin monotherapy group, and a temozolomide and metformin combination therapy group. Each group had three replicates, and the corresponding culture medium or drug solution was added and incubated for 24 hours. The cells to be lysed were then collected by cell scraping and placed in a 15 mL centrifuge tube. The cells were centrifuged at 500 g for 5 min at 4 °C, and the supernatant was discarded. The cells were resuspended in 5 mL of pre-cooled PBS, centrifuged at 500 g for 5 min at 4 °C, and the supernatant was discarded. The cells were resuspended in 1 mL of pre-cooled PBS, centrifuged at 5000 g for 2 min at 4 °C, and the supernatant was discarded. An appropriate amount of cell lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCI, 10% glycerol, 1% NP-40 and protease inhibitors); incubate on ice for 30 minutes, then centrifuge at 15,000g for 20 minutes at 4°C. Collect the supernatant (which can be quickly frozen in liquid nitrogen and stored at -80°C until needed). Cellular protein was extracted, and Western blot analysis was performed to determine the expression of γH2AX, a marker of DNA damage. The relative expression of γH2AX protein relative to β-actin in each well of each group was statistically analyzed and plotted as a bar graph.

[0050] Results see Figure 4, it can be seen that compared with the control group and the single drug group, the combination of drugs significantly promoted the DNA damage process of cells and increased the expression of γH2AX.

[0051] Example 5 In this example, immunofluorescence was used to detect the effect of drugs on the expression of marker molecules of DNA damage in liver cancer cells. The specific steps are as follows: HepG2 liver cancer cells were plated at a density of 50,000 cells / dish in confocal microscopy dishes. After overnight attachment, the cells were divided into control, temozolomide alone, metformin alone, and temozolomide and metformin combination groups. Each group was treated with triplicate wells of the corresponding culture medium or drug solution. The cells were incubated for 24 hours, washed with 1× PBS, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were then washed twice with 1× PBS and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. The cells were washed three times with 1× PBS, blocked in immunofluorescence blocking buffer for 15 minutes at room temperature, and incubated with γH2AX antibody (CST, #9718) overnight at 4°C. After washing with 1× PBS, the cells were incubated with DAPI for 7 minutes at room temperature. The cells were washed with 1× PBS and imaged using a ZEISS LSM980 confocal microscope. The expression of γH2AX protein in each group was counted and a bar graph was drawn. Results see Figure 5 Compared with the control group and the single-drug group, the combination of drugs significantly promoted the DNA damage process of cells and increased the expression of γH2AX.

[0052] Example 6 In this example, a tumor-bearing mouse model was constructed to study the effect of the combined drug regimen on the growth of liver cancer cells in mice. The specific steps are as follows: 6.1 Material Preparation Intraperitoneal injection of control solution: 10 μL DMSO + 80 μL corn oil + 10 μL PBS.

[0053] Met gavage solution: 65 mg / mL (concentration 503 mM; dissolved in PBS). Dissolve 2.5 g of Met in 38 mL of PBS and administer 100 μL directly to each mouse.

[0054] Preparation of TMZ intraperitoneal injection solution: Prepare TMZ stock solution at a concentration of 1.3 mg / mL (6.7 mM); inject 100 μL intraperitoneally.

[0055] TMZ stock solution preparation: 19.5 mg TMZ dissolved in 1.5 mL DMSO.

[0056] Mice: C57BL / 6 wild-type (WT) mice (8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were housed in a specific pathogen-free facility with a 12-h light-dark cycle at a temperature of 20–26°C and a humidity of 50–70%.

[0057] Hepatocellular carcinoma cells: Mouse hepatocellular carcinoma cells Hepa1-6 were purchased from the Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0058] 6.2 Construction of mouse model: Through an orthotopic transplantation process, mouse liver cancer cells were directly injected into the subcapsular area of the left liver lobe parenchyma of C57BL / 6 male mice to establish an orthotopic liver cancer model in mice. After the model was successfully established, the mice were divided into four groups and treated with medication at intervals of one day. During the medication period, they were fed normally and fed with irradiated sterilized mouse diet (WQJXBIO Technology).

[0059] The specific experimental groups and drug treatments are as follows: Control group (PBS): 100 μL PBS was administered orally once a day; 100 μL control solution was injected intraperitoneally every 2 days.

[0060] Temozolomide monotherapy group (5 mg / kg / 2 d): 100 μL PBS was administered orally once a day; 100 μL TMZ was injected intraperitoneally every 2 days.

[0061] Metformin monotherapy group (250 mg / kg / d): 100 μL Met gavage solution was administered orally once a day; 100 μL control solution was injected intraperitoneally every 2 days.

[0062] Temozolomide and metformin combination group (Met 250 mg / kg / d + TMZ 5 mg / kg / 2d): 100 μL Met solution was administered orally once a day; 100 μL TMZ solution was injected intraperitoneally every 2 days.

[0063] Each group was given the drug for a total of 21 days. After the mice were killed, the tumors were removed, and organs such as the liver, spleen, and kidneys were also removed to observe the growth of the tumors in the mice. The removed spleen and kidneys of the mice were placed in 4% neutral buffered formalin, and HE staining experiments were subsequently commissioned to a third-party company, Xinle.

[0064] See the results Figure 6 .in, Figure 6 Figure A shows the results of in situ liver cancer tumor growth. Compared with the control group and the single-drug group, the combination drug has a significant inhibitory effect on tumor growth. The tumor in the combination drug group has the slowest growth rate and the smallest volume. Figure 6B in the figure shows that the control group, single drug group and combination drug group had no significant effect on the body weight of mice. Figure 6 C in the figure shows that the ratio of liver to body weight of mice in the final combination drug group was the smallest. Figure 6 D and E in the figure showed that there was no significant difference in the ratio of spleen and kidney to body weight of mice in the control group, single drug group and combination drug group. Figure 6 F in the figure shows the HE staining results of spleen and kidney of mice in the control group, single drug group and combination drug group, which proves that single drug group and combination drug group do not produce obvious liver and kidney toxicity.

[0065] The examples demonstrate that the combined use of temozolomide and metformin has a synergistic effect in the treatment of liver cancer. The combination enhances the inhibitory effect on liver cancer cells, has no effect on mouse body weight, and produces no significant hepatotoxicity or renal toxicity. This demonstrates that the combination of temozolomide and metformin has a high safety profile and minimal side effects, providing a new medication regimen for the clinical treatment of liver cancer.

[0066] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. Use of an effective amount of temozolomide and metformin in combination in the preparation of a drug or pharmaceutical composition for treating liver cancer.

2. The use according to claim 1, characterized in that The dosage ratio of temozolomide to metformin is 0.05:(0.5~8).

3. The use according to claim 1, characterized in that The medicine or pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

4. The use according to claim 1, wherein The dosage form of the medicine or pharmaceutical composition is oral preparation or injection.

5. The use according to claim 1, characterized in that The drug or pharmaceutical composition for treating liver cancer includes at least one of the following manifestations: a: Inhibit the proliferation of liver cancer cells; b: Inhibit or delay the growth of liver cancer cells; c: Promote the damage of liver cancer cell DNA.

6. A method for inhibiting or delaying the proliferation or growth of liver cancer cells in vitro for non-therapeutic purposes, characterized in that: The method comprises the step of administering effective amounts of temozolomide and metformin to liver cancer cells.

7. The method according to claim 6, wherein The concentration of the temozolomide is 0.05 mM, and the concentration of the metformin is 0.5-8 mM.

8. The method according to claim 6 or 7, wherein: The liver cancer cells are liver cancer cell line HepG2.

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