Application of ametinib in preparation of medicine for treating liver cancer

Abstract

The invention discloses an application of ametinib in preparation of a medicine for treating liver cancer, belongs to the technical field of new application of medicines, and verifies the potential of third generation EGFR-TKI ametinib in inhibiting liver cancer progression in vivo and in vitro, and cell function experiments prove that ametinib can effectively inhibit proliferation, migration and invasion ability of liver cancer cells; the exact type of ametinib for inducing liver cancer cell death is determined, and ametinib inhibits survival and proliferation of liver cancer cells by activating autophagy and inducing ferroptosis; the application proves that autophagy activated by the ametinib can promote the occurrence of ferroptosis by regulating and controlling the degradation of GPX4 (glutathione peroxidase 4) and the accumulation of reactive oxygen species (ROS), and the ametinib inhibits the progress of liver cancer by inducing autophagy-dependent ferroptosis, so that the ametinib is used for preparing the medicine for treating liver cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of new uses of drugs, and particularly relates to application of amatinib in preparation of a liver cancer treatment drug. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third leading cause of cancer-related death in humans. Due to the lack of specific clinical manifestations in the early stage, most patients are diagnosed at an advanced stage. Although targeted therapy has provided new treatment options for patients with advanced HCC, current clinical data show that the overall survival of patients receiving targeted therapy is only 10.7-14.6 months, and the therapeutic effect is still very limited. In addition, targeted therapy also faces many challenges, such as poor prognosis of patients, high tumor recurrence rate, rapid disease progression, and easy drug resistance, etc. Therefore, it is urgent to develop new treatment strategies and drugs to improve the prognosis and quality of life of patients.

[0003] The pathogenesis of HCC is related to many important events, such as autophagy and ferroptosis. Autophagy, as a highly conserved intracellular degradation system, plays a key role in cells coping with various stress conditions, and can degrade damaged proteins and cytoplasmic components in lysosomes, thereby maintaining cellular homeostasis and providing substrates for energy production. In HCC, there have been reports on the molecular mechanism of autophagy, and the PI3K / Akt1 / mTOR signaling pathway plays an important role in HCC autophagy. Ferroptosis, as a kind of iron-dependent regulatory cell death, is different from apoptosis, necrosis, pyroptosis and autophagy, and is caused by iron-dependent cell death mediated by active oxygen-induced lipid peroxide accumulation, and plays an important role in the occurrence and development of tumors. There are various antioxidant systems in cells resistant to ferroptosis, among which the SLC7A11 / glutathione (GSH) / GPX4 axis is the most widely studied.

[0004] The SLC7A11 / GSH / GPX4 antioxidant axis is often in a state of disorder and plays a key regulatory role in the occurrence and development of tumors. Accumulating research evidence shows that ferroptosis presents an autophagy-dependent feature in various types of human malignancies. Autophagy-dependent ferroptosis is a regulated form of cell death that depends on autophagy-induced ferroptosis to ultimately inhibit tumor progression. Therefore, inducing autophagy-dependent ferroptosis is a promising strategy to inhibit the progression of cancer cells.

[0005] Amatinib is a third-generation EGFR small molecule NSCLC inhibitor, which inhibits the growth and proliferation of NSCLC cells by inducing apoptosis and autophagy. So far, there is no report on the correlation between amatinib and autophagy-dependent ferroptosis in hepatocellular carcinoma in existing scientific research. In view of the key regulatory role of autophagy and ferroptosis in hepatocellular carcinoma, and the potential pleiotropism of amatinib as a targeted drug, it is explored whether it can inhibit the progression of hepatocellular carcinoma by inducing autophagy-dependent ferroptosis, which can provide new theoretical basis and potential drug selection for the treatment of hepatocellular carcinoma. SUMMARY

[0006] The technical problem solved by the present application is that the present application provides an application of amatinib in the preparation of a hepatocellular carcinoma treatment drug, which solves the technical problems of poor patient prognosis, high tumor recurrence rate, rapid disease progression and easy drug resistance in the prior art.

[0007] To achieve the above-mentioned object, the present application is realized by the following technical scheme: An application of amatinib in the preparation of a hepatocellular carcinoma treatment drug, wherein the structural formula of the amatinib is shown as formula (I): (I).

[0008] Further, the hepatocellular carcinoma treatment drug exerts an effect by inducing autophagy-dependent ferroptosis of hepatocellular carcinoma cells.

[0009] Further, the autophagy-dependent ferroptosis is that amatinib activates the autophagy pathway and induces ferroptosis dependent on the activation of autophagy; the ferroptosis can be reversed by using the autophagy inhibitor chloroquine CQ or knocking down the autophagy key gene ATG5.

[0010] Further, the activation of the autophagy pathway is to reduce the phosphorylation level of PI3K, Akt or mTOR in the PI3K / Akt / mTOR signaling pathway, to up-regulate the ratio of autophagy marker LC3-II / LC3-I, or to promote the degradation of autophagy substrate p62.

[0011] Further, the ferroptosis is to inhibit the SLC7A11-GSH-GPX4 antioxidant axis, specifically including reducing the expression of SLC7A11, reducing the content of intracellular glutathione GSH, inhibiting the activity of GPX4, i.e., glutathione peroxidase 4, or increasing the accumulation of intracellular free Fe 2+ level and lipid peroxidation products.

[0012] A drug for treating hepatocellular carcinoma, comprising amatinib and a pharmaceutically acceptable excipient.

[0013] Further, the drug for treating hepatocellular carcinoma is in oral or non-oral dosage form.

[0014] Further, the drug for treating liver cancer is a pharmaceutically acceptable injection, powder, paste or transdermal patch.

[0015] Further, the excipient includes one or more of a filler, a stabilizer, a diluent, an adjuvant, an excipient, a binder, a humectant, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, and a lubricant.

[0016] Further, the drug for treating liver cancer is a tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository, inhaler or spray.

[0017] The original explanation: Amatinib induces autophagy in liver cancer cells, Amatinib activates autophagy in liver cancer cells by inhibiting the PI3K / Akt1 / mTOR pathway, thereby inhibiting the proliferation of liver cancer cells, Amatinib inhibits the SLC7A11 / GSH / GPX4 axis to induce ferroptosis in liver cancer cells, inhibits the proliferation of liver cancer cells, Amatinib-induced ferroptosis is related to the activation of autophagy, inhibiting autophagy will cause the expression level of GPX4 to rise and the level of ROS to drop, the effective dose of Amatinib is 1-20mg / kg, in summary, Amatinib can inhibit the survival, proliferation, migration and invasion of liver cancer cells.

[0018] The application provides an application of Amatinib in the preparation of a liver cancer treatment drug, which has the following beneficial effects compared with the prior art: 1. New indication expansion: the application uses Amatinib to prepare a liver cancer treatment drug, breaking through the limitation of its traditional indication of non-small cell lung cancer, and providing a new drug selection for liver cancer patients; 2. New mechanism of action: it is revealed that Amatinib inhibits the progression of liver cancer through "autophagy-dependent ferroptosis", which is different from the EGFR inhibition mechanism of Amatinib in lung cancer; this mechanism cooperatively regulates the autophagy and ferroptosis pathways, avoiding the drug resistance problem of single pathway targeted therapy and improving the antitumor effect; 3. High efficiency and specificity: Amatinib can specifically induce liver cancer cell death with low toxicity to normal liver cells; in vivo and in vitro experiments prove that it can significantly inhibit the proliferation, migration, invasion and tumor growth of liver cancer cells, and the safety of the drug approved for marketing is controllable, and the clinical conversion potential is high; 4. The application Amatinib can inhibit the survival, proliferation, migration and invasion of liver cancer cells, and the effective dose of Amatinib is 1-20mg / kg. Preferably, the dose is 5mg / kg. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1CCK-8 result graph of HepG2 and MHCC-97H cells after amatinib treatment of the present application, wherein the left graph is the CCK-8 result graph of HepG2 after amatinib treatment, and the right graph is the CCK-8 result graph of MHCC-97H cells after amatinib treatment; Figure 2 CCK-8 result graph of normal liver cells THL2-2 after amatinib treatment of the present application; Figure 3 CCK-8 result graph of HepG2 and MHCC-97H cells after sorafenib treatment of the present application, wherein the left graph is the CCK-8 result graph of HepG2 after sorafenib treatment, and the right graph is the CCK-8 result graph of MHCC-97H cells after sorafenib treatment; Figure 4 EdU staining result graph of the present application, Figure 4 The upper graph is the EdU fluorescence graph of HepG2 and MHCC-97H cells, and the lower left graph is the fluorescence statistical graph of HepG2 cells, and the lower right graph is the fluorescence statistical graph of MHCC-97H cells; Figure 5 Cell migration and invasion result graph of the present application, Figure 5 The upper graph is the HepG2 cell graph, the lower left graph is the HepG2 cell migration statistical graph, and the lower right graph is the HepG2 cell invasion statistical graph; Figure 6 Cell migration and invasion result graph of the present application, Figure 6 The upper graph is the MHCC-97H cell graph, the lower left graph is the MHCC-97H cell migration statistical graph, and the lower right graph is the MHCC-97H cell invasion statistical graph; Figure 7 Western blot detection graph of migration and invasion related proteins E-cad, N-cad and Vimentin of the present application, Figure 7 The left graph is the Western blot detection graph of HepG2 cells, and the right graph is the Western blot detection graph of MHCC-97H cells; Figure 8 CCK-8 result graph after treatment of apoptosis inhibitor Z-VAD-FMK of the present application, Figure 8 The left graph is the CCK-8 result graph of HepG2 cells after treatment of apoptosis inhibitor Z-VAD-FMK, and the right graph is the CCK-8 result graph of MHCC-97H cells after treatment of apoptosis inhibitor Z-VAD-FMK; Figure 9 CCK-8 result graph after treatment of autophagy inhibitor chloroquine CQ of the present application, Figure 9CCK-8 results of HepG2 cells after treatment with autophagy inhibitor chloroquine CQ, and CCK-8 results of MHCC-97H cells after treatment with autophagy inhibitor chloroquine CQ; Figure 10 Transmission electron microscopy diagram of the present application, Figure 10 The upper left diagram is the transmission electron microscopy diagram of HepG2 and MHCC-97H cells, the lower left diagram is the autophagosome statistics diagram of HepG2 cells, and the right diagram is the autophagosome statistics diagram of MHCC-97H cells; Figure 11 Western blot detection diagram of autophagy-related proteins LC3, p62, ATG5 and Beclin-1 of the present application, Figure 11 The left diagram is the Western blot detection diagram of HepG2 cells, and the right diagram is the Western blot detection diagram of MHCC-97H cells; Figure 12 Acridine orange AO staining diagram of the present application, Figure 12 The upper left diagram is the AO fluorescence diagram of HepG2 and MHCC-97H cells, the lower left diagram is the fluorescence statistics diagram of HepG2 cells, and the right diagram is the fluorescence statistics diagram of MHCC-97H cells; Figure 13 Western blot detection diagram of LC3 protein after treatment with autophagy inhibitor chloroquine CQ of the present application, Figure 13 The left diagram is the Western blot detection diagram of HepG2 cells, and the right diagram is the Western blot detection diagram of MHCC-97H cells; Figure 14 Amatinib regulates hepatocellular carcinoma cell autophagy through the PI3K / Akt1 / mTOR pathway result diagram of the present application, Figure 14 The left diagram is the detection diagram of HepG2 cells, and the right diagram is the detection diagram of MHCC-97H cells; Figure 15 Fe detection diagram of the present application in the process of amatinib inducing ferroptosis in hepatocellular carcinoma cells, 2+ Detection schematic diagram, Figure 15 The left diagram is the Fe detection diagram of HepG2, and the right diagram is the Fe detection diagram of MHCC-97H; 2+ Detection schematic diagram, 2+ Detection schematic diagram; Figure 16 Malondialdehyde MDA detection schematic diagram of the present application, Figure 16 The left diagram is the malondialdehyde MDA detection schematic diagram of HepG2, and the right diagram is the malondialdehyde MDA detection schematic diagram of MHCC-97H; Figure 17 Glutathione GSH detection schematic diagram of the present application,Figure 17 The left graph is the glutathione GSH detection schematic of HepG2, and the right graph is the glutathione GSH detection schematic of MHCC-97H; Figure 18 The ROS detection schematic of the present application is shown in the figure below, Figure 18 The ROS detection schematic of HepG2 is shown in the left graph, and the ROS detection schematic of MHCC-97H is shown in the right graph; Figure 19 The Western blot detection of iron death-related proteins GPX4, SLC7A11 and ACSL4 in the induction of ferroptosis of liver cancer cells by amatinib is shown in the figure below; Figure 20 The ROS detection schematic of CQ treatment is shown in the figure below, Figure 20 The ROS detection schematic of HepG2 after CQ treatment is shown in the left graph, and the ROS detection schematic of MHCC-97H after CQ treatment is shown in the right graph; Figure 21 The Western blot detection of GPX4 protein after siATG5 treatment is shown in the figure below, Figure 21 The Western blot detection of GPX4 protein after siATG5 treatment of HepG2 is shown in the left graph, and the Western blot detection of GPX4 protein after siATG5 treatment of MHCC-97H is shown in the right graph; Figure 22 The Western blot detection of LC3 and ATG5 proteins after Fer-1 treatment is shown in the figure below, Figure 22 The Western blot detection of LC3 and ATG5 proteins after Fer-1 treatment of HepG2 is shown in the left graph, and the Western blot detection of LC3 and ATG5 proteins after Fer-1 treatment of MHCC-97H is shown in the right graph; Figure 23 The mouse tumor formation experiment model of the present application is shown in the figure below; Figure 24 The mouse tumor of the control group and the amatinib treatment group of the present application is shown in the figure below; Figure 25 The mouse tumor weight and volume of the control group and the amatinib treatment group of the present application are shown in the figure below; Figure 25 The left graph is a mouse tumor weight statistical graph, and the right graph is a mouse tumor volume statistical graph; Figure 26 The Western blot detection of GPX4, SLC7A11 and ACSL4 proteins in tumor tissues of the present application is shown in the figure below; Figure 27Figure of Western blot detection of LC3, p62, ATG5 and Beclin-1 proteins in tumor tissues of the present application; Figure 28 Figure of detection of Fe 2+ , MDA and GSH in tumor tissues of the present application; Figure 29 Figure of immunohistochemical IHC results of Ki67, GPX4 and LC3 in tumor tissues of the present application, wherein the upper graph is the IHC detection result graph of Ki67, GPX4 and LC3 in mouse tumors, and the scale is 50 μm, and the lower graph is the IHC statistical result graph of Ki67, GPX4 and LC3 in mouse tumors. DETAILED DESCRIPTION

[0020] The present application will be further described in conjunction with specific examples. The following examples are only used to illustrate the present application, and are not used to limit the scope of application. Modifications or replacements of the methods, steps or conditions of the present application without departing from the spirit and essence of the present application shall fall within the scope of the present application.

[0021] Unless otherwise specified, the experimental methods in the following examples of the present application are conventional methods; the materials, reagents and the like used in the examples are commercially available products, unless otherwise specified.

[0022] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as understood by a person skilled in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific examples, and are not used to limit the present application.

[0023] Example 1, application of amatinib in preparation of a liver cancer treatment drug, the structural formula of amatinib is shown as formula (I): (I).

[0024] The liver cancer treatment drug exerts an effect by inducing autophagy-dependent ferroptosis of liver cancer cells, and the autophagy-dependent ferroptosis is specifically that amatinib induces ferroptosis by activating the autophagy pathway and depending on the activation of autophagy; the ferroptosis can be reversed by using the autophagy inhibitor chloroquine CQ or knocking down the autophagy key gene ATG5; the activated autophagy pathway is to reduce the phosphorylation level of PI3K, Akt or mTOR in the PI3K / Akt / mTOR signaling pathway, to increase the ratio of autophagy marker LC3-II / LC3-I, or to promote the degradation of autophagy substrate p62; and the induced ferroptosis is to inhibit the SLC7A11-GSH-GPX4 antioxidant axis, specifically including reducing the expression of SLC7A11, reducing the content of intracellular glutathione GSH, inhibiting the activity of GPX4, i.e. glutathione peroxidase 4, or increasing the content of intracellular free Fe 2+Accumulation of horizontal and lipid peroxidation products.

[0025] I. Experimental materials and settings: 1. Cell culture: Hepatoma cell HepG2 and MHCC-97H were purchased from American Type Culture Collection. According to the guidelines of American Type Culture Collection (ATCC), the cells used in this application were cultured in DMEM medium containing 10% FBS and 1% antibiotic, and the culture conditions were 37℃, 5% carbon dioxide humidified environment.

[0026] 2. Raising of nude mice: BALB / c male nude mice were raised in SPF barrier environment, using independent ventilation cage (IVC), maintaining temperature 25℃, humidity 50%, 12 hours light cycle. The feed and litter were sterilized by high temperature / irradiation, and sterile drinking water was provided.

[0027] II. Experimental methods: 1. Cell viability detection: 3000 cells were inoculated into 96-well plates, with 6 replicates. CCK-8 detection kit was used to detect cell viability.

[0028] 2. Edu detection: HepG2 and MHCC-97H cells were inoculated in 96-well plates at a density of 3×10 3 cells per well, and incubated in a 37℃ constant temperature incubator for 24 hours under the condition of containing 100 μL complete culture medium, while the cells were treated with amuvratinib (4 and 8 μM) respectively. Each concentration of treatment was repeated in three wells. After incubation for 24 hours, the cells were incubated in EdU staining reaction solution for 2 hours, fixed with 4% formaldehyde for 15 minutes, then the cell nucleus was stained with Hoechst 33342 staining solution, and the image was captured using fluorescence microscope.

[0029] 3. Transwell detection: The cells were digested, centrifuged and resuspended in DMEM medium at a concentration of 2×10 5 cells / mL, then 200 μL of cell suspension was used for migration and invasion assay. For invasion assay, the upper chamber was coated with 1:5 diluted matrigel before the cells were inoculated into the chamber, and the subsequent steps were the same as migration assay. For migration, incubate for 24h, for invasion, incubate for 48h. After incubation, the bottom of the chamber was fixed with 4% polyformaldehyde for 15 min, then stained with 0.1% crystal violet for 15 min, the cells in the upper chamber were wiped off with a wet cotton swab, and the cell migration and invasion status was observed under a microscope.

[0030] 4. Western blotting: Total proteins were extracted from cultured hepatocarcinoma cells or tissues, washed twice with 1x PBS, and then lysed thoroughly on ice by RIPA lysis buffer. Protein samples were electrophoretically separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and the gel was transferred to an NC membrane. The transferred NC membrane was then blocked in 5% skim milk for 1 hour and incubated with the corresponding primary antibody at 4°C overnight, including N-cad (HuaBIO, ET1607-37, 1:1000), E-cad (HuaBIO, M1405-3, 1:1000), Vimentin (92547, Abeam, 1:1000), Beclin-1 (CST, 3495S, 1:1000), ATG5 (CST, 12994S, 1:1000), LC3 (HUABIO, ET1701-65, 1:1000), p62 (Selleck, F0106, 1:5000), PI3K (HUABIO, HA721672, 1:1000), p-PI3K (CST, 12994, 1:1000), Akt (CST, 2938S, 1:1000), p-Akt (CST, 4060S, 1:1000), mTOR (CST, 2972S, 1:1000), p-mTOR (CST, 5536S, 1:1000), SLC7A11 (HUABIO, A7C6, 1:1000), GPX4 (Selleck, F1580, 1:3000), ACSL4 (HUABIO, ET7111-43, 1:1000), GAPDH (HUABIO, ET1601-4, 1:10000). Finally, the membrane was incubated with the corresponding secondary antibody for 1 h to obtain the image.

[0031] 5. AO detection: HepG2 and MHCC-97H cells were treated with or without amivantan for 24 hours, and stained with 5 pg / mL of AO at 37°C for 20 minutes in the presence of complete culture medium. Images were captured using a fluorescence microscope.

[0032] 6. Transmission electron microscopy experiment: After treatment with amivantan, cells were collected, fixed with 2.5% glutaraldehyde and 4% formaldehyde overnight, added with 1% osmium chromate, and dehydrated with a series of graded ethanol and acetone. Then the sample was rinsed and added with 812 epoxy resin. After polymerization, ultrathin sections were prepared using a Leica EM UC7 microtome and stained with 3% lead acetate and 2% uranyl acetate. Finally, autophagy in the cells was observed by transmission electron microscopy.

[0033] 7. Tumor-bearing experiment: 5 x 10 6One MHCC-97H cell was suspended in 200 μL of PBS and injected subcutaneously into the back of a 4-week-old nude mouse. The tumor volume was increased to approximately 100 mg / m³. 3 Mice were randomly divided into three groups and administered saline or amitinib (1 mg / kg or 5 mg / kg) by gavage daily. Body weight and tumor volume were measured every two days. After 12 days of treatment, the mice were euthanized. Tumor tissue was removed from the mice, photographed, weighed, and fixed in 4% paraformaldehyde or immediately frozen in liquid nitrogen. All experimental procedures involving mice were performed in accordance with guidelines approved by the Ethics Review Committee of Jiangsu Normal University.

[0034] 8. Immunohistochemical assay: Tissue samples were paraffin-embedded and cut into 5 μm sections. The slides were then heated at 65°C for 120 minutes, followed by dewaxing, hydration, and antigen retrieval. Endogenous peroxidase was blocked with 3% peroxide for 15 minutes, then with 5% BSA for 1 hour, and incubated overnight at 4°C with the designated primary antibody. Subsequently, anti-rabbit / mouse secondary antibody was applied and incubated at 37°C for 60 minutes. Signal was visualized using freshly prepared DAB substrate solution at room temperature for 5 minutes. Finally, sections were counterstained with Mayer hematoxylin, dehydrated, and mounted. Staining results were captured using a microscope.

[0035] 9. Statistical Data: The statistical analyses used in this application include t-tests and Pearson correlation coefficients applied to the publicly available data. Data are expressed as mean ± standard deviation of mean (SD), * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0036] III. Experimental Results: 1. Ametinib inhibits the proliferation, migration, and invasion of liver cancer cells: such as... Figure 1 As shown, amitinib has an effect on the growth of liver cancer cells. Amitinib effectively inhibits the proliferation of HepG2 and MHCC-97H cells. The IC50 of HepG2 cells treated with amitinib for 24 h is 6.5 μM, and that of MHCC-97H cells is [not specified in the original text]. 50 It is 7.7 μM. For example... Figure 2 As shown, compared with normal hepatocytes THLE-2, hepatocellular carcinoma lines HepG2 and MHCC-97H were more susceptible to amitinib after 24 hours of treatment. Figure 3 As shown, under the same drug concentration, ametinib had a higher killing effect on liver cancer cells than sorafenib.

[0037] like Figure 4 As shown, in the EdU proliferation assay, ametinib reduced the incorporation of EdU in liver cancer cells. Figure 5 and Figure 6As shown, with increasing ametinib concentration, the migration and invasion abilities of both HepG2 and MHCC-97H cells were significantly inhibited. Figure 7 As shown, the expression of migration and invasion-related proteins E-cadherin was upregulated, while the expression of N-cadherin and Vimentin proteins was downregulated. These data indicate that amitinib significantly inhibits the survival, proliferation, migration, and invasion of HepG2 and MHCC-97H cells, demonstrating that amitinib has a certain killing effect on liver cancer cells in vitro.

[0038] 2. Amitinib activates autophagy in liver cancer cells: such as Figure 8 and Figure 9 As shown, amitinib activated autophagy in liver cancer cells. The autophagy inhibitor chloroquine (CQ) effectively reversed amitinib-induced cell death, but the apoptosis inhibitor Z-VAD-FMK did not. Figure 10 As shown, the number of autophagosomes increased in the ametinib treatment group compared to the control group. Figure 11 As shown, amitinib treatment enhanced the transformation of the autophagy marker protein LC3 from LC3-I to LC3-II, and also increased the expression of autophagy-related proteins ATG5 and Beclin1, while p62 expression was suppressed in HepG2 and MHCC-97H cells. Figure 12 As shown, after acridine orange (AO) staining, autophagic vesicles significantly increased in hepatocellular carcinoma cells in the ametinib-treated group. Figure 13 As shown, treatment with the autophagy inhibitor chloroquine (CQ) reduced the LC3 II / I ratio in hepatocellular carcinoma cells treated with amitinib. These data indicate that amitinib can induce autophagy in hepatocellular carcinoma cells. 3. Amitinib regulates autophagy in hepatocellular carcinoma cells through the PI3K / Akt1 / mTOR pathway: such as Figure 14 As shown, the phosphorylation levels of PI3K, Akt1, and mTOR were significantly reduced in hepatocellular carcinoma cells treated with amitinib, indicating that amitinib alters the expression of autophagy-related proteins by inhibiting the PI3K / Akt1 / mTOR signaling pathway.

[0039] 4. Ametinib induces ferroptosis in liver cancer cells: such as... Figure 15 As shown, the ferroptosis inhibitor (Fer-1) can also reverse ametinib-induced cell death. Figure 16 As shown, Fe in liver cancer cells in the ametinib-treated group 2+ Ion levels were higher than in the control group. For example... Figure 17 and Figure 18 As shown, with increasing ametinib concentration, MDA and ROS levels increased in a dose-dependent manner, while GSH levels decreased. Figure 19As shown, the expression of iron death related proteins GPX4 and SLC7A11 decreased after amatinib treatment, while ACSL4 expression increased in HepG2 and MHCC-97H cells. These data suggest that amatinib can induce ferroptosis in liver cancer cells by inhibiting the SLC7A11-GSH-GPX4 axis. Figure 20 As shown, ROS levels decreased after co-treatment with amatinib and CQ compared to the amatinib treatment group. As shown, Figure 21 As shown, the expression level of GPX4 increased after co-treatment with amatinib and siATG5 compared to the amatinib treatment group, indicating that the activation of autophagy in HepG2 and MHCC-97H cells was blocked, and ferroptosis was inhibited. As shown, Figure 22 As shown, Fer-1 was added to inhibit ferroptosis. Western blot was used to detect the expression of autophagy proteins LC3 and ATG5. The results showed that ferroptosis was inhibited by Fer-1, while autophagy was not significantly inhibited.

[0040] 6. Amatinib inhibits the growth of mouse tumors: As shown, Figure 23 MHCC-97H cells were subcutaneously inoculated into nude mice to establish a mouse xenograft tumor model. As shown, Figure 24 and Figure 25 As shown, compared with the control group, a significant decrease in tumor size and weight was observed in the amatinib treatment group. The tumor growth curve showed that the tumor growth rate in the amatinib group was significantly reduced compared with the control group. As shown, Figure 26 and Figure 27 As shown, in the tumor tissue, the expression of ferroptosis-related protein ACSL4 increased in the amatinib treatment group, while the expression of SLC7A11 and GPX4 decreased, and the expression of autophagy-related proteins Beclin-1, autophagy protein 5 (ATG5) and LC3 II / I significantly increased. As shown, Figure 28 As shown, Fe 2+ and MDA levels increased, and GSH decreased. As shown, Figure 29 As shown, Ki67 was weaker in the amatinib treatment group than in the control group, indicating that amatinib can also inhibit the growth of liver cancer cells in vivo; at the same time, it also confirmed that the expression level of LC3 in the amatinib group was significantly increased, and the expression level of GXP4 was significantly decreased. The above data further verified that amatinib can induce autophagy and ferroptosis in liver cancer cells in vivo, and inhibit the progression of liver cancer.

[0041] The above is an exemplary description of the present application, it should be noted that without departing from the core of the present application, any simple modification, modification or other equivalent replacement which can not cost creative labor for those skilled in the art falls within the protection scope of the present application.

Claims

1. Use of amatinib in the preparation of a medicament for treating liver cancer, characterized in that, The structural formula of the amatinib is shown as formula (I): (I). 2.The use of lenvatinib according to claim 1 in the preparation of a drug for treating liver cancer, characterized in that: The liver cancer treatment drug exerts an effect by inducing autophagy-dependent ferroptosis of liver cancer cells. 3.The use of lenvatinib according to claim 2 in the preparation of a drug for treating liver cancer, characterized in that: The autophagy-dependent ferroptosis is specifically amatinib-induced ferroptosis by activating the autophagy pathway and depending on the activation of autophagy; the ferroptosis can be reversed by using the autophagy inhibitor chloroquine CQ or knocking down the autophagy key gene ATG5. 4.The use of lenvatinib according to claim 3 in the preparation of a drug for treating liver cancer, characterized in that: The activation of the autophagy pathway is to reduce the phosphorylation level of PI3K, Akt or mTOR in the PI3K / Akt / mTOR signaling pathway, to up-regulate the ratio of autophagy marker LC3-II / LC3-I, or to promote the degradation of autophagy substrate p62. 5.The use of lenvatinib according to claim 3 in the preparation of a drug for treating liver cancer, characterized in that: The inducing ferroptosis is inhibiting SLC7A11-GSH-GPX4 antioxidant axis, specifically including reducing the expression of SLC7A11, reducing the content of intracellular glutathione GSH, inhibiting the activity of GPX4, i.e., glutathione peroxidase 4, or increasing the content of intracellular free Fe 2+ level and accumulation of lipid peroxidation products.

6. A medicament for treating liver cancer, characterized by comprising the compound according to claim 1. The drug comprises amatinib and pharmaceutically acceptable excipients.

7. The medicament for treating liver cancer according to claim 6, wherein the medicament is a pharmaceutical composition comprising the compound of the formula (I) as an active ingredient. The liver cancer treatment drug is in an oral or non-oral dosage form.

8. The medicament for treating liver cancer according to claim 6, wherein: The liver cancer treatment drug is in a pharmaceutically acceptable injection, powder, paste or transdermal patch dosage form.

9. The drug for treating liver cancer according to claim 6, characterized in that: The excipients include one or more of fillers, stabilizers, diluents, adjuvants, excipients, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants.

10. The medicament for treating liver cancer according to claim 6, wherein: The liver cancer treatment drug is in a tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository, inhaler or spray.

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