Use of hsp70 inhibitors in combination with sorafenib in the treatment of mtor-activated hepatocarcinoma

Abstract

The application provides application of an HSP70 inhibitor combined with sorafenib in treatment of mTOR-activated liver cancer, and belongs to the technical field of combined medication. The application uses the HSP70 inhibitor as a sensitizer of sorafenib, overcomes drug resistance of liver cancer cells to sorafenib caused by mTOR activation, improves the sensitivity of mTOR-activated liver cancer cells to sorafenib, promotes occurrence of ferroptosis, and thus enhances the anti-tumor effect of sorafenib.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of combination drug, and particularly relates to application of HSP70 inhibitor combined with sorafenib in treatment of mTOR-activated hepatocellular carcinoma. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is a highly malignant tumor and the sixth most common cancer and the third leading cause of cancer death worldwide. Nearly half of the global cases of HCC diagnosed are in China due to persistent infection with hepatitis B virus (HBV) as a major risk factor. It is the fourth most common cancer and the second leading cause of cancer death in China. Therefore, the diagnosis, prevention and treatment of HCC have become a serious challenge to global health. Sorafenib is the first choice for the treatment of advanced HCC, which can relieve the symptoms of HCC and improve the prognosis of patients by inducing intracellular ferroptosis. However, the therapeutic effect of sorafenib is easily affected by drug resistance, especially in HBV-positive HCC patients, sorafenib does not show therapeutic effect. Therefore, finding new targets for sorafenib resistance and combination drug will be a new strategy for the treatment of HCC.

[0003] Mechanistic target of rapamycin (mTOR) is a key signaling molecule that regulates biological processes such as cell proliferation, metabolism and autophagy, and is closely related to the occurrence of HCC. About 50% of HCC samples have activation of the mTOR pathway, and the mutation of the tumor suppressor gene tuberous sclerosis complex 2 (TSC2) on the upstream of the mTOR signaling pathway leads to the activation of mTOR. It is particularly noteworthy that in HBV-positive HCC samples, up to 20% of patients have inactivating mutations of TSC2, which is associated with aggressive behavior and early recurrence after liver resection.

[0004] Our research shows that the activation of mTOR may be the key reason for the drug resistance of HCC cells to sorafenib. However, although mTOR inhibitors have achieved remarkable results in the treatment of benign tumors, their therapeutic effect in the treatment of malignant tumors is relatively limited. In the clinical trial of combination of sorafenib and mTOR inhibitor everolimus, the overall survival rate of HCC patients did not significantly improve, so it is necessary to find new targeted drugs to solve the problem of drug resistance of mTOR-activated HCC to sorafenib.

[0005] Pifithrin-μ, also known as 2-phenylethyl sulfonamide, as a HSP70 function inhibitor, can inhibit the activity of HSP70, weaken its protective effect on tumor cells, increase the sensitivity of tumor cells to environmental stimuli, thereby inhibiting the growth and proliferation of tumor cells, and improving the prognosis of tumors. Studies have shown that Pifithrin-μ has an inhibitory effect on various types of tumors, including primary effusion lymphoma, acute leukemia, bladder cancer cells, breast cancer, colon cancer, etc. Therefore, the combination of Pifithrin-μ and traditional anticancer drugs will be a potential strategy to reduce tumor cell drug resistance and improve treatment efficiency. However, Pifithrin-μ has rarely been reported to inhibit human hepatocellular carcinoma cells, and there is also a lack of research on whether Pifithrin-μ has a chemosensitizing effect on human hepatocellular carcinoma cells. SUMMARY

[0006] To solve the above technical problems, the present application provides a combination therapy method using HSP70 inhibitors as sensitizers for anti-hepatoma drugs, especially in the treatment of mTOR-activated hepatoma, which achieves significant combined therapeutic effect.

[0007] The present application provides the use of HSP70 inhibitors in the preparation of sensitizers for anti-hepatoma drugs.

[0008] In a preferred mode of the present application, the anti-hepatoma drug comprises sorafenib.

[0009] The present application also provides the use of HSP70 inhibitors in combination with anti-hepatoma drugs in the preparation of drugs for treating mTOR-activated hepatoma.

[0010] In a preferred mode of the present application, the HSP70 inhibitor is selected from at least one of Pifithrin-μ, Apoptozole and VER-155008.

[0011] In a preferred mode of the present application, the anti-hepatoma drug comprises sorafenib.

[0012] The present application also provides a drug for treating mTOR-activated hepatoma, which contains HSP70 inhibitors and sorafenib of the same or different specifications for common or separate administration.

[0013] In a preferred mode of the present application, the HSP70 inhibitor is selected from at least one of Pifithrin-μ, Apoptozole and VER-155008.

[0014] In a preferred mode of the present application, when the HSP70 inhibitor is Pifithrin-μ, the mass ratio of Pifithrin-μ to sorafenib is 1:2.

[0015] In a preferred embodiment of the present application, the Pifithrin-μ and Sorafenib are co-administered.

[0016] In a preferred embodiment of the present application, the Pifithrin-μ and Sorafenib are co-administered.

[0017] Beneficial effects: mTOR activation makes liver cancer cells resistant to Sorafenib, the present application uses HSP70 inhibitors as sensitizers of Sorafenib, such as using HSP70 inhibitor Pifithrin-μ or VER-155008 or other HSP70 inhibitors to improve the sensitivity of mTOR-activated liver cancer cells to Sorafenib, promote the occurrence of ferroptosis, thereby enhancing the anti-tumor effect of Sorafenib; HSP70 inhibitor Pifithrin-μ can also enhance the inhibitory effect of Sorafenib in mTOR-activated liver cancer cell xenografts in nude mice. In another embodiment of the present application, the application of Pifithrin-μ in nude mouse xenograft and primary liver cancer models further verifies its ability to enhance the anti-tumor effect of Sorafenib, indicating that Pifithrin-μ not only enhances the efficacy of Sorafenib at the cellular level, but also achieves good combination therapy effect in in vivo models, supporting its potential for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 is a diagram showing the verification results of mTOR-activated liver cancer cell resistance to Sorafenib, where A: Tsc2 - / - Figure 1 is a diagram showing the verification results of mTOR-activated liver cancer cell resistance to Sorafenib, where A: Tsc2 - / - Figure 1 is a diagram showing the verification results of mTOR-activated liver cancer cell resistance to Sorafenib, where A: Tsc2

[0019] Figure 2Figure for the result of Pifithrin-μ inhibiting the drug resistance of liver cancer cells to sorafenib, in which A: diagram of the influence of Pifithrin-μ (PES) and sorafenib (Sora) on the cell viability of mTOR-activated liver cancer cells; B: diagram of the immunofluorescence result of PES and Sora on mTOR-activated SNU886 cells; C: diagram of the influence of PES and Sora on the ROS production in mTOR-activated SNU886 cells; D: diagram of the lipid peroxidation level in mTOR-activated SNU886 cells treated by PES and Sora;

[0020] Figure 3 Figure for the treatment effect of Pifithrin-μ combined with sorafenib on the transplanted tumor of nude mice, in which A: diagram of the influence on the body weight during the treatment; B: diagram of the tumor volume of each experimental group over time; C: diagram of the tumors of each experimental group; D: diagram of the tumor weight of each experimental group;

[0021] Figure 4 Figure for the construction result of the liver-specific Tsc2 knockout mouse model, in which A: diagram of the Western Blot detection result of TSC2 protein in different organs; B: diagram of the protein level detection result of the downstream molecules of the mTOR signaling pathway in liver tissues;

[0022] Figure 5 Figure for the result of Pifithrin-μ combined with sorafenib inhibiting the occurrence and development of mTOR-activated liver cancer, in which A: tumor formation of 8-month-old Tsc2 - / - mice, showing representative liver images; B: H&E, Heppar1 and CK19 staining diagrams of liver tissues of 8-month-old Tsc2 - / - mice, scale bar = 100 μm; C: diagram of the treatment scheme, showing the treatment methods of Pifithrin-μ (PES, 10 mg / kg), sorafenib (Sora, 20 mg / kg), PES combined with Sora treatment, and the control group; D: representative liver images of each experimental group; E: H&E and Ki67 liver tissue staining results of each experimental group, scale bar = 100 μm; F: diagram of the number of liver tumors of each experimental group; G: diagram of the liver weight and body weight ratio of each experimental group; H: diagram of the serum ALT level; I: diagram of the serum AST level. DETAILED DESCRIPTION

[0023] The application also provides the use of the HSP70 inhibitor in the preparation of a sorafenib sensitizer.

[0024] The HSP70 inhibitor in the present application is Pifithrin-μ in one embodiment, and Pifithrin-μ is combined with sorafenib in one embodiment, which proves that the HSP70 inhibitor has the effect of increasing the sensitivity of mTOR-activated hepatocarcinoma cells to sorafenib, and can be used as a sensitizer of sorafenib in the treatment of mTOR-activated hepatocarcinoma.

[0025] The present application provides the use of a HSP70 inhibitor combined with an anti-hepatocarcinoma drug in the preparation of a drug for treating mTOR-activated hepatocarcinoma.

[0026] The HSP70 inhibitor in the present application is an agent that can enhance the intracellular ferroptosis induced by sorafenib, and is closely related to the antioxidant level and intracellular iron metabolism. The HSP70 inhibitor in the present application can be combined with sorafenib to further promote the ferroptosis reaction of hepatocarcinoma cells under the activation of mTOR and enhance the sensitivity to sorafenib.

[0027] The HSP70 inhibitor in the present application can also be a drug that inhibits the antioxidant capacity of cells, wherein the drug that inhibits the antioxidant capacity of cells can enhance the oxidative stress reaction in cells, thereby enhancing the killing effect of sorafenib on mTOR-activated hepatocarcinoma cells. The HSP70 inhibitor in the present application can be a substance that inhibits the functional activity of HSP70, such as Pifithrin-μ, VER-155008 or other HSP70 inhibitors, which are combined with sorafenib to inhibit the effect of HSP70 and further promote the effect of sorafenib on hepatocarcinoma cells.

[0028] The anti-hepatocarcinoma drug in the present application can be sorafenib, and in one embodiment, Pifithrin-μ is combined with sorafenib, which verifies that Pifithrin-μ increases the sensitivity of mTOR-activated hepatocarcinoma cells to sorafenib and overcomes the common drug resistance problem in existing treatment methods; Pifithrin-μ not only enhances the efficacy of sorafenib at the cellular level, but also achieves good combined treatment effect in the in vivo xenograft model and primary cancer model, supporting its potential for clinical treatment.

[0029] The present application also provides a drug for treating mTOR-activated hepatocarcinoma, which contains HSP70 inhibitors and sorafenib of the same or different specifications for simultaneous or separate administration.

[0030] The present application does not have special limitations on the dosage form and administration method of the HSP70 inhibitor, and can induce cell ferroptosis after administration by the corresponding dosage form and administration method, such as the combined use of Pifithrin-μ intraperitoneal injection combined with sorafenib gavage in one embodiment.

[0031] In a preferred mode of the present application, when the HSP70 inhibitor is Pifithrin-μ, the mass ratio of Pifithrin-μ and sorafenib is 1:2, and the Pifithrin-μ and sorafenib are co-administered. The dosages of the Pifithrin-μ and sorafenib according to the present application are 10 mg / kg Pifithrin-μ and 20 mg / kg sorafenib in mice.

[0032] In order to further illustrate the present application, the application of the HSP70 inhibitor combined with sorafenib in the treatment of mTOR-activated hepatocarcinoma provided by the present application is described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0033] The sorafenib and Pifithrin-μ used in the examples of the present application are both purchased from MCE company, the CCK-8 kit is purchased from Shanghai Yisheng Biotechnology Co., Ltd., BODIPY TM 581 / 591 C11 and Phen Green TM SK is purchased from Invitrogen company, bovine serum albumin BSA is purchased from Hyclone company, mounting medium (containing DAPI) is purchased from Abeam company, DMEM or 1640 culture medium and fetal bovine serum are purchased from Gibco company, 4% paraformaldehyde fixing solution is purchased from Wuhan Savel Biotechnology Co., Ltd., reactive oxygen species detection kit is purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., Tsc2 - / -The MEF cells of the control group were provided by Zhang Hongbing's research group of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, the HCCLM3, HepG2 and MHCC97H cells were provided by the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, the SNU886 cells were purchased from Nanjing Kebai Biological Technology Co., Ltd., the SNU398 and Li-7 cells were purchased from Wuhan Shangen Biological Technology Co., Ltd., the 293FT cells were purchased from Wuhan Aibote Biological Technology Co., Ltd., and the above-mentioned cells are common cells in the art and have been disclosed in articles, such as: 1. Liu F, Gai X, Wu Y, Zhang B, Wu X, Cheng R, et al. Oncogenic beta-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer. Proc Natl Acad Sci U S A 2022, 119(39): e2202157119.; 2. Liu F, Wu Y, Zhang B, Yang S, Shang K, Li J, et al. Oncogenic beta-catenin-driven liver cancer is susceptible to methotrexate-mediated disruption of nucleotide synthesis. Chin Med J (Engl) 2023.; 3. Li C, Chen H, Lan Z, He S, Chen R, Wang F, et al. mTOR-dependent upregulation of xCT blocks melanin synthesis and promotes tumorigenesis. Cell Death Differ 2019, 26(10): 2015-2028.; 4-6-week-old BALB / c female nude mice were purchased from Beijing Huafukang Biological Technology Co., Ltd., TSC2 flox / flox mice were purchased from the Jackson Laboratory Company in the United States, and liver-specific expression Cre recombinase (Alb-Cre) mice were purchased from Shanghai South Model Organism Technology Development Co., Ltd.

[0034] The test methods used in the embodiments of the present application can be carried out according to the conventional means or kit instructions in the art, unless otherwise specified:

[0035] I. Cell activity detection

[0036] (1) Cell counting

[0037] Cells in logarithmic growth phase were digested, 10 μL of cell suspension was mixed with 10 μL of trypan blue dye after the digestion of DMEM or 1640 complete medium, then 10 μL of the mixture was added to the side notch of the counting plate, and the Luna automatic cell counter was used for cell counting with a dilution factor of 2. According to the calculation results of the relative number of living cells, the cell suspension was diluted, so that the cell density in each well of the 96-well plate was 3000-5000 / 200 μL, and the wells without cells were supplemented with PBS solution to prevent evaporation of the culture medium. Continue to culture at 37℃, 5% CO2 incubator.

[0038] (2) CCK8 method for detecting cell proliferation

[0039] After the cells adhered, the original culture medium was removed and drug treatment was performed. Set the concentration gradient, dilute the drug with the corresponding DMEM or 1640 complete medium, the culture volume in each well is 200 μL, and 5 replicate wells are set for each treatment. After the drug treatment time is over, add 20 μL of CCK8 solution to each well, and return it to the cell culture box for 2-4 h. Detect the absorbance at 450 nm with a microplate reader, record the values for data processing (note: avoid bubbles in the wells to prevent affecting the OD value reading).

[0040] II. Construction of shRNA stable expression cell line

[0041] (1) Lentivirus packaging

[0042] After digestion, 293FT cells were plated in a 10 cm dish. The next day, the cell density was observed under a microscope at 70%-80%, the supernatant was removed, and fresh serum-free medium was added for transfection using lipo2000 reagent. Add 35 μL of lipo2000 to 900 μL of opti-MEM and mix well, incubate at room temperature for 5 min; add 4 μg of PMD2G, 8 μg of PSPAX2 and 8 μg of the target plasmid to 900 μL of opti-MEM and mix well, incubate at room temperature for 5 min; mix the above two mixtures and blow gently, incubate at room temperature for 30 min, then add dropwise to the cell supernatant, incubate at 37℃ for 6-8 h, replace with fresh complete medium and continue to culture; collect the supernatant containing virus after 48 h, add fresh complete medium to the culture dish again and continue to culture for 24 h, collect the supernatant containing virus; mix all the virus liquid, filter through a 0.45 μm filter, and directly transfect cells or store at -80℃ for long-term storage.

[0043] The primers used for shRNA were synthesized by Beijing Qikang Biological Technology Co., Ltd., and the target sequence is as follows:

[0044] shmTOR-1 (SEQ ID No. 1): 5'-CCGCTAGTAGGGAGGTTTATT-3';

[0045] shmTOR-2 (SEQ ID No. 2): 5'-CCTGGCAACAATAGGAGAATT-3'.

[0046] (2) Infection of cells

[0047] Cells to be infected with the virus were plated in a six-well plate, and the next day, when the cell density was about 30% under a microscope, the cells were infected with the virus liquid. The supernatant was removed, and 900 μL of complete culture medium, 450 μL of virus liquid, and 2 μL of 10 mg / ml polybrene were added to each well. After 48-72 h of infection, the stable cell line was screened.

[0048] (3) Screening of stable mTOR low-expression cells

[0049] 2 μL of 10 mg / ml puromycin was added to 50 ml of complete culture medium. The supernatant of the virus-infected cells was removed, and the culture medium containing puromycin was added to kill the cells that were not successfully infected. After 48 h of continuous culture, the surviving cells were the cells that were successfully infected with the virus, and the knockdown efficiency of the stable cell line was verified by Western blot.

[0050] III. Transient transfection of cells

[0051] Cells to be infected with the virus were plated in a six-well plate, and the next day, when the cell density was about 30% under a microscope, the cells were infected with the virus liquid. The supernatant was removed, and 900 μL of complete culture medium, 450 μL of virus liquid, and 2 μL of 10 mg / ml polybrene were added to each well. After 48-72 h of infection, the stable cell line was screened.

[0052] IV. Detection of intracellular reactive oxygen species

[0053] The intracellular reactive oxygen species detection kit from Shanghai Biyun Tian Biotechnology Co., Ltd. was used, and the general steps were as follows:

[0054] (1) Cell treatment

[0055] After 24h of sorafenib treatment, remove the culture medium, wash once with PBS, trypsinize the cells, terminate the digestion when the cells are just digested but not completely detached from the culture dish, collect the cells in a 1.5ml centrifuge tube, and centrifuge at 1000rpm for 5min.

[0056] (2) Incubate the probe

[0057] Dilute the DCFH-DA probe with serum-free DMEM or 1640 medium at a ratio of 1:1000 to a final concentration of 10μM; remove the cell supernatant, resuspend the cell pellet with 500μL of the diluted DCFH-DA solution, incubate in a 37℃ cell incubator for 20min in the dark, invert and mix every 3-5min to ensure that the probe and cells are in full contact; centrifuge at 1000rpm for 5min, remove the supernatant, and wash the cells with PBS 2-3 times to remove the DCFH-DA that has not entered the cells.

[0058] (3) Flow detection

[0059] Resuspend the cell pellet with 300μL of PBS, filter the cell suspension through a filter screen to form single cells, and then detect using a CytoFLEX flow cytometer with a FITC-A channel; collect at least 10000 cells per sample, and detect the fluorescence values before and after sorafenib treatment in real time, and analyze the statistical differences in terms of mean fluorescence intensity values.

[0060] Five, intracellular lipid peroxidation detection

[0061] (1) Cell treatment

[0062] After 24h of sorafenib treatment, remove the culture medium, wash once with PBS, trypsinize the cells, terminate the digestion when the cells are just digested but not completely detached from the culture dish, collect the cells in a 1.5ml centrifuge tube, and centrifuge at 1000rpm for 5min.

[0063] (2) Incubate the probe

[0064] Dilute the BODIPY TM 581 / 591 C11 with serum-free DMEM or 1640 medium at a ratio of 1:1000 to a final concentration of 2μM; remove the cell supernatant, resuspend the cell pellet with 500μL of the diluted BODIPY TM 581 / 591 C11 solution, incubate in a 37℃ cell incubator for 30min in the dark, invert and mix every 5min; centrifuge at 1000rpm for 5min, remove the supernatant, and wash the cells with PBS 3 times to remove the BODIPY TM 581 / 591 C11 that has not entered the cells.

[0065] (3) Flow cytometry detection method same as above.

[0066] VI. Intracellular iron content detection

[0067] (1) Cell treatment

[0068] Put the cell slides into 6-well plates, inoculate 2 x 10 5 cells per well, shake to spread evenly, and incubate in a 37°C 5% CO2 incubator; after the cells adhere, give sorafenib treatment for 24 h, remove the culture medium, and wash the cells with PBS 2-3 times.

[0069] (2) Incubate the probe

[0070] Dilute Phen Green TM SK to a concentration of 2 μM with serum-free DMEM or 1640 culture medium, add the diluted Phen Green TM SK to the cells, place in the incubator under light-protected conditions for 15-30 min; remove the culture medium, wash with PBS 2-3 times, and then add 4% paraformaldehyde for fixation for 30 min, wash with PBS 3 times, 5 min each time.

[0071] (3) Fluorescence imaging

[0072] Take out the cell slides in the 6-well plates with tweezers, air dry, drop a drop of mounting medium containing DAPI on a glass slide, store in a 4°C refrigerator under light protection, and perform imaging detection under a laser confocal microscope the next day, excitation light: 507 nm, emission light: 532 nm.

[0073] VII. Tumorigenesis experiment in nude mice

[0074] (1) Preparation of nude mice

[0075] Select 24 female BALB / c nude mice aged 4-6 weeks, randomly divide into 4 groups, 6 mice per group, weigh and number, and perform subcutaneous inoculation after feeding in a SPF animal room for 1 week.

[0076] (2) Cell preparation

[0077] Substantially expand SNU886 cells in several 15 cm dishes, trypsinize, resuspend with complete culture medium, and collect in a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min; wash the cells with PBS 2 times, and finally resuspend the cells with about 1 ml PBS, perform cell counting; dilute the cell suspension to 3 x 10 6 cells / 100 μL.

[0078] (3) Subcutaneous inoculation

[0079] Blow the cells, take 100 μL of cell suspension with a disposable insulin needle, and inject it subcutaneously on the back of the nude mouse (100 μL per mouse) after the air bubbles are exhausted. The cells form a circular protrusion under the skin. Note: Before inoculation, the skin of the nude mouse is pinched to form more wrinkles; the needle tip is lifted as much as possible to the skin, and the needle is inserted subcutaneously about 1 cm.

[0080] (4) Treatment and sampling

[0081] After inoculation, observe the tumor formation of the nude mice, and after the mice grow subcutaneous visible lumps, the following treatments are performed:

[0082] 1) Control group: equal amount of solvent gavage treatment + equal amount of solvent intraperitoneal injection treatment;

[0083] 2) Experimental group 1: 20 mg / kg sorafenib gavage treatment + equal amount of solvent intraperitoneal injection treatment;

[0084] 3) Experimental group 2: equal amount of solvent gavage treatment + 10 mg / kg Pifithrin-μ intraperitoneal injection treatment;

[0085] 4) Experimental group 3: 20 mg / kg sorafenib gavage treatment + 10 mg / kg Pifithrin-μ intraperitoneal injection treatment.

[0086] Treatment once every 2 days, and during the treatment period, the mice are weighed every 2 days, and the length and width of the tumor are measured with a vernier caliper, and the tumor volume is calculated, the formula is as follows: Tumor volume (V) = 1 / 2 x length (L) x width (W) 2 When the tumor volume of the control group reaches 1000 mm 3 , the mice are anesthetized with sodium pentobarbital, the tumor is separated, weighed and photographed. The tumor tissue is cut open, part is fixed in 4% paraformaldehyde, and part is stored in a -80°C refrigerator.

[0087] Eight, treatment and sampling of primary liver cancer model in mice

[0088] (1) Drug treatment

[0089] The 8-month-old Tsc2 flox / flox; Alb-Cre mice were randomly divided into 4 groups, 8 in each group, and the following treatments were performed: 1) Control group: equal amount of solvent gavage treatment + equal amount of solvent intraperitoneal injection treatment;

[0090] 2) Experimental group 1: 20 mg / kg sorafenib gavage treatment + equal amount of solvent intraperitoneal injection treatment;

[0091] 3) Experimental group 2: equal amount of solvent gavage treatment + 10 mg / kg Pifithrin-μ intraperitoneal injection treatment;

[0092] 4) Experimental group 3: 20 mg / kg sorafenib gavage treatment + 10 mg / kg Pifithrin-μ intraperitoneal injection treatment.

[0093] Treatment once every 2 days, weight the mice every week, and take samples after 8 weeks of continuous administration.

[0094] (2) Mouse sampling

[0095] 1) Weigh the mice, anesthetize them with sodium pentobarbital, and then fix them. First, perform heart blood sampling, open the chest cavity, fully expose the heart, and use a 1 ml syringe soaked in sodium heparin to take blood at the ventricle at 45°C. The blood taken is placed in a 1.5 ml centrifuge tube and left in a 4°C refrigerator overnight. The next day, centrifuge at 3000 rpm for 10 min at 4°C, collect the upper serum, and then perform ALT and AST detection in the laboratory.

[0096] 2) After blood sampling, the liver is fully exposed and removed from the abdominal cavity, and the morphology and tumor formation are observed. The liver is weighed and photographed. A portion of the liver is fixed in 4% paraformaldehyde, and another portion is quickly frozen in liquid nitrogen and stored in a -80°C freezer. An appropriate amount of liver tissue is placed in a 2 ml tissue grinding tube containing grinding beads, and a tissue grinder is used to fully grind it. Centrifuge at 12000 rpm for 10 min at 4°C, collect the supernatant into a new centrifuge tube, and detect the protein concentration. Then, add the corresponding volume of loading buffer and incubate at 98°C for 10 min. Directly use for Western blot detection or store in a -20°C freezer for later use.

[0097] Example 1 mTOR activation of hepatocellular carcinoma cells confers resistance to sorafenib

[0098] Loss of tumor suppressor TSC2 leads to constitutive mTOR activation. To investigate the sensitivity of mTOR-activated cells to sorafenib, the present application first treated WT and Tsc2 - / - MEF with sorafenib. Tsc2 - / - MEF have lower sensitivity to sorafenib Figure 1 A and B). Then, 3 Tsc2 -deleted human hepatocellular carcinoma cell lines (SNU886, SNU398 and Li7) and 3 Tsc2 WT cell lines (HCCLM3, HepG2 and MHCC97H) were used to evaluate the effect of mTOR on the sorafenib resistance of human hepatocellular carcinoma cells. The changes in mTOR pathway-related molecules in hepatocellular carcinoma cells were detected by Western Blot experiment, and the results are shown in Figure 1The phosphorylation levels of P70S6k and 4EBP1 in TSC2-deficient hepatoma cells (SNU886, SNU398 and Li7) were significantly higher than those in TSC2 WT hepatoma cells. The sensitivity of mTOR-activated cells to sorafenib was lower than that of WT cells Figure 1 Middle D).

[0099] In addition, overexpression of mTOR can make HCCLM3 and HepG2 cells resistant to sorafenib Figure 1 Middle E and F). Conversely, knockdown of mTOR can impair the resistance of SNU886 and SNU398 cells to sorafenib Figure 1 Middle G and H). In summary, mTOR confers resistance to sorafenib in cells.

[0100] Example 2 HSP70 inhibitor Pifithrin-μ increases sorafenib-induced ferroptosis in mTOR-activated hepatoma cells

[0101] The present application adds Pifithrin-μ on the basis of sorafenib treatment. The results of CCK8 experiment show that the cell viability of the combination drug group is significantly lower than that of the single drug group Figure 2 Middle A). Next, the effect of Pifithrin-μ and sorafenib used in combination on ferroptosis in hepatoma cells was evaluated. In mTOR-activated SNU886 cells, Pifithrin-μ and sorafenib were used in combination for treatment, and indicators related to ferroptosis were detected. The results of immunofluorescence showed that the fluorescence level of PGSK in the combination treatment group was significantly reduced, indicating that the use of Pifithrin-μ and sorafenib in combination led to a significant increase in intracellular Fe 2+ level Figure 2 Middle B). The results of flow cytometry showed that the production of ROS and the level of lipid peroxidation in hepatoma cells induced by sorafenib were significantly increased Figure 2 Middle C and D).

[0102] Example 3 HSP70 inhibitor Pifithrin-μ promotes the inhibitory effect of sorafenib on mTOR-activated hepatoma

[0103] The present application first established a nude mouse transplantation model, a total of 24, SNU886 cells were subcutaneously inoculated into nude mice. After the tumor volume grew to 100 mm 3 The mice were randomly divided into 4 groups:

[0104] The solvent group, the Pifithrin-μ single treatment group, the sorafenib single treatment group and the Pifithrin-μ and sorafenib combined treatment group, each group of 6.

[0105] Mice were weighed every three days to monitor weight changes and tumor volume was measured. Tumors were harvested and weighed on day 24 of treatment. Compared to the solvent group, the combined treatment group showed no significant change in mouse weight, suggesting that the drug combination did not cause significant toxic side effects. The combination treatment group of Pifithrin-μ and sorafenib showed significantly greater inhibition of tumor growth compared to the single-drug treatment group. Figure 3 ).

[0106] Example 4: HSP70 inhibitor Pifithrin-μ combined with sorafenib inhibits the development and progression of mTOR-activated hepatocellular carcinoma.

[0107] To mimic the activation mutation of mTOR in liver cancer, this invention utilizes gene targeting technology to construct a liver-specific Tsc2-deficient mouse model. Using the Cre-loxp system, Tsc2-flox mice are crossed with transgenic mice expressing liver-specific cre (Alb-Cre) to establish a Tsc2-deficient mouse model. flox / flox Alb cre mice (Tsc2) - / - Mice).

[0108] First, an 8-week-old control group and Tsc2 were collected. - / - Western blot analysis was performed on the expression of TSC2 protein in the heart, liver, spleen, lungs, kidneys, and brain tissues of mice. The results showed that only TSC2 protein was expressed in these tissues. 2- / - TSC2 was knocked out in the liver tissue of mice. Figure 4 (A). Subsequently, the protein levels of downstream molecules of the mTOR signaling pathway in liver tissue were detected, and the results showed that Tsc2... - / - The levels of phosphorylated P70S6K and 4EBP1 in the liver tissue of mice were significantly increased, indicating that the liver-specific Tsc2-deficient mouse model was successfully constructed. Figure 4 (B)

[0109] To investigate liver tumor formation in mice, 8-month-old mice were used, and Tsc2 was observed. - / - The mice exhibited visible liver tumors, while the control group mice had essentially normal liver morphology. Figure 5 (A) Fixation and hematoxylin-eosin staining (HE staining) of mouse liver tissue were performed. The results showed that the liver tissue structure of the control group mice was intact and the liver lobules were clearly visible, while Tsc2... - / - The liver tissue of the mice showed significant disorganization, with a lack of clearly defined hepatic lobules. Immunohistochemical staining results showed Heppar1 positivity and CK19 negativity, suggesting Tsc2. - / - The tumor in the mouse was hepatocellular carcinoma. Figure 5B). The spontaneous hepatocarcinoma mouse model with mTOR activation due to TSC2 deletion was successfully constructed.

[0110] To investigate whether Pifithrin-μ can synergize with sorafenib to treat mTOR-activated hepatocarcinoma, 8-month-old Tsc2 - / - mice were randomly divided into 4 groups: solvent group, Pifithrin-μ single treatment group (10 mg / kg, intraperitoneal injection), sorafenib single treatment group (20 mg / kg, gavage), and Pifithrin-μ (10 mg / kg, intraperitoneal injection) combined with sorafenib (20 mg / kg, gavage) treatment group, with 8 mice in each group. The mice in the solvent group were given the same dose of normal saline, and the change in body weight of the mice was monitored. After 8 weeks of treatment, the mice were subjected to heart blood sampling and liver tissue sampling Figure 5 C). Macroscopic observation found that the livers of the control mice had obvious tumor formation, while the combined treatment group showed obvious remission. The statistical results of the number of liver tumors showed that although the volume was smaller after sorafenib or Pifithrin-μ single treatment, there was no statistically significant difference in the number compared with the control group Figure 5 D and F); the HE staining results of the mouse liver suggested that the liver tissue structure of the mice in the combined treatment group was restored. In addition, the results of immunohistochemical detection of the cell proliferation marker Ki-67 showed that Ki-67 was positive in the liver tissue of the control mice, while it was almost not expressed in the combined treatment group Figure 5 E). However, after combined treatment, the number and volume of liver tumors were significantly reduced, which was significantly different from the control and single treatment groups, and during the treatment process, the body weight of the mice did not change significantly, indicating that the two drugs had no toxic side effects Figure 5 G).

[0111] After liver cells are damaged, alanine transaminase (ALT) and aspertate aminotransferase (AST) are easily released into the blood and are usually used as indicators reflecting liver cell function. Therefore, the inventors detected the ALT and AST levels in the serum of the mice and found that the combined treatment group of Pifithrin-μ and sorafenib was significantly lower than the single treatment group, suggesting that the combined treatment regimen had a significant remission effect on liver cell damage in mice Figure 5 H and I).

[0112] In summary, the combined treatment of Pifithrin-μ and sorafenib can effectively inhibit the occurrence and development of mTOR-activated hepatocarcinoma.

[0113] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained on the basis of the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. Use of an HSP70 inhibitor for the preparation of a drug sensitizer against mTOR-activated hepatocarcinoma, characterized in that, The anti-mTOR activating liver cancer drug is sorafenib; the HSP70 inhibitor is Pifithrin-μ.

2. Use of the HSP70 inhibitor combined with an anti-hepatoma drug in the preparation of a drug for treating mTOR-activated hepatoma, characterized in that, The HSP70 inhibitor is Pifithrin-μ; the anti-liver cancer drug is sorafenib.

3. A medicament for treating mTOR-activated hepatocarcinoma, characterized by, HSP70 inhibitor and sorafenib, the HSP70 inhibitor being Pifithrin-μ, for co-administration or separate administration.

4. The medicament for treating mTOR-activated hepatocarcinoma according to claim 3, wherein the compound is a compound of the following formula: ###0002### or a pharmaceutically acceptable salt thereof. The mass ratio of the Pifithrin-μ and sorafenib is 1:

2.

5. The medicament for treating mTOR-activated hepatocarcinoma according to claim 4, wherein the compound is ###0002### or a pharmaceutically acceptable salt thereof. The Pifithrin-μ and sorafenib are co-administered.

6. The medicament for treating mTOR-activated hepatocarcinoma according to claim 4, wherein The administration amount of the Pifithrin-μ and sorafenib, in terms of mice, is 10 mg / kg Pifithrin-μ and 20 mg / kg sorafenib.

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