Application of combination of HSP70 inhibitor and sorafenib in treatment of mTOR activated liver cancer

Abstract

The invention provides application of combination of an HSP70 inhibitor and sorafenib in treatment of mTOR activated liver cancer, and belongs to the technical field of drug combination. According to the invention, the HSP70 inhibitor is used as a sorafenib sensitizer, so that the drug resistance of hepatoma carcinoma cells to sorafenib caused by activation of mTOR is overcome, the sensitivity of the mTOR activated hepatoma carcinoma cells to sorafenib is improved, and the occurrence of ferroptosis is promoted, thereby enhancing the anti-tumor effect of sorafenib.

Description

Technical Field

[0001] The present invention belongs to the technical field of combined drug administration, and specifically relates to the application of an HSP70 inhibitor combined with sorafenib in the treatment of mTOR-activated liver cancer. Background Art

[0002] Liver cancer, as a highly malignant tumor, is the sixth most common cancer and the third most deadly cancer in the world. Since persistent infection with the hepatitis B virus (HBV) is a major risk factor, nearly half of the liver cancer cases diagnosed worldwide occur in China. It is the fourth most common cancer in China and the second leading cause of cancer death. Therefore, the diagnosis, prevention and treatment of liver cancer have become a serious challenge facing global health. Sorafenib is the first choice for the treatment of advanced liver cancer. By inducing intracellular ferroptosis, it can alleviate liver cancer symptoms and improve patient prognosis. However, the therapeutic effect of sorafenib is easily affected by drug resistance, especially in HBV-positive liver cancer patients, sorafenib has not shown a therapeutic effect. Therefore, finding new targets for sorafenib resistance and combination therapy will be a new strategy for the treatment of liver cancer.

[0003] The mechanistic target ofrapamycin (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 liver cancer. The mTOR pathway is activated in about 50% of liver cancer samples, and mutations in the upstream tumor suppressor gene tuberous sclerosis complex 2 (TSC2) in the mTOR signaling pathway lead to mTOR activation. It is particularly noteworthy that in HBV-positive liver cancer samples, up to 20% of patients have inactivating mutations in TSC2, which are associated with aggressive behavior and early recurrence after liver resection.

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

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

[0006] In order to solve the above technical problems, the present invention provides a combined drug method, using HSP70 inhibitors as sensitizers for anti-liver cancer drugs, especially in the treatment of mTOR-activated liver cancer to achieve significant combined therapeutic effects.

[0007] The present invention provides the use of an HSP70 inhibitor in the preparation of an anti-liver cancer drug sensitizer.

[0008] In a preferred embodiment of the present invention, the anti-liver cancer drug includes sorafenib.

[0009] The present invention also provides the use of an HSP70 inhibitor in combination with an anti-liver cancer drug in the preparation of a drug for treating mTOR-activated liver cancer.

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

[0011] In a preferred embodiment of the present invention, the anti-liver cancer drug includes sorafenib.

[0012] The present invention also provides a drug for treating mTOR-activated liver cancer, comprising an HSP70 inhibitor and sorafenib in unit preparations of the same or different specifications for co- or separate administration.

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

[0014] In a preferred embodiment of the present invention, 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 invention, the Pifithrin-μ and Sorafenib are co-administered.

[0016] In a preferred embodiment of the present invention, the dosage of Pifithrin-μ and Sorafenib is 10 mg / kg Pifithrin-μ and 20 mg / kg Sorafenib, calculated on a mouse basis.

[0017] Beneficial effects: mTOR activation makes liver cancer cells resistant to sorafenib. The present invention uses HSP70 inhibitors as sensitizers for sorafenib, such as using HSP70 inhibitors Pifithrin-μ or VER-155008 or other HSP70 inhibitors to increase the sensitivity of mTOR-activated liver cancer cells to sorafenib, promote the occurrence of ferroptosis, and thus enhance the anti-tumor effect of sorafenib; HSP70 inhibitor Pifithrin-μ can also enhance the inhibitory effect of sorafenib in nude mouse transplanted tumors of mTOR-activated liver cancer cells. In another embodiment of the present invention, the application of Pifithrin-μ in nude mouse transplanted tumors and primary liver cancer models was further verified to further verify its ability to enhance the anti-tumor effect of sorafenib, indicating that Pifithrin-μ can not only enhance the efficacy of sorafenib at the cellular level, but also achieve good combined therapeutic effects in in vivo models, supporting its potential as a clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the results of the drug resistance of mTOR-activated liver cancer cells to sorafenib. A in the figure: Tsc2 - / - Western blot results of embryonic fibroblasts (MEF); B: Tsc2 after 24h treatment with different concentrations of sorafenib - / - MEF survival results; C: Human liver cancer cell line immunoblotting results; D: Sorafenib treatment of liver cancer cell survival results for 24 hours; EH: Sorafenib treatment for 24 hours of HCCLM3 (E) or HepG2 (F) cells transfected with vector or mTOR plasmid, SNU886 (G) or SNU398 (H) cells transfected with control or mTOR shRNA viability effect diagram;

[0019] Figure 2The results of Pifithrin-μ inhibiting the drug resistance of liver cancer cells to Sorafenib are shown in Figure A: the effect of Pifithrin-μ (PES) and Sorafenib (Sora) on the cell viability of mTOR-activated liver cancer cells; B: the immunofluorescence results of PES and Sora on mTOR-activated SNU886 cells; C: the effect of PES and Sora on ROS production in mTOR-activated SNU886 cells; D: the effect of PES and Sora on lipid peroxidation levels in mTOR-activated SNU886 cells;

[0020] Figure 3 The figure is a diagram showing the therapeutic effect of Pifithrin-μ combined with sorafenib on nude mouse transplanted tumors. In the figure, A: the effect on body weight during treatment; B: the statistical diagram of tumor volume over time in each experimental group; C: the tumor diagram of each experimental group; D: the tumor weight diagram of each experimental group;

[0021] Figure 4 The results of constructing a liver-specific Tsc2 knockout mouse model are shown in Figure A: Western Blot detection results of TSC2 proteins in different organs; B: Protein level detection results of downstream molecules of the mTOR signaling pathway in liver tissue;

[0022] Figure 5 The results of Pifithrin-μ combined with sorafenib in the treatment of mTOR-activated liver cancer are shown in Figure A: 8-month-old Tsc2 - / - Tumor formation in mice, showing representative liver images; B: 8-month-old Tsc2 - / - H&E, Heppar1 and CK19 staining of mouse liver tissue, scale bar = 100 μm; C: Schematic diagram of the treatment plan, showing Pifithrin-μ (PES, 10 mg / kg), sorafenib (Sora, 20 mg / kg), combined treatment with PES and Sora, and the treatment method of 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: Statistical graph of the number of liver tumors in each experimental group; G: Statistical graph of the ratio of liver weight to body weight in each experimental group; H: Statistical graph of serum ALT levels; I: Statistical graph of serum AST levels. DETAILED DESCRIPTION

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

[0024] In one embodiment, the HSP70 inhibitor of the present invention is Pifithrin-μ. In one example, Pifithrin-μ is combined with sorafenib to prove that the HSP70 inhibitor has the effect of increasing the sensitivity of mTOR-activated liver cancer cells to sorafenib, and can be used as a sensitizer of sorafenib in the treatment of mTOR-activated liver cancer.

[0025] The present invention provides the use of an HSP70 inhibitor in combination with an anti-liver cancer drug in the preparation of a drug for treating mTOR-activated liver cancer.

[0026] The HSP70 inhibitor referred to in the present invention is an agent that can enhance sorafenib-induced intracellular ferroptosis, which is closely related to the antioxidant level and intracellular iron metabolism. The HSP70 inhibitor described in the present invention can be used in combination with sorafenib to further promote the ferroptosis reaction of liver cancer cells under mTOR activation and enhance the sensitivity to sorafenib.

[0027] The HSP70 inhibitor of the present invention 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 killing effect of sorafenib on mTOR-activated liver cancer cells by enhancing the intracellular oxidative stress response. The HSP70 inhibitor of the present invention can be a substance that inhibits the functional activity of HSP70, such as Pifithrin-μ, VER-155008 or other HSP70 inhibitors, which are used in combination with sorafenib to inhibit the action of HSP70 and further promote the effect of sorafenib on liver cancer cells.

[0028] The anti-liver cancer drug of the present invention can be sorafenib. In one embodiment, Pifithrin-μ is combined with sorafenib to verify that Pifithrin-μ increases the sensitivity of mTOR-activated liver cancer cells to sorafenib, overcoming the common drug resistance problem in existing treatment methods; Pifithrin-μ can not only enhance the efficacy of sorafenib at the cellular level, but also achieve good combined therapeutic effects in in vivo transplanted tumor models and primary cancer models, supporting its potential as a clinical treatment.

[0029] The present invention also provides a drug for treating mTOR-activated liver cancer, comprising an HSP70 inhibitor and sorafenib in unit preparations of the same or different specifications for simultaneous or separate administration.

[0030] The present invention does not specifically limit the dosage form and administration method of the HSP70 inhibitor. After administration in the corresponding dosage form and administration method, it can induce cell ferroptosis. For example, in one embodiment, the combined medication effect of intraperitoneal injection of Pifithrin-μ combined with oral administration of Sorafenib is used.

[0031] In a preferred embodiment of the present invention, when the HSP70 inhibitor is Pifithrin-μ, the mass ratio of Pifithrin-μ to Sorafenib is 1:2, and the Pifithrin-μ and Sorafenib are co-administered. The dosage of Pifithrin-μ and Sorafenib in the present invention, in terms of mice, is 10 mg / kg Pifithrin-μ and 20 mg / kg Sorafenib in one embodiment.

[0032] To further illustrate the present invention, the application of the HSP70 inhibitor provided by the present invention in combination with sorafenib in the treatment of mTOR-activated liver cancer is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Sorafenib and Pifithrin-μ used in the examples of the present invention were purchased from MCE, CCK-8 kit was purchased from Shanghai Yisheng Biotechnology Co., Ltd., BODIPY TM 581 / 591 C11 and Phen Green TM SK was purchased from Invitrogen, bovine serum albumin (BSA) was purchased from Hyclone, mounting medium (containing DAPI) was purchased from Abcam, DMEM or 1640 culture medium and fetal bovine serum were purchased from Gibco, 4% paraformaldehyde fixative was purchased from Wuhan Saiweier Biotechnology Co., Ltd., reactive oxygen species detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., Tsc2 - / -MEF cells and control group were provided by Zhang Hongbing's research group, Institute of Basic Medicine, Chinese Academy of Medical Sciences. HCCLM3, HepG2 and MHCC97H cells were provided by the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. SNU886 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd., SNU398 and Li-7 cells were purchased from Wuhan Shangen Biotechnology Co., Ltd., and 293FT cells were purchased from Wuhan Abotek Biotechnology Co., Ltd. The above cells are common cells in this field and have been disclosed in articles, such as: ①Liu 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 USA 2022, 119(39): e2202157119.; ②Liu 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.; ③Li 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 Biotechnology Co., Ltd., TSC2 flox / flox mice were purchased from Jackson Laboratory, USA, and mice expressing liver-specific Cre recombinase (Alb-Cre) were purchased from Shanghai Southern Model Organisms Technology Development Co., Ltd.

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

[0035] 1. Cell Viability Detection

[0036] (1) Cell counting

[0037] Digest the cells in the logarithmic growth phase. After terminating the digestion with DMEM or 1640 complete medium, take 10 μL of the cell suspension and mix it evenly with 10 μL of trypan blue dye. Then take 10 μL of the mixture and add it to the groove on the side of the counting plate. Use Luna automatic cell counter to count the cells. The dilution factor is 2. Dilute the cell suspension according to the calculation results of the relative viable cell number so that the cell density in each well of the 96-well plate is 3000-5000 cells / 200 μL. The surrounding wells without cells are supplemented with PBS solution to prevent the volatilization of the culture medium. Incubate at 37°C, 5% CO 2 Continue culturing in the incubator.

[0038] (2) CCK8 assay to detect cell proliferation

[0039] After the cells adhere to the wall, remove the original culture medium and add drugs. Set the concentration gradient, use the corresponding DMEM or 1640 complete culture medium to dilute the drug, 200μL of culture medium volume per well, and set 5 replicates for each treatment. After the drug treatment time is over, add 20μL of CCK8 solution to each well, return to the cell culture incubator to react for 2 to 4 hours, and detect the absorbance at 450nm with an enzyme reader, and record the value for data processing (Note: Avoid bubbles in the wells to prevent affecting the OD value reading).

[0040] 2. Construction of shRNA Stable Expression Cell Line

[0041] (1) Lentiviral packaging

[0042] After digestion, 293FT cells were plated on a 10 cm dish. The next day, the cell density was observed under a microscope at 70% to 80%. The supernatant was removed, and fresh serum-free culture medium was added and transfected using lipo2000 reagent. 35 μL lipo2000 was added to 900 μL opti-MEM and mixed, and placed at room temperature for 5 min; 4 μg PMD2G, 8 μg PSPAX2 and 8 μg target plasmid were added to 900 μL opti-MEM and mixed, and placed at room temperature for 5 min; the above two mixed solutions were mixed and gently blown, and the cell supernatant was added dropwise after standing at room temperature for 30 min, and cultured in a 37°C incubator for 6 to 8 h, and then replaced with fresh complete culture medium and continued to culture; after 48 h, the supernatant containing the virus was collected, and fresh complete culture medium was added to the culture dish again, and culture was continued for 24 h, and the supernatant containing the virus was collected; after mixing all the virus solutions, they were filtered through a 0.45 μm filter, and directly transfected into cells or stored in a -80°C refrigerator for a long time.

[0043] The primers used for shRNA were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the targeting sequences are as follows:

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

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

[0046] (2) Infected cells

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

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

[0049] Add 2 μL of 10 mg / ml puromycin to 50 ml of complete culture medium; remove the supernatant of virus-infected cells and add culture medium containing puromycin to kill uninfected cells; after continuing to culture for 48 hours, the surviving cells are the cells successfully infected with the virus, and use Western blot to verify the knockdown efficiency of the stable cell line.

[0050] 3. Transient Transfection of Cells

[0051] The cells to be infected with the virus were spread into a six-well plate. When the cell density was about 30% under a microscope the next day, the supernatant was removed, and fresh serum-free culture medium was added and transfected using lipo2000 reagent. 8μL lipo2000 was added to 150μL opti-MEM and mixed, and placed at room temperature for 5min; 2μg pcDNA3-Flag-mTOR plasmid (purchased from Addgene, USA) was added to 150μL opti-MEM and mixed, and placed at room temperature for 5min; the above two mixtures were mixed and gently blown, and the cells were added after standing at room temperature for 30min, and cultured in a 37°C incubator. After transfection for 6 to 8h, fresh complete culture medium was replaced, and after continuing to culture for 48h, the overexpression efficiency of mTOR was verified by Western blot.

[0052] IV. Intracellular Reactive Oxygen Detection

[0053] The intracellular reactive oxygen species detection kit of Shanghai Bio-Tech Biotechnology Co., Ltd. is used. The general steps are as follows:

[0054] (1) Cell treatment

[0055] After treating the cells with sorafenib for 24 hours, the culture medium was removed, the cells were washed once with PBS, and the cells were digested with trypsin. The digestion was terminated just before the cells were completely digested but not completely detached from the bottom of the culture dish. The cells were collected in a 1.5 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes.

[0056] (2) Incubation of probe

[0057] The DCFH-DA probe was diluted with serum-free DMEM or 1640 culture medium at a ratio of 1:1000 to a final concentration of 10 μM. The cell supernatant was removed, and the cell pellet at the bottom of the tube was resuspended with 500 μL of the diluted DCFH-DA solution, and then incubated in a 37°C cell culture incubator in the dark for 20 minutes, and mixed by inversion every 3 to 5 minutes to ensure full contact between the probe and the cells. The tube was centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the cells were washed 2 to 3 times with PBS to remove the DCFH-DA that did not enter the cells.

[0058] (3) Flow cytometry

[0059] The cell pellet was resuspended in 300 μL PBS, and the cell suspension was filtered through a filter to form single cells, which were then detected by a CytoFLEX flow cytometer with the FITC-A channel. At least 10,000 cells were collected for each sample, and the fluorescence values ​​before and after the action of sorafenib were detected in real time, and the statistical differences were analyzed using the average fluorescence intensity values.

[0060] 5. Intracellular lipid peroxidation detection

[0061] (1) Cell treatment

[0062] After treating the cells with sorafenib for 24 hours, the culture medium was removed, the cells were washed once with PBS, and the cells were digested with trypsin. The digestion was terminated just before the cells were completely digested but not completely detached from the bottom of the culture dish. The cells were collected in a 1.5 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes.

[0063] (2) Incubation of probe

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

[0065] (3) The flow cytometry method is the same as before.

[0066] 6. Intracellular iron content detection

[0067] (1) Cell treatment

[0068] The cells were placed in a 6-well plate and 2 × 10 5 After shaking and spreading, incubate at 37°C with 5% CO 2 Culture in an incubator; after the cells adhere to the wall, treat with sorafenib for 24 hours, remove the culture medium, and wash the cells 2 to 3 times with PBS.

[0069] (2) Incubation of probe

[0070] Phen Green was cultured in serum-free DMEM or 1640 medium. TM SK was diluted to a concentration of 2 μM and the diluted Phen Green TM SK was added to the cells and placed back in the incubator for incubation for 15-30 min in the dark. The culture medium was removed, the cells were washed 2-3 times with PBS, and then fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS, each time for 5 min.

[0071] (3) Fluorescence imaging

[0072] Use tweezers to remove the cell slides from the 6-well plate, dry them, drop a drop of mounting medium containing DAPI on the slide, and store them in a 4°C refrigerator away from light. The next day, perform imaging detection under laser confocal microscopy, with excitation light: 507nm, emission light: 532nm.

[0073] VII. Nude Mouse Tumor Experiment

[0074] (1) Preparation of nude mice

[0075] Twenty-four female BALB / c nude mice aged 4 to 6 weeks were selected and randomly divided into 4 groups, with 6 mice in each group. The mice were weighed and numbered, and were raised in an SPF animal room for one week before being subcutaneously inoculated.

[0076] (2) Cell preparation

[0077] SNU886 cells were expanded in several 15 cm dishes. After trypsin digestion, they were resuspended in complete medium and collected in 15 ml centrifuge tubes. Centrifugation was performed at 1000 rpm for 5 min. The cells were washed twice with PBS and resuspended in about 1 ml PBS for cell counting. The cell suspension was diluted to 3 × 10 6 pcs / 100μL.

[0078] (3) Subcutaneous inoculation

[0079] Blow the cells evenly, draw 100μL of cell suspension with a disposable insulin needle, expel the air bubbles and inject it into the subcutaneous tissue of the nude mouse (100μL / mouse). The cells can be seen forming round protrusions under the skin. Note: Before inoculation, pull up the skin of the nude mouse to form more wrinkles; lift the skin upwards as much as possible with the needle tip, and insert the needle about 1cm under the skin.

[0080] (4) Treatment and material collection

[0081] After inoculation, observe the tumor formation in nude mice. When a visible mass grows under the skin of the mice, perform the following treatments:

[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 volume 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 was performed once every 2 days. During the treatment period, the mice were weighed every 2 days, and the length and width of the tumor were measured with a vernier caliper to calculate the tumor volume. The formula is as follows: tumor volume (V) = 1 / 2 × length (L) × width (W) 2 When the tumor volume of the control group reaches 1000mm 3 Around 20 minutes later, mice were anesthetized with sodium pentobarbital, and the tumors were separated, weighed, and photographed. Tumor tissues were cut open, partly fixed with 4% paraformaldehyde, and partly frozen in a -80°C refrigerator.

[0087] 8. Treatment and sampling of mouse primary liver cancer model

[0088] (1) Drug treatment

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

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

[0091] 3) Experimental group 2: equal volume 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] The mice were treated every 2 days, weighed weekly, and samples were collected after 8 weeks of continuous administration.

[0094] (2) Mouse sampling

[0095] 1) Weigh the mice, anesthetize them with sodium pentobarbital, and fix them. First, draw blood from the heart. Open the chest cavity to fully expose the heart. Use a 1 ml syringe soaked in sodium heparin to insert the needle into the ventricle at 45°C to draw blood. Place the drawn blood in a 1.5 ml centrifuge tube and let it stand in a 4°C refrigerator overnight. The next day, centrifuge at 3000 rpm for 10 min at 4°C. Collect the upper serum and complete the detection of ALT and AST indicators in the laboratory.

[0096] 2) After blood was drawn from the mice, the liver in the abdominal cavity was fully exposed and removed, and the morphology and tumor formation were observed. The liver was weighed and photographed for record; part of the liver was fixed in 4% paraformaldehyde, and part of the liver was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator; an appropriate amount of liver tissue was placed in a 2 ml tissue grinding tube containing grinding beads, and it was fully ground using a tissue grinder. Centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was collected into a new centrifuge tube and the protein concentration was detected, and then the corresponding volume of loading buffer was added, incubated at 98°C for 10 min, and used directly for Western blot detection or frozen in a -20°C refrigerator for later use.

[0097] Example 1 mTOR activated liver cancer cells are resistant to sorafenib

[0098] Loss of the tumor suppressor TSC2 leads to constitutive mTOR activation. To investigate the sensitivity of mTOR-activated cells to sorafenib, we first treated WT and Tsc2 cells with sorafenib. - / - MEF.Tsc2 - / - MEFs are less sensitive to sorafenib ( Figure 1 A and B). Then, three Tsc2-deficient human liver cancer cell lines (SNU886, SNU398, and Li7) and three Tsc2 WT cell lines (HCCLM3, HepG2, and MHCC97H) were used to evaluate the effect of mTOR on sorafenib resistance in human liver cancer cells. The changes in mTOR pathway-related molecules in liver cancer cells were detected by Western Blot experiments. The results are shown in Figure 1C, the phosphorylation levels of P70S6k and 4EBP1 in TSC2-deficient hepatocellular carcinoma cells (SNU886, SNU398, and Li7) were significantly higher than those in TSC2WT hepatocellular carcinoma cells. mTOR-activated cells were less sensitive to sorafenib than WT cells ( Figure 1 (middle D).

[0099] In addition, overexpression of mTOR can induce drug resistance in HCCLM3 and HepG2 cells ( Figure 1 In contrast, knockdown of mTOR impaired the resistance of SNU886 and SNU398 cells to sorafenib ( Figure 1 In conclusion, mTOR confers resistance to sorafenib in cells.

[0100] Example 2 HSP70 inhibitor Pifithrin-μ increases sorafenib-induced ferroptosis of mTOR-activated liver cancer cells

[0101] The present invention adds Pifithrin-μ on the basis of sorafenib treatment. The CCK8 experimental results show that the cell viability of the combined drug group is significantly lower than that of the single drug group ( Figure 2 Next, the effect of Pifithrin-μ and sorafenib on ferroptosis in liver cancer cells was evaluated. In mTOR-activated SNU886 cells, Pifithrin-μ and sorafenib were used to detect ferroptosis-related indicators. Immunofluorescence results showed that the PGSK fluorescence level in the combined treatment group was significantly reduced, indicating that the combined use of Pifithrin-μ and sorafenib led to the intracellular Fe 2+ A significant increase in the level of Figure 2 (B). Flow cytometry results showed that sorafenib-induced ROS production and lipid peroxidation levels in liver cancer cells were significantly increased ( Figure 2 C and D).

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

[0103] The present invention first established a nude mouse transplantation model, with a total of 24 mice, and subcutaneously inoculated SNU886 cells into the nude mice. 3 Afterwards, the mice were randomly divided into 4 groups:

[0104] There were 6 rats in each of the solvent group, Pifithrin-μ alone treatment group, Sorafenib alone treatment group and Pifithrin-μ combined with Sorafenib treatment group.

[0105] The mice were weighed every three days, their weight changes were monitored, and the tumor volume was measured. Samples were taken and the tumor weight was weighed after the 24th day of treatment. Compared with the solvent group, the weight of the mice in the combined treatment group did not change significantly, indicating that the drug combination did not cause obvious toxic side effects, and the Pifithrin-μ and sorafenib combined treatment group showed more significant performance in inhibiting tumor growth compared with the single drug treatment group ( Figure 3 ).

[0106] Example 4 HSP70 inhibitor Pifithrin-μ combined with sorafenib inhibits the occurrence and development of mTOR activated liver cancer

[0107] To simulate the activation mutation of mTOR in liver cancer, the present invention uses gene targeting technology to construct a mouse model of liver-specific Tsc2 deficiency. Through the Cre-loxp system, Tsc2-flox mice are hybridized with liver-specific cre (Alb-Cre) transgenic mice to establish Tsc2 flox / flox ;Alb cre Mouse (Tsc2 - / - mice).

[0108] First, 8-week-old control group and Tsc2 - / - The expression of TSC2 protein in the heart, liver, spleen, lung, kidney and brain tissues of mice was detected by Western Blot. The results showed that only Tsc 2- / - TSC2 was knocked out in the liver tissue of mice ( Figure 4 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 increased significantly, indicating that mice with liver-specific Tsc2 deficiency were successfully constructed ( Figure 4 (B).

[0109] In order to investigate the liver tumorigenesis in mice, 8-month-old mice were selected and Tsc2 - / - The mice showed visible liver tumors, while the liver morphology of the control mice was basically normal ( Figure 5 The liver tissue of mice was fixed and sectioned and stained with hematoxylin-eosin (HE). The results showed that the liver tissue structure of the control group mice was intact and the liver lobules were clearly visible, while the liver tissue of Tsc2 - / - The liver tissue structure of mice was obviously disordered, showing the lack of clear liver lobules. Immunohistochemical staining results showed that Heppar1 was positive and CK19 was negative, suggesting that Tsc2 - / - The tumors in the mice were hepatocellular carcinoma ( Figure 5Middle B). This demonstrates that the TSC2-deficient, mTOR-activated spontaneous liver cancer mouse model was successfully established.

[0110] To investigate whether Pifithrin-μ can synergize with sorafenib in the treatment of mTOR-activated liver cancer, 8-month-old Tsc2 - / - Mice were randomly divided into 4 groups: solvent group, Pifithrin-μ treatment group (10 mg / kg, intraperitoneal injection), sorafenib treatment group (20 mg / kg, gavage), and Pifithrin-μ (10 mg / kg, intraperitoneal injection) and sorafenib (20 mg / kg, gavage) combined treatment group, 8 mice in each group. The mice in the solvent group were given the same dose of normal saline, treated once every two days, and the changes in the weight of the mice were monitored. After 8 weeks of treatment, heart blood and liver tissue samples were taken from the mice ( Figure 5 C). Macroscopic observation revealed that the livers of mice in the control group had obvious tumor formation, while the combined treatment group showed significant remission. The statistical results of the number of liver tumors showed that although the size of the tumors was reduced after sorafenib or Pifithrin-μ alone, the number was not statistically different from that of the control group ( Figure 5 D and F); HE staining results of mouse livers indicated that the liver tissue structure of mice in the combined treatment group was restored. In addition, the results of immunohistochemical detection of cell proliferation marker Ki-67 showed that Ki-67 was positive in the liver tissue of mice in the control group, while it was almost not expressed in the combined treatment group ( Figure 5 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. During the treatment, the body weight of the mice did not change significantly, indicating that the two drugs had no toxic side effects ( Figure 5 Middle G).

[0111] After liver cells are damaged, alanine transaminase (ALT) and aspertate aminotransferase (AST) are easily released into the blood, which are usually used as indicators of liver cell function. Therefore, the present invention detects the ALT and AST levels in mouse serum and finds that the Pifithrin-μ and sorafenib combined treatment group is significantly lower than the single treatment group, indicating that the combined treatment has a significant effect on alleviating liver cell damage in mice ( Figure 5 H and I).

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

[0113] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of HSP70 inhibitors in the preparation of anti-liver cancer drug sensitizers.

2. The application according to claim 1, characterized in that: The anti-liver cancer drug includes sorafenib.

3. Application of HSP70 inhibitors combined with anti-liver cancer drugs in the preparation of drugs for the treatment of mTOR-activated liver cancer.

4. The use according to claim 3, characterized in that: The HSP70 inhibitor is selected from at least one of the following: Pifithrin-μ, Apoptozole and VER-155008.

5. The application according to claim 3, characterized in that: The anti-liver cancer drug includes sorafenib.

6. A drug for treating mTOR-activated liver cancer, characterized in that: Contains HSP70 inhibitor and sorafenib in unit preparations of the same or different strengths for co- or separate administration.

7. The drug for treating mTOR-activated liver cancer according to claim 6, characterized in that: The HSP70 inhibitor is selected from at least one of the following: Pifithrin-μ, Apoptozole and VER-155008.

8. The drug for treating mTOR-activated liver cancer according to claim 7, characterized in that: When the HSP70 inhibitor is Pifithrin-μ, the mass ratio of Pifithrin-μ to Sorafenib is 1:

2.

9. The drug for treating mTOR-activated liver cancer according to claim 8, characterized in that: The Pifithrin-μ and Sorafenib are co-administered.

10. The drug for treating mTOR-activated liver cancer according to claim 8, characterized in that: The dosage of Pifithrin-μ and Sorafenib, calculated on a mouse basis, is 10 mg / kg Pifithrin-μ and 20 mg / kg Sorafenib.

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