CN112870194B - Composition for treating liver cancer and application thereof - Google Patents

Abstract

The invention belongs to the field of biomedicine and specifically discloses that Crizo and Dox synergistically induce cancer cell death. Compared with single-agent treatment, the combination of the two drugs increased cell death by at least 50%, and was much stronger than the combination of sorafenib and Dox, the first-line treatment for advanced liver cancer, in the induction of apoptosis in liver cancer cells. The present invention clarifies the mechanism of the above action from the cellular level, and reveals that Crizo combined with Dox can overcome chemotherapeutic drug resistance by inhibiting the expression of MDR1 and inducing autophagic cell death. Combined with in vivo experiments, it is shown that Crizo combined with Dox can effectively inhibit tumor growth in animal models with less toxic and side effects, providing a potential therapeutic strategy for the treatment of cancer, especially advanced liver cancer.

Description

Composition for treating liver cancer and application thereof

technical field

The present invention relates to the technical field of biomedicine, more particularly, to a composition for treating liver cancer, specifically a pharmaceutical composition containing crizotinib and doxorubicin and its application in the preparation of a drug for treating liver cancer.

Background technique

Liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related death. According to the risk factors for liver cancer, the incidence and mortality of liver cancer were significantly associated with age, sex, and geographic region. Primary liver cancer includes hepatocellular carcinoma (HCC), intrahepatic ductal carcinoma and other rare types. Among them, hepatocellular carcinoma with the highest degree of malignancy accounts for more than 80% and is the main cause of death in patients with liver cancer. Surgical resection and liver transplantation are the most effective treatments for hepatocellular carcinoma. However, the survival rate of hepatocellular carcinoma is low, because hepatocellular carcinoma is not easy to find, and most of them are diagnosed at an advanced stage. Chemotherapy is the conventional treatment for advanced liver cancer.

Chemotherapy drugs anthracyclines, especially doxorubicin (dox), have long been the mainstay of cancer treatment. Of particular note is the cardiotoxicity it causes, which limits the clinical use of traditional doxorubicin (Carvalho et al., 2009). Cardiotoxicity associated with traditional doxorubicin can be broadly classified into acute or chronic cardiotoxicity (Carvalho et al., 2009; Pai and Nahata, 2000; Waterhouse et al., 2001). This cardiotoxicity has been shown to correlate with peak plasma doxorubicin concentrations and cumulative lifetime doses of dosing (Maluf and Spriggs, 2002; Von Hoff et al., 1979). This side effect is particularly pronounced in patients with advanced disease requiring increased drug doses (Theodoulou and Hudis, 2004). In addition, dox resistance often occurs in advanced tumors, leading to poor prognosis (Chen et al., 2018). Therefore, it is necessary to seek a new combination therapy to improve the anticancer effect. At present, almost all successful cancer chemotherapy regimens mostly use a multidrug combination therapy strategy to achieve better therapeutic effect and minimize side effects (Valero et al., 2001). Therefore, the development of new therapeutic strategies is crucial for improving the survival of HCC patients, especially those with advanced HCC.

SUMMARY OF THE INVENTION

The present invention aims to overcome the above-mentioned defects of the prior art, and provides a pharmaceutical composition containing crizotinib and doxorubicin.

Another object of the present invention is to provide the application of the above-mentioned pharmaceutical composition in the preparation of a medicine for treating liver cancer.

The technical scheme adopted by the present invention is as follows:

A pharmaceutical composition comprising crizotinib and doxorubicin.

As a preferred solution, the pharmaceutical composition includes a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate or a pharmaceutically acceptable carrier.

As a preferred solution, the dosage form of the pharmaceutical composition is injection, tablet, pill, capsule, suspension or emulsion.

The invention also protects the application of the pharmaceutical composition in the preparation of cancer-inhibiting drugs.

Further, the cancer includes malignant tumors such as melanoma, lung cancer, oral squamous cell carcinoma or liver cancer.

In particular, the present invention especially protects the application of the pharmaceutical composition in the preparation of drugs for inhibiting liver cancer.

As a preferred solution, the present invention protects the application of the pharmaceutical composition in reducing the chemoresistance of liver cancer.

Further, crizotinib is used to reduce the side effects of doxorubicin therapy, including the cardiotoxicity of cancer, especially liver cancer chemotherapy.

The use of Doxorubicin (Dox), which has potent antitumor activity, not only makes patients resistant to chemotherapy, but also causes strong side effects, such as cardiotoxicity. Based on this, our goal was to find a regimen that could reduce Dox resistance and its toxic side effects, and to elucidate its mechanism of action.

The abnormal expression of multiple drug resistant protein 1 (MDR1) is an important cause of multidrug resistance in cancer treatment. The accumulation of Dox in the nucleus is the main reason for its toxic effect, and Crizo increased the accumulation of Dox in the nucleus of HCC cells by 3- to 16-fold. We detected various proteins that lead to drug accumulation and efflux and found that the drug efflux pump MDR1 was significantly decreased after combined administration. Further studies found that the combination of Crizo and Dox could significantly reduce MDR1 at the protein level but not its nucleic acid (mRNA) level. Inhibiting the protein degradation pathway found that the decrease in MDR1 was not due to increased protein degradation. Finally, we confirmed that the reduction of MDR1 was due to ER stress-activated protein kinase RNA-like ER kinase (PERK) caused by the combination of the two drugs. Activation of PERK leads to phosphorylation of eIF2α, thereby inhibiting protein translation and synthesis, and the reduction of MDR1 Inhibits Dox efflux, thereby increasing nuclear accumulation, resulting in enhanced cell death. Meanwhile, Crizo combined with Dox overcomes HCC resistance by inducing autophagic cell death. Finally, the results of Crizo combined with Dox in the treatment of xenograft models showed that the combination of the two drugs could significantly inhibit tumor growth.

In conclusion, it is revealed that Crizo combined with Dox can overcome chemotherapeutic drug resistance by inhibiting the expression of MDR1 and inducing autophagic cell death. The combination of Crizo and Dox provides a potential therapeutic strategy for the treatment of advanced HCC.

The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form, and is physiologically Compatible with receptors.

The terms "salt", "acceptable salt" and "pharmaceutically acceptable salt" refer to the above-mentioned compounds or their stereoisomers, acid and/or base salts formed with inorganic and/or organic acids and bases, Also included are zwitterionic salts (inner salts), as well as quaternary ammonium salts such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound, or a stereoisomer thereof, with a certain amount of acid or base as appropriate (for example, an equivalent amount). These salts may form a precipitate in solution and be collected by filtration, or recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium.

Compared with the prior art, the beneficial effects of the present invention are:

The combination of Crizo and Dox provided by the invention synergistically induces the death of liver cancer cells. Compared with single-agent treatment, the combination of the two drugs increased cell death by at least 50%, and was much stronger than the combination of sorafenib and Dox, the first-line treatment for advanced liver cancer, in the induction of HCC cell apoptosis. The present invention clarifies the mechanism of the above effects from the cellular level: including Crizo combined with Dox induces more cleavage of PARP-1 leading to more HCC cell death, Crio combined with Dox significantly promotes dox in the nucleus of HCC cells by downregulating the expression of MDR1 Accumulation and the combination of the two drugs induce the occurrence of endoplasmic reticulum stress, activate the PERK-p-eIF2α signaling pathway, thereby inhibiting the protein translation machine, reducing the translation of MDR1, and reducing the synthesis of MDR1, which leads to the reduction of Dox efflux and the continuous accumulation of Dox in the nucleus. , resulting in more severe DNA damage and cell death, Crizo combined with Dox activates autophagosome formation through the PERK-p-eIF2α-JNK signaling pathway cascade. Crizo combined with Dox inhibits the fusion of autophagosome and lysosome, leading to HCC cell death. In vivo experiments show that Crizo combined with Dox can effectively inhibit tumor growth in animal models with less toxic and side effects, which shows the therapeutic prospect of the combination of the two drugs.

Description of drawings

Figure 1 is a graph of the combined use of Dox and Crizo to synergistically induce liver cancer cell death.

FIG. 2 is a comparison diagram showing that Dox+Crizo is more effective in inducing cell death than Dox+sorafenib.

Figure 3 is a graph showing that low-dose Dox+Crizo significantly inhibited the migration of HCC cells in a scratch healing experiment.

Figure 4 is a graph showing the synergistic killing of cancer cells by Crizo combined with Dox by inducing the cleavage of PARP-1.

Figure 5 is a graph showing the morphological characteristics of drug-treated HCC cells. Hepatoma cells were treated with Vehicle, Dox, Crizo and D+C for 24 hours, and the cell morphology and the formation of apoptotic bodies were observed under an inverted phase contrast microscope.

Figure 6 is a graph showing the synergistic effect of Crizo combined with Dox on killing cancer cells by inducing the cleavage of PARP-1.

Figure 7 is a graph showing that Crizo+Dox induces nuclear accumulation of Dox by reducing the level of MDR1 protein.

Figure 8 is a graph showing the mRNA level of MDR1 detected by real-time quantitative PCR.

Figure 9 is a graph showing that Crizo+Dox treatment inhibits the expression of MDR1 in HCC cells by increasing the phosphorylation of PERK-eIF2α.

Figure 10 is a graph of Dox+Crizo inhibiting protein translation.

Figure 11 shows that Crizo+Dox can activate PERK-eIF2α-JNK signaling cascade and induce autophagosome formation.

Figure 12 is a graph showing the formation of more autophagosomes induced by Crizo+Dox.

Figure 13 is a graph of GSK attenuating Dox+Crizo-induced cell death.

Figure 14 is a graph of the induction of PARP-1-dependent cell death by Dox+Crizo treatment.

Figure 15 is a comparison graph of the reduction of C-Met activity by Dox+Crizo treatment compared with pure Dox or Crizo treatment. No consistency was shown in reducing C-Met activity.

Figure 16 is a graph showing that Crizo+Dox significantly inhibited tumor growth in xenografts.

Figure 17 is a graph showing the synergistic killing effect of Dox+Crizo on other tumor cells.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described in detail below in conjunction with specific embodiments and comparative examples. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

Experimental example 1:

1. Cell culture, passage and cryopreservation

(1) Preparation of cell culture-related reagents and cell culture:

1X PBS preparation (1L): Weigh 8g NaCl, 0.2g KCl, 3.58g Na ₂ HPO ₄ ·12H ₂ O ₂ , 0.24g KH ₂ PO ₄ , dissolve in 800ml deionized water, stir evenly, and dilute to volume with a measuring cylinder to 1L. Use after autoclaving.

0.25% pancreatin preparation (0.02% EDTA) (1L): add 8g NaCl, 0.4g KCl, 0.12g Na ₂ HPO ₄ ·12H ₂ O ₂ , 0.06g KH ₂ PO ₄ , 0.35g in about 800ml ultrapure water NaHCO ₃ , 1 g glucose, 0.01 g phenol red, 2.5 g pancreatin, and then 1.07 ml of 0.5 mol/L EDTA were added, fully stirred to dissolve, the volume was adjusted to 1 L, and the pH was adjusted to 7.2.

DMEM basal culture preparation (1L): Dissolve a pack of DMEM powder (Gibco, 12100-046) in about 800mL of ultrapure water, stir well with a magnetic stirrer for 2 hours, weigh 3g HEPES and 3.17g NaHCO ₃ respectively. , until completely dissolved, make up to 1L.

The cells used in this study were cultured in DMEM high glucose complete medium: 90% DMEM basal medium + 10% fetal bovine serum FBS (Biological Industries, 04-001-1ACS) + 1% double antibody PS (Biological Industries, 03-031 -1B).

(2) Cell passage

The above cells were cultured in a 37°C cell incubator (Thermo) containing 5% CO ₂ , and subculture was generally performed when the cell confluence was 75% to 90%.

Cell digestion: Pour off the original medium and carefully wash the cells with sterilized 1x PBS; add an appropriate amount of 0.25% trypsin (0.02% EDTA) according to the cell type, and gently tilt and tap the Petri dish to make the trypsin and The cells were fully contacted and placed in a 37°C cell incubator to digest for 3-5 minutes; finally, an appropriate amount of DMEM complete medium was added to terminate the digestion, and the cells were gently pipetted into a single-cell suspension, centrifuged at 1000 rpm for three minutes, resuspended, and continued to culture. .

(3) Cell cryopreservation

The digested cell suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded to obtain a cell mass, an appropriate amount of cell freezing solution (90% serum + 10% DMSO) was added to blow the cells evenly, and 1 ml per tube was added to the freezing tube. The freezing box is placed in a -80°C refrigerator for gradient cooling, and the cells can be transferred to a liquid nitrogen tank after 3-4 hours for long-term storage.

(4) Cell recovery:

Adjust the temperature of the water bath to 37°C in advance, take out the corresponding cells from the liquid nitrogen tank, and quickly fully thaw them in warm water at 37°C; then centrifuge at 1000 rpm for 5 min, discard the upper freezing solution, and reconstitute the lower cell mass with the corresponding medium. Suspended, transferred to a petri dish, and cultured in a 37°C cell incubator.

2. Cell Proliferation and Cell Survival Experiments

First, take well-grown cells and digest them into a single-cell suspension. Count with a hemocytometer. The cells were diluted with DMEM complete medium to 5× ^{10 4} /ml, and then 100 μl per well was evenly plated into a 96-well plate, and each cell was plated in three duplicate wells, and cultured overnight in a 37°C constant temperature incubator. Dilute the drug dox or crizotinib into different concentration gradients, then aspirate the old medium, replace with the diluted drug, add 100 microliters to each well, and treat for different times (0, 24, 48, 72h) as required, After the treatment, 20 microliters of MTS solution was added to each well, carefully shaken and mixed, and incubated in a constant temperature incubator at 37 degrees Celsius for 2 hours. Finally, the absorbance value at a wavelength of 490 nm was read with a microplate reader, which was the OD value, indicating cell viability.

3. Cell morphology observation

7402, HLF, HepG2 cells were evenly plated into 12-well plates at 2.5× ^{10 5} cells/well, and each cell was plated in 4 wells. The medium was replaced with the diluted Vehicle, 1 μM dox, 3 μM crizo, 1 μM dox+3 μM crizo, respectively. After 24 hours of drug addition and culture, observed and photographed under an inverted phase contrast microscope.

4. Clone Formation Experiments

7402, HLF, HepG2 cells were evenly plated into 6-well plates at 5000 cells/well, and each cell was plated in 4 wells. After 12 hours of culture, the medium in the wells was replaced with fresh medium supplemented with Vehicle and final concentrations of 20nM dox, 0.6μM crizo or 20nM dox + 0.6μM crizo, and the medium was changed every three days for continuous culture. The experiment was terminated after 10 days. Next, the medium was removed and the cells were rinsed twice with 1X PBS. It was then fixed with 4% paraformaldehyde PFA for 20 minutes, followed by staining with 0.1% crystal violet for 30 minutes. Rinse off the remaining dye with running water, and finally, take pictures and count.

5. Detect the median inhibitory concentration (IC50) of dox and crizo and determine the drug combination index (CI)

7402, Sk-hep1, HLF, HepG2 cells in logarithmic growth phase were digested and counted with a hemocytometer, the cell density was diluted to 5x10 ⁴ /ml, and then plated at 100 μl per well into a 96-well plate and cultured in an incubator After 12 hours, the medium was removed, and different concentrations of dox or crizo, and the corresponding concentration of dox+crizo diluted medium were added. After culturing for 48 hours, the MTS experiment was carried out, and the OD490 value was measured with a microplate reader. Calculate the inhibition rate of cell survival = (OD(vehicle)-OD(experimental group))/OD(vehicle), then according to the theory of Chou et al. (Chou and Talalay, 1984), use the software to calculate the maximum half-inhibitory concentration IC50 of the drug and analyze dox and crizo combination index combination index (CI). CI>1 indicates that the drug combination has an antagonistic effect, CI=1 indicates an additive effect, and CI<1 indicates a synergistic effect.

6. Apoptosis detection

Drug-treated cells were detected using annexin V-FITC/PI Apoptosis Detection Kit, followed by flow cytometry for apoptotic cell analysis. Briefly, 2x10 ⁵ cells were plated with HLF, Sk-hep1, 7402 on a six-well plate, and after 12 hours of culture, drug treatment was added for 24 hours: vehicle, dox (0.85μM), crizo (5μM), dox (0.85μM) )+crizo (5 μM). After the apoptosis stimulation was completed, the cells were digested with trypsin without EDTA, centrifuged at 1000g for 5 minutes, the supernatant was discarded, the cells were collected, and the cells were gently resuspended with PBS and counted; Centrifuge at 1000g for 5 minutes, discard the supernatant, add 195 μl Annexin V-FITC binding solution and gently resuspend the cells; add 5 μl Annexin V-FITC and mix gently; add 10 μl propidium iodide staining solution (PI) and mix gently ; Incubate in the dark at room temperature for 10-20 minutes, then place in an ice bath. Resuspend cells 2-3 times during incubation to improve staining. Finally, it was detected by flow cytometry. 10,000 cells were collected per treated sample and the data obtained were analyzed using software.

7. Dox accumulation experiment

Cells were seeded into confocal glass dishes (NEST, China) at 5× ^{10 4} /ml and cultured overnight. On the next day, dosing treatment, 0.85μM dox or dox (0.85μM)+crizo (5μM) were treated for 24 hours respectively. Pour off the medium, rinse twice with PBS, then immediately fix the cells with 4% paraformaldehyde PFA for 20 minutes at room temperature; wash twice with PBS, then dilute with the prepared blocking solution (10% goat serum + 1% BSA in Cells were blocked for 1 hour at room temperature with 1x PBS); nuclei were counterstained with 500ng/ml DAPI dye for 5 minutes, protected from light at room temperature. After staining, rinsed 3 times with PBS, and added two drops of fluorescent anti-quencher. Then observe under the microscope and take pictures to record. The accumulation of Dox in the nucleus was defined as the intensity of red fluorescence, which was quantified with software.

8. Quantitative real-time PCR (qRT-PCR) to detect the expression of MDR1

Total RNA was extracted from cells 24 hours after drug treatment using an RNA extraction kit (Novizan). The SYBR Green method was used for qRT-PCR in the experiment, and each gene was tested in three groups in parallel. The primers used were all confirmed by melting curve analysis that the product was single and no primer-dimer existed. The primer sequences of each analyzed gene are shown in the sequence listing, and the Actin gene is used as an internal reference.

Green Select Master Mix的操作说明书，反应体系如下：Reaction solution formula basis

The operation manual of Green Select Master Mix, the reaction system is as follows:

PCR amplification reaction conditions were: 50°C for 2 min, 95°C for 2 min; 95°C for 15 sec, 60°C for 1 min, 40 cycles; and finished at 4°C.

After the experiment, select the appropriate position as the threshold value to obtain the Ct value of each well, use Actin as the internal reference to calculate the ΔCt value, and then subtract the ΔCt value of the control group from the ΔCt value of the experimental group to obtain the ΔΔCt value, and then follow 2 ^- The relative expression of each gene was calculated by ^ΔΔCt method. Real-time quantitative PCR primers are as follows:

9. Western blotting: (1) Prepare cell lysate. The formula of cell lysate is as follows:

(1) Extraction, concentration determination and denaturation of total cell protein

Add PMSF and protease inhibitor cocktail before using the lysate. Protein extraction should be performed in a refrigerator at 4°C or on ice. Proceed as follows:

1) Discard the medium and rinse twice with pre-cooled PBS.

2) Add an appropriate amount of cell lysis solution, scrape off the cells and transfer them to a 1.5mL EP tube, disrupt the cells by ultrasonic 5 times for 5 seconds each time, and incubate on ice for 30 minutes.

3) Centrifuge at 14000g at 4°C for 10 minutes, transfer the supernatant to a new EP tube, and obtain a protein sample.

4) Determination of protein concentration was performed according to Bio-Rad protein assay kit. Then, according to the measured protein concentration, the protein concentration of each sample was adjusted to be consistent with cell lysate, 4×SDS loading buffer was added, and boiled at 100° C. for 10 min to completely denature the protein.

(2) SDS-PAGE electrophoresis and membrane transfer

1) Preparation of buffers required for WB:

4xSDS loading buffer (100 mL): 40 mL glycerol, 25 mL 1M Tris (pH 6.8), 20 mM β-mercaptoethanol, 8 g SDS, 0.001% (W/V) bromophenol blue.

Running buffer (1L): Tris 3.03g, Glycine 14.4g, SDS 1g, add 800mL of water to dissolve, and dilute to 1L.

Transfer buffer (1L): Tris 3.03g, Glycine 14.4g, add water to dissolve to a total volume of 850mL, and add 150mL methanol before use.

TBST (0.1% Tween-20): add 800 mL of water to dissolve 1.22 g of Tris and 8.8 g of NaCl, adjust the pH to 7.6 with concentrated hydrochloric acid, add 0.1% of Tween 20, and dilute to 1 L.

2) Electrophoresis: According to the molecular weight of the protein to be detected, prepare a separation gel with an appropriate concentration, assemble an electrophoresis device, add electrophoresis buffer, load equal amounts of each histone, and add a pre-stained protein marker as a protein size reference. Electrophoresis conditions: 70V, 30min; 120V, 1h.

3) Transfer membrane: after electrophoresis, the separated proteins are transferred to nitrocellulose membrane (NC), and the transfer conditions are 120mA, 1h 20min.

(3) closed

After transfer, the membrane was stained with Ponceau to check the transfer effect. After washing with 1x PBST for 5 min. Block with 5% nonfat dry milk (dissolved in PBST) and shake slowly on a shaker for 1 h at room temperature.

(4) Primary antibody incubation

After blocking, wash twice with 1×PBST for 5 min each time, add the diluted primary antibody (diluted with 5% BSA), and incubate overnight at 4°C on a shaker.

(5) Secondary antibody incubation

Discard the primary antibody incubation solution and wash the membrane 3 times with 1×PBST for 10 min each time. Then use 1:5000-1:10000 diluted secondary antibody (diluted with 5% nonfat milk powder) to incubate at room temperature for 1 h, and then wash with 1 × PBST for 3 times, 10 min each time.

(6) Chemiluminescence detection

Add horseradish peroxidase substrate to detect the signal, mix solution A and solution B at a ratio of 1:1, put the membrane in the luminescent solution for 1 min, and place it in a LAS4000 for imaging.

10. Immunofluorescence

1) Seeding: The cells were seeded in confocal dishes (BD Biosciences, CA, USA) at 5×10 ⁴ /well.

2) Fixation: cells were fixed with 4% paraformaldehyde (adjusted to pH about 7.4) at 500 μl/well for 10 min, and then washed three times with 1×PBS for 5 min each time.

3) Permeabilization: The cells were permeabilized with 1 ml/well of 0.1% Triton X-100-PBS for 10 minutes, and then washed three times with 1×PBS, soaking for 5 minutes each time.

4) Blocking: cells were blocked with 10% goat serum + 1% BSA solution at a volume of 500 μL/well at room temperature for 60 min.

5) Primary antibody incubation: Aspirate the blocking solution, dilute the primary antibody MDR1 (rabbit) or LC3A/B (rabbit) directly with 1×PBS solution at a ratio of 1:100, and add 200 μl of primary antibody dilution to each well of cells , incubate at 4°C overnight or at room temperature for 1-2hr. After incubation, wash three times with 1×PBS, soaking for 5 min each time.

6) Secondary antibody incubation: Dilute the secondary antibody with 1×PBS solution at a ratio of 1:2000, and incubate with 200 μl/well at room temperature for 1 h in the dark. Then wash three times with 1×PBS, soaking for 5 min each time.

7) Nuclei staining: DAPI staining, prepared with 1×PBS at 1:2000, stained at room temperature for 5 minutes, washed 3 times with 1×PBS, and soaked for 5 minutes each time.

8) Add a sufficient amount of anti-quencher dropwise, store in the dark, and take pictures.

11. Ponceau Staining

To determine the effect of combined use of dox and crizo on overall protein synthesis, the same number of cells were lysed after different treatments, as described above. The proteins in the lysate were separated by SDS-PAGE and transferred to the NC membrane. After the transfer was completed, the membrane was immediately immersed in Ponceau red solution for staining for 10 min. Then rinse with clean water and take a photo scan.

12. Detection of Autophagosomes by Transmission Electron Microscopy

To determine autophagosome formation during drug treatment. We carried out transmission electron microscope observation experiments. Proceed as follows:

1) Material and fixing

After the cells in the 10cm dish were treated with different drug combinations for 24 hours, the culture medium was poured off, 1 mL of pre-cooled 2.5% glutaraldehyde was added, and the cells were fixed at 4°C for 15 minutes, and then the cells were scraped off with a cell scraper to collect 1.5 mL centrifuge tube, centrifuge, retain the fixative, and store at 4°C. Fixed for at least 4 hours.

2) Osmic acid fixation

Cells fixed with glutaraldehyde were rinsed 3 times with 0.1M PBS (pH7.4) for 15 minutes each; then fixed with 1% osmic acid.0.1M PBS at room temperature for two hours; PH7.4) rinsed 3 times for 15 minutes each time.

3) Dehydration

The samples were dehydrated by 30%, 50%, 70%, 80%, 85%, 90%, 95%, and 100% alcohol gradients (twice) for 15-20 minutes each time.

4) Penetration

The penetrating agent is acetone: epoxy resin (2:1), acetone: epoxy resin (1:1), epoxy resin, in a 37°C thermostat, penetrating for 8 to 12 hours each time.

5) Embedding

Put the infiltrated samples into capsules or embedding plates, add embedding agent epoxy resin, and polymerize in a 60°C incubator for 48 hours.

6) Ultrathin section: section thickness: 80-100nm.

7) Double staining

Uranium-lead double staining (2% uranyl acetate saturated aqueous solution, lead citrate, staining at room temperature for 15 minutes), sections were dried overnight at room temperature, and observed by transmission electron microscope (Tecnai G2 20TWIN, FEI, America).

13. Autophagy flow detection

To assess autophagic flux, we transfected GFP-LC3 plasmid (2 μg) into 7402, HLF and HepG2 cells in six-well plates. Twelve hours after transfection, cells were trypsinized, resuspended, and seeded in glass-bottom confocal dishes for 24 hours. The old medium in the petri dish was replaced with fresh complete medium containing 0.85 μM Dox or/and 5 μM Crizo, and the culture medium was continued for 12 hours and then photographed and observed under a fluorescence microscope. Fluorescence intensity was analyzed with Image J software.

To investigate whether the two-drug combination therapy affects autophagosome accumulation and inhibits autophagolysosome formation, mCherry-GFP-LC3 plasmid (2 μg) was transfected into 7402 cells and HLF cells, respectively. Twelve hours after transfection, the transfected cells were reseeded into glass bottom confocal dishes for an additional 24 hours. The normal culture was replaced with fresh complete medium containing 0.85 μM Dox or/and 5 μM Crizo, and the culture was continued for 6 hours. Cells were fixed and observed by Nikon two-photon super-resolution fluorescence microscope (Nikon A1 MP STORM). Both EGFP and mCherry positive indicated autophagosomes, and only mCherry positive indicated autophagolysosomes, which were the fusion products of autophagosomes and lysosomes.

14. Inhibition of protein degradation assay

To investigate whether Dox combined with Crizo treatment could degrade MDR1 protein, vehicles, 10 μM MG132, 50 μM CQ or 4 mM 3-MA were diluted in complete medium containing 0.85 μM Dox and 5 μM Crizo, and then added to 6-well plates plated with HCC cells. Continue processing for 4 hours. To assess the effect of inhibitors, cells were seeded in 12-well plates overnight and treated with fresh medium containing 0.85 [mu]M Dox and 5 [mu]M Crizo for 18 hours. The indicated concentrations of inhibitors (GSK, SB or INO) or controls were added to fresh medium containing 0.85 [mu]M Dox and 5 [mu]M Crizo to replace the original medium and the culture was continued for 2 hours. Cell lysates were then prepared and proteins were quantified. Isolated proteins were immunoblotted with specific antibodies.

15. Xenograft experiments

6-8 week old BALB/c male nude mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. were used for in vivo xenotransplantation experiments. The experimental animals were kept under SPF-II conditions and the animal experiment protocol (WIVA28201703) was approved by the Animal Care and Use Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (Wuhan, China). 1×10 ⁷ HepG2 or 7402 cells were suspended in 100 μL of PBS, respectively, and then seeded subcutaneously on the right back of mice. When the average tumor volume reached 150-200 mm, the mice were randomly divided into ⁴ groups according to different treatment methods: for HepG2 cells, divided into a solvent control group (0.5% sodium carboxymethyl cellulose by gavage and intraperitoneal injection of PBS) , Dox group (5mg/kg intraperitoneal injection every two days), Crizo group (50mg/kg/day gavage), Dox+Crizo group (5mg/kg intraperitoneal injection every two days and 50mg/kg daily gavage dose); For 7402 cells, the conditions were the same as in the HepG2 group except that the concentration of Dox was reduced to 2.5 mg/kg. Body weight and tumor volume of each mouse were measured every other day. Tumor volume (V) was estimated according to the following formula: V = 0.5 x length x width ² . After two weeks of treatment, mice were euthanized, and the tumors of mice in each group were excised and weighed. Tumors from different treatment groups were aligned and photographed. Note: Dox was dissolved in PBS for ip injection; Crizo was powdered in a mortar and suspended in 0.5% sodium carboxymethylcellulose to prepare a gavage suspension.

16. Statistical analysis of data

All experiments were repeated at least three times. Quantitative data errors are shown as mean ± standard error. One-way analysis of variance (ANOVA) was used to analyze differences between groups. Other statistical analyses were performed using a two-tailed Student's t-test. *p<0.05 means significant difference; **p<0.01, ***p<0.001 means extremely significant difference; ns means no statistically significant difference.

Experimental results:

1. The combined use of Dox and Crizo synergistically induces liver cancer cell death:

The liver cancer cell lines HLF, Sk-hep1, HepG2, 7402 and HLE were selected as the research objects. First, we observed the effect of the combination of the two drugs on cell morphology. The results of phase contrast microscopy are shown in Figure 1B. The number of cells in the combined treatment group was significantly less than that in the single treatment group, and a large number of cells shrank, became round and fell off, and the cells formed Apparently dead.

Next, we examined the effect of the two-drug combination on the survival of liver cancer cells at the indicated concentrations. The results showed that the combination therapy significantly increased cytotoxicity and reduced cell survival by at least 50% compared to Dox or Crizo monotherapy (Figure 1D). Further clone formation experiments showed that lower concentrations of Dox+Crizo significantly inhibited the proliferation of 7402, HepG2, and HLF cells (Fig. 1C). We then determined the Dox+Crizo combination index (CI) by disproportionately changing the concentration combination of the two drugs to determine synergistic or additive effects. The drug combination and CI index are shown in Table 1. According to the theory of Chou et al., CI<1 indicates synergy, and CI=1 indicates additive effect. Consistent with the expected results, in all cells tested, the concentration combinations used had a CI < 1, thus Dox+Crizo had a synergistic effect (Fig. 1E). In addition, it is surprising that compared with the combination of the first-line drug sorafenib for advanced liver cancer, the combination of Crizo and Dox has a significantly stronger inhibitory effect on the activity of liver cancer cells (Figure 2).

Among them, the description of Figure 1 is as follows: Figure 1 (A) shows that HFL is relatively resistant to Crizo. Quantification with MTS was performed after cells were treated with different concentrations of Dox or Crizo for 24 hours. The survival rate of cells in the control group was considered to be 100%, and the survival rate was the ratio of the number of cells in the Dox or Crizo-treated group to the number of cells in the control group under the same conditions. (B) After treatment of Sk-hep1 and 7402 cells with different concentrations of Dox or Crizo, the relative proliferation of cells at different time points was detected by MTS. The drugs were dose-dependent in killing the cancer cells tested. The number of cells at time 0 of drug treatment was considered to be defined as 1. Relative cell proliferation refers to the number of cells treated with the indicated drug at the indicated time normalized to the number of cells at zero drug treatment. (C) Treatment with Crizo+Dox at IC50 concentrations induces significantly more cell death compared to treatment with Dox or Crizo alone, respectively. HCC cells were treated with Vehicle, Dox, Crizo or Crizo+Dox at the indicated concentrations for 24 hours, respectively, and viable cells were detected by MTS. Percent cell viability was calculated as in 1(A). (D) The colony formation results of different hepatoma cells showed that the surviving cells were significantly reduced after Dox + Crizo treatment. Cells were treated as indicated by Vehicle, Dox (20 nM), Crizo (0.6 μM), and Dox (20 nM)+Crizo (0.6 μM). (E) Calculate the combination index (CI) (using CompuSyn software), treat liver cancer cells with different concentrations of Crizo, Dox or Crizo+Dox combination for 48h, MTS to monitor cell survival, the results show that under certain combinations of Dox and Crizo , even on HLF and HepG2 cells, Crizo+Dox has synergistic killing effect on HCC cells.

Table 1 Concentration and CI value of drug combination

2. Low-dose Dox+Crizo significantly inhibited HCC cell migration and infiltration

Appropriate drug concentrations were selected, namely 0.15μM Dox and 2μM Crizo, and the results of MTS experiments showed that the lower drug concentrations had no significant effect on cell proliferation (Fig. 4B). Then, through scratch experiments, it was found that Dox+Crizo significantly inhibited the migration of 7402, HLF cells (Fig. 3A, 3C). The statistical results of three independent repeated experiments are shown in Fig. 3B, 3D.

Further Transwell experiments found that Dox+Crizo significantly weakened the ability of cell migration in 7402, SK-hep1, and HLF cells (Fig. 4A). The statistical results of three independent replicate experiments are shown in Figure 4C. To detect cell infiltration, we found that Dox+Crizo significantly inhibited 7402, SK-hep1, HLF cell infiltration by adding Matrigel in Transwell assay (Fig. 4D). The statistical results of three independent replicate experiments are shown in Figure 4E. Obviously, Dox or Crizo alone can slightly inhibit the migration and infiltration of HCC cells, but the combined use of the two drugs has a significantly better inhibitory effect on the migration and infiltration of HCC cells than the single drug. Therefore, low-dose Dox+Crizo is promising for the treatment of advanced liver cancer to prevent cancer metastasis.

3. Crizo+Dox significantly increases apoptosis of liver cancer cells by inducing PARP-1 cleavage

Three HCC cell lines were treated with Vehicle, Dox (0.85 μM), Crizo (5 μM) or Crizo (5 μM)+Dox (0.85 μM), respectively, and apoptosis was quantified by flow cytometry. The results showed that Crizo+Dox induced significantly more apoptosis with significantly more representative apoptotic bodies in all treated cell lines (Figure 5). In addition, representative flow cytometry also yielded consistent results, as shown in Figure 6A. A summary analysis of the results of three independent replicate experiments is shown in Figure 6B.

et al.,2009)。接下来，我们研究了PARP-1是否参与了Crizo+Dox诱导的 细胞死亡。HCC细胞系分别用Vehicle、Dox、Crizo或Crizo+Dox处理。然后制备细胞裂解 液并进行免疫印迹。与所有其他对照相比，使用Crizo+Dox处理的细胞裂解液中检测到更 多小的89KD片段，这表明更多的PARP-1发生了切割。在三个被测细胞系中，Crizo+Dox 处理组切割的PARP-1增加了16倍至30.8倍(图6C)。Apoptotic signaling involves the activation of caspase proteases. Caspase 3 is one of the most well-characterized caspases as an apoptotic executor (Green and Llambi, 2015). Therefore, we can detect whether Crizo+Dox activates caspase 3. The results are shown in Figure 6C, Crizo or Dox itself seems to be able to slightly increase the cleavage of caspase 3, but it is not obvious that Crizo+Dox has a synergistic increase in caspase 3 cleavage. Therefore, the synergistic effect of Crizo+Dox-induced HCC cell death is unlikely to be due to the activation of caspase 3. Dox has been reported to induce autophagic death of 3T3 cells by activating poly ADP-ribosome transferase (PARP-1) without involving caspases (

et al., 2009). Next, we investigated whether PARP-1 is involved in Crizo+Dox-induced cell death. HCC cell lines were treated with Vehicle, Dox, Crizo or Crizo+Dox, respectively. Cell lysates were then prepared and immunoblotted. Compared to all other controls, more small 89KD fragments were detected in cell lysates treated with Crizo+Dox, indicating that more PARP-1 was cleaved. In the three tested cell lines, the Crizo+Dox treatment group increased cleavage of PARP-1 by 16- to 30.8-fold (Fig. 6C).

Among them, (A) in Figure 6 represents that the apoptosis induced by Crizo+Dox was significantly increased compared with Vehicle, Dox or Crizo alone. The detection of HLF, Sk-hep1 and 7402 cells by flow cytometry showed that Crizo (5 μM) + Dox (0.85 μM) treatment compared with HCC cells treated with vehicle, Dox (0.85 μM), Crizo (5 μM) alone Induction of early and late apoptosis (Q3 and Q2) was significantly more active in these cells. (B) The results of statistical analysis of three different experiments showed that Crizo+Dox induced significantly more apoptosis on HCC cells compared with Dox or Crizo. Percent apoptosis was defined as the combination of early and late apoptosis (Q2+Q3) (filled histograms represent mean and standard deviation). (C) Crizo+Dox induced more cleaved PARP-1 compared to control, Dox or Crizo only when treating HCC cells. Crizo+Dox induced more cleaved PARP-1 compared to treatment of HCC cells with Vehicle, Dox or Crizo alone. Immunoblotting of 7402, Sk-hep1, HLF cell lysates treated with Vehicle, Dox, Crizo or Crizo+Dox showed that the Crizo+Dox treatment group had more PARP-1 cleavage. In contrast, active caspase-3 (cl-caspase-3) was not significantly increased in the Dox+Crizo treatment group. Cell treatment was the same as A.

4. Crizo+Dox significantly increases the accumulation of Dox in the nucleus of liver cancer cells by regulating the expression of MDR-1:

Dox must be able to enter and stay in the nucleus to mediate its cytotoxic effects. Therefore, we investigated whether the synergistic effect of Crizo + Dox was due to the increased accumulation of Dox in the nucleus of treated HCC cells. Since Dox itself fluoresces red, we can directly observe it by fluorescence microscopy, and the fluorescence images show that only a weak background signal of Dox can be seen in the nuclei of HCC cells treated with Dox (Figure 7A, top panel). . However, in the presence of Crizo+Dox, more Dox signal was detected in the nucleus, 3-16-fold depending on the cell line (Fig. 7B, bottom panel). The quantitative results of three independent experiments are shown in Figure 7B. Therefore, Crizo promotes the accumulation of Dox in the nucleus.

The accumulation of Dox in the nucleus suggests that this phenomenon may be due to enhanced uptake or reduced efflux of Dox, or both. Therefore, we examined whether proteins involved in drug absorption or efflux were affected by Dox + Crizo treatment. We found that the expression of MDR1 (ABCB1), a key protein of the drug efflux pump, was altered when HCC cells were treated with Crizo + Dox. Immunofluorescence staining and confocal imaging showed that the expression level of MDR1 was significantly reduced in HCC cells treated with Crizo+Dox compared with control cells (Fig. 7C). In addition to membrane staining, partial MDR1 nuclear staining was also seen in HepG2 and Sk-hep1 cells (Fig. 5, MDR1: green, red arrows indicate: nucleus: blue). The reduction in MDR1 expression was further confirmed by immunoblotting of lysates from HCC cells treated with vehicle, Crizo, Dox or Crizo + Dox. Only Crizo + Dox treatment significantly reduced MDR1 protein levels by 40% - 80% in a cell line-dependent manner (Fig. 7D). A summary of the results of three independent replicate experiments is shown in Figure 7E. Taken together, these results suggest that the synergistic effect of Crizo + Dox-induced HCC cell death is due to reduced MDR1 expression levels, resulting in Dox accumulation in the nucleus, which perpetuates downstream responses.

Among them, in Figure 7 (A) compared with pure Dox (0.85μM) treatment, Crizo (5μM) + Dox (0.85μM) treatment of liver cancer cells resulted in more Dox (red) accumulation in the nucleus (blue). Immunofluorescence images were taken 24h after the cells were treated. (B) A summary of the results of three independent replicate experiments shows that Dox + Crizo results in significantly more nuclear accumulation of Dox. Open and filled histograms represent mean and standard deviation. (C) Immunofluorescence staining of MDR1 with specific antibodies showed that Crizo+Dox treatment inhibited MDR1 expression compared to Vehicle, Dox, or Crizo alone. Cells were fixed for immunofluorescence staining after 24 h of treatment. In HepG2 cells, Dox treatment appeared to alter the distribution of MDR1. (D) MDR1 detected from lysates of HCC cells treated with Crizo+Dox was significantly lower than cells treated with Vehicle, Dox or Crizo alone. (E) Statistical analysis of the results of three repeated experiments showed that Dox+Crizo significantly reduced the level of MDR1 protein in hepatoma cells. Filled bar charts represent mean and standard deviation. After 24 hours of cell treatment, cell lysates were prepared and immunoblotted with antibodies specific for MDR1 or actin. Nuclei were counterstained with DAPI.

5. Crizo+Dox reduces the expression of MDR1 by regulating the translation mechanism.

First, the mRNA expression levels of MDR1 in two liver cancer cells treated with Vehicle, Dox, Crizo and Dox+Crizo were quantified using 3 different pairs of primers against MDR1 mRNA ranging from 5' to 3'. The results are shown in Figure 8, in HepG2 and Sk-hep1 cells, detected by all primer pairs, cells treated with Dox alone showed a significant increase in MDR1 mRNA. This effect was undetectable in cells treated with Crizo alone. On the other hand, the synergistic effect of Crizo+Dox on MDR1 mRNA levels is less clear. It seems to be primer dependent and also dependent on the cellular environment. Since Crizo+Dox did not significantly reduce MDR1 mRNA levels, it is unlikely that the down-regulation of MDR1 protein expression was at the transcriptional level.

We then investigated whether MDR1 protein degradation was enhanced when hepatoma cells were treated with Crizo+Dox. To this end, in addition to adding Crizo+Dox to the medium, we also added MG132, a general inhibitor of the proteasome, or chloroquine (CQ), an inhibitor of lysosomes, or 3MA, an inhibitor of autophagy. If it is hypothesized that the decrease in MDR1 expression is due to increased degradation by the proteasome, lysosome or autophagy, then one or more of these inhibitors should be able to reverse the effects of Crizo+Dox by upregulating MDR1 levels. We found that addition of these inhibitors did not consistently reverse the effects of Crizo and Dox on MDR1 expression (Figure 9A). These results suggest that downregulation of MDR1 protein levels is unlikely to be the result of enhanced protein degradation. Therefore, Crizo+Dox may affect the translation of MDR1 protein.

We performed immunoblotting of HCC cell lysates treated with Crizo, Dox or Crizo+Dox. The results showed that p-PERK levels were significantly increased in all three cell lines after Crizo+Dox treatment, ranging from 2.7-19-fold (Fig. 9B). A downstream target of PERK is (p)-eukaryotic initiation factor 2 (eIF2). PERK phosphorylates eIF2α thereby inhibiting protein synthesis (Harding et al., 2000; Shi et al., 1998). We then investigated whether Crizo+Dox could also alter eIF2α expression. We observed that p-eIF2α protein levels were also significantly up-regulated in all three treated cell lines, by 1.9-3.1-fold (Fig. 9B). Upregulation of p-eIF2α is known to lead to the inhibition of some protein synthesis, but to allow selective translation of activated transcription factor 4 (ATF4) (Harding et al., 2000; Shi et al., 1998). As expected, we detected the inhibitory effect of Crizo+Dox on protein synthesis in three liver cancer cell lines by Ponceau staining assay (Fig. 10).

GSK2606414 (GSK) is a specific inhibitor of PERK (Kim et al., 2008). If our hypothesis is correct, GSK will mitigate the impact of Crizo+Dox. In fact, the effect of Crizo+Dox on MDR1 downregulation was partially reversed when HCC cell lines were cultured with Crizo+Dox as well as GSK (Figure 9C). The quantitative results of three independent experiments are shown in Figure 9D. Correspondingly, p-eIF2α levels were significantly reduced (Fig. 9C & 9D). Therefore, the decrease in MDR1 protein levels may be due to the inhibition of protein translation through the PERK-eIF2α signaling cascade.

To further support our interpretation that reduced MDR1 expression leads to Dox accumulation in the nucleus, we performed immunofluorescence observations of Dox in liver cancer cell lines treated with Crizo+Dox, in addition, we added GSK or Vehicle as controls. As expected, we found reduced accumulation of Dox in the nucleus of GSK-cultured cell lines with a corresponding increase in MDR1 immunoreactivity (Fig. 9E). The quantitative results of three independent experiments are shown in Figure 9F. Therefore, Crizo+Dox induces endoplasmic reticulum (ER) stress, which activates the PERK-p-eIF2α signaling pathway, resulting in reduced MDR1 translation, which in turn leads to reduced Dox efflux, and Dox accumulation in the nucleus, leading to more DNA damage and cellular die.

Among them, Figure 9(A) HCC cells treated with Crizo and Dox were simultaneously incubated with proteasome, autophagy or lysosomal inhibitors, and cell lysates were blotted with MDR1 or actin-specific antibodies. After various inhibitors inhibited protein degradation, MDR1 was not significantly elevated. (B) Phosphorylation levels of PERK and eIF2 were significantly higher in cells treated with Crizo+Dox compared to Vehicle, Dox alone, or Crizo-treated cells. Significantly more p-PERK and p-eIF2α signals were observed by immunoblotting of 24-hour drug-treated cell lysates with specific protein antibodies. (C) More MDR1 was detected from cell lysates after cells were treated with GSK, a specific inhibitor of PERK, for 2 h. The effect of p-eIF2 small molecule on PERK was detected by immunoblotting. Cells were first treated with Crizo+Dox for 22h, then vehicle or GSK was added for 2h before cell lysate preparation. (D) Open and filled histograms represent the mean and standard deviation of three experiments. (E) MDR1 immunofluorescence staining showed that significantly more MDR1 could be detected in cells treated with Crizo+Dox after 2 h of GSK addition (fourth column). In contrast, MDR1 was rarely detected in HCC cells treated with vehicle under the same conditions (column 3). Clearly, more Dox accumulation could be detected in cells treated with vehicle (column 1), while less Dox accumulation was observed in cells treated with GSK (column 2). (F) Open and filled column graphs represent the mean and standard deviation of three experiments. V: vehicle, G: GSK. P-values are displayed as specified values. Scale bar: 1 μm.

Figure 10 (A) Ponceau staining of total cell lysates of hepatoma cells treated with culture medium, Dox, Crizo or Dox+Crizo showed that normal protein synthesis was significantly inhibited when cells were treated with Dox+Crizo. The same number of cells were counted, and cell lysates were prepared and separated by 10% SDS-PAGE. After NC membrane transfer, the isolated proteins were ponceau stained according to Materials and methods. These experiments were repeated three times with similar results. (B) Summary of three experiments, mean and standard deviation from three independent experiments.

6. Crizo+Dox activates autophagosome formation through PERK-p-eIF2α-JNK signaling cascade

Since Dox+Crizo did not appear to induce caspase-3 to become more active, but more PARP-1 cleavage (Fig. 4C), we set out to investigate how Dox+Crizo kills HCC cells. We investigated whether Crizo + Dox does activate JNK. To this end, we treated HCC cells with Vehicle, Crizo, Dox or Crizo+Dox, respectively, and then detected cell lysates by immunoblotting. We found that JNK phosphorylation levels were significantly increased after treatment with Crizo+Dox in the three liver cancer cell lines (Fig. 11A).

LC3-II and p62 play important roles in autophagosome formation (Kabeya et al., 2000). LC3B is an autophagy-related protein that undergoes post-translational modification leading to its lipidation. The lipidation of LC3-I produces lipidated LC3-II, which can be docked with specific cargoes, while p62 is a receptor for cargo destined to be degraded by autophagy (Kabeya et al., 2000). Next, we investigated whether Crizo+Dox modulates the conversion of LC3-I to LC3-II and the expression level of p62. We found that LC3-II levels were significantly higher in HCC cell lines treated with Crizo+Dox compared to Vehicle, Dox or Crizo controls in all four cell lines tested (Figure 11B). The quantitative results of three independent experiments are shown in Figure 11C. On the other hand, the effect of Crizo+Dox on the expression level of p62 was more complicated. It appears that Dox alone does not significantly affect p62 expression. In contrast, Crizo alone stimulated p62 expression in all 4 cell lines compared to the Vehicle group. Unexpectedly, the addition of Dox actually slightly mitigated the effect of Crizo on p62 expression (Fig. 11B). Nonetheless, the effect of Crizo+Dox was still significantly higher than that of Vehicle or Dox-treated cells. The quantitative results of four independent experiments are shown in Figure 11C. Therefore, Crizo+Dox also up-regulated the expression of p62. The results of immunoblotting suggested that Dox+Crizo treatment might promote the formation of autophagosomes.

To confirm that autophagosome formation is indeed activated by Crizo+Dox, we transfected exogenous LC3-GFP into HCC cells, treated cells with Crizo+Dox or each drug alone and monitored autophagosome-associated punctate formation. We observed that when the transfected hepatoma cells were treated with Crizo+Dox, the formation of autophagosomes was significantly increased, which was manifested by a marked increase in green fluorescent dots (Fig. 12A). The quantitative results of three independent experiments are shown in Figure 12B. In addition, we performed transmission electron microscopy on cells treated with Dox, vehicle, or Dox+Crizo. We observed that Dox+Crizo-treated cells had more vesicles, and most of these vesicles contained content (Fig. 12C). Collectively, these results suggest that Dox+Crizo activates endoplasmic reticulum stress, thereby increasing autophagosome formation.

Due to the important role of PERK-p-eIF2α-JNK signaling in the formation of autophagosomes, combined with the previous results, we have reason to hypothesize that Dox+Crizo can activate this signaling pathway, thereby inducing the formation of autophagosomes. To investigate whether the enhanced autophagosome formation stems from the PERK-eIF2α-JNK signaling cascade, in addition to Crizo+Dox, we also added the inhibitor GSK or the JNK-specific inhibitor SB203580(SB) to the cell culture medium. Immunoblotting results showed that both inhibitors significantly reduced the level of 14KD lipidated LC3-II protein (Fig. 11D). The quantitative analysis results of three different experiments are shown in Figure 11E. Correspondingly, LC3 immunofluorescence results showed that the level of autophagosome formation in HCC cells treated with inhibitors GSK or SB was significantly reduced (Fig. 11F). The quantitative results of three different experiments are shown in Figure 11G. In addition, light microscope observation found that the PERK inhibitor GSK significantly alleviated Dox+Crizo-induced HCC cell death (Figure 13). Therefore, Crizo + Dox can activate ER stress and also stimulate autophagosome formation through the PERK-eIF2α-JNK signaling cascade.

Figure 11(A) Blotting of cell lysates with antibodies specific for JNK or phosphorylated JNK, significant upregulation of p-JNK was detected in cells treated with Crizo+Dox, while in cells treated with vehicle, Dox or Crizo alone The up-regulation of p-JNK was not detected in the cells of . (B) LC3-II was significantly upregulated in HCC cells treated with Crizo+Dox compared to Vehicle, Dox alone or Crizo treated HCC cells. Immunoblotting of cell lysates with antibodies specific for p62 or LC3 showed that LC3-II content increased when cells were treated with Crizo+Dox. Furthermore, p62 was also up-regulated when cells were treated with Crizo+Dox or Crizo alone. (C) Analysis of the results of four experiments. Open and filled histograms show the mean and standard deviation of the four experiments. (D) PERK and JNK inhibitors significantly reduced LC3-II formation in Crizo+Dox-treated cells compared to Vehicle-treated cells. Cell lysates treated with Crizo+Dox and Vehicle, GSK or SB203580 were immunoblotted using antibodies specific for LC3. (E) The results of three experiments were analyzed. Open and filled histograms show the mean and standard deviation of the three experiments. (F) Treatment of cells with GSK or SB203580 in the presence of Crizo+Dox reduces autophagosome formation. Immunofluorescence staining of LC3 in cells treated with Crizo+Dox and vehicle, GSK or SB203580 showed that inhibition of PERK or JNK activity attenuated autophagosome formation. Cells were treated with Crizo+Dox for 22h, followed by vehicle or GSK or SB203580 for 2h. (G) Analysis of three experiments. Quantitative results. Open and filled histograms show the mean and standard deviation of the three experiments. Fixed cells were stained for LC3A/B, and nuclear counterstained and photographed. Relative immunofluorescence intensities or protein levels were processed with the free software Image J as indicated. : V: vehicle, G: GSK. SB: SB203580. P values are displayed.

Figure 12(A) Dox+Crizo treatment induces more autophagosomes than Vehicle, Dox or Crizo treatment. The cells transfected with LC3-GFP were treated with the above drug combinations for 24 hours, and the cells were fixed and imaged using confocal immunofluorescence microscopy. (B) A summary of the three experiments is shown as a bar graph. Means and standard deviations are from three independent experiments. P values are displayed. (C) Transmission electron microscopy results showed that Dox+Crizo-treated HCC cells had more vesicles with contents inside the vesicles than Dox- or Vehicle-treated cells. V: vehicle; D: Dox; C: Crizo; D+C: Dox+Crizo. 7. Crizo+Dox stimulates the formation of autophagosomes and unfused autophagosomes leading to cell death

In addition to enhanced levels of LC3-II and autophagosomes, Crizo+Dox-treated cells significantly increased p62 (Figure 14B). Therefore, we investigated whether Dox+Crizo affects the fusion of autophagosome and lysosome. Cells treated with Crizo+Dox were found to have more autophagosomes, which were GFP-positive and mCherry-positive, compared to control-treated cells (Figure 14A). In addition, the number of unfused autophagosomes in Dox+Crizo-treated cells was significantly higher than in cells treated with Crizo or Dox alone (Fig. 14A, unfused autophagosomes are shown as yellow dots; red dots indicate autolysosomes). The quantitative results of three different experiments are shown in Figure 14B. Thus, Crizo+Dox did activate autophagosome formation, but inhibited autophagosome-lysosome fusion.

We then investigated whether the synergistic effect of Dox and Crizo in killing HCC cells is dependent on cleavage of PARP-1. For this, in addition to Dox+Crizo, we also added the following inhibitors to the medium, including: Vehicle control, GSK, PARP-1 inhibitor INO, lysosomal inhibitor Chloroquine CQ. After immunoblotting of cell lysates, we found that GSK and INO consistently reduced levels of LC3-II (Figure 14A, lane 1). In addition, this treatment also resulted in reduced levels of cleaved caspase-3 and cleaved PARP-1 (Figure 14C, lanes 2, 3, 4). Therefore, the synergistic effect of Dox+Crizo on induction of apoptosis in HCC cells is due to attenuated translation of MDR1 and is dependent on the cleavage of PARP-1. The significant increase in PARP-1 cleavage after cells were further treated with CQ suggested that PARP-1 cleavage may also be the result of enhanced autophagosome formation. Because when CQ was added to cell culture medium containing Crizo+Dox, this treatment did not alter the levels of MDR1 (Fig. 9A), but greatly increased the levels of LC3-II, cleaved caspase-3 and cleaved PARP-1 (Fig. 14C). ).

We further treated HCC cells with inhibitors of PERK, caspase-3 (C3-I), PARP-1 or lysosomes in the presence of Dox+Crizo and we observed that when cells were co-treated with CQ , cell death was significantly increased, while co-treatment with GSK, C3-I or INO, cell death was significantly reduced (Figure 14D), suggesting that cleavage of caspase-3 and PARP-1 contributes to Dox+Crizo-induced cell death. Furthermore, GSK or INO rescued significantly more HCC cells than C3-I, suggesting that unfused autophagosomes may play a more important role in inducing HCC cell death through cleavage of PARP-1, which is in agreement with our earlier work. The observed conclusions were consistent (Fig. 6C).

Next, we investigated whether the synergistic effect of Crizo+Dox was due to the effect on the two best characterized targets of Crizo, C-Met and ALK (Heigener and Reck, 2014). The results showed that the effects of Crizo, Dox or Crizo+Dox varied widely among the three cell lines tested (Figure 15). Indeed, p-MET levels were greatly reduced in HepG2 cells treated with Crizo and Dox. This effect was at best weak in treated HLF cells and not observed at all in treated Sk-hepl cells (Figure 15). We detected no significant ALK expression in all HCC cell lines (results not shown). Therefore, in HCC cells, the inhibition of protein translation by Crizo+Dox was not dependent on its function of inhibiting ALK or/and C-Met activity.

Figure 14(A) Dox+Crizo induces autophagosome formation but reduces autophagosome and lysosomal fusion. Cells transfected with the LC3-GFP-mcherry plasmid were treated with vehicle, Dox, Crizo or Dox+Crizo for 24 hours and observed by confocal microscopy. (B) The results of three experiments were analyzed. (C) In the case of Dox+Crizo, HCC cells were treated with vehicle, GSK, INO or CQ as indicated. GSK and INO treatments consistently reduced LC3-II, activation of caspase-3 (cl-caspase-3) and cleavage of PARP-1 (cl-PARP-1), whereas CQ activated LC3-II, cleaved caspase-3 and cleavedPARP-1. The experiment was repeated three times with similar results. (D) According to MTS assay results, GSK, INO and caspase-3 inhibitor (C3-I) treatment reduced the toxicity of Dox+Crizo to HCC cells, while CQ treatment enhanced the induction of apoptosis of HCC cells by Dox+Crizo. Three seeded cells were treated with vehicle (V), GSK, INO, caspas3 inhibitor (C3-I) or CQ in the presence of Dox+Crizo for 24 hours. The experiment was repeated three times with similar results.

8. Crizo+Dox significantly inhibited tumor growth in HCC xenograft in vivo model.

HepG2 or 7402 cells (BALB/c) were subcutaneously transplanted in nude mice. After 7 days, when the tumors grew to about 150-200 mm ³ , the mice were randomly divided into 4 groups, and all inoculated mice were treated with Vehicle, Dox, Crizo or Crizo+Dox. We also assessed the general health of all mice by monitoring their body weight (Figure 16A). Clearly, Crizo+Dox showed a synergistic effect of inhibiting tumor growth in vivo as measured by tumor volume (Figure 16B). At the end of the experiment, tumors were surgically excised and weighed (FIG. 16C) and quantified (FIG. 16D). Indeed, mean tumor weights were reduced by 30% or 65% with Dox+Crizo compared to HepG2 cells treated with Dox or Crizo alone; and 55% compared with 7402 cells treated with Dox or Crizo alone. Therefore, Crizo significantly enhanced the tumor growth inhibition effect of Dox both in vitro and in vivo. Figure 16(A) Dox or Dox plus Crizo has a slight effect on mouse body weight. (B) Dox+Crizo treatment significantly reduced xenograft volume compared to Dox or Crizo treatment alone. (C&D) Dox plus Crizo treatment resulted in a significant reduction in xenograft weight compared with Dox and Crizo treatment alone. Each point represents the weight of the xenograft tumor. Ns: no statistical significance; X: tumor disappeared after treatment.

9. Dox+Crizo synergistically induce other cancer cells to die

Dox+Crizo also had a synergistic killing effect on melanoma cells M2 and A375-MA2, lung cancer cells A549 and H460, and oral squamous cell carcinoma cells cal27 (Figure 17). Among them, Figure 17 In order to calculate the combination index (CI), we treated different cancer cells with different concentrations of Crizo, Dox or Crizo+Dox for 48hs, and monitored cell survival by MTS. The results showed that under the combined action of Dox and Crizo, Crizo+Dox had synergistic killing effect on different cancer cells.

Obviously, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

sequence listing

<110> Guangzhou Medical University Affiliated Cancer Hospital

<120> Composition for treating liver cancer and application thereof

<130> ZM211009CS

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Mouse

<400> 1

agaaaatctg gcaccacacc 20

<210> 2

<211> 20

<212> DNA

<213> Mouse

<400> 2

tgatctgggt catcttctcg 20

<210> 3

<211> 20

<212> DNA

<213> Mouse

<400> 3

ccgctgttcg tttcctttag 20

<210> 4

<211> 20

<212> DNA

<213> Mouse

<400> 4

tcaagatcca ttccgacctc 20

<210> 5

<211> 20

<212> DNA

<213> Mouse

<400> 5

tggttggaag ctaacccttg 20

<210> 6

<211> 20

<212> DNA

<213> Mouse

<400> 6

tatctttgcc cagacagcag 20

<210> 7

<211> 20

<212> DNA

<213> Mouse

<400> 7

ctttttggatg aagccacgtc 20

<210> 8

<211> 20

<212> DNA

<213> Mouse

<400> 8

ggtgagcaat cacaatgcag 20

Claims (1)

1. the application of a pharmaceutical composition in the preparation of a drug for inhibiting liver cancer, characterized in that the pharmaceutical composition is crizotinib and doxorubicin.

Images