APG-1252 combined with Cabozantinib inhibits hepatocellular carcinoma through MEK/ERK and CREB/Bcl-xl pathways

APG-1252 combined with Cabozantinib inhibits hepatocellular carcinoma through MEK/ERK and CREB/Bcl-xl pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article APG-1252 combined with Cabozantinib inhibits hepatocellular carcinoma through MEK/ERK and CREB/Bcl-xl pathways Tian Di, qiuyun Luo, Jiang-tao Song, Xiang-lei Yan, Lin Zhang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4206490/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Purpose Liver cancer is the fourth leading cause of cancer-related death worldwide, and hepatocellular carcinoma (HCC) is the most common primary liver cancer. APG-1252 is a small molecule inhibitor of Bcl-2/Bcl-xl, and the anti-tumor effect of APG-1252 in HCC, or its anti-tumor effects in combination with cabozantinib, has not been researched. Experimental Approach: TCGA database analysis was used to analysis the gene expression levels of Bcl-2 and Bcl-xl in HCC tissues. Western Blot was used to detect the proteins’ expression level. And the inhibitory effects of APG-1252 and Cabozantinib on the proliferation of HCC cell lines was detected by CCK-8. The effect on the migration and invasion of HCC cells was verified by Transwell assay. Huh7 xenograft model in nude mice was used to detect the combined effect in vivo. Key Results: We found that APG-1252 monotherapy could inhibit the proliferation and migration of HCC cells and promote apoptosis of HCC cells. APG-1252 combined with Cabozantinib could inhibit the proliferation, migration and invasion of HCC cells and promote the apoptosis of hepatocellular carcinoma cells and exerted synergistic effect in vivo. The combination could significantly downregulate MEK/ERK phosphorylation levels. Besides, the treatment of Cabozantinib could cause the protein level of phosphorylation CREB and BCL-XL increased, while combined with APG-1252 could impair this effect. Conclusion and Implications: Our data suggest that APG-1252 in combination with Cabozantinib can provide more effective treatment strategies for HCC patients and deserve further clinical investigation. Hepatocellular carcinoma Apoptosis APG-1252 Bcl-2/Bcl-xL inhibitor Cabozantinib Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hepatocellular carcinoma (HCC) accounts for about 90% of primary liver cancers, and the main known risk factors associated with HCC are viruses (chronic hepatitis B and C), metabolism (diabetes and non-alcoholic fatty liver disease or nonalcoholic fatty liver disease (NAFLD)), toxicity (alcohol and aflatoxin), and immune system-related disorders( 1 ). Globally, the incidence of HCC in middle-aged people aged 30–59 years has decreased significantly due to the successful implementation of the hepatitis B virus (HBV) vaccination program( 2 ). However, overall morbidity and mortality of HCC continue to rise, and the global mortality rate of HCC is expected to increase by another 41% by 2040 due to the increased incidence of NAFLD due to the obesity pandemic( 3 ). When HCC is diagnosed early or the tumor size < 5 cm, liver transplantation and surgical resection are treatment options for HCC( 4 ). However, due to the insidious onset of HCC, the lack of specificity of the clinical manifestations, and the lack of early markers of specificity, most patients with HCC have progressed to advanced stages by the time of diagnosis. Advanced HCC is treated with RFA, TACE, TKI, and immunotherapy, but these modalities do not significantly prolong life as the occurrence of treatment resistance and disease recurrence( 5 ). Therefore, exploring new combination drug regimens is an urgent clinical problem for HCC patients. Cabozantinib is a tyrosine kinase inhibitor that targets VEGFR-1, VEGFR-2 and VEGFR-3, MET and AXL( 6 ). In a randomized, double-blind, placebo-controlled phase 3 clinical trial, overall survival and progression-free survival were longer in the Cabozantinib group than in the placebo group in patients with previously treated advanced HCC( 7 ). The Bcl-2 family of proteins includes pro-apoptotic proteins and anti-apoptotic proteins( 8 ). The structure of Bcl-2 family proteins contains a short conserved sequence, known as the Bcl-2 homology (BH) domain( 9 ). Anti-apoptotic proteins include Bcl-2, Bcl-xl, Mcl-1, Bcl-w, and Bfl-1, which can inhibit cell apoptosis, and tumor cells can evade apoptosis by upregulating one or more anti-apoptotic proteins( 10 ). Pro-apoptotic proteins are divided into two types, BH3-only proteins and effector proteins( 8 ). BH3-only proteins contain only one BH3 domain, including Bim, Bid, Puma, Bad, Noxa, Bik, Bmf, and Hrk( 11 ). Bim, Bid, and Puma can directly activate effector proteins, while other BH3-only proteins can bind to anti-apoptotic proteins, thus inhibiting the binding of anti-apoptotic proteins to Bim, Bid, and Puma, and activating effector proteins( 10 ). Effector proteins include Bax and Bak, which can initiate cell apoptosis by forming mitochondrial outer membrane permeabilization (MOMP) complexes( 11 , 12 ). The balance between the anti-apoptotic proteins and pro-apoptotic proteins of the Bcl-2 protein family can inhibit or activate MOMP and ultimately determine the fate of the cell( 13 ). Due to the crucial role of Bcl-2 family proteins in cell apoptosis, drugs targeting Bcl-2 family proteins have been developed. The first specific small molecule inhibitor of the Bcl-2 family, ABT-737, targets Bcl-2, Bcl-xl, and Bcl-w. It inhibits the binding of anti-apoptotic proteins to BH3-only proteins, thereby inducing tumor cell apoptosis( 14 ). ABT-263 has a high affinity for Bcl-2 family anti-apoptotic proteins (i.e., Bcl-xl, Bcl-2, Bcl-w) and shows better oral bioavailability( 15 ). It can bind to anti-apoptotic proteins, releasing the apoptotic effector proteins Bax and Bak from the anti-apoptotic proteins, thereby inducing cell apoptosis( 8 ). Previous studies have found that the combination of ABT737 and sorafenib can inhibit tumor growth in hepatocellular carcinoma( 16 ). ABT263 can sensitize the anti-tumor effect of sorafenib in hepatocellular carcinoma( 17 ). As for the combined effect of Bcl-2 family proteins and cabozantinib, there are currently no research reports. The novel small molecule Bcl-2/Bcl-xl dual-target inhibitor APG-1252 used in our laboratory has shown monotherapy effectiveness in gastric cancer, nasopharyngeal carcinoma, and acute myeloid leukemia (AML)( 18 – 20 ). APG-1252 is a novel BH3 mimetic that specifically binds the hydrophobic pocket of Bcl-2 or Bcl-xl. It is converted to its active metabolite APG-1252-M1 in vivo, which then exerts potent antitumor effects. APG-1252 has shown single-agent effectiveness in gastric cancer, nasopharyngeal carcinoma, and acute myeloid leukemia (AML), however, its antitumor effects in hepatocellular carcinoma and its possible combination regimen have not been reported in studies. Method Cells culture The human HCC cell lines Huh7, PLC/PRF/5, SK-Hep-1, HLE, HLF, BEL7402, Hep3B were purchased from Cobioer Biosciences Co. LTD (Nanjing, China). Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco Life Technologies) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) was used to culture Huh7, SK-Hep-1, HLE, HLF, Hep3B cells in a humidified incubator containing 5% CO2 at 37°C, and PLC/PRF/5, BEL7402 were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 IU/mL penicillin and 100 mg/mL streptomycin and in a humidified atmosphere containing 5% CO2 at 37 ◦C. Regents and antibodies APG-1252 and APG-1252-M1 were kindly provided by Ascentage Pharma Group Inc (Taizhou, China). For the in vitro experiments APG-1252M1 was dissolved in dimethyl sulfoxide (DMSO) at 10 µM and kept at − 20°C. For the in vivo experiments, APG-1252 was dissolved in 10% polyethylene glycol 4000 (PEG400)/5% Castor oil ethoxylated (EL)/85% phosphate-buffered saline (PBS). Cabozantinib was purchased from TargetMol and z-VAD-fmk was purchased from Selleck Chemicals (Houston, TX, USA). All compounds were dissolved in dimethylsulfoxide (DMSO; Sigma Aldrich, MO, USA) at a stock concentration of 40 mM, stored at − 20 ◦C. The final concentration of DMSO to dilute compound in culture media did not exceed 0.1%. The antibodies against Bcl-2, Bcl-xl, Mcl-1, Bad, Bim, Puma, Noxa, CREB, p-CREB, ERK, p-ERK and p-MEK were purchased from Cell Signaling Technology (MA, USA). The antibody against GAPDH was purchased from ABGENT (San Diego, USA). The secondary anti-mouse and antirabbit antibodies were purchased from Santa Cruz Biotechnology. Cell proliferation detection Cell viability was determined using Cell Counting Kit-8 purchased from EZ Bioscience (Beijing, China) according to the manufacturer’s instructions. Briefly, HCC cells were seeded at 3000 to 4000 cells/well in 96-well plates for 72 h in the presence of DMSO, APG-1252, Cabozantinib or combination therapy of APG-1252 and Cabozantinib. After 72 h, CCK-8 reagent (10 µL/well) was added and incubated at 37◦ C for 1–2 h and their absorbance readings were taken at 450 nm. Their IC50 values were calculated by using GraphPad Prism version 6.0.0 (GraphPad Software, San Diego, California USA) for Windows. Colony formation assay HCC cells were seeded in a 6-well plate at approximately 500 cells/well and were treated with a single drug or combination of drugs, a negative control group was added with an equal volume of DMSO. Fresh culture medium was replaced every 3–4 days. After 14 days, the cells were fixed in methanol and stained with 0.5% crystal violet for 15 min at room temperature, after which the number of colonies were counted. Transwell assay The HCC cells (8 × 104 cells) were suspended in 200 µL medium containing no FBS with indicated drugs and were then aspirated into a transwell chamber (PC membrane, pore size 8.0 µm, Corning, NY, USA). The transwell chamber was placed in a 24-well plate containing 750 uL of 50% FBS culture medium. After 24–30 h, the chamber was taken out, the inner membrane of the chamber was washed with a cotton swab to remove adherent cells, and then fixed in methanol and stained with 0.5% crystal violet for 15 min. Then, the chambers were dried at room temperature and imaged using a microscope. Wound healing assay Wound healing assays were applied to test cell migration ability. HCC cells were seeded in six-well plates and fused to 100%. Then, a 200-µL pipette tube was used to create an artificial wound. Fresh medium containing 1% FBS with indicated chemicals was added. The wound closure was photographed immediately and 24 h later under a microscope. The Image J software was used to calculate the area between cells. Cell apoptosis assays HCC cells were plated at 1 × 105 cells/well in 12-well plates and treated with indicated chemicals for 72 h. The cells were then collected and stained with Annexin V-FITC/PI according to the instructions of the Apoptosis Detection Kit purchased from Sizhengbo Biotechnology (Beijing, China). Cell apoptosis were detected using an ACEA NovoCyte™ flow cytometer (ACEA Biosciences Inc. China). Western blot analysis Cells were treated with DMSO and indicated dose of drugs for 24 h, and then harvested and washed once with pre-cooled PBS. The cells were lysed on ice using a cell lysis buffer (#9803) purchased from Cell Signaling Technology (MA, USA), containing 1% protease inhibitor (PMSF), 1% phosphokinase inhibitor for 30 min, centrifuged at 12000 rpm for 15 min at 4 ◦C, and the supernatant protein lysate was collected. Protein concentration was measured by a Pierce BCA Protein Assay kit (Thermo Scientific). Cellular protein lysates were separated using 8–12% SDS-PAGE gel and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% BSA buffer for 1 h at room temperature and then incubated with indicated primary antibody at 4 ◦C overnight. 1xTBST (washing buffer) washing the membrane 3 times with 10 min each time. The protein membrane was incubated with secondary antibody for 1 h at room temperature and washed with 1xTBST for 3 times for 10 min each time. Signal generation and detection were performed using an ECL chemiluminescence hypersensitive colorimetric kit and a chemiluminescent imaging system. Proteome profiler analysis The Human Phospho-Kinase Array Kit (#ARY003C, R&D Systems, Wiesbaden-Nordenstadt, Germany) was applied to detect the levels of 43 specific kinase phosphorylation sites in Huh7 cells with different treatments. For the preparation of total protein extracts, 1x106 /mL Huh7 cells were seeded in 6 cm dish. After 24 h, the cells were treated DMSO, 1 µM APG-1252, 2 µM Cabozantinib and combination treatment for 24 h. The preparation of cellular extracts and the proteome profiling were carried out according to the manufacturer’s instructions. Briefly, the membranes were incubated with blocking buffer before the experiment. The cell lysates were diluted in blocking buffer with a total protein amount of 600 µg then incubated overnight with detected membranes. After several washing steps, the membranes were incubated in the provided detection antibody cocktail for 2 h, then washed again and incubated for 30 min in streptavidin-horseradish peroxidase (HRP). The unbound HRP antibody was washed away, and then the signal was analyzed with a chemiluminescent substrate and detected with an X-ray array. Animal experiment Huh7 xenograft models were used to evaluate the anti-tumor effects of APG-1252 and Cabozantinib monotherapy and combination in vivo. Four-week-old female BALB/c athymic nude mice were purchased from Beijing Vital River Laboratory Technology Co. Ltd. Huh7 cells (5 × 106) suspended in 100 µL cold PBS were subcutaneously injected into the dorsal flank of the mice. When the tumor volume reached approximately 100 mm3, the mice were randomly assigned into different groups. For the APG-1252-treated mice, 50 mg/kg of APG-1252 was injected via the caudal vein twice a week. For the Cabozantinib-treated mice, 50 mg/kg of Cabozantinib was delivered orally twice a week. Tumor sizes and animal weights were recorded twice per week and tumor volumes were calculated as V (mm3) = 1/2 × (length × width2). All animal experiments were performed under the guidance of Sun Yat-Sen University Committee for Use and Care of Laboratory Animals and were approved by the animal experimentation ethics committee. Animal studies are also reported in compliance with the ARRIVE guidelines( 21 ) and with the recommendations made by the British Journal of Pharmacology( 22 ). Statistical analysis The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology( 23 ). Statistical analyses were performed in GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, California USA). Unless indicated, all results were presented as mean ± SD of three independent experiments. Differences between two groups were analyzed using unpaired sample t-test. Comparison among more than two groups was analyzed by One-way ANOVA and Two-way ANOVA. p 1 for antagonism effect, CI = 1 for additive effect, 0.8 ≤ CI < 1 for low synergy, 0.6 ≤ CI < 0.8 for moderate synergy, 0.4 ≤ CI < 0.6 for high synergy, and 0.2 ≤ CI < 0.4 for strong synergy effect. Result APG-1252 elicits antitumor effect on HCC cell lines Firstly, we analyzed the expression levels of Bcl-2 and Bcl-xl in hepatocellular carcinoma tissues in the TCGA database and found that the gene expression levels of Bcl-2 and Bcl-xl in hepatocellular carcinoma tissues were higher than those in normal tissues (Fig. 1 A-B), and high expression of Bcl-xl was associated with poor prognosis (Fig. 1 D), further suggesting the possibility of Bcl-xl as a therapeutic target for HCC. Next, to study the sensitivity of hepatocellular carcinoma cell lines to the Bcl-2/Bcl-xl inhibitor APG-1252, we detected the basic expression of Bcl-2 family proteins and the IC 50 value of APG-1252 in eight hepatocellular carcinoma cell lines (Supplementary Fig. 1A-C). The IC50 of APG1252-M1 is relatively low in Hep3B and BEL-7402, which have high levels of Bcl2 protein expression, while the IC50 of APG1252-M1 is lowest in PLC/PRF/5, which has high levels of Bcl-xl protein expression (supplementary Fig. 4A). To further explore the effect of APG-1252 on the proliferation of hepatocellular carcinoma cell lines, we selected three hepatocellular carcinoma cell lines (Huh7, PLC/PRF/5, SK-Hep-1) for cell colony formation experiments, and the results showed that as the drug concentration increased, the number of cell colonies gradually decreased (Fig. 1 E, G-I). This indicates that APG-1252 can inhibit the proliferation of hepatocellular carcinoma cell lines in a concentration-dependent manner. Considering that hepatocellular carcinoma is prone to metastasis, we used the wound healing assay to detect the changes in the migration ability of hepatocellular carcinoma cell lines after 12 hours of APG-1252 drug treatment. The results showed that as the drug concentration increased, the speed of scratch healing gradually decreased (Fig. 1 F, J-L). This suggests that APG1252 can inhibit the migration of hepatocellular carcinoma cells in a concentration-dependent manner. The above results indicate that APG1252 alone can inhibit the proliferation and migration ability of hepatocellular carcinoma cells. Next, we used Annexin V/PI staining to analyze the effect of APG-1252 on the apoptosis of hepatocellular carcinoma cell lines. The results showed that as the drug action time prolonged, the percentage of apoptotic cells gradually increased (Figure A, C-E); as the drug concentration increased, the percentage of apoptotic cells gradually increased (Fig. 2 B, F-H). Then we also observed that APG-1252 treatment could significantly increase the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-PARP (Fig. 2 I-J, supplementary Fig. 4B-C, supplementary Fig. 5A-B). This indicates that APG-1252 can promote the apoptosis of hepatocellular carcinoma cell lines in a concentration-dependent and time-dependent manner. APG-1252 combined with Cabozantinib exerts synergistic antitumor effects in hepatocellular carcinoma in vitro. Sorafenib is the first targeted drug proven effective in patients with advanced liver cancer and has been the standard treatment for over a decade( 24 , 25 ). Lenvatinib was subsequently approved, further consolidating the role of multi-kinase inhibitors in the first-line treatment of advanced hepatocellular carcinoma( 26 ). However, the therapeutic effects of both drugs are far from satisfactory. Compared with placebo, sorafenib only has a survival advantage of 2.8 months in liver cancer patients( 24 ). Although it has a high response rate, Lenvatinib only shows non-inferiority compared to sorafenib, and the extension of overall survival is limited( 26 ). In previous studies, Bcl-xl was found to be upregulated in tumor tissues of HCC patients( 27 ), and overexpression of Bcl-xl was associated with poor overall survival and progression-free survival after surgical resection of tumors in HCC patients( 28 ). Through analysis of the TCGA database, we found that the main targets of Cabozantinib, VEGFR1, 2, 3, have a strong positive correlation with the expression of Bcl-2 and Bcl-xl (Fig. 3 A-F). This suggests that the combination of APG-1252 and Cabozantinib may have a synergistic anti-tumor effect in hepatocellular carcinoma. Therefore, we began to explore the anti-tumor effect of APG-1252 combined with Cabozantinib in hepatocellular carcinoma in vitro. First, we used the CCK8 method to detect the IC50 of Cabozantinib in seven hepatocellular carcinoma cell lines (supplementary Fig. 2A-B). Then, we evaluated the effect of APG-1252 combined with Cabozantinib on the growth and proliferation of hepatocellular carcinoma cell lines (Fig. 3 G-I, supplementary Fig. 2C-F). The results showed that the combination of APG-1252 and Cabozantinib for 72 hours can significantly inhibit cell proliferation in PLC/PRF/5, Huh7, HepG2 three cell lines, and can reduce cell viability by more than 50% at lower concentrations; while in HLE, SK-Hep-1, BEL7402, HLF, Hep3B cell lines, significant inhibitory effect on cell proliferation can be observed at higher drug concentrations after combination therapy. At the same time, the combination index CI value also shows that APG-1252 and Cabozantinib combined have a significant synergistic anti-tumor effect in hepatocellular carcinoma cell lines (Fig. 3 J). Next, we selected PLC/PRF/5, Huh7, SK-Hep-1 three cell lines to use the plate clone experiment to detect the effect of APG-1252 combined with Cabozantinib on the long-term growth of hepatocellular carcinoma cells. The experimental results showed that the formation of cell colonies in the combination group significantly decreased after 14 days of drug action (Fig. 4 A-D). At the same time, we used CCK8 to detect the effect of APG-1252 combined with Cabozantinib on the inhibition of tumor cell growth in hepatocellular carcinoma cells PLC/PRF/5, Huh7, SK-Hep-1 at different times. The experimental results showed that as the drug action time prolonged, the vitality of the cells in the combination group gradually decreased (Fig. 4 E-G). These results indicate that the combination of APG-1252 and Cabozantinib can significantly inhibit the proliferation of hepatocellular carcinoma cell lines in vitro, and this inhibitory effect was time-dependent, thus exerting a synergistic anti-tumor effect. Then, we used Annexin V/PI staining to analyze the effect of the combination of the two drugs on cell apoptosis. We selected PLC/PRF/5, Huh7, SK-Hep-1 three cell lines to treat with corresponding concentrations and times to detect cell apoptosis by flow cytometry. The results showed that in Huh7 cells, after 72 hours of drug treatment, the apoptosis rate of APG-1252 single drug group and Cabozantinib single drug group were 28.93% and 7.12% respectively, while the apoptosis rate of the combination group significantly increased to 89.75%, and with the prolongation of drug action time, the apoptosis rate of the combination group cells significantly increased (Fig. 5 A-B); at the same time, we also observed similar results in PLC/PRF/5 and SK-Hep-1 cell lines (Fig. 5 C-D, S3A-B). These results indicate that the combination of APG-1252 and Cabozantinib can significantly promote tumor cell apoptosis, and this effect shows a significant time dependence. Western Blot was used to detect the effect of APG-1252 and Cabozantinib on the expression of Cleaved-PARP and Cleaved-caspase3 in three cell lines after 72 hours of combined treatment. The experimental results showed that the expression of Cleaved-PARP and Cleaved-caspase3 in the combination group was significantly up-regulated (Fig. 5 F, supplementary Fig. 6A, C, E). At the same time, the changes of apoptosis-related proteins in tumor cells were also detected at different drug action times. The experimental results showed that with the prolongation of drug action time, the expression of Cleaved-PARP and Cleaved-caspase3 gradually increased (Fig. 5 G, supplementary Fig. 6B, D, F), indicating that the combination of APG-1252 and Cabozantinib in hepatocellular carcinoma cell lines can significantly up-regulate the expression of apoptosis proteins Cleaved-PARP and Cleaved-caspase3 in a time-dependent manner. APG-1252 combined with Cabozantinib inhibited the metastasis and invasion of hepatocellular cancer cells in vitro. Due to the high propensity of hepatocellular carcinoma to metastasize, we further tested the effect of APG-1252 combined with Cabozantinib on the migration and invasion capabilities of hepatocellular carcinoma cells in PLC/PRF/5, Huh7, and SK-Hep-1 cells using Transwell experiments. The results showed that the number of cells migrating and invading after combined medication was significantly reduced (Fig. 6 A-H). This suggests that APG-1252 combined with Cabozantinib exerts a synergistic anti-tumor effect in hepatocellular carcinoma by inhibiting cell migration and invasion capabilities. The reason for tumor cell metastasis and invasion is often due to epithelial-mesenchymal transition (EMT), the hallmark of which is the reduction in the expression of cell adhesion molecules (such as E-cadherin), and the transformation of the cytokeratin cytoskeleton to a vimentin-based cytoskeleton. Therefore, we used Western Blot to detect changes in the expression levels of EMT-related proteins in PLC/PRF/5, Huh7, and SK-Hep-1 cells after 24 hours of drug treatment. The experimental results showed that the expression levels of E-cadherin and ZO-1 in the combined drug group were significantly upregulated, while the expression levels of Vimentin and α-catenin were significantly downregulated (Fig. 6 I, supplementary Fig. 7). This suggests that APG-1252 combined with Cabozantinib affects the migration and invasion capabilities of hepatocellular carcinoma cells by affecting the expression levels of EMT-related proteins E-cadherin, ZO-1, Vimentin, and α-catenin. APG-1252 combined with Cabozantinib exerts a synergistic anti-tumor effect in hepatocellular carcinoma in vivo To further validate the synergistic antitumor effect of APG-1252 and Cabozantinib in hepatocellular carcinoma in vivo, we constructed a nude mouse xenograft model using Huh7 cells. The results showed that APG-1252 and Cabozantinib alone could inhibit the growth of hepatocellular carcinoma in vivo, while the combination group showed a synergistic effect in suppressing the tumor growth (Fig. 7 A-D). We next detected the expression of apoptosis proteins cleaved-PARP and cleaved-caspase-3 in the tumor tissues. The results showed that compared with the single drug group, the expression levels of cleaved-PARP and cleaved-caspase-3 in the combination group were significantly upregulated (Fig. 7 E, supplementary Fig. 8A). Furthermore, compared with other groups, the expression of cleaved-caspase-3 in the combination group was significantly increased by IHC assay, and the number of TUNEL positive cells in the combination group was significantly more than the other groups (Fig. 7 F). These results suggest that APG-1252 combined with Cabozantinib exerted synergistic anti-tumor effect in hepatocellular carcinoma in vivo. The synergistic anti-tumor effect of APG-1252 combined with Cabozantinib was mainly through inhibiting MRK/ERK and CREB/Bcl-xl pathways. To further explore the underlying mechanism of the synergistic anti-tumor effect of APG-1252 combined with Cabozantinib in hepatocellular carcinoma, we applied a phosphorylated protein array to investigate the potential molecules that may change (Fig. 8 A). The results showed that Cabozantinib single drug treatment can cause a significant increase in p-CREB level, while the expression level of p-CREB in the combination group of APG-1252 and Cabozantinib can return to the baseline level comparable to the control group. We further verified the expression level of p-CREB in PLC/PRF/5, Huh7 and SK-Hep-1 cells, the results showed that compared with APG-1252 and Cabozantinib single drug group, the expression level of p-CREB in the combination group was significantly reduced (Fig. 8 B, supplementary Fig. 8B, C, D). According to previous studies, p-MEK/p-ERK can phosphorylate CREB( 29 ), activating it to p-CREB, thus causing the transcription of downstream target genes Bcl-2/Bcl-xl( 30 ). To further explore the changes in the expression of upstream and downstream proteins of p-CREB, we detected the expression of p-MEK, p-ERK, Bcl-2, Bcl-xl in HCC cells with indicated treatment. The results showed that the expression levels of p-MEK, p-ERK, Bcl-xl in the combination group of APG-1252 and Cabozantinib were significantly reduced (Fig. 8 B, supplementary Fig. 8B, C, D). However, the p-MEK, p-ERK in the Cabozantinib single drug group did not significantly increase, so we suggest that APG-1252 combined with Cabozantinib may be through inhibiting MEK/ERK pathway to exert synergistic anti-tumor effects. To further explore the changes in other proteins of the Bcl-2 family in PLC/PRF/5, Huh7, SK-Hep-1 three strains of cells after 72 hours of drug treatment, we used Western Blot to detect the expression of Bcl-2 family proteins (Fig. 8 B, supplementary Fig. 8B, C, D). The results showed that the expression level of Bax in the combination group of APG-1252 and Cabozantinib was significantly upregulated, while the expression level of Mcl-1 was significantly downregulated. Next, we treated SK-Hep-1 cells with different concentrations of Cabozantinib and found that Cabozantinib decreased the level of p-CREB and Bcl-xl (Fig. 8 C-D, supplementary Fig. 9A-B). We also used the apoptosis inhibitor Z-VAD for recovery, and found that after adding the apoptosis inhibitor Z-VAD, the expression of apoptosis-related molecules Cleaved-PARP and Cleaved-caspase3 was significantly downregulated(Fig. 8 E, supplementary Fig. 8B, C, D), although the level of p-CREB was upregulated to some extent, but compared with the Cabozantinib single drug group, the level of p-CREB was still reduced, these results indicate that the downregulation of p-CREB is not caused by apoptosis, but is caused by the combined action of APG-1252 and Cabozantinib. Therefore, p-CREB may be a key mechanism of the combined action of APG-1252 and Cabozantinib. Discussion As the most common cause of cancer-related deaths globally, hepatocellular carcinoma is the only one among the top five most lethal cancers that sees an annual increase in incidence( 31 ). Although the relative survival rate of cancer patients in our country has continually improved compared to the past, there has been no significant progress in the diagnosis and treatment of hepatocellular carcinoma. Therefore, for patients with advanced hepatocellular carcinoma, pursuing combination drug therapy is a very promising treatment strategy. Cabozantinib was approved by the FDA in 2019 for second-line treatment of patients with advanced hepatocellular carcinoma who failed sorafenib treatment( 26 ). However, due to the the multi-drug resistance, the effectiveness of single-drug therapy is limited. Bcl-2 family protein inhibitors ABT737 and ABT263 have been found to exert anti-tumor effects in hepatocellular carcinoma when combined with Sorafenib( 16 , 17 ). In previous studies, Bcl-xl was found to be upregulated in tumor tissues of HCC patients( 27 ), and overexpression of Bcl-xl was associated with poor overall survival and progression-free survival after surgical resection of tumors in HCC patients( 28 ).Moreover, through the analysis of the TCGA database, we found a strong positive correlation between the main targets of Cabozantinib, VEGFR1, 2, 3, and the expression of Bcl-2 and Bcl-xl. This suggests that the combination of APG-1252 and Cabozantinib may have a synergistic anti-tumor effect in hepatocellular carcinoma. To seek more effective treatment strategies, this study combined the Bcl-2/Bcl-xl inhibitor APG-1252 and the tyrosine kinase inhibitor Cabozantinib to investigate their synergistic anti-tumor effects in hepatocellular carcinoma. In this study, we initially investigated whether the Bcl-2/Bcl-xl inhibitor APG-1252 could exert an anti-tumor effect on hepatocellular carcinoma in vitro and its potential mechanism. CCK8 and colony formation assay results indicated that APG-1252 could inhibit the proliferation of hepatocellular carcinoma cells in vitro. The flow cytometry apoptosis assay and Western Blot results showed that APG-1252 could significantly induce apoptosis in hepatocellular carcinoma cells, and this effect exhibited a clear time-dependence and dose-dependence. The subsequent wound healing assay results showed that APG-1252 could inhibit the migration of hepatocellular carcinoma cells in vitro. Next, we investigated whether the Bcl-2/Bcl-xl inhibitor APG-1252 combined with Cabozantinib could exert a synergistic anti-tumor effect on hepatocellular carcinoma both in vitro and in vivo, and its potential mechanism. The in vitro CCK8 experiment and cell colony formation experiment results showed that APG-1252 combined with Cabozantinib could significantly inhibit tumor cell proliferation, demonstrating a synergistic anti-tumor effect. This provides potential for the clinical combination application of Cabozantinib and the Bcl-2/Bcl-xl inhibitor APG-1252 in hepatocellular carcinoma. Then, we tried to explore the mechanism of the synergistic effect of APG-1252 combined with Cabozantinib in hepatocellular carcinoma. The flow cytometry apoptosis detection results showed that APG-1252 combined with Cabozantinib could significantly promote apoptosis in hepatocellular carcinoma cells, and this effect showed significant time-dependence. And the western Blot results also showed that in the combined drug group, the expression levels of cell apoptosis markers Cleaved-PARP and Cleaved-caspase3 were significantly upregulated, and this effect showed clear time-dependence. Next, considering the characteristic of hepatocellular carcinoma that it is prone to metastasis, we used Transwell experiments to verify the effect of combined drug treatment on the migration and invasion ability of hepatocellular carcinoma cells. The experimental results showed that the number of tumor cells migrating and invading significantly decreased after combined drug treatment. Meanwhile, the results of Western Blot showed that the combination could cause changes in the expression of EMT-related proteins. The expression levels of E-cadherin and ZO-1 in the combination group were significantly upregulated, while the expression levels of Vimentin and α-catenin were significantly downregulated. These experimental results indicate that the combination exerts a synergistic anti-tumor effect by inhibiting the migration and invasion of hepatocellular carcinoma cells. We think that the changes in EMT-related markers might have an impact on the tumor microenvironment. And the data of this part is not covered in our study. Next, we further elucidated the mechanism of the anti-tumor effect of APG-1252 combined with Cabozantinib through phosphoprotein chip experiments. The phosphorylated protein array results showed that the combination might exert a synergistic anti-tumor effect through p-CREB. cAMP-response-element-binding protein (CREB) is a transcriptional cofactor that triggers multiple transcriptional cascade responses and target gene expression( 32 ). Activation of CREB involves the reversible phosphorylation of serine residues located at 129 (S129), 133 (S133) and 142 (S142), which is triggered by multiple cellular effector kinases stimulated by growth factors or extracellular stresses( 33 , 34 ). With the activation of phosphorylation at site 133, CREB binds to its coactivator CREB-binding protein (CBP) and is thus able to recruit additional transcriptional machinery elements to drive the malignantly progressive transcriptional program( 35 ).The subsequent Western Blot results further indicated that APG-1252 combined with Cabozantinib might exert a synergistic anti-tumor effect through the MEK/ERK pathway and the CREB/Bcl-xl pathway. At the same time, our Western Blot experimental results showed that APG-1252 combined with Cabozantinib could further downregulate the expression level of anti-apoptotic protein Mcl-1 and upregulate the expression of pro-apoptotic protein Bax. Notably, the Western Blot experimental results showed that the p-CREB level significantly increased in the Cabozantinib treatment group, and according to previous research reports, the upregulation of CREB expression can inhibit the anti-tumor effect of Sorafenib in kidney cancer( 36 ). Therefore, we speculate that the increase of p-CREB level induced by Cabozantinib in hepatocellular carcinoma may inhibit the anti-tumor effect of Cabozantinib in hepatocellular carcinoma, but due to the limited length, we did not study and discuss this part of the content in the project. Mitogen-activated protein kinases (MAPKs) consist of three main subfamilies: the extracellular-signal regulated kinases (ERK), the c-jun N-terminal kinase or stress-activated protein kinases (JNK or SAPK), and MAPK14( 37 ). The ERK signaling pathway, including kinase RAS, RAF, MEK, and ERK, is a three- or four-layer phosphorylation cascade that transmits upstream signals from membrane receptors to a series of downstream effector substrates( 38 ). Extracellular signaling proteins bind to specific cell surface receptors, such as cytokine receptors, receptor tyrosine kinase (RTK), and G protein-coupled receptors, and activate a series of signaling cascades involving RAS, RAF, and MEK( 39 ). Activated MEK can phosphorylate the conserved threonine and tyrosine residues within the activation loop of ERK, which then modulate other protein kinases and transcription factors involved in cell proliferation, cell survival, cell migration, and cell differentiation( 40 , 41 ). The Western Blot results further indicated that APG-1252 combined with Cabozantinib might exert a synergistic anti-tumor effect through the MEK/ERK pathway. Finally, further verification in the in vivo mouse tumor model showed that the combined application of APG-1252 and Cabozantinib in hepatocellular carcinoma mice could significantly inhibit the growth of mouse tumors, indicating the good application effect of this drug combination. At the same time, this combination had no significant effect on the weight of the mice, which indicates the safety of the combined application of APG-1252 and Cabozantinib in hepatocellular carcinoma. Our research indicates that the combined application of APG-1252 and Cabozantinib in vitro and in vivo can exert a strong synergistic anti-tumor effect in hepatocellular carcinoma. The mechanism of this synergistic anti-tumor effect is primarily through promoting apoptosis of hepatocellular carcinoma cells, inhibiting the proliferation, migration, and invasion of hepatocellular carcinoma cells. At the same time, our experimental results also show that the combined application of APG-1252 and Cabozantinib in hepatocellular carcinoma exerts a synergistic anti-tumor effect by inhibiting the MEK/ERK pathway and the CREB/Bcl-xl pathway, and also exerts an anti-tumor effect by upregulating the pro-apoptotic protein Bax and downregulating the anti-apoptotic protein Mcl-1. As for the pathway through which APG-1252 combined with Cabozantinib inhibits the MEK/ERK pathway, due to time and space constraints, we did not expand on this part of the research; likewise, for the changes in the upstream kinases of CREB/Bcl-xl, our current research has not found any significant changes, which can be further researched. This study provides new ideas and strategies for the treatment of hepatocellular carcinoma, and further clinical trials are needed in the future to verify the effectiveness and safety of this combined therapy. However, our research still has some limitations. Firstly, the effect of the combination of APG-1252 and Cabozantinib on the proliferation, migration, and invasion abilities of hepatocellular carcinoma cells may also be due to its promotion of apoptosis. We did not further verify this phenomenon in the experiment, which is a flaw in the design of our study and an area for improvement in future research. Secondly, the mechanism of the combined action of APG-1252 and Cabozantinib in hepatocellular carcinoma still needs more in-depth research. Thirdly, we need to further verify the role of CREB/Bcl-xl in clinical patient samples, to provide more definite theoretical basis and support for clinical application. Abbreviations HCC: hepatocellular carcinoma. HBV: hepatitis B virus. NAFLD: nonalcoholic fatty liver disease. MAPKs: Mitogen-activated protein kinases. ERK: extracellular-signal regulated kinases. JNK: c-jun N-terminal kinase. SAPK: stress-activated protein kinases. RTK: receptor tyrosine kinase. CREB: cAMP-response-element-binding protein. CBP: CREB-binding protein. AML: acute myeloid leukemia. MOMP: mitochondrial outer membrane permeabilization. VEGFR2: vascular endothelial growth factor receptor. VEGFR3: vascular endothelial growth factor receptor. EMT: Epithelial-Mesenchymal Transition. Declarations Acknowledgement This study was supported by the National Natural Science Foundation of China (NSFC grant numbers 82073377, 82003268, and 81772587); the Natural Science Foundation of Guangdong (grant number 2021A1515012439); the Science and Technology Program of Guangdong (grant number 2019B020227002). Conflict of interest statement DJ. Y has an ownership interest (including patents) in Ascentage Pharma Group Corp. Limited. WT.P. is an employee of Ascentage Pharma Group Corp. Limited. All other authors declare no competing financial interests. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Author contributions Tian Di,Qiu-yun Luo performed the in vitro experiments, in vivo experiments and wrote the main manuscript. Jiang-tao Song analyzed research data. Xiang-lei Yan assisted with experiments in vivo, and prepared Fig. 6. Lin Zhang is involved in discussion. Wen-tao Pan is involved in discussion. Fei-teng Lu contributed reagents or other essential material. Yu-ting Sun contributed reagents or other essential material. Zeng-fei Xia contributed reagents or other essential material. Li-qiong Yang contributed reagents or other essential material.Miao-zhen Qiu concept and supervised. Da-jun Yangprovide the funding acquisition. Jian Sun oncept and supervised. 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Huang S, Cui P, Lin S, Yao X, Wang X, Ren Y, et al. The Transcription Factor Creb is Involved in Sorafenib-Inhibited Renal Cancer Cell Proliferation, Migration and Invasion. Acta Pharm. 2018;68(4):497-506. Fang JY, Richardson BC. The MAPK signalling pathways and colorectal cancer. Lancet Oncol. 2005;6(5):322-7. Cargnello M, Roux PP. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol Mol Biol Rev. 2011;75(1):50-83. Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors. 2006;24(1):21-44. Lavoie H, Gagnon J, Therrien M. ERK signalling: a master regulator of cell behaviour, life and fate. Nat Rev Mol Cell Biol. 2020;21(10):607-32. von Kriegsheim A, Baiocchi D, Birtwistle M, Sumpton D, Bienvenut W, Morrice N, et al. Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol. 2009;11(12):1458-64. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigureslegends.docx figures1d1.tif figures2d1.tif figures3d1.tif supplementaryfigure4d1.tif supplementaryfigure5d1.tif supplementaryfigure6d1.tif supplementaryfigure7d1.tif supplementaryfigure8d1.tif supplementaryfigure9d1.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4206490","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":286973661,"identity":"01fc0dc9-a317-45bb-8faf-7c51ea5bb23f","order_by":0,"name":"Tian Di","email":"","orcid":"","institution":"Sun Yat-Sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Di","suffix":""},{"id":286973663,"identity":"4be28f4e-920d-45b2-bae0-36a35663ba6a","order_by":1,"name":"qiuyun Luo","email":"","orcid":"","institution":"Sun Yat-Sen 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11:53:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4206490/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4206490/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54313814,"identity":"ed3901f9-32e8-41c1-be2a-31354fe18fe8","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":391836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPG-1252 elicits antitumor effect on HCC cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, B: The gene expression levels of Bcl-2 and Bcl-xl in hepatocellular carcinoma tissues in TCGA. C, D：The relationship between the expression level of Bcl-xl and Bcl-2 with prognosis of HCC patients in TCGA. E: Cell colony formation experiment in Huh7, PLC/PRF/5, SK-Hep-1. G-I: Statistical result of cell colony formation experiment. F: Cell scratch test in Huh7, PLC/PRF/5, SK-Hep-1. J-L: Statistical result of cell scratch test.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/326edf207ba5a0c4b7bab826.png"},{"id":54313810,"identity":"4bc24824-22ae-4c54-a73b-12287a48c337","added_by":"auto","created_at":"2024-04-08 17:30:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":231889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of APG-1252 on the apoptosis of hepatocellular carcinoma cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, B: Annexin V/PI staining to detect the effect of APG-1252 on the apoptosis of hepatocellular carcinoma cell lines. C-E, F-H: Statistical result of Annexin V/PI staining. I, J: Western Blot method to detect the expression of apoptosis-related proteins Cleaved-caspase3 and Cleaved-PARP.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/4e10dc121cad64d8a43c00eb.png"},{"id":54313819,"identity":"ccc2818a-4d23-4cae-b269-ae993db844b8","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":256611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of APG-1252 combined with Cabozantinib on the growth and proliferation of hepatocellular carcinoma cell lines in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-F: The relationship between the expression level of VEGFR1, 2, 3 with Bcl-xl and Bcl-2 in TCGA. G-I: CCK8 to detect the effect of APG-1252 combined with Cabozantinib on the growth and proliferation of hepatocellular carcinoma cell lines. J: The combination index value of APG-1252 and Cabozantinib.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/d7876e16f1d4064a963787cd.png"},{"id":54313811,"identity":"6e599ca4-2459-4421-b0ef-8682be4d75c1","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of APG-1252 combined with Cabozantinib on the long-term growth of hepatocellular carcinoma cells in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Plate clone experiment to detect the effect of APG-1252 combined with Cabozantinib on the long-term growth of hepatocellular carcinoma cells. B-D: Statistical result of Plate clone experiment. E-G: Statistical result of CCK8.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/ea71b9da52154a117d9786b8.png"},{"id":54313816,"identity":"fbe052a8-0ecb-479d-96a9-7145da6d8125","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":432016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of the combination of APG-1252 and Cabozantinib on cell apoptosis in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Annexin V/PI staining to analyze the effect of the combination of the two drugs on cell apoptosis. B-D: Statistical result of Annexin V/PI staining. F-G: western Blot to detect the effect of APG-1252 and Cabozantinib on the expression of Cleaved-PARP and Cleaved-caspase3.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/f19d3956623096733b9fb51e.png"},{"id":54313817,"identity":"20b80127-b5be-49cc-b2b1-4d6a8e657a69","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":468953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of APG-1252 combined with Cabozantinib on the migration and invasion capabilities of hepatocellular carcinoma cells in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B: Transwell experiment to test the effect of APG-1252 combined with Cabozantinib on the migration and invasion capabilities of hepatocellular carcinoma cells. C-H: Statistical result of Transwell experiment. I: Western Blot to detect changes in the expression levels of EMT-related proteins in PLC/PRF/5, Huh7, and SK-Hep-1 cells after 24 hours of drug treatment.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/45d0705101d6cf372a7a7d5d.png"},{"id":54313818,"identity":"2f1e4bc0-5f94-4699-8dba-495d5c95b342","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":443761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPG-1252 combined with Cabozantinib exerts a synergistic anti-tumor effect in hepatocellular carcinoma in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-D: The tumour volume, mouse weight, tumour weight of nude mouse xenograft model using Huh7 cells (n=8). E: Western Blot to detect the changes in the expression of apoptosis proteins Cleaved-PARP and Cleaved-caspase3 in the mouse tumor tissues. F: immunohistochemistry and TUNEL assay to detect the apoptosis of mouse tumor tissues.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/92f3c3bd8a3dab5e358308ba.png"},{"id":54314626,"identity":"6aa1e5b6-d513-4e1f-9281-31009e284585","added_by":"auto","created_at":"2024-04-08 17:38:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":320394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe synergistic anti-tumor effect of APG-1252 combined with Cabozantinib was mainly through Mek/Erk and CREB/Bcl-xl pathways.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003ePhosphorylated protein array to investigate the potential molecules that may change. B: Western Blot to detect the expression level of associated proteins in PLC/PRF/5, Huh7, SK-Hep-1. C-D: Western Blot to detect the p-CREB and Bcl-xl with different concentrations or time of Cabozantinib. E: Western Blot to detect the p-CREB and Bcl-xl with apoptosis inhibitor Z-VAD.\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/9c6b89d42a5955a7375e692b.png"},{"id":55858249,"identity":"32fc2ede-fabf-4b55-bc10-2968e52defe7","added_by":"auto","created_at":"2024-05-05 02:25:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3701665,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/ef062aec-1277-46cd-852d-daa150e0db30.pdf"},{"id":54313812,"identity":"9c320883-76e7-4f90-912d-d184d352f1af","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23068,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureslegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/3b7b10a97fa10e476c874d3f.docx"},{"id":54315968,"identity":"83e2e065-47fb-473c-9a49-d1f9285216c3","added_by":"auto","created_at":"2024-04-08 17:46:09","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":471604,"visible":true,"origin":"","legend":"","description":"","filename":"figures1d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/603645b2b73201b96a091a13.tif"},{"id":54313813,"identity":"a6192445-f564-4fde-80af-5353cae1b173","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":431806,"visible":true,"origin":"","legend":"","description":"","filename":"figures2d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/ea2f828fda2b0620b5e5912a.tif"},{"id":54313822,"identity":"bec33920-6d23-4519-90b5-0447ea7588c0","added_by":"auto","created_at":"2024-04-08 17:30:10","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":279830,"visible":true,"origin":"","legend":"","description":"","filename":"figures3d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/0cde8be404a3f87994acd859.tif"},{"id":54313825,"identity":"3c48844e-9f8c-43c5-9f5c-aba5baaca40e","added_by":"auto","created_at":"2024-04-08 17:30:10","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":511884,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure4d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/4ccb973f8e65877bd65c1b02.tif"},{"id":54313873,"identity":"4141195f-b377-4c77-a03e-14eaee31dc01","added_by":"auto","created_at":"2024-04-08 17:30:11","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":303590,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure5d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/27885dcf19413e7541d8143a.tif"},{"id":54313824,"identity":"cd5387bc-a6e4-4e63-bd0f-9fa1744d211d","added_by":"auto","created_at":"2024-04-08 17:30:10","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":683536,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure6d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/927f725deff59019dc9f0614.tif"},{"id":54313823,"identity":"a589ac2e-6220-4e0f-8125-0a66836a9025","added_by":"auto","created_at":"2024-04-08 17:30:10","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":517688,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure7d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/161a70792b3d0496b1c55a0e.tif"},{"id":54314628,"identity":"da94e04f-9747-4434-bf27-efb8639761d0","added_by":"auto","created_at":"2024-04-08 17:38:10","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":549978,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure8d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/b0989061e8e9f3a53b397309.tif"},{"id":54313815,"identity":"7f39d5ec-5b48-43e6-a277-7763f950f10f","added_by":"auto","created_at":"2024-04-08 17:30:09","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":402628,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure9d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4206490/v1/22f215b79a9c2499f0fe36e5.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"APG-1252 combined with Cabozantinib inhibits hepatocellular carcinoma through MEK/ERK and CREB/Bcl-xl pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) accounts for about 90% of primary liver cancers, and the main known risk factors associated with HCC are viruses (chronic hepatitis B and C), metabolism (diabetes and non-alcoholic fatty liver disease or nonalcoholic fatty liver disease (NAFLD)), toxicity (alcohol and aflatoxin), and immune system-related disorders(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Globally, the incidence of HCC in middle-aged people aged 30\u0026ndash;59 years has decreased significantly due to the successful implementation of the hepatitis B virus (HBV) vaccination program(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, overall morbidity and mortality of HCC continue to rise, and the global mortality rate of HCC is expected to increase by another 41% by 2040 due to the increased incidence of NAFLD due to the obesity pandemic(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). When HCC is diagnosed early or the tumor size\u0026thinsp;\u0026lt;\u0026thinsp;5 cm, liver transplantation and surgical resection are treatment options for HCC(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, due to the insidious onset of HCC, the lack of specificity of the clinical manifestations, and the lack of early markers of specificity, most patients with HCC have progressed to advanced stages by the time of diagnosis. Advanced HCC is treated with RFA, TACE, TKI, and immunotherapy, but these modalities do not significantly prolong life as the occurrence of treatment resistance and disease recurrence(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Therefore, exploring new combination drug regimens is an urgent clinical problem for HCC patients.\u003c/p\u003e \u003cp\u003eCabozantinib is a tyrosine kinase inhibitor that targets VEGFR-1, VEGFR-2 and VEGFR-3, MET and AXL(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In a randomized, double-blind, placebo-controlled phase 3 clinical trial, overall survival and progression-free survival were longer in the Cabozantinib group than in the placebo group in patients with previously treated advanced HCC(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Bcl-2 family of proteins includes pro-apoptotic proteins and anti-apoptotic proteins(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The structure of Bcl-2 family proteins contains a short conserved sequence, known as the Bcl-2 homology (BH) domain(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Anti-apoptotic proteins include Bcl-2, Bcl-xl, Mcl-1, Bcl-w, and Bfl-1, which can inhibit cell apoptosis, and tumor cells can evade apoptosis by upregulating one or more anti-apoptotic proteins(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Pro-apoptotic proteins are divided into two types, BH3-only proteins and effector proteins(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). BH3-only proteins contain only one BH3 domain, including Bim, Bid, Puma, Bad, Noxa, Bik, Bmf, and Hrk(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Bim, Bid, and Puma can directly activate effector proteins, while other BH3-only proteins can bind to anti-apoptotic proteins, thus inhibiting the binding of anti-apoptotic proteins to Bim, Bid, and Puma, and activating effector proteins(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Effector proteins include Bax and Bak, which can initiate cell apoptosis by forming mitochondrial outer membrane permeabilization (MOMP) complexes(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The balance between the anti-apoptotic proteins and pro-apoptotic proteins of the Bcl-2 protein family can inhibit or activate MOMP and ultimately determine the fate of the cell(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the crucial role of Bcl-2 family proteins in cell apoptosis, drugs targeting Bcl-2 family proteins have been developed. The first specific small molecule inhibitor of the Bcl-2 family, ABT-737, targets Bcl-2, Bcl-xl, and Bcl-w. It inhibits the binding of anti-apoptotic proteins to BH3-only proteins, thereby inducing tumor cell apoptosis(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). ABT-263 has a high affinity for Bcl-2 family anti-apoptotic proteins (i.e., Bcl-xl, Bcl-2, Bcl-w) and shows better oral bioavailability(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). It can bind to anti-apoptotic proteins, releasing the apoptotic effector proteins Bax and Bak from the anti-apoptotic proteins, thereby inducing cell apoptosis(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Previous studies have found that the combination of ABT737 and sorafenib can inhibit tumor growth in hepatocellular carcinoma(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). ABT263 can sensitize the anti-tumor effect of sorafenib in hepatocellular carcinoma(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). As for the combined effect of Bcl-2 family proteins and cabozantinib, there are currently no research reports.\u003c/p\u003e \u003cp\u003eThe novel small molecule Bcl-2/Bcl-xl dual-target inhibitor APG-1252 used in our laboratory has shown monotherapy effectiveness in gastric cancer, nasopharyngeal carcinoma, and acute myeloid leukemia (AML)(\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). APG-1252 is a novel BH3 mimetic that specifically binds the hydrophobic pocket of Bcl-2 or Bcl-xl. It is converted to its active metabolite APG-1252-M1 in vivo, which then exerts potent antitumor effects. APG-1252 has shown single-agent effectiveness in gastric cancer, nasopharyngeal carcinoma, and acute myeloid leukemia (AML), however, its antitumor effects in hepatocellular carcinoma and its possible combination regimen have not been reported in studies.\u003c/p\u003e"},{"header":"Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCells culture\u003c/h2\u003e \u003cp\u003eThe human HCC cell lines Huh7, PLC/PRF/5, SK-Hep-1, HLE, HLF, BEL7402, Hep3B were purchased from Cobioer Biosciences Co. LTD (Nanjing, China). Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco Life Technologies) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) was used to culture Huh7, SK-Hep-1, HLE, HLF, Hep3B cells in a humidified incubator containing 5% CO2 at 37°C, and PLC/PRF/5, BEL7402 were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 IU/mL penicillin and 100 mg/mL streptomycin and in a humidified atmosphere containing 5% CO2 at 37 ◦C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRegents and antibodies\u003c/h2\u003e \u003cp\u003eAPG-1252 and APG-1252-M1 were kindly provided by Ascentage Pharma Group Inc (Taizhou, China). For the in vitro experiments APG-1252M1 was dissolved in dimethyl sulfoxide (DMSO) at 10 µM and kept at − 20°C. For the in vivo experiments, APG-1252 was dissolved in 10% polyethylene glycol 4000 (PEG400)/5% Castor oil ethoxylated (EL)/85% phosphate-buffered saline (PBS). Cabozantinib was purchased from TargetMol and z-VAD-fmk was purchased from Selleck Chemicals (Houston, TX, USA). All compounds were dissolved in dimethylsulfoxide (DMSO; Sigma Aldrich, MO, USA) at a stock concentration of 40 mM, stored at − 20 ◦C. The final concentration of DMSO to dilute compound in culture media did not exceed 0.1%. The antibodies against Bcl-2, Bcl-xl, Mcl-1, Bad, Bim, Puma, Noxa, CREB, p-CREB, ERK, p-ERK and p-MEK were purchased from Cell Signaling Technology (MA, USA). The antibody against GAPDH was purchased from ABGENT (San Diego, USA). The secondary anti-mouse and antirabbit antibodies were purchased from Santa Cruz Biotechnology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation detection\u003c/h2\u003e \u003cp\u003eCell viability was determined using Cell Counting Kit-8 purchased from EZ Bioscience (Beijing, China) according to the manufacturer’s instructions. Briefly, HCC cells were seeded at 3000 to 4000 cells/well in 96-well plates for 72 h in the presence of DMSO, APG-1252, Cabozantinib or combination therapy of APG-1252 and Cabozantinib. After 72 h, CCK-8 reagent (10 µL/well) was added and incubated at 37◦ C for 1–2 h and their absorbance readings were taken at 450 nm. Their IC50 values were calculated by using GraphPad Prism version 6.0.0 (GraphPad Software, San Diego, California USA) for Windows.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eHCC cells were seeded in a 6-well plate at approximately 500 cells/well and were treated with a single drug or combination of drugs, a negative control group was added with an equal volume of DMSO. Fresh culture medium was replaced every 3–4 days. After 14 days, the cells were fixed in methanol and stained with 0.5% crystal violet for 15 min at room temperature, after which the number of colonies were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eThe HCC cells (8 × 104 cells) were suspended in 200 µL medium containing no FBS with indicated drugs and were then aspirated into a transwell chamber (PC membrane, pore size 8.0 µm, Corning, NY, USA). The transwell chamber was placed in a 24-well plate containing 750 uL of 50% FBS culture medium. After 24–30 h, the chamber was taken out, the inner membrane of the chamber was washed with a cotton swab to remove adherent cells, and then fixed in methanol and stained with 0.5% crystal violet for 15 min. Then, the chambers were dried at room temperature and imaged using a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eWound healing assays were applied to test cell migration ability. HCC cells were seeded in six-well plates and fused to 100%. Then, a 200-µL pipette tube was used to create an artificial wound. Fresh medium containing 1% FBS with indicated chemicals was added. The wound closure was photographed immediately and 24 h later under a microscope. The Image J software was used to calculate the area between cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis assays\u003c/h2\u003e \u003cp\u003eHCC cells were plated at 1 × 105 cells/well in 12-well plates and treated with indicated chemicals for 72 h. The cells were then collected and stained with Annexin V-FITC/PI according to the instructions of the Apoptosis Detection Kit purchased from Sizhengbo Biotechnology (Beijing, China). Cell apoptosis were detected using an ACEA NovoCyte™ flow cytometer (ACEA Biosciences Inc. China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCells were treated with DMSO and indicated dose of drugs for 24 h, and then harvested and washed once with pre-cooled PBS. The cells were lysed on ice using a cell lysis buffer (#9803) purchased from Cell Signaling Technology (MA, USA), containing 1% protease inhibitor (PMSF), 1% phosphokinase inhibitor for 30 min, centrifuged at 12000 rpm for 15 min at 4 ◦C, and the supernatant protein lysate was collected. Protein concentration was measured by a Pierce BCA Protein Assay kit (Thermo Scientific). Cellular protein lysates were separated using 8–12% SDS-PAGE gel and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% BSA buffer for 1 h at room temperature and then incubated with indicated primary antibody at 4 ◦C overnight. 1xTBST (washing buffer) washing the membrane 3 times with 10 min each time. The protein membrane was incubated with secondary antibody for 1 h at room temperature and washed with 1xTBST for 3 times for 10 min each time. Signal generation and detection were performed using an ECL chemiluminescence hypersensitive colorimetric kit and a chemiluminescent imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProteome profiler analysis\u003c/h2\u003e \u003cp\u003eThe Human Phospho-Kinase Array Kit (#ARY003C, R\u0026amp;D Systems, Wiesbaden-Nordenstadt, Germany) was applied to detect the levels of 43 specific kinase phosphorylation sites in Huh7 cells with different treatments. For the preparation of total protein extracts, 1x106 /mL Huh7 cells were seeded in 6 cm dish. After 24 h, the cells were treated DMSO, 1 µM APG-1252, 2 µM Cabozantinib and combination treatment for 24 h. The preparation of cellular extracts and the proteome profiling were carried out according to the manufacturer’s instructions. Briefly, the membranes were incubated with blocking buffer before the experiment. The cell lysates were diluted in blocking buffer with a total protein amount of 600 µg then incubated overnight with detected membranes. After several washing steps, the membranes were incubated in the provided detection antibody cocktail for 2 h, then washed again and incubated for 30 min in streptavidin-horseradish peroxidase (HRP). The unbound HRP antibody was washed away, and then the signal was analyzed with a chemiluminescent substrate and detected with an X-ray array.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiment\u003c/h2\u003e \u003cp\u003eHuh7 xenograft models were used to evaluate the anti-tumor effects of APG-1252 and Cabozantinib monotherapy and combination in vivo. Four-week-old female BALB/c athymic nude mice were purchased from Beijing Vital River Laboratory Technology Co. Ltd. Huh7 cells (5 × 106) suspended in 100 µL cold PBS were subcutaneously injected into the dorsal flank of the mice. When the tumor volume reached approximately 100 mm3, the mice were randomly assigned into different groups. For the APG-1252-treated mice, 50 mg/kg of APG-1252 was injected via the caudal vein twice a week. For the Cabozantinib-treated mice, 50 mg/kg of Cabozantinib was delivered orally twice a week. Tumor sizes and animal weights were recorded twice per week and tumor volumes were calculated as V (mm3) = 1/2 × (length × width2). All animal experiments were performed under the guidance of Sun Yat-Sen University Committee for Use and Care of Laboratory Animals and were approved by the animal experimentation ethics committee. Animal studies are also reported in compliance with the ARRIVE guidelines(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and with the recommendations made by the British Journal of Pharmacology(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Statistical analyses were performed in GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, California USA). Unless indicated, all results were presented as mean ± SD of three independent experiments. Differences between two groups were analyzed using unpaired sample t-test. Comparison among more than two groups was analyzed by One-way ANOVA and Two-way ANOVA. p \u0026lt; 0.05 was considered as statistically significant. Combination Index (CI) was calculated using the CalcuSyn software (BIOSOFT, MO, USA). The criteria for the CI value are as follows: CI \u0026gt; 1 for antagonism effect, CI = 1 for additive effect, 0.8 ≤ CI \u0026lt; 1 for low synergy, 0.6 ≤ CI \u0026lt; 0.8 for moderate synergy, 0.4 ≤ CI \u0026lt; 0.6 for high synergy, and 0.2 ≤ CI \u0026lt; 0.4 for strong synergy effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" type=\"Results\" class=\"Section2\"\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003ch2\u003eAPG-1252 elicits antitumor effect on HCC cell lines\u003c/h2\u003e\u003cp\u003eFirstly, we analyzed the expression levels of Bcl-2 and Bcl-xl in hepatocellular carcinoma tissues in the TCGA database and found that the gene expression levels of Bcl-2 and Bcl-xl in hepatocellular carcinoma tissues were higher than those in normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B), and high expression of Bcl-xl was associated with poor prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), further suggesting the possibility of Bcl-xl as a therapeutic target for HCC. Next, to study the sensitivity of hepatocellular carcinoma cell lines to the Bcl-2/Bcl-xl inhibitor APG-1252, we detected the basic expression of Bcl-2 family proteins and the IC 50 value of APG-1252 in eight hepatocellular carcinoma cell lines (Supplementary Fig.\u0026nbsp;1A-C). The IC50 of APG1252-M1 is relatively low in Hep3B and BEL-7402, which have high levels of Bcl2 protein expression, while the IC50 of APG1252-M1 is lowest in PLC/PRF/5, which has high levels of Bcl-xl protein expression (supplementary Fig.\u0026nbsp;4A). To further explore the effect of APG-1252 on the proliferation of hepatocellular carcinoma cell lines, we selected three hepatocellular carcinoma cell lines (Huh7, PLC/PRF/5, SK-Hep-1) for cell colony formation experiments, and the results showed that as the drug concentration increased, the number of cell colonies gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, G-I). This indicates that APG-1252 can inhibit the proliferation of hepatocellular carcinoma cell lines in a concentration-dependent manner. Considering that hepatocellular carcinoma is prone to metastasis, we used the wound healing assay to detect the changes in the migration ability of hepatocellular carcinoma cell lines after 12 hours of APG-1252 drug treatment. The results showed that as the drug concentration increased, the speed of scratch healing gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, J-L). This suggests that APG1252 can inhibit the migration of hepatocellular carcinoma cells in a concentration-dependent manner. The above results indicate that APG1252 alone can inhibit the proliferation and migration ability of hepatocellular carcinoma cells.\u003c/p\u003e\u003cp\u003eNext, we used Annexin V/PI staining to analyze the effect of APG-1252 on the apoptosis of hepatocellular carcinoma cell lines. The results showed that as the drug action time prolonged, the percentage of apoptotic cells gradually increased (Figure A, C-E); as the drug concentration increased, the percentage of apoptotic cells gradually increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, F-H). Then we also observed that APG-1252 treatment could significantly increase the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-PARP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J, supplementary Fig.\u0026nbsp;4B-C, supplementary Fig.\u0026nbsp;5A-B). This indicates that APG-1252 can promote the apoptosis of hepatocellular carcinoma cell lines in a concentration-dependent and time-dependent manner.\u003c/p\u003e\u003cp\u003e \u003cb\u003eAPG-1252 combined with Cabozantinib exerts synergistic antitumor effects in hepatocellular carcinoma in vitro.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eSorafenib is the first targeted drug proven effective in patients with advanced liver cancer and has been the standard treatment for over a decade(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Lenvatinib was subsequently approved, further consolidating the role of multi-kinase inhibitors in the first-line treatment of advanced hepatocellular carcinoma(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, the therapeutic effects of both drugs are far from satisfactory. Compared with placebo, sorafenib only has a survival advantage of 2.8 months in liver cancer patients(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Although it has a high response rate, Lenvatinib only shows non-inferiority compared to sorafenib, and the extension of overall survival is limited(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In previous studies, Bcl-xl was found to be upregulated in tumor tissues of HCC patients(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and overexpression of Bcl-xl was associated with poor overall survival and progression-free survival after surgical resection of tumors in HCC patients(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Through analysis of the TCGA database, we found that the main targets of Cabozantinib, VEGFR1, 2, 3, have a strong positive correlation with the expression of Bcl-2 and Bcl-xl (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F). This suggests that the combination of APG-1252 and Cabozantinib may have a synergistic anti-tumor effect in hepatocellular carcinoma. Therefore, we began to explore the anti-tumor effect of APG-1252 combined with Cabozantinib in hepatocellular carcinoma in vitro. First, we used the CCK8 method to detect the IC50 of Cabozantinib in seven hepatocellular carcinoma cell lines (supplementary Fig.\u0026nbsp;2A-B). Then, we evaluated the effect of APG-1252 combined with Cabozantinib on the growth and proliferation of hepatocellular carcinoma cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I, supplementary Fig.\u0026nbsp;2C-F). The results showed that the combination of APG-1252 and Cabozantinib for 72 hours can significantly inhibit cell proliferation in PLC/PRF/5, Huh7, HepG2 three cell lines, and can reduce cell viability by more than 50% at lower concentrations; while in HLE, SK-Hep-1, BEL7402, HLF, Hep3B cell lines, significant inhibitory effect on cell proliferation can be observed at higher drug concentrations after combination therapy. At the same time, the combination index CI value also shows that APG-1252 and Cabozantinib combined have a significant synergistic anti-tumor effect in hepatocellular carcinoma cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003eNext, we selected PLC/PRF/5, Huh7, SK-Hep-1 three cell lines to use the plate clone experiment to detect the effect of APG-1252 combined with Cabozantinib on the long-term growth of hepatocellular carcinoma cells. The experimental results showed that the formation of cell colonies in the combination group significantly decreased after 14 days of drug action (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). At the same time, we used CCK8 to detect the effect of APG-1252 combined with Cabozantinib on the inhibition of tumor cell growth in hepatocellular carcinoma cells PLC/PRF/5, Huh7, SK-Hep-1 at different times. The experimental results showed that as the drug action time prolonged, the vitality of the cells in the combination group gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). These results indicate that the combination of APG-1252 and Cabozantinib can significantly inhibit the proliferation of hepatocellular carcinoma cell lines in vitro, and this inhibitory effect was time-dependent, thus exerting a synergistic anti-tumor effect.\u003c/p\u003e\u003cp\u003eThen, we used Annexin V/PI staining to analyze the effect of the combination of the two drugs on cell apoptosis. We selected PLC/PRF/5, Huh7, SK-Hep-1 three cell lines to treat with corresponding concentrations and times to detect cell apoptosis by flow cytometry. The results showed that in Huh7 cells, after 72 hours of drug treatment, the apoptosis rate of APG-1252 single drug group and Cabozantinib single drug group were 28.93% and 7.12% respectively, while the apoptosis rate of the combination group significantly increased to 89.75%, and with the prolongation of drug action time, the apoptosis rate of the combination group cells significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B); at the same time, we also observed similar results in PLC/PRF/5 and SK-Hep-1 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D, S3A-B). These results indicate that the combination of APG-1252 and Cabozantinib can significantly promote tumor cell apoptosis, and this effect shows a significant time dependence.\u003c/p\u003e\u003cp\u003eWestern Blot was used to detect the effect of APG-1252 and Cabozantinib on the expression of Cleaved-PARP and Cleaved-caspase3 in three cell lines after 72 hours of combined treatment. The experimental results showed that the expression of Cleaved-PARP and Cleaved-caspase3 in the combination group was significantly up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, supplementary Fig.\u0026nbsp;6A, C, E). At the same time, the changes of apoptosis-related proteins in tumor cells were also detected at different drug action times. The experimental results showed that with the prolongation of drug action time, the expression of Cleaved-PARP and Cleaved-caspase3 gradually increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, supplementary Fig.\u0026nbsp;6B, D, F), indicating that the combination of APG-1252 and Cabozantinib in hepatocellular carcinoma cell lines can significantly up-regulate the expression of apoptosis proteins Cleaved-PARP and Cleaved-caspase3 in a time-dependent manner.\u003c/p\u003e\u003cp\u003e \u003cb\u003eAPG-1252 combined with Cabozantinib inhibited the metastasis and invasion of hepatocellular cancer cells in vitro.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eDue to the high propensity of hepatocellular carcinoma to metastasize, we further tested the effect of APG-1252 combined with Cabozantinib on the migration and invasion capabilities of hepatocellular carcinoma cells in PLC/PRF/5, Huh7, and SK-Hep-1 cells using Transwell experiments. The results showed that the number of cells migrating and invading after combined medication was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-H). This suggests that APG-1252 combined with Cabozantinib exerts a synergistic anti-tumor effect in hepatocellular carcinoma by inhibiting cell migration and invasion capabilities.\u003c/p\u003e\u003cp\u003eThe reason for tumor cell metastasis and invasion is often due to epithelial-mesenchymal transition (EMT), the hallmark of which is the reduction in the expression of cell adhesion molecules (such as E-cadherin), and the transformation of the cytokeratin cytoskeleton to a vimentin-based cytoskeleton. Therefore, we used Western Blot to detect changes in the expression levels of EMT-related proteins in PLC/PRF/5, Huh7, and SK-Hep-1 cells after 24 hours of drug treatment. The experimental results showed that the expression levels of E-cadherin and ZO-1 in the combined drug group were significantly upregulated, while the expression levels of Vimentin and α-catenin were significantly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, supplementary Fig.\u0026nbsp;7). This suggests that APG-1252 combined with Cabozantinib affects the migration and invasion capabilities of hepatocellular carcinoma cells by affecting the expression levels of EMT-related proteins E-cadherin, ZO-1, Vimentin, and α-catenin.\u003c/p\u003e\u003ch2\u003eAPG-1252 combined with Cabozantinib exerts a synergistic anti-tumor effect in hepatocellular carcinoma in vivo\u003c/h2\u003e\u003cp\u003eTo further validate the synergistic antitumor effect of APG-1252 and Cabozantinib in hepatocellular carcinoma in vivo, we constructed a nude mouse xenograft model using Huh7 cells. The results showed that APG-1252 and Cabozantinib alone could inhibit the growth of hepatocellular carcinoma in vivo, while the combination group showed a synergistic effect in suppressing the tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). We next detected the expression of apoptosis proteins cleaved-PARP and cleaved-caspase-3 in the tumor tissues. The results showed that compared with the single drug group, the expression levels of cleaved-PARP and cleaved-caspase-3 in the combination group were significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, supplementary Fig.\u0026nbsp;8A). Furthermore, compared with other groups, the expression of cleaved-caspase-3 in the combination group was significantly increased by IHC assay, and the number of TUNEL positive cells in the combination group was significantly more than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These results suggest that APG-1252 combined with Cabozantinib exerted synergistic anti-tumor effect in hepatocellular carcinoma in vivo.\u003c/p\u003e\u003cp\u003e \u003cb\u003eThe synergistic anti-tumor effect of APG-1252 combined with Cabozantinib was mainly through inhibiting MRK/ERK and CREB/Bcl-xl pathways.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTo further explore the underlying mechanism of the synergistic anti-tumor effect of APG-1252 combined with Cabozantinib in hepatocellular carcinoma, we applied a phosphorylated protein array to investigate the potential molecules that may change (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The results showed that Cabozantinib single drug treatment can cause a significant increase in p-CREB level, while the expression level of p-CREB in the combination group of APG-1252 and Cabozantinib can return to the baseline level comparable to the control group. We further verified the expression level of p-CREB in PLC/PRF/5, Huh7 and SK-Hep-1 cells, the results showed that compared with APG-1252 and Cabozantinib single drug group, the expression level of p-CREB in the combination group was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, supplementary Fig.\u0026nbsp;8B, C, D).\u003c/p\u003e\u003cp\u003eAccording to previous studies, p-MEK/p-ERK can phosphorylate CREB(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), activating it to p-CREB, thus causing the transcription of downstream target genes Bcl-2/Bcl-xl(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). To further explore the changes in the expression of upstream and downstream proteins of p-CREB, we detected the expression of p-MEK, p-ERK, Bcl-2, Bcl-xl in HCC cells with indicated treatment. The results showed that the expression levels of p-MEK, p-ERK, Bcl-xl in the combination group of APG-1252 and Cabozantinib were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, supplementary Fig.\u0026nbsp;8B, C, D). However, the p-MEK, p-ERK in the Cabozantinib single drug group did not significantly increase, so we suggest that APG-1252 combined with Cabozantinib may be through inhibiting MEK/ERK pathway to exert synergistic anti-tumor effects.\u003c/p\u003e\u003cp\u003eTo further explore the changes in other proteins of the Bcl-2 family in PLC/PRF/5, Huh7, SK-Hep-1 three strains of cells after 72 hours of drug treatment, we used Western Blot to detect the expression of Bcl-2 family proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, supplementary Fig.\u0026nbsp;8B, C, D). The results showed that the expression level of Bax in the combination group of APG-1252 and Cabozantinib was significantly upregulated, while the expression level of Mcl-1 was significantly downregulated.\u003c/p\u003e\u003cp\u003eNext, we treated SK-Hep-1 cells with different concentrations of Cabozantinib and found that Cabozantinib decreased the level of p-CREB and Bcl-xl (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D, supplementary Fig.\u0026nbsp;9A-B). We also used the apoptosis inhibitor Z-VAD for recovery, and found that after adding the apoptosis inhibitor Z-VAD, the expression of apoptosis-related molecules Cleaved-PARP and Cleaved-caspase3 was significantly downregulated(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, supplementary Fig.\u0026nbsp;8B, C, D), although the level of p-CREB was upregulated to some extent, but compared with the Cabozantinib single drug group, the level of p-CREB was still reduced, these results indicate that the downregulation of p-CREB is not caused by apoptosis, but is caused by the combined action of APG-1252 and Cabozantinib. Therefore, p-CREB may be a key mechanism of the combined action of APG-1252 and Cabozantinib.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs the most common cause of cancer-related deaths globally, hepatocellular carcinoma is the only one among the top five most lethal cancers that sees an annual increase in incidence(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Although the relative survival rate of cancer patients in our country has continually improved compared to the past, there has been no significant progress in the diagnosis and treatment of hepatocellular carcinoma. Therefore, for patients with advanced hepatocellular carcinoma, pursuing combination drug therapy is a very promising treatment strategy. Cabozantinib was approved by the FDA in 2019 for second-line treatment of patients with advanced hepatocellular carcinoma who failed sorafenib treatment(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, due to the the multi-drug resistance, the effectiveness of single-drug therapy is limited. Bcl-2 family protein inhibitors ABT737 and ABT263 have been found to exert anti-tumor effects in hepatocellular carcinoma when combined with Sorafenib(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In previous studies, Bcl-xl was found to be upregulated in tumor tissues of HCC patients(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and overexpression of Bcl-xl was associated with poor overall survival and progression-free survival after surgical resection of tumors in HCC patients(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).Moreover, through the analysis of the TCGA database, we found a strong positive correlation between the main targets of Cabozantinib, VEGFR1, 2, 3, and the expression of Bcl-2 and Bcl-xl. This suggests that the combination of APG-1252 and Cabozantinib may have a synergistic anti-tumor effect in hepatocellular carcinoma. To seek more effective treatment strategies, this study combined the Bcl-2/Bcl-xl inhibitor APG-1252 and the tyrosine kinase inhibitor Cabozantinib to investigate their synergistic anti-tumor effects in hepatocellular carcinoma.\u003c/p\u003e \u003cp\u003eIn this study, we initially investigated whether the Bcl-2/Bcl-xl inhibitor APG-1252 could exert an anti-tumor effect on hepatocellular carcinoma in vitro and its potential mechanism. CCK8 and colony formation assay results indicated that APG-1252 could inhibit the proliferation of hepatocellular carcinoma cells in vitro. The flow cytometry apoptosis assay and Western Blot results showed that APG-1252 could significantly induce apoptosis in hepatocellular carcinoma cells, and this effect exhibited a clear time-dependence and dose-dependence. The subsequent wound healing assay results showed that APG-1252 could inhibit the migration of hepatocellular carcinoma cells in vitro.\u003c/p\u003e \u003cp\u003eNext, we investigated whether the Bcl-2/Bcl-xl inhibitor APG-1252 combined with Cabozantinib could exert a synergistic anti-tumor effect on hepatocellular carcinoma both in vitro and in vivo, and its potential mechanism. The in vitro CCK8 experiment and cell colony formation experiment results showed that APG-1252 combined with Cabozantinib could significantly inhibit tumor cell proliferation, demonstrating a synergistic anti-tumor effect. This provides potential for the clinical combination application of Cabozantinib and the Bcl-2/Bcl-xl inhibitor APG-1252 in hepatocellular carcinoma.\u003c/p\u003e \u003cp\u003eThen, we tried to explore the mechanism of the synergistic effect of APG-1252 combined with Cabozantinib in hepatocellular carcinoma. The flow cytometry apoptosis detection results showed that APG-1252 combined with Cabozantinib could significantly promote apoptosis in hepatocellular carcinoma cells, and this effect showed significant time-dependence. And the western Blot results also showed that in the combined drug group, the expression levels of cell apoptosis markers Cleaved-PARP and Cleaved-caspase3 were significantly upregulated, and this effect showed clear time-dependence.\u003c/p\u003e \u003cp\u003eNext, considering the characteristic of hepatocellular carcinoma that it is prone to metastasis, we used Transwell experiments to verify the effect of combined drug treatment on the migration and invasion ability of hepatocellular carcinoma cells. The experimental results showed that the number of tumor cells migrating and invading significantly decreased after combined drug treatment. Meanwhile, the results of Western Blot showed that the combination could cause changes in the expression of EMT-related proteins. The expression levels of E-cadherin and ZO-1 in the combination group were significantly upregulated, while the expression levels of Vimentin and α-catenin were significantly downregulated. These experimental results indicate that the combination exerts a synergistic anti-tumor effect by inhibiting the migration and invasion of hepatocellular carcinoma cells. We think that the changes in EMT-related markers might have an impact on the tumor microenvironment. And the data of this part is not covered in our study.\u003c/p\u003e \u003cp\u003eNext, we further elucidated the mechanism of the anti-tumor effect of APG-1252 combined with Cabozantinib through phosphoprotein chip experiments. The phosphorylated protein array results showed that the combination might exert a synergistic anti-tumor effect through p-CREB. cAMP-response-element-binding protein (CREB) is a transcriptional cofactor that triggers multiple transcriptional cascade responses and target gene expression(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Activation of CREB involves the reversible phosphorylation of serine residues located at 129 (S129), 133 (S133) and 142 (S142), which is triggered by multiple cellular effector kinases stimulated by growth factors or extracellular stresses(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). With the activation of phosphorylation at site 133, CREB binds to its coactivator CREB-binding protein (CBP) and is thus able to recruit additional transcriptional machinery elements to drive the malignantly progressive transcriptional program(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).The subsequent Western Blot results further indicated that APG-1252 combined with Cabozantinib might exert a synergistic anti-tumor effect through the MEK/ERK pathway and the CREB/Bcl-xl pathway. At the same time, our Western Blot experimental results showed that APG-1252 combined with Cabozantinib could further downregulate the expression level of anti-apoptotic protein Mcl-1 and upregulate the expression of pro-apoptotic protein Bax. Notably, the Western Blot experimental results showed that the p-CREB level significantly increased in the Cabozantinib treatment group, and according to previous research reports, the upregulation of CREB expression can inhibit the anti-tumor effect of Sorafenib in kidney cancer(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Therefore, we speculate that the increase of p-CREB level induced by Cabozantinib in hepatocellular carcinoma may inhibit the anti-tumor effect of Cabozantinib in hepatocellular carcinoma, but due to the limited length, we did not study and discuss this part of the content in the project.\u003c/p\u003e \u003cp\u003eMitogen-activated protein kinases (MAPKs) consist of three main subfamilies: the extracellular-signal regulated kinases (ERK), the c-jun N-terminal kinase or stress-activated protein kinases (JNK or SAPK), and MAPK14(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The ERK signaling pathway, including kinase RAS, RAF, MEK, and ERK, is a three- or four-layer phosphorylation cascade that transmits upstream signals from membrane receptors to a series of downstream effector substrates(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Extracellular signaling proteins bind to specific cell surface receptors, such as cytokine receptors, receptor tyrosine kinase (RTK), and G protein-coupled receptors, and activate a series of signaling cascades involving RAS, RAF, and MEK(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Activated MEK can phosphorylate the conserved threonine and tyrosine residues within the activation loop of ERK, which then modulate other protein kinases and transcription factors involved in cell proliferation, cell survival, cell migration, and cell differentiation(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The Western Blot results further indicated that APG-1252 combined with Cabozantinib might exert a synergistic anti-tumor effect through the MEK/ERK pathway.\u003c/p\u003e \u003cp\u003eFinally, further verification in the in vivo mouse tumor model showed that the combined application of APG-1252 and Cabozantinib in hepatocellular carcinoma mice could significantly inhibit the growth of mouse tumors, indicating the good application effect of this drug combination. At the same time, this combination had no significant effect on the weight of the mice, which indicates the safety of the combined application of APG-1252 and Cabozantinib in hepatocellular carcinoma.\u003c/p\u003e \u003cp\u003eOur research indicates that the combined application of APG-1252 and Cabozantinib in vitro and in vivo can exert a strong synergistic anti-tumor effect in hepatocellular carcinoma. The mechanism of this synergistic anti-tumor effect is primarily through promoting apoptosis of hepatocellular carcinoma cells, inhibiting the proliferation, migration, and invasion of hepatocellular carcinoma cells. At the same time, our experimental results also show that the combined application of APG-1252 and Cabozantinib in hepatocellular carcinoma exerts a synergistic anti-tumor effect by inhibiting the MEK/ERK pathway and the CREB/Bcl-xl pathway, and also exerts an anti-tumor effect by upregulating the pro-apoptotic protein Bax and downregulating the anti-apoptotic protein Mcl-1. As for the pathway through which APG-1252 combined with Cabozantinib inhibits the MEK/ERK pathway, due to time and space constraints, we did not expand on this part of the research; likewise, for the changes in the upstream kinases of CREB/Bcl-xl, our current research has not found any significant changes, which can be further researched. This study provides new ideas and strategies for the treatment of hepatocellular carcinoma, and further clinical trials are needed in the future to verify the effectiveness and safety of this combined therapy.\u003c/p\u003e \u003cp\u003eHowever, our research still has some limitations. Firstly, the effect of the combination of APG-1252 and Cabozantinib on the proliferation, migration, and invasion abilities of hepatocellular carcinoma cells may also be due to its promotion of apoptosis. We did not further verify this phenomenon in the experiment, which is a flaw in the design of our study and an area for improvement in future research. Secondly, the mechanism of the combined action of APG-1252 and Cabozantinib in hepatocellular carcinoma still needs more in-depth research. Thirdly, we need to further verify the role of CREB/Bcl-xl in clinical patient samples, to provide more definite theoretical basis and support for clinical application.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHCC: hepatocellular carcinoma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHBV: hepatitis B virus.\u003c/p\u003e\n\u003cp\u003eNAFLD: nonalcoholic fatty liver disease.\u003c/p\u003e\n\u003cp\u003eMAPKs: Mitogen-activated protein kinases.\u003c/p\u003e\n\u003cp\u003eERK: extracellular-signal regulated kinases.\u003c/p\u003e\n\u003cp\u003eJNK: c-jun N-terminal kinase.\u003c/p\u003e\n\u003cp\u003eSAPK: stress-activated protein kinases.\u003c/p\u003e\n\u003cp\u003eRTK: receptor tyrosine kinase.\u003c/p\u003e\n\u003cp\u003eCREB: cAMP-response-element-binding protein.\u003c/p\u003e\n\u003cp\u003eCBP: CREB-binding protein.\u003c/p\u003e\n\u003cp\u003eAML: acute myeloid leukemia.\u003c/p\u003e\n\u003cp\u003eMOMP: mitochondrial outer membrane permeabilization.\u003c/p\u003e\n\u003cp\u003eVEGFR2: vascular endothelial growth factor receptor.\u003c/p\u003e\n\u003cp\u003eVEGFR3: vascular endothelial growth factor receptor.\u003c/p\u003e\n\u003cp\u003eEMT: Epithelial-Mesenchymal Transition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (NSFC grant numbers 82073377, 82003268, and 81772587); the Natural Science Foundation of Guangdong (grant number 2021A1515012439); the Science and Technology Program of Guangdong (grant number 2019B020227002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDJ. Y has an ownership interest (including patents) in Ascentage Pharma Group Corp. Limited. WT.P. is an employee of Ascentage Pharma Group Corp. Limited. All other authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTian Di,Qiu-yun Luo performed the in vitro experiments, in vivo experiments and wrote the main manuscript. Jiang-tao Song analyzed research data. Xiang-lei Yan assisted with experiments in vivo, and prepared Fig. 6. Lin Zhang is involved in discussion. Wen-tao Pan is involved in discussion. Fei-teng Lu contributed reagents or other essential material. Yu-ting Sun contributed reagents or other essential material. Zeng-fei Xia contributed reagents or other essential material. Li-qiong Yang contributed reagents or other essential material.Miao-zhen Qiu concept and supervised. Da-jun Yangprovide the funding acquisition. Jian Sun oncept and supervised. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding authors upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRinaldi L, Guarino M, Perrella A, Pafundi PC, Valente G, Fontanella L, et al. Role of Liver Stiffness Measurement in Predicting HCC Occurrence in Direct-Acting Antivirals Setting: A Real-Life Experience. 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Nat Cell Biol. 2009;11(12):1458-64.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, Apoptosis, APG-1252, Bcl-2/Bcl-xL inhibitor, Cabozantinib","lastPublishedDoi":"10.21203/rs.3.rs-4206490/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4206490/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Purpose\u003c/h2\u003e \u003cp\u003eLiver cancer is the fourth leading cause of cancer-related death worldwide, and hepatocellular carcinoma (HCC) is the most common primary liver cancer. APG-1252 is a small molecule inhibitor of Bcl-2/Bcl-xl, and the anti-tumor effect of APG-1252 in HCC, or its anti-tumor effects in combination with cabozantinib, has not been researched.\u003c/p\u003e\u003ch2\u003eExperimental Approach:\u003c/h2\u003e \u003cp\u003eTCGA database analysis was used to analysis the gene expression levels of Bcl-2 and Bcl-xl in HCC tissues. Western Blot was used to detect the proteins\u0026rsquo; expression level. And the inhibitory effects of APG-1252 and Cabozantinib on the proliferation of HCC cell lines was detected by CCK-8. The effect on the migration and invasion of HCC cells was verified by Transwell assay. Huh7 xenograft model in nude mice was used to detect the combined effect in vivo.\u003c/p\u003e\u003ch2\u003eKey Results:\u003c/h2\u003e \u003cp\u003eWe found that APG-1252 monotherapy could inhibit the proliferation and migration of HCC cells and promote apoptosis of HCC cells. APG-1252 combined with Cabozantinib could inhibit the proliferation, migration and invasion of HCC cells and promote the apoptosis of hepatocellular carcinoma cells and exerted synergistic effect in vivo. The combination could significantly downregulate MEK/ERK phosphorylation levels. Besides, the treatment of Cabozantinib could cause the protein level of phosphorylation CREB and BCL-XL increased, while combined with APG-1252 could impair this effect.\u003c/p\u003e\u003ch2\u003eConclusion and Implications:\u003c/h2\u003e \u003cp\u003eOur data suggest that APG-1252 in combination with Cabozantinib can provide more effective treatment strategies for HCC patients and deserve further clinical investigation.\u003c/p\u003e","manuscriptTitle":"APG-1252 combined with Cabozantinib inhibits hepatocellular carcinoma through MEK/ERK and CREB/Bcl-xl pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 17:30:03","doi":"10.21203/rs.3.rs-4206490/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd60b2f9-7fdd-49b9-968a-2eaa27ee89cb","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-05T02:25:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-08 17:30:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4206490","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4206490","identity":"rs-4206490","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}