The Role And Molecular Mechanism Of Apatinib Combined With RG7112 In Inducing Hepatocellular Carcinoma Apoptosis Through Endoplasmic Reticulum Stress

Objective: Hepatocellular carcinoma(HCC),one of the most common malignancies in the world,is characterized by insidious onset,easy metastasis and recurrence,and there is no effective treatment for it.Apatinib,a domestic small molecule anti-angiogenic targeted drug,has been proven to be effective and safe in the treatment of HCC.However,patients often experience various adverse reactions and are prone to drug resistance during the course of taking the drug,which greatly limits the clinical application of Apatinib.RG7112 is the first MDM2 inhibitor approved for clinical trials,which activates p53 by binding to MDM2,thereby inhibiting tumor growth.Endoplasmic reticulum stress(ERS)plays an important role in the restoration of endoplasmic homeostasis,cell survival,and cell death.The ERS is mainly regulated by PKR-like endoplasmic reticulum kinase(PERK),inositolrequiring enzyme 1α(IRE-1α),activating transcription factor-6(ATF-6),binding immunoglobulin protein(Bi P),Calnexin and other proteins.In preliminary experiments,we found that Apatinib in combination with RG7112 could exert a synergistic effect.The combination of the two drugs can induce apoptosis by altering the ERS pathway,and significantly inhibiting HCC.Methods: Part I.In vitro culture the human HCC cell lines HEPG2 and QGY7701,human normal hepatocytes HHL-5,to investigate the effect and molecular mechanism of Apatinib combined with RG7112 on HCC inhibition at the cellular level.A mouse xenograft tumor model was constructed using HEPG2 to verify the inhibitory effect of the combination of the two drugs on HCC at the animal level.The effect of Apatinib combined with RG7112 on cellular activity was examined using Cell Counting Kit-8 kit.Hoechst 33258 staining solution was used to observe the alteration of cell nuclei by the combination of the two drugs.Flow cytometry was used to quantify the effect of Apatinib in combination with RG7112 to induce apoptosis.The levels of apoptosis-related protein expression in cells and tissues were detected using Western blotting.The effect of the combination of the two drugs on the metastatic ability was examined by transwell metastasis assay.The effect of Apatinib combined with RG7112 on cell invasion ability was examined by transwell invasion assay.The effect of Apatinib combined with RG7112 on the wound healing ability of HCC cells was examined by scratch assay.The effect of the combination of the two drugs on the ability of cell clone formation was examined by colony formation assay.The amount of epithelialmesenchymal transition(EMT)related protein expression in the cells was examined by using Western blotting.Part II.Human HCC cell lines HEPG2 and QGY7701 were cultured in vitro to investigate the mechanism of Apatinib combined with RG7112 to induce apoptosis through the endoplasmic reticulum stress pathway.Endoplasmic reticulum fluorescence probe was used to observe the alteration of endoplasmic reticulum by Apatinib combined with RG7112.Use calcium fluorescence probe to observe the alteration of intracellular calcium ion by the combination of two drugs.The expression levels of ERS-related proteins in cells were detected using Western blotting.Inhibition of ERS in cells using the small molecule inhibitor 4-PBA to assess the effect of ERS inhibition on apoptosis induced by RG7112 in combination with Apatinib.The expression level of PERK,a key protein of ERS,was down-regulated using PERK si RNA to assess the effect of PERK expression down-regulation on apoptosis induced by the combination of the two drugs.Results: Part I.Apatinib combined with RG7112 significantly reduced the cellular activity of HCC in a time-dependent manner compared with Apatinib or RG7112 alone.Apatinib combined with RG7112 acting on HCC cell lines QGY7701 and HEPG2 significantly induced apoptotic changes in the nucleus.The results of flow cytometry showed that the percentage of apoptotic cells was significantly increased after Apatinib combined with RG7112 treatment compared with Apatinib or RG7112 alone,and had a time-dependent effect.Western blotting results showed that p53 was significantly activated after the combination of the two drugs compared with the control and alone treatment groups.And the downstream anti-apoptotic proteins Bcl-2,Survivin and Mcl-1 were significantly downregulated,while the pro-apoptotic proteins Cleaved PARP,Bak,Bax,Bad and were significantly upregulated.This suggests that Apatinib combined with RG7112 can induce activation of endogenous apoptotic pathway through activation of p53.Moreover,the growth of xenograft tumors in the Apatinib combined with RG7112-treated group was significantly inhibited.And the expression of p53,Cleaved Caspase-3 and Cleaved PARP in the tumor tissues were significantly increased compared with the blank group,Apatinib alone group and RG7112 alone group.This suggests that Apatinib combined with RG7112 can also inhibit tumor growth by inducing apoptosis in vivo.The results of transwell metastasis assay and scratch healing assay showed that the combination of the two drugs could inhibit the migration ability of HCC cells.The results of transwell invasion assay showed that Apatinib combined with RG7112 could inhibit the invasive ability of the cells.The results of colony formation assay showed that the combination of Apatinib and RG7112 significantly inhibited the clonogenic ability of QGY7701 and HEPG2 compared with Apatinib or RG7112 alone.Western blotting results further confirmed that the epithelial marker E-Cadherin expression was significantly upregulated,while the expression of mesenchymal markers N-Cadherin and Vimentin was significantly downregulated in the combination groups compared with the control group and the treatment group alone.This suggested that the combination of Apatinib and RG7112 could inhibit the invasive and metastatic ability of cells by blocking EMT.The effect of the combination of the two drugs on normal human hepatocytes was evaluated by culturing normal human hepatocytes HHL-5 at effective concentrations.The results showed that the number and morphology of normal human hepatocytes HHL-5 did not change after treating the cells with the same drug concentration compared with QGY7701,and neither apoptosis-related protein Cleaved Caspase-3 nor Cleaved PARP was induced to activate.Part II.Staining of cells by endoplasmic reticulum fluorescent probe showed that Apatinib combined with RG7112 significantly activated the membrane potential of the endoplasmic reticulum,indicating the important role of ERS in the combination of the two drugs.The results of calcium fluorescence probe staining showed that the concentration of calcium ions in the cytoplasm increased significantly after treatment of cells with Apatinib and RG7112.This indicates that the combined treatment of cells with the two drugs will release large amounts of calcium ions from the endoplasmic reticulum to the cytoplasm,further confirming the important role of endoplasmic reticulum alterations in Apatinib combined with RG7112.Western blotting results showed that compared with Apatinib or RG7112 alone,Apatinib combined with RG7112 increased expression of ATF-4 and CHOP and decreased the expression of ATF-6,Bi P,Calnexin,IRE-1α,and PERK in a time-dependent manner.PERK plays an important role in ERS-dependent apoptosis.Direct inhibition of PERK expression using si RNA enhanced the apoptotic effect induced by Apatinib combined with RG7112.Conclusions: In short,treatment of HCC cells with Apatinib and RG7112 will induce ERS-dependent apoptosis by targeted the p53-PERK signaling pathway,and ultimately inhibiting the development of HCC.In addition,Apatinib combined with RG7112 could inhibit cell invasion,metastasis and clone formation ability by altering EMT,suggesting that the combination of the two drugs could reduce the invasiveness and metastasis of HCC.More importantly,the effective concentration of Apatinib in combination with RG7112 is not toxic to cultured normal human hepatocytes,indicating the safety of Apatinib in combination with RG7112 for HCC.In a word,this study reveals the effect and mechanism of the anti-angiogenic targeting drug Apatinib combined with the small molecule p53 activator RG7112 in the treatment of HCC,which may provide new ideas and methods for the treatment of HCC.