Dracohodin Perochlorate Induced Apoptosis Through Activating Endoplasmic Reticulum Stress In HCC

Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant tumor and ranked sixth worldwide,incidence of more than 840,000 new cases every year.Although a various methods were used for the treatment of HCC at present,including surgery,interventional therapy,ablation,chemical drug therapy and molecular targeted therapy,but the overall treatment effect is not satisfied,the five-year survival rate is18%,more than 780,000 death due to HCC each year.Therefore,how to improve the prognosis of HCC is an urgent problem to be solved.Surgical resection is the most basic clinical treatment for HCC,but due to its high degree of malignancy,hidden development and other characteristics,the vast majority of cases can not be performed radical surgery at the time of diagnosis,and postoperative recurrence and metastasis are easy to occur,so drug therapy has become an important means of anti-HCC treatment.Although there have been a variety of anti-HCC drugs represented by sorafenib,but the effect is not ideal,the development of new anti-HCC drugs has become the focus of researchers.Apoptosis is one of the most important ways of programmed cell death.It is an active death that occurs through an orderly and coordinated cellular program under physiological and pathological conditions,and its process is tightly regulated.Under normal physiological conditions,apoptosis can remove senile and abnormal cells,which is of great significance for evolution,maintenance of homeostasis and organ development.However,under certain pathological conditions,abnormal regulation of apoptosis can also lead to a variety of diseases,including tumors and neurodegeneration.According to the different pathways of apoptosis signal initiation,cell apoptosis is mainly divided into exogenous apoptosis and endogenous apoptosis,also known as death receptor apoptosis pathway and mitochondrial apoptosis pathway.Both of them ultimately perform apoptosis changes by activating Caspase-3 and other proteins through different cascading signal transduction.Inducing tumor cell apoptosis through various ways is an important way to achieve the purpose of anti-tumor,among which,the development of novel drugs directly acting on hepatocellular carcinoma cells and selectively inducing hepatocellular carcinoma cell apoptosis is an important direction for the research and development of anti-hepatocellular carcinoma drugs.Dracohodin perochlorate is a synthetic flavonoid like flavonoid and its medicinal value has been confirmed by many studies,including certain effects on tissue healing,wound repair and anti-diabetes.However,its antitumor effect has become the focus of its drug research.At present,some in vitro experiments have preliminarily confirmed that it has inhibitory effect on human cervical cancer,lung cancer and breast cancer in vitro,and the main mechanism of its anti-tumor effect is its induction of tumor cell apoptosis,however,the current of dracohodin perochlorate anti-tumor mechanism of action research is not thorough,and whether its existing in HCC and effective anticancer clearly confirmed that there is no research result,we will through the experiments in vivo and in vitro to explorting its anticancer effect and mechanism,which could be benefit for the development of novel anti-HCC drug.Methods:1.MTT assay,Hoechst33342,and flow cytometry were used to detect the effects of dracohodin perchlorate on HCC cells and normal liver cells.2.The morphologic changes of organelles in hepatocellular carcinoma cells were observed by electron microscopy under the action of dracohodin perchlorate.3.The transcriptome sequencing was used to analyze the changes in the transcriptional level of HCC cells under the action of dracohodin perchlorate.4.MTT assay,Western Blot assay,flow cytometer apoptosis assay,flow cytometer mitochondrial membrane potential assay and other methods were used to detect the effects of dracohodin perchlorate on the apoptosis pathway,endoplasmic reticulum stress pathway and mTORC1 pathway of liver cancer cells,and the upstream and downstream relationship of each pathway was analyzed by adding corresponding signaling pathway inhibitors.5.The model of xenograft tumor in nude mice with liver cancer was established,and the volume changes of tumor under the action of dracohodin perchlorate were measured and recorded,and the body weight changes of mice were recorded.6.HE staining was used to observe the pathological structure changes of the transplanted tumor and other important organs.8.TUNEL staining was used to detect the apoptosis of the transplanted tumor tissue.7.The expressions of p-4EBP1,p-PERK and CHOP in the transplanted tumor were detected by immunohistochemistry.Results:1.The activity of human liver cancer cell lines SMMC-7721,Hep G2,Hep3 B and Huh-7 was significantly decreased in vitro,and the cytotoxicity to human normal liver cell lines L02 was significantly lower than that of liver cancer cell lines.2.Hoechst staining,flow cytometry apoptosis detection and Western Blot showed that dracohodin perchlorate could induce apoptosis of HCC cells in vitro,and the inhibition of cell viability and apoptosis rate could be reversed by the apoptosis inhibitor Z-VAD-FMK.3.dracohodin perchlorate induces significant changes in hepatoma cell lines at the transcriptome level,which involves a variety of biological processes and signaling pathways,including endoplasmic reticulum stress,endogenous apoptosis,and mTORC1 signal activation,etc.4.It was observed under electron microscope that dracohodin perchlorate could induce mitochondrial swelling,endoplasmic reticulum dilatation,lysosome increase and other changes of hepatoma cells.5.Flow cytometry and Western Blot analysis showed that dracohodin perchlorate could induce the decrease of mitochondrial membrane potential of liver cancer cells,and induce the imbalance of expression ratio of Bax and Bcl-2 and the activation of Caspase-9.6.Western Blot analysis showed that dracohodin perchlorate activated ER stress of liver cancer cells,and the PERK-ATF4-CHOP signaling pathway was significantly activated.When 4-PBA inhibited ER stress signal,the apoptosis signal of mitochondrial pathway induced by it was reversed.7.It was found by Western Blot that dracohodin perchlorate could activate mTORC1 signal in hepatoma cells.By inhibiting mTORC1 activation with Torin-1,it could reverse the endoplasmic reticulum stress and downstream apoptosis signal induced by it.8 and dragon's blood,perchlorate can inhibit the growth of liver cancer cells in the body,and had no obvious effect on body weight in nude mice,by HE staining to observe the pathological structure of the tumor and important organs found that dragon's blood,perchlorate can induce liver transplantation tumor tissue structure disorder,necrosis degree aggravating,and to the liver,lung,kidney,heart,brain has no obvious toxic effect.9.The TUNEL method was used to detect the tumor sections,and it was found that the TUNEL apoptosis staining of the transplanted tumor tissues was significantly enhanced by dracohodin perchloric.10.Through immunohistochemical detection,it was found that dracohodin perchlorate could up-regulate the expression of key proteins in mTORC1 pathway and endoplasmic reticulum stress pathway in the transplanted tumor.Conclusion:1.Dracohodin perchlorate can significantly inhibit the growth of HCC cells in vivo and in vitro,and has little toxicity to normal cells and tissues.2.Dracohodin perchlorate can activate the endoplasmic reticulum stress signaling pathway and induce endogenous apoptosis in hepatoma cells.3.Dracohodin perchlorate can activate mTORC1 and mediate downstream apoptotic signal by activating endoplasmic reticulum stress branch signaling pathway PERK-ATF4-CHOP.