Effect And Mechanism Of Rh2-O Induced Immunogenic Cell Death In Hepatoma Cells

Background : Ginsenoside Rh2 not only has antitumor effect,but also has immunomodulatory function.In the pre-phase work,we synthesized octyl ester derivative of ginsenoside Rh2(Rh2-O)from Rh2 and octyl chloride,and found that Rh2-O is more favorable to cell absorption and has stronger pharmacological activity than its prototype of Rh2.Immunogenic cell death is a special type of cell death manifested by the release of damage-related molecular patterns(DAMPs)by dying tumor cells stimulated by chemotherapy or radiotherapy drugs.DAMPs transmit antigenic signals and activate the adaptive immune response.Based on the previous work,we once again proposed the scientific hypothesis that Rh2-O can induce immunogenic cell death in cancer cells,and explored the mechanism of Rh2-O inducing immunogenic cell death.Objective: To investigate the effect of Rh2-O on Huh-7 hepatoma cell immunogenic cell death and its possible mechanism by cell and animal experiments.Methods: 1.Huh-7 cells were cultured in vitro,and were treated with Rh2-O or mitoxantrone(positive control).The viability and apoptosis of cells were detected by CCK8 assay and flow cytometry,respectively.The concentrations of high mobility family protein 1(HMGB1)and adenosine triphosphate(ATP)in supernatant were detected by ELISA and chemiluminescence assay,respectively.Membrane eversion of calreticulin(CRT)was determined by immunofluorescence.In the animal vaccine experiment,C57BL/6 mice were randomly divid ed into vaccine group and PBS group.Dying Hepa1-6 hepatocellular carcinoma cells treated with Rh2-O were injected subcutaneously into the inclined costris of mice as vaccine,and the other group of mice were injected with PBS as control group.Normal Hepa1-6hepatocellular carcinoma cells without drug treatment were injected into the other oblique costal abdomen for reattack.The tumor size of mice was recorded with vernier caliper every three days,and the tumor-free survival time,tumor growth and body weight of mice were calculated.2.ROS levels in cells were detected by fluorescence probe DCFH-DA,expression of autophagy related genes was measured through QPCR and the expression levels of proteins associated with endoplasmic reticulum stress and autophagy related proteins were detected by Western blot.The effect of endoplasmic reticulum stress inhibitor 4-Phenylbutyric acid(4-PBA)on Huh-7 cell membrane ectropion induced by Rh2-O was detected by immunofluorescence assay.The effect of autophagy inhibitor 3-Methyladenine(3-MA)on Rh2-O-induced ATP secretion in Huh-7 cells was detected by chemiluminescence.Mitochondrial morphology was observed by its infrared fluorescence probe..The co-localization of mitochondria and LC3 B protein was detected by immunofluorescence method.C57BL/6 mice were randomly divided into Rh2-O,Rh2-O+4-PBA and normal saline groups,with 6 mice in each group.C57BL/6 murine Hepa1-6 hepatocellular carcinoma cells were injected subcutaneously into the slanting costris of mice to establish a mouse hepatocellular carcinoma model,and were administered intragastric the next day.The dosage of Rh2-O group was 10mg/kg/d,and the dosage of Rh2-O+4-PBA group was 100mg/kg/d of 4-PBA in addition to the dosage of Rh2-O of 10mg/kg/d by intragastric administration.The gavage volume was 200 μL.The tumor size of mice was recorded with vernier caliper every three days,and organ indexes were counted after the experiment.Results: 1.The cell experiment results showed that when compared with the control group,Rh2-O treatment significantly reduced the cell viability,increased the apoptosis rate,the secretion levels of HMGB1 and ATP(P<0.05).And the membrane-evagination levels of CRT.The results of animal experiments showed that4 of the 11 mice in the vaccine group developed tumors after being attacked again by live cancer cells,and all of the 7 mice in the PBS group developed tumors after being attacked again by live cancer cells.The weight of mice in the PBS group dropped sharply in the late observation period.2.Accumulation levels of ROS in cells were elevated after Rh2-O treatment(P<0.05).Moreover,the expression levels of ER stress-related proteins(PERK、e IF2a、p-e IF2a)were enhanced by Rh2-O administration(P<0.05).After the addition of endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA),the translocation of CRT induced by Rh2-O was significantly inhibited in Huh-7 cells(P <0.05).After the establishment of mouse liver cancer model,the drug was given intragastric administration.The spleen index of Rh2-O group was significantly higher than that of Rh2-O+4-PBA and control group,and Rh2-O significantly inhibited the growth of tumor.3.Rh2-O also disrupted the morphology of mitochondria and increased the expression levels of autophagy related genes(P62,LC3,Atg3)and autophagy proteins(LC3A,LC3B).Pretreatment with the autophagy inhibitor 3-MA significantly reduced the amount of ATP secretion induced by Rh2-O in Huh-7cells.Rh2-O also promotes the colocalization of mitochondria and autophagy marker protein LC3 B.Conclusion: Rh2-O can induce immunogenic cell death in hepatoma cells,and the mechanism may be related to the induction of endoplasmic reticulum stress by Rh2-O to promote CRT membrane ectropion and mitochondrial autophagy.Rh2-O can induce immunogenic cell death of cancer cells and has certain anti-tumor immune function...