Compound K Induces Endoplasmic Reticulum Stress-Apoptosis In Human Liver Cancer Cells By Regulating STAT3

Background:Hepatocellular carcinoma(HCC)is the most commonly diagnosed cancer worldwide.The latest national cancer statistics of 2018 showed the new death toll of liver cancer is about 318.000 in China,accounting for the second leading cause of cancer-related mortality.Because of late diagnosis and lack of safe and effective drugs,HCC has a poor prognosis.Meanwhile,systemic chemotherapy is still the mainstay of HCC treatment.Thus,there is an urgent need to develop the effective but less-toxic chemotherapeutic drugs targeting for HCC.Recently,natural products have been used to treat cancer and become a research hotspot in anti-tumor chemotherapy.Ginsenoside compound K(20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol,CK)is an intestinal bacterial metabolite of ginseng protopanaxadiol saponin that has been reported to induce apoptosis in many cancer cells;however,the precise mechanisms of its action in human HCC cells remain unclear.Signal transducers and activators of transcription 3(STAT3)has been observed to be hyperactive in cancer and involved in the regulation of cell growth,apoptosis and transduction of signaling pathways.Endoplasmic reticulum stress(ERS)is a response of cells to relieve stress caused by accumulation of abnormal proteins in the endoplasmic reticulum.When ERS is severe or prolonged,it promotes ERS-associated signaling cascades that stimulate apoptosis.Both STAT3 and ERS are.Therefore,the STAT3-and ERS-related apoptosis is closely related to the anti-tumor mechanisms.Objective:In this study,we investigated the effect of ginsenoside CK on the growth of hepatoma cells in vitro and in vivo,and its regulation on STAT3,ERS and apoptosis.We also explored the regulatory mechanism of ginsenoside CK on ERS and apoptosis via STAT3,so as to provide the experimental basis for the application of ginsenoside CK for treatment of liver cancer.Methods:1.Experiments in vitro:Cell viabilities of human HCC cells(HepG2,SMMC-7721,Hep3B,Huh7)and normal liver cell L02 were detected by MTT assay.Colony formation assay was used to test the proliferation abilities of HepG2 and SMMC-7721 cells.Hoechst staining and flow cytometry were used to detect apoptosis of HepG2 and SMMC-7721 cells.Immunocytochemistry(ICC)and immunofluorescence(IF)were used to examine the levels and sub-cellular localizations of STAT3 and p-STAT3,and EMSA was used to detect the binding activity of STAT3 to its target DNA.The expressions of apoptotic proteins,ERS-related proteins,and STAT3 protein were detected by western blot.Furthermore,STAT3-knockout or overexpressed HepG2 and SMMC-7721 cell lines were established using CRISPR/Cas9 technology or plasmid transfection,and used to explore the regulatory mechanism of ginsenoside CK on ERS and apoptosis via STAT3.2.Experiments in vivo:The BALB/c nude mice bearing human HCC xenografts were randomized to 2 groups(n=6),treatment group with intravenous CK(5,10,or 20 mg/kg)injection and normal control(vehicle control)group with saline injection once a day for 15 days.The length and width of each tumor were measured by caliper to estimate relative tumor volume.At the end of experiment,the animals were sacrificed,the tumor volume and weight were measured,and hematoxylin and eosin(HE),TUNEL,immunohistochemical(IHC)staining and western blot assay were performed.Results:1.Experiments in vitro:The results showed that ginsenoside CK decreased the viabilities of HepG2,SMMC-7721,Hep3B and Huh7 cells in a dose-and time-dependent manner,while it showed little inhibition effect in L02 cells at 24 and 48 h.Western blot results showed that p-STAT3 levels were decreased in these HCC cell lines following treatment with ginsenoside CK.while the most significant reduction was observed in HepG2 and SMMC-7721 cells.ICC and IF results clearly indicated that STAT3 was localized in the cytosol while p-STAT3 was localized in the nucleus,and p-STAT3 levels were significantly reduced in a dose-dependent manner in response to ginsenoside CK treatment.EMSA results showed that ginsenoside CK inhibited STAT3 DNA-binding activity in a dose-dependent manner in HepG2 and SMMC-7721 cells.Meanwhile,western blot assay showed that apoptosis-associated proteins,including cleaved PARP,cleaved caspase 3,and ERS-related proteins,namely GRP78,CHOP,cleaved caspase 4,p-PERK,p-IRE1,p-JNK,p-eIF2a were markedly increased following ginsenoside CK treatment.The results using STAT3-knockout and overexpressed HepG2 and SMMC-7721 cells showed that STAT3 regulated the ERS and apoptosis in response to ginsenoside CK.2.Experiments in vivo:The results showed that ginsenoside CK inhibited tumor growth in mice.Compared with vehicle control,tumor weights and volumes were significantly reduced following ginsenoside CK treatment.Additionally,there were no significant differences in body weight among the vehicle,5 and 10 mg/kg ginsenoside CK-treated groups.Furthermore,HE staining and TUNEL assay showed that the number of necrotic and apoptotic cells in tumor tissues was significantly increased following ginsenoside CK treatment as compared with the vehicle control.Meanwhile,IHC staining and western blot results showed that ginsenoside CK treatment upregulated the expression of ERS-related proteins and downregulated p-STAT3 level.Conclusion:1.Ginsenoside CK inhibited the proliferation and induced apoptosis of liver cancer cells in vitro and vivo.2.Ginsenoside CK inhibited the phophorylation of STAT3,decreased STAT3 DNA-binding capacity.3.Ginsenoside CK induced ERS in liver cancer cells.4.Blocking STAT3 phophorylation enhanced ERS and apoptosis in CK-treated liver cancer cells.