Study On Xanthotoxin Inducing HepG2 Apoptosis Through Endoplasmic Reticulum Pathway

Xanthotoxin is a kind of natural compound furan coumarin which exist widely in Chinese herbs.With the deeply researching on xanthotoxin,its antitumor activity is gradually known by people,but its mechanism of inhibiting tumor cell proliferation is not very clear.The apoptosis induced by endoplasmic reticulum pathway has become a hot topic in recent years at home and abroad in the field of cell.There were research shown that variation of Ca2+existed in endoplasmic reticulum could lead endoplasmic reticulum stress (ERS) to induce apoptosis.Therefore,on the basis of related literature,the study explored the mechanism of HepG2 cells apoptosis induced by xanthotoxin from the perspective of endoplasmic reticulum stress to provide new evidence about the regulation mechanism of ERS in apoptosis,and may provide the basis for the design and development of targeted anti-tumor drugs.The subject investigated xanthotoxin in HepG2 cells proliferation inhibition by MTT and SRB methods, and matching the results of two methods;Observing morphological changes and each phase of apoptosis of HepG2 cells effected by xanthotoxin after 48h with Hoechst 33258 staining method;PI staining to detect the influence of HepG2 cell cycle effected by xanthotoxin;Detecting xanthotoxin on the effect of Ca2+ concentration in HepG2 cells with Fluo-3/AM tag by laser confocal scanning microscope;Detecting Caspase-3?Caspase-12 activity in HepG2 cells by colorimetry method.The study found that xanthotoxin has inhibition of proliferation in HepG2 cells,IC50 values of two methods were 65.21?g/mL and 52.14?g/mL;By Hoechst staining study,we found adherent cells reduce and apoptotic body increase after the treatment,and as the higher the concentration of xanthotoxin, the more obvious of the phenomenon;The results of PI staining showed that as the dosage of xanthotoxin increased,the cell population percentage of treatment group in Go/Gi phase raised gradually,cell population percentage were (33.433±1.002)%? (37.233±1.168)%?(71.467±0.833)% compared with the control group (22.100±1.513)%; Fluo-3/AM fluorescent probe shown that with increasing dosage intracellular calcium ion concentration [Ca2+], also increased,control group [Ca2+]i was 13.003±0.942,treatment group [Ca2+]i were 21.785±1.231?37.090±2.056?73.787+ 2.700;Colorimetry method results shown that the activity of Caspase-3 and Caspase-12 could be raised by increasing dosage,compared with the control group with significant difference (P < 0.01).The results above indicated that xanthotoxin has proliferation inhibition in HepG2 cells and the suppression was in a dose dependent manner;Different doses of xanthotoxin can cause varying degrees of apoptosis;Xanthotoxin also had an effect on HepG2 cells cycle,by blocking cells in Go/Gi phase to induce apoptosis;Xanthotoxin may induce HepG2 cells apoptosis through ERS,by raising Ca2+ concentration in HepG2 cells and activating Caspase-12 and Caspase-3 downstream to induce apoptosis.