Trichloromethane fraction of Incarvillea compacta induces lytic cytotoxicity and apoptosis in Epstein-Barr virus-positive gastric cancer AGS cells

BACKGROUND Incarvillea compacta Maxim. has been used to treat stomach disease in Tibet for many years. The objectives of this study were to explore the anti-cancer ability of trichloromethane fraction of I. compacta Maxim. roots (IC-TCL, R2) in EBV positive AGS cancer cells and its effects on cell cycle arrest, apoptosis and lytic induction. METHODS MTT and trypan blue assays were to detect the inhibitory effects of different fraction in different cell lines. Hoechst 33342 staining, Annexin V-PE/7-AAD staining and DIOC6 staining were used to detect the apoptosis induction effects of R2. Western blot experiments were used to detect the expression of apoptosis related proteins BAX and Bcl-2, EBV lytic related proteins BZLF1 and BMRF1, cell cycle regulation related proteins Cyclin D1 and RB after R2 treatment. Cell cycle arrest was analyzed by flow cytometry. RESULTS MTT and trypan blue assays revealed that R2 could significantly reduce cell viability in a dose-dependent manner in EBV positive AGS cells compared with non-EBV infected AGS and other cancer cell lines, whereas n-BuOH and H2O fractions showed non-inhibitory effects in tested cancer cells. R2 could decrease mitochondrial membrane potential and the expression of Bcl-2, while increase the expression of BAX. R2 could also induce EBV lytic replication by activating mRNA levels of BZLF1, BRLF1 and BMRF1. Protein expressions of BZLF1 and BMRF1 were also increased after R2 treatment. Cell cycle analysis showed that R2 treatment could induce G0/G1 phase arrest. The expression of Cyclin D1 decreased, while Rb increased. CONCLUSIONS These results demonstrated that R2 could inhibit the proliferation of AGS-EBV cancer cells by inducing EBV lytic replication, apoptosis and G0/G1 arrest, through the regulation of related proteins. Therefore, R2 could be used as a potential treatment in AGS-EBV cells.