Nerigoside suppresses colorectal cancer cell growth and metastatic potential through inhibition of ERK/GSK3β/β-catenin signaling pathway

Background: Nerigoside (NG), a cardenolide isolated from a commonfolk medicine, Nerium oleander Linn. (Apocynaceae), has not been explored for its biological effects. To date, cardenolides have received considerable attention in pharmacology studies due to their direct effects of apoptosis-induction or growth-inhibitory against tumor in vitro and in vivo. Whether and how NG exerts anticancer effects against colorectal cancer remains to be elucidated. Purpose: The aim of this study was to investigate the anticancer effect of NG in human colorectal cancer cells. Methods: To test anticancer effect, we compared potency of NG in two colorectal cancer cell lines, HT29 and SW620 by WST-1 and colony proliferation assays. And we investigated mechanism of anticancer activities by analyzing players in apoptotic and ERK/GSK3 beta/beta-catenin signaling pathways in HT29 and SW620 cells treated with NG. Results: In this study, we showed that NG markedly suppressed the cell viability and colony formation of colorectal cancer cells HT29 and SW620, with no significant toxic effect on non-cancer cells NCM460. Annexin VFITC/PI and CFSE labeling results revealed that NG suppressed cell proliferation in low concentration, along with reducing expression of PCNA, while NG induced apoptosis in high concentration, Meanwhile, NG significantly arrested cell migration by reversal of EMT and cell cycle on G2/M. Then, we found that the ERK and GSK3 beta/beta-catenin signaling pathway were noticeably blocked in CRC cells after treatment with NG. According to western blot, NG upregulated the expression of p-GSK3 beta/GSK3 beta and decreased especially the expression of beta-catenin in nuclear. In addition, Wnt signaling and its target genes were suppressed in response to NG. Then, the Ser9 phosphorylation of GSK3 beta can be reduced / raised by GO 6983 / LiCl, respectively. Thus, we further confirmed that the GSK3 beta/beta-catenin axis is involved in NG-prevented cell proliferation. Conclusion: NG inhibited the growth of colorectal cancer cells by suppressing ERK/GSK3 beta/beta-catenin signaling pathway. And the GSK3 beta/beta-catenin axis is involved in preventing cell proliferation and migration by NG-treatment. These results suggest that NG may be used to treat colorectal cancer, with better outcome by combining with GSK3 beta inhibitor to block Wnt pathway.