Cyperus amuricus induces G1 arrest and apoptosis through endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells

Abstract

Ethnopharmacology relevance: Cyperus amuricus (C. amuricus), belongs to the family Cyperaceae, was used to exert wound healing, diuretic, astringent and other intestinal problems in oriental medicine. However, only a few studies have reported its anticancer activities.

Aim of the study: In this study, we determined the activity of C. amuricus on ER stress-induced cell death and G1 cell cycle arrest in human hepatocellular carcinoma (HCC) Hep3B cells.

Materials and methods: The in vitro cell proliferation assay of C. amuricus was tested on Hep3B and human embryonic kidney HEK293 cells. Subsequently, the cell cycle distribution in the indicated stages using flow cytometric analysis, the expression of cell cycle-related proteins by western blot analysis and immunofluorescence detection of p21^CIP1/WAF1 were determined for the comprehensive identification of cell cycle arrest in Hep3B cells. The effect of C. amuricus on the expression of apoptosis-related proteins in Hep3B cells was also performed by western blot analysis. Furthermore, induction of the ER stress mediators in C. amuricus-treated Hep3B cells were observed by western blot analysis, intracellular Ca²⁺ mobilization assay and immunofluorescence detection of caspase-12.

Results: C. amuricus strongly exhibited cytotoxic activity on Hep3B cells, but not on HEK293 cells. C. amuricus affected the phosphorylation levels of endoplasmic reticulum sensors and increased the expression of GRP78/BiP and CHOP, resulting in the accumulation of unfolded proteins in the ER and triggering the unfolded protein response. These changes occurred by the elevation of intracellular Ca²⁺ concentrations, which contributed to ER stress-induced apoptosis in C. amuricus-treated Hep3B cells. C. amuricus also coordinated the stimulation of ER chaperones, which initiated G1 cell cycle arrest through the induction of CDKIs and the inhibition of cyclins and CDKs. Furthermore, C. amuricus triggered apoptosis through the activation of mitochondrial-dependent pathway in Hep3B cells.

Conclusions: Our results suggest that C. amuricus is an effective apoptosis inducing agent for Hep3B cells via the G1 arrest, ER stress and mitochondrial mediated apoptotic pathways.

Keywords: Apoptosis; Cell cycle arrest; Cyperus amuricus; ER stress; Hep3B cells; Mitochondria.