Cyperus amuricus Induces Cell Death in Human Hepatocellular Carcinoma Hep3B cells

Abstract

Cyperus amuricus (C. amuricus), belongs to Cyperaceae family, has been widely used to treat astringent, diuretic, wound healing and other intestinal problems for centuries. Recent studies have demonstrated that C. amuricus retains potent pharmacological efficiency in anti-lipase, antioxidant and antineoplastic capabilities. However, the molecular mechanisms of C. amuricus on anticancer activities remain unclear. Therefore, the present study was carried out to investigate the precise mechanisms of C. amuricus-induced cell death in human hepatocellular carcinoma cells (HCC). C. amuricus were comparably and significantly cytotoxic to Hep3B cells, but not to A549, HaCaT and HEK293 cells. C. amuricus obviously elicited G1 cell cycle arrest in Hep3B cells concomitant with the upregulation of p21CIP1/WAF1, p27KIPI and p16INK4a proteins and the downregulation of cdc25A, cyclin D1 and cyclin E, CDK4 and 2 as well as E2F-1, phospho-Rb. C. amuricus also sequentially activated the various caspases (cleaved of caspase-8, -9, -3, -7, and -6, and cleaved PARP) and increased ratio of pro-versus anti-apoptotic Bcl-2-related proteins, prompting the permeability change of mitochondrial membranes and the release of cytochrome c from mitochondria. Based on these results, C. amuricus induces apoptosis via the activation of both extrinsic death receptor- and intrinsic mitochondria-mediated pathways in HCC Hep3B cells. The potentials of C. amuricus on endoplasmic reticulum (ER) stress-mediated apoptosis and G1 cell cycle arrest in Hep3B cells was further evaluated. C. amuricus profoundly triggered ER stress through the activation of unfolded protein response (UPR), leading to the alteration of the phosphorylation levels of ER sensors, the dissociation of GRP78/BiP, the reduction of p-PERK and the increase ATF6 and IRE1α. These consequences were accompanied by the increment of cytosolic Ca2+ levels and the activation of caspase-12 and CHOP, which could incite ER stress-induced apoptosis in C. amuricus-treated Hep3B cells. In addition, the effect of C. amuricus-mediated G1 arrest was clarified by the induction of ER chaperones on modulating cell cycle regulatory molecules. Consistent with the above results, C. amuricus is an efficient apoptosis-inducing agent for Hep3B cells, via the G1 arrest, ER stress and mitochondrial-dependent intrinsic pathways. Next, the detailed mechanism of C. amuricus-induced apoptosis associated with autophagy were examined. During early exposure (3-12 h), C. amuricus induced autophagy via the accumulation of acidic vesicular organelle (AVO)-positive cells and the upregulation of Atg5-Atg12 conjugate, Atg7, Beclin-1, LC3-II and DAPK3 proteins. Interestingly, C. amuricus suppressed the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) and upregulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), stimulating the activation of apoptosis. Especially, pre-treatment of 3-methyladenine (3-MA) blocked C. amuricus-induced increase of Atg7, Beclin-1, LC3-II and AMPK phosphorylation, revealing the crosstalk between C. amuricus-induced apoptosis and autophagy. Collectively, this is the first study indicating the effects of C. amuricus on cell cycle arrest, ER stress, apoptosis and autophagy in HCC Hep3B cells. It could be informative to elucidate the precise mechanism and biological efficacy of C. amuricus on cellular response in other cancer types to chemo-sensitization.