Modulation of apoptosis in human hepatocellular carcinoma (HepG2 cells) by a standardized herbal decoction of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes with anti- hepatocarcinogenic effects

Evaluation of apoptosis in HepG2 cells was carried out by (a) microscopic observations of cell morphology, (b) DNA fragmentation analysis, (c) activities of caspase 3 and 9, as well as by (d) analysis of the expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins associated with cell death.The results demonstrated that in HepG2 cells, the decoction can induce (a) DNA fragmentation and (b) characteristic morphological changes associated with apoptosis (nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies). The decoction could also, in a time and dose dependent manner, up regulate the expression of the pro-apoptotic gene Bax and down regulate expression of anti-apoptotic Bcl-2 gene (as evident from RT-PCR analysis, immunohistochemistry and western blotting). Further, the decoction significantly (p < .001) enhanced the activities of caspase-3 and caspase-9 in a time and dose dependent manner.Overall findings provide confirmatory evidence to demonstrate that the decoction may mediate its reported anti-hepatocarcinogenic effect, at least in part, through modulation of apoptosis.A decoction prepared from a mixture of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes has traditionally been used for many years by a particular family of Ayurveda physicians in Sri Lanka [1] for the treatment of cancer patients. Findings of recent in vivo investigations carried out by Iddamaldeniya et al., (2003 and 2006) indicate that this poly-herbal decoction has the potential to significantly inhibit chemically induced hepatocarcinogenesis [1,2]. The decoction has been standardized considering its High Performance Liquid Chromatography (HPLC) profile and physico-chemical parameters according to WHO guidelines [3]. Further in vivo and in vitro investigations have demonstrated that cytotoxicity, [3,4] antioxidant activity, anti-inflammatory activity [5] and up regulation of p53 and p21 activities [6] are some of the possibl