Dose-dependent apoptotic effect of Woodfordia fruticosa on hepatocellular carcinoma (HepG2) cells by upregulating bax and caspase-9.

Abstract

This study investigated the dose-dependent apoptotic effect of Woodfordia fruticosa (WF) in HepG2 hepatocellular carcinoma cells, focusing on Bax and caspase-9 upregulation. The ethanol extracts, WF70 (70% ethanol) and WF30 (30% ethanol), were tested for cytotoxicity, pro-apoptotic properties, and cell cycle effects. MTT assays revealed a dose- and time-dependent reduction in cell viability, with IC50 values of 14.39 µg/mL and 15.96 µg/mL for WF70 and WF30, respectively. Flow cytometry analysis showed that WF70 induced significantly higher apoptosis rates (54.82% at 30 µg/mL) than WF30 (27.84%), confirming its stronger pro-apoptotic potential. Cell cycle analysis demonstrated a dose-dependent increase in G₂/M phase arrest, reaching 30.69% and 24.53% with 30 µg/mL WF70 and WF30, respectively. Gene expression analysis revealed a significant upregulation of pro-apoptotic markers (Bax: 3.65-fold, Caspase-9: 4.32-fold, Caspase-3: 2.12-fold) and a marked downregulation of an anti-apoptotic marker (Bcl-2: 0.08-fold) after treatment with 30 µg/mL WF70. Thus, WF70 exerts a stronger apoptotic effect through both intrinsic (mitochondrial) and extrinsic apoptotic pathways, primarily driven by Bax-mediated mitochondrial membrane destabilization and Caspase-9 activation. This study highlights the potential of WF as an anticancer agent for hepatocellular carcinoma, although this warrants further in vivo validation and optimization for therapeutic applications.