Reactive oxygen species generation and mitochondrial dysfunction for the initiation of apoptotic cell death in human hepatocellular carcinoma HepG2 cells by a cyclic dipeptide Cyclo(-Pro-Tyr).

Abstract

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulatingPI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.