ABSTRACT
Bioactive compounds extracted from leaves and twigs of Goniothalamus griffithii include pinocembrin (PCN) and goniothalamin (GTN). The objectives of this study were to investigate the cytotoxic activities of PCN and GTN and their influence on molecular signaling for cell death in several human cancer cell lines compared to normal murine fibroblast NIH3T3 cells. GTN exhibited the most potent cytotoxicity against MCF7 > HeLa > HepG2 > NIH3T3 cells with IC50 values of 7.33, 14.8, 37.1 and 65.4 µM, respectively, whereas PCN was cytotoxic only to HepG2 cells with IC50 values of ~80 µM. Apoptotic cell death was confirmed by staining the cells with annexin VFITC and propidium iodide (PI) employing flow cytometry. Apoptosis was shown by externalization of phosphatidylserine in goniothalamintreated MCF7 cells in a dose response manner. Positive PIstained cells with the typical morphology of apoptotic cells were increased dosedependently. Furthermore, reduction of mitochondrial transmembrane potential was found in goniothalamintreated MCF7, HepG2 and HeLa cells. GTN treatment in MCF7 increased caspase3, 8 and 9 activities while GTNinduced HeLa cells showed an increase of both caspase3 and 9 activities. But an increased caspase8 activity was demonstrated in GTN and PCNtreated MCF7 and HepG2 cells, respectively. Taken together, GTN and PCNinduced human cancer cell apoptosis was through different molecular mechanisms or signaling pathways, which might be due to different machineries in different types of cancer cells, as evidenced by the compoundmodulated caspase activities in both intrinsic and/or extrinsic pathways.
Subject(s)
Apoptosis/drug effects , Goniothalamus/chemistry , Neoplasms/drug therapy , Phytochemicals/pharmacology , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Caspases/metabolism , Cell Death/drug effects , Cell Line, Tumor , Flavanones/pharmacology , HeLa Cells , Hep G2 Cells , Humans , MCF-7 Cells , Membrane Potential, Mitochondrial/drug effects , Mice , NIH 3T3 Cells , Neoplasms/metabolism , Pyrones/pharmacology , Signal Transduction/drug effectsABSTRACT
Naringin, a bioflavonoid found in Citrus seeds, inhibits proliferation of cancer cells. The objectives of this study were to investigate the mode and mechanism(s) of hepatocellular carcinoma HepG2 cell death induced by naringin. The cytotoxicity of naringin towards HepG2 cells proved dosedependent, measured by MTT assay. Naringintreated HepG2 cells underwent apoptosis also in a concentration related manner, determined by annexin Vfluorescein isothiocyanate (FITC) and propidium iodide (PI) employing flow cytometry. Mitochondrial transmembrane potential (MTP) measured using 3,3'dihexyloxacarbocyanine iodide (DiOC6) and flow cytometer was reduced concentrationdependently, which indicated influence on the mitochondrial signaling pathway. Caspase3, 8 and 9 activities were enhanced as evidenced by colorimetric detection of paranitroaniline tagged with a substrate for each caspase. Thus, the extrinsic and intrinsic pathways were linked in human naringintreated HepG2 cell apoptosis. The expression levels of proapoptotic Bax and Bak proteins were increased whereas that of the antiapoptotic BclxL protein was decreased, confirming the involvement of the mitochondrial pathway by immunoblotting. There was an increased expression of truncated Bid (tBid), which indicated caspase8 proteolysis activity in Bid cleavage as its substrate in the extrinsic pathway. In conclusion, naringin induces human hepatocellular carcinoma HepG2 cell apoptosis via mitochondriamediated activation of caspase9 and caspase8mediated proteolysis of Bid. Naringin anticancer activity warrants further investigation for application in medical treatment.
