Lignans and polyphenols of Phyllanthus amarus Schumach & Thonn induce apoptosis in HCT116 human colon cancer cells through caspases-dependent pathway

Background: The anticancer effects of Phyllanthus amarus extract on various cancer cells have been investigated, however, effect of its major constituents on HCT116 human colorectal cancer cells has not been reported.

Objective: In the present study, we investigated the cytotoxic effect of 80% ethanol extract of P. amarus and its marker constituents (phyllanthin, hypophyllanthin, gallic acid, niranthin, greraniin, phyltetralin, isolintetralin, corilagin and ellagic acid) on HCT116 and their underlying mechanisms of action.

Method: Their anti-proliferative and apoptotic effects on HCT 116 were performed using MTT assay and flow cytometric analysis, respectively, while caspases 3/7, 8 and 9 activities were examined using colorimetric method. The expression of cleaved poly ADP ribose polymerase enzyme (PARP) and cytochrome c proteins was investigated by immune-blot technique.

Results: HPLC and LC-MS/MS analyses demonstrated the extract contained mainly lignans and polyphenols. The plant samples markedly suppressed the growth and expansion of HCT116 cells concentration- and time-dependently with no toxicity against normal human fibroblast CCD18 Co. P. amarus extract, phyllanthin and gallic acid induced mode of cell death primarily via apoptosis as confirmed by the exteriorization of phosphatidylserine. Caspases 3/7, 8, and 9 activities increased concentration-dependently following 24 h treatment. The expressions of cleaved PARP (Asp 214) and cytochrome c were markedly upregulated.

Conclusion: P. amarus extract, phyllanthin and gallic acid exhibited apoptotic effect on HCT116 cells via caspases-dependent pathway.

Keywords: HCT116 cells; Phyllanthus amarus; apoptosis; caspases; cleaved PARP; phyllanthin.