Enterocarpam-III Induces Human Liver and Breast Cancer Cell Apoptosis via Mitochondrial and Caspase-9 Activation

Abstract

An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6-tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancercell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigatecytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells.Cell viability was determined by MTT assay to determine IC10, IC20 and IC50 levels, reactive oxygen species(ROS) production with 2’,7’-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities usingspecific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA andemploying a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by stainingwith 3, 3’-dihexyloxacarbocyanine iodide (DiOC6) and using flow cytometry. The compound was cytotoxic toHepG2 and MDA-MB-231 cells with the IC50 levels of 26.0±4.45 and 51.3±2.05 μM, respectively. For murinenormal fibroblast NIH3T3 cells, the IC50 concentration was 81.3±10.1 μM. ROS production was reduced in adose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependentmanner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-IIIdecreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting thatthe compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-IIIinhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosisvia the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.