Enterocarpam-III induces human liver and breast cancer cell apoptosis via mitochondrial and caspase-9 activation.

Ratana Banjerdpongchai,Benjawan Wudtiwai,Wilart Pompimon

doi:10.7314/apjcp.2015.16.5.1833

Ratana Banjerdpongchai, Benjawan Wudtiwai + Show 1 more

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https://doi.org/10.7314/apjcp.2015.16.5.1833

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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 cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and 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 using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC6) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the IC50 levels of 26.0±4.45 and 51.3±2.05 μM, respectively. For murine normal fibroblast NIH3T3 cells, the IC50 concentration was 81.3±10.1 μM. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.

Highlights

There has been a report of enterocarpam-I and –II isolated from Orophea enterocarpa (Kamaliah et al, 1986)
The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells
Cytotoxicity of enterocarpam-III Enterocarpam-III was cytotoxic to both HepG2 and MDA-MB-231 cells but less toxic to normal murine fibroblasts with the IC50 of 26.0±4.45, 51.3±2.05 and 81.3±10.1 μM, respectively

Summary

Introduction

There has been a report of enterocarpam-I and –II isolated from Orophea enterocarpa (Kamaliah et al, 1986). The structure of enterocarpam-I (10-amino-3hydroxy-4,8,9-trimethoxyphenanthrene-1-carboxylic acid lactam) and enterocarpam-II (10-amino-3-hydroxy-4,8dimethoxyphenanthrene-1-carboxylic acid lactam) are different from that of enterocarpam-III (10-amino-2, 3, 4, 6-tetramethoxyphenanthrene-1-carboxylic acid lactam). Their core structures are classified as aristolactam-type alkaloids. (Annonacaea) are isolated and identified as 1,2,3-trimethoxy-5-oxonoraporphine and ouregidione Both ouregidione and 1, 2, 3-trimethoxy-5oxonoraporphine induce cytotoxicity against human leukemic HL-60, U937, and K562 cells whereas 1, 2, 3-trimethoxy-5-oxonoraporphine is more active than ouregidione. Both compounds increase significantly the proportion of the cells in S phase, whereas 1, 2, 3-trimethoxy-5-oxonoraporphine induces HL-60 cell line arrest in G2/M and ouregidione does the G1 phase arrest (Uadkla et al, 2013)

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