Regulation of apoptotic effects of Erythrocarpine E, a new Cytotoxic limonoid extracted from Chisocheton Erythrocarpus / Norliza bt Shah Jehan Muttiah
The aim of this study is to determine the cytotoxic and apoptotic effects of erythrocarpine E (CEB4), a new limonoid extracted from Chisocheton erythrocarpus.
MTT assay, Live/Dead® Viability/Cytotoxicity assay, cell cycle analysis, annexin V analysis, PARP cleavage analysis, DNA fragmentation assay, sandwich ELISA assay
and Western blot analysis were performed on five CEB4 treated cancer cell lines; HSC4COX2- and HSC2 (oral), CaSki (cervical), HepG2 (liver), MCF7 (breast) and NHBE (normal human bronchial epithelial cell line). CEB4 treated HSC4COX2-, HSC2, CaSki, HepG2 and MCF7 cells demonstrated a cytotoxic effect and inhibited cell proliferation in a dose and time dependent manner. CEB4 had minimal effect on NHBE.
Cell cycle analysis of treated cancer cells detected a hypodiploid sub-G1 peak in all the cancer cell lines expect in the HSC2 cells confirming CEB4 induced apoptosis. The
shift in percentage in the G0/G1 phase in HSC4COX2-, CaSki, and HepG2 cells suggested a possible G0/G1 arrest of the cancer cells upon treatment with CEB4 at 12 hours.
Annexin V analysis by flow cytometry, PARP cleavage analysis, and DNA fragmentation results showed that CEB4 induces apoptosis in CEB treated cancer cell
lines. The results of the ELISA analysis suggest that CEB4 induced apoptosis may be mediated by p53, a tumour suppressor gene. Western blot results demonstrated that
CEB4 induce growth arrest and apoptosis in HSC4COX2- cells appeared to be mediated through regulation of p53 signalling pathway as there is an increase in expression of
phosphorylated p53 and decrease in expression of the p53 inhibitor, Mdm2. CEB4 upregulates pro-apoptotic gene, Bax and at the same time down-regulate the anti-apoptotic
gene, Bcl-2. It also activates activator caspase 9 and executioner caspase 3, which are downstream molecules to induce apoptosis. These results demonstrated the cytotoxic
and apoptotic ability of this compound and suggest its potential use as a cancer chemopreventive agent.