Regulation of Apoptotic Effects by Erythrocarpine E, a Cytotoxic Limonoid from Chisocheton erythrocarpus in HSC-4 Human Oral Cancer Cells

The aim of this study was to determine the cytotoxic and apoptotic effects of erythrocarpine E (CEB4), a limonoid extracted from Chisocheton erythrocarpus on human oral squamous cell carcinoma. Based on preliminary dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, CEB4 treated HSC-4 cells demonstrated a cytotoxic effect and inhibited cell proliferation in a time and dose dependent manner with an IC50 value of 4.0±1.9 µM within 24 h of treatment. CEB4 was also found to have minimal cytotoxic effects on the normal cell line, NHBE with cell viability levels maintained above 80% upon treatment. Annexin V-fluorescein isothiocyanate (FITC), poly-ADP ribose polymerase (PARP) cleavage and DNA fragmentation assay results showed that CEB4 induces apoptosis mediated cell death. Western blotting results demonstrated that the induction of apoptosis by CEB4 appeared to be mediated through regulation of the p53 signalling pathway as there was an increase in p53 phosphorylation levels. CEB4 was also found to up-regulate the pro-apoptotic protein, Bax, while down-regulating the anti-apoptotic protein, Bcl-2, suggesting the involvement of the intrinsic mitochondrial pathway. Reduced levels of initiator procaspase-9 and executioner caspase-3 zymogen were also observed following CEB4 exposure, hence indicating the involvement of cytochrome c mediated apoptosis. These results demonstrate the cytotoxic and apoptotic ability of erythrocarpine E, and suggest its potential development as a cancer chemopreventive agent

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

Comparison of total relative cell viability (%) between various human tumor cell lines and normal human bronchial epithelial cells (NHBE) after treatment with CEB4 at different concentrations (0 to 40.0 µM) over 24 h.

<p>(A) Dose dependent MTT assay graph upon 24 h of CEB4 exposure. (B) Time dependent MTT assay graph upon treatment with 40.0 µM CEB4. All results were expressed as total percentage of viable cells with mean ±SD of three independent determinations. Solvent controls using DMSO was performed on HSC-4 cells as a representative of all other cell lines.</p

Chemical structure of erythrocarpine E (CEB4) extracted from <i>Chisocheton erythrocarpus.</i>

<p>Chemical structure of erythrocarpine E (CEB4) extracted from <i>Chisocheton erythrocarpus.</i></p

Summary of IC₅₀ values and percentage cell viability of CEB4 treated tumor cell lines and NHBE cells as obtained from MTT cell viability assays after 24 h of exposure.

†<p>IC₅₀ values at 24 h exposure.</p>††<p>Viability upon 24 h exposure with 40 µM CEB4.</p><p>All results were expressed as total percentage of viable cells with mean ±SD of three independent determinations.</p

Detection of apoptosis using annexin V-FITC and PI dual staining on (A) NHBE cells and (B) HSC-4 cells.

<p>Untreated cells (upper panel) and treated cells (lower panel) after CEB4 treatment at IC₅₀ concentrations for 12 h. Quadrants were designed as follows, I: non-stained cells indicating viable cells; II: annexin V-FITC stained cells indicating early apoptosis; III: annexin V-FITC and PI stained cells indicating late apoptosis; and IV: PI stained cells indicating secondary necrosis. All dot plots are a representation of equal cell populations (n = 10,000).</p