Abstract
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.
Highlights
Various natural phytocompounds have been screened and investigated extensively as potential anti-cancer agents
The relative stereostructure of erythrocarpin E was established through nuclear Overhauser effect spectroscopy (NOESY)
Due to the diverse manner and hallmarks in which the process of apoptosis can be manifested, we examined the induction of apoptosis in HSC-4 cells through the occurrence of DNA fragmentation and cleavage of poly-ADP ribose polymerase (PARP) proteins
Summary
Various natural phytocompounds have been screened and investigated extensively as potential anti-cancer agents. Most of natural anti-cancer agents available to date are derived from plants, animals, marine organisms and microorganisms. Some currently used examples of plant-derived compounds with potential anti-cancer activities are 19S-19acetoxyeugenol acetate and 19S-19-acetoxychavicol acetate [2], vincristine, irinotecan, etoposide and paclitaxel, while bleomycin and doxorubicin are microbial-derived compounds, and citarabine, derived from marine sources [3]. Several citrus limonoid aglycones such as limonin, nomilin, obacunone, isoobacunoic acid and ichangin have recently been subjected to anti-cancer screening procedures, and were found to induce significant glutathione-S-transferase (GST) and quinine reductase (QR) activity in the liver and intestinal mucosa of mice and rats respectively [5,6]. Limonoids from the ethanolic extract of Azadirachta indica (neem bark, leaves and seed oil) were found to cause cell death of prostate cancer cells (PC-3) by inducing apoptosis through a reduction in Bcl-2 expression levels corresponding with an increase in Bax levels [8]
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