Mechanism of apoptosis: comparative analyses of selected polyphenols and synthetic stilbene / Mohd Saad Zamani

Polyphenols such as curcumin, resveratrol and gallic acid have anticancer potential to suppress proliferation of variety of tumour cells. Nowadays, research and development of new cancer chemotherapeutic drugs has escalated tremendously. Therefore, the need for new chemotherapy with less or no side effects are much sought after. For the same objective, this study was aimed to investigate comparatively the mechanism of apoptosis induced by these pure polyphenols and a novel synthetic stilbene, 3, 4, 10-trimethoxystilbene (S2) on different types of cancerous cell lines. The antiproliferative effects were determined via MTS assay followed by annexin V and PI staining in flow cytometry to determine the stages of cell death resulting from either apoptotic or necrotic processes and also their cell cycle arrest. The results were further supported by the apoptosis gene expression via RTPCR and the levels of caspase (caspase-3/7, -8 and -9) to determine their pathway of apoptosis. Cytochrome c release was also measured to elucidate the apoptotic pathway of the compounds and Bid (BH3 Interacting Domain Death Agonist) levels was determined on HepG2 cells treated with gallic acid. Results of the MTS assay showed that the IC50 value of S2 against human hepatocelullar carcinoma (HepG2) cells was lower than curcumin, gallic acid and resveratrol with 0.14 ± 0.01 |M, 35.8 ± 2.5 |M, 33.4 ± 1.6 |M and 51.2 ± 1.1 |M, respectively. S2 even demonstrated high selectivity index (WRL68/HepG2) of 8.7, compared to others. Lower doses of S2 were able to induce higher percentage of early apoptosis compared to the other compounds. Curcumin, resveratrol and S2 have been shown to induce cell cycle arrest at S phase while HepG2 treated with gallic acid were arrested at G1/G0 phase. Activities of caspases-3 and -9 were also detected on HepG2 cells treated with S2, curcumin and resveratrol while caspases-3 and -8 were detected on HepG2 treated with gallic acid. All of the tested compounds were able to upregulate the expression of the apoptotic genes (p53, Bax and caspase 3) and downregulate the anti-apoptotic gene, bcl-2. S2 showed a 3-fold higher ratio of Bax/Bcl-2 compared to resveratrol indicating higher potential of S2 in inhibiting proliferation of hepatoma cells. Cytochrome c was detected in HepG2 cells treated with curcumin, resveratrol, gallic acid and S2 and significant (p<0.05) increase of Bid was found in the cells of HepG2 treated with gallic acid. As a conclusion, curcumin, resveratrol and S2 have the potential to induce apoptosis on HepG2 cells via mitochondrial pathway and S phase cell cycle arrest. On the other hand, gallic acid was able to trigger apoptosis through Fas signaling pathway (caspase-8 activation) involving Bid and cytochrome c which induced G1/G0 phase-arrested cells.