SYNERGISTIC EFFECT OF TOCOTRIENOLS AND OTHER PHYTONUTRIENTS FROM PALM OIL IN A CELL-BASED SYSTEM

Abstract

Palm oil contains biologically active phytonutrients that are important antioxidants used in various ailments and chemoprevention. This study was conducted to compare the antiproliferative effect of various tocotrienol isomers [tocotrienol-rich fraction (TRF), tocotrienol-enrich fraction (TEF), and (α-,δ-,γ-) T3 fractions] with other palm phytonutrients such as carotenoids, squalene and Coenzyme-Q10 on two human breast cancer cell lines, namely the MDA-MB-231 (oestrogen-independent) and MCF-7 (oestrogen-dependent) cells. Alpha-tocopherol was also included in this study as this was a phytonutrient that is present in palm oil. We were able to obtain a time dependent and dose dependent partial inhibition concentrations (IC50) for all the phytonutients studied with the exception of α-tocopherol. The range of concentrations used to determine the IC50 value was 0-20 g/ml. None of the palm phytonutrients with the exception of the tocotrienol isomers could completely suppress the growth of the two human breast cancer cells. Cellular uptake of tocotrienols was found to be better than -tocopherol; hence this could explain its biopotency. Next, we looked at strategies involving combinations of tocotrienols at IC50 with the other palm phytonutrients as this could provide a much wider range of anti-proliferative mechanisms, which could result in higher efficacy than a single agent alone. Although the combination treatments reduced the growth of these cells in the exponential stage, the treatments could not completely suppress cell proliferation. With most combinations, it was not possible to determine the IC50 values as cell growth reached a plateau. In addition, no additive or synergistic effects were observed. In order to assess the underlying cell death mechanisms, the respective IC50 concentrations were used. Based on DNA fragmentation analysis, the results showed that tocotrienols selectively enhanced induction of apoptosis as compared to Paclitaxel, which was used as a positive control. This activity was also associated with the time-dependent cleavage of the poly-(ADP-ribose) polymerase (PARP), a DNA repair protein, demonstrating an apoptotic cell death signalling pathway. Programmed cell death has emerged as a potential target for cancer treatment at various stages of cancer progression. The cell lines studied are aggressive human breast cancer cell lines that have been shown to have high levels of activated transcription factor nuclear factor-kappa-B (NF-B) and PARP expression. The ability of these compounds to inhibit NF-κB increases the sensitivity of cancer cells to apoptotic action. The levels of cyclooxygenase-2 (COX-2) are reported to be elevated in aggressive breast cancers. Elevated levels of COX-2 could result in certain kinds of cancer cells becoming resistant to programmed cell death mostly due to the impaired ability of the cell to undergo intrinsic cell death. In this study, COX-2, an NF-B regulated gene product associated with proliferation was found to be down-regulated with all treatments. Our current observation indicates tocotrienols, carotenoids, squalene and Coenzyme-Q10 possesses anti-proliferative effect on MDA-MB-231 and MCF-7 human breast cancer cells, and the mechanism could be through inhibition of inflammatory factors COX-2 and NF-B. Besides, the evidence of the significant anti-proliferative and apoptotic effects of tocotrienols not only reveals but also reinforces the notion that tocotrienols are indeed the hallmark nutrient in palm oil as potent anti-tumour agent in inhibiting breast cancer.