ELUCIDATING THE ROLE OF PALM GAMMA-TOCOTRIENOL IN REGULATING AUTOPHAGY IN MDA-MB231 HUMAN BREAST CANCER CELLS

Abstract

Crude palm oil (CPO) contains several phytonutrients such as carotenoids, tocopherols (TP), tocotrienols (T3), sterols, phospholipids, squalene, and tripterpenic and aliphatic hydrocarbons. Tocopherols (α, β, γ, δ) along with tocotrienols (α, β, γ, δ) represent the two naturally-occurring sub-classes of vitamin E. Both sub-classes exhibit multiple antioxidant and immunity-enhancing properties. However, only tocotrienols have been shown to possess potent anticancer activities using in vivo and in vitro studies. Several studies have reported that the delta- (δ) and gamma- (γ) isoforms of T3 possess more potent anti-proliferative and anticancer effects compared to the other isoforms (α or β). The anti-cancer effects of T3 have been demonstrated in many types of solid cancers, including breast cancer (BC). Recently several studies showed that T3s exert their anti-cancer effects through targeting multiple cell signalling pathways. To date, the ability of γT3 inducing autophagy in breast cancer cells has not been fully explored. Therefore, in this study we investigated the ability of palm γT3 to differentially regulate genes involved in autophagy in the MDA-MB-231 human breast cancer cells using different culture conditions. Cell proliferation was quantified using the WST cell proliferation kit. The half maximal inhibitory concentration (IC50) of γT3 was calculated for two culture condition; (a) complete media with 10% FBS [IC50 at 24 hrs: 13.25 μg/mL; 48 hrs: 7.1μg/mL and 72 hrs: 6.5 μg/mL] and media supplemented with 1% FBS [IC50 at 24 hrs: 1.9 μg/mL; 48 hrs: 1.6 μg/mL and 72 hrs: 1.2 μg/mL]. For detection of autophagy genes, RNA was extracted post 48 hrs of exposure. The purified RNA was analysed using a commercial quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) array annotated with genes involved in human autophagy to identify differentially regulated genes associated with autophagy in the MDA-MB-231 cells. Results from the human autophagy qPCR array showed that a total of 54 genes were differentially regulated by γT3 in the MDA-MB-231 human breast cancer. Ten of these genes were significantly up-regulated. Most of the differentially regulated genes were from: (i) genes involved in autophagic vacuole formation [from genes categorised as belonging to the autophagic machinery components] (ii) co-regulators of autophagy and apoptosis [from genes categorised as belonging to the regulation of autophagy genes]. One of the major genes involved in the autophagy pathway inducers i.e. the BECN1 gene was found markedly up-regulated along with the EIF2AK3, GABARAP and GABARAP2 genes. The results from this study suggest that the potent cytotoxic effects observed when the MDA-MB-231 human breast cancer cells were treated with γT3 at 7.1 μg/mL for 48 hours could also be via regulation of several autophagy-inducing genes.