ELUCIDATION OF MOLECULAR TARGETS REGULATED BY TOCOTRIENOLS (DELTA AND GAMMA) AND BETA CAROTENE IN HUMAN CHRONIC MYELOGENOUS LEUKAEMIA (K562) CELLS

Abstract

Palm oil is rich in various phytonutrients, in particular, carotenoids and vitamin E. Carotenoids and vitamin E are fat soluble micronutrients that have important physiological functions. The vitamin E family is comprised of two major families, (i) the tocopherols and (ii) the tocotrienols. Among the tocotrienol sub-family, the delta- (δ-T3) and gamma- (γ-T3) isoforms reportedly possess exceptional in vitro anticancer activity. In addition, there is also strong evidence from in vitro and in vivo studies that beta-carotene has potential anti-cancer benefits. Chronic myeloid leukaemia (CML) is a BCR-ABL positive form of leukaemia characterised by overproliferation of partially mature myeloid cells. The effects of δ-T3, γ-T3 or β- carotene have not been well characterised in a leukemic system. Therefore, in this study, the pro-apoptotic and anti-leukemic effects of these three palm phytonutrients (δ-T3, γ-T3 and β-carotenes) were compared using cell-based approaches with the K562 CML cell line that was derived from a CML patient. Cell proliferation assay using the MTT showed that δ-T3 was most cytotoxic (8.04 μg/ml) followed by γ-T3 (8.77 μg/ml) and finally β-carotene (18.51 μg/ml). This observation was based on the 72 hours MTT IC50 values. However, the cytotoxic effects of these three test agents were found to be significantly (p<0.05) lower in human peripheral blood mononuclear cells (PBMCs). A similar pattern was obtained with the BrdU assay approach where it was observed that these test compounds exerted dose-dependent and time-dependent anti-proliferative effects on the K562 cells. Then, the K562 cells were cultured in the presence of δ-T3, γ-T3 or β-carotene at the respective 72 hours IC50 values obtained from the MTT assay. Following 72 hours of exposure, RNA was extracted from these cells and purified. The purified RNA was analysed using two commercial quantitative reverse transcriptase polymerase chain reaction (qRTPCR) arrays (human apoptosis and human leukaemia) to identify differentially regulated genes associated with apoptosis or leukaemia in the K562 cells. Results from the human apoptosis qPCR array showed that δ- and γ-tocotrienols differentially up-regulated 24 genes each in the apoptotic pathway where most genes regulated by tocotrienols were found to be from the (i) BCL-2 family, (ii) CARD domain members, (iii) Caspases, (iv) GADD family, (v) P53 family and (vi) the TNF receptor and (vii) the TNFR ligand super-families. Beta-carotene differentially regulated four genes that belong to the BCL-2 family as well as the TNF receptor and ligand super-families. Results from the leukemic array indicate all three isoforms differentially regulated leukemic genes including genes encoding lymphocyte proliferation, myeloid cell differentiation, promoters and transcriptional regulators. Following the results obtained from the apoptosis array, five apoptotic genes were selected to be validated via real-time PCR and ELISA. The real time expression data revealed a direct correlation with the data obtained from the human apoptosis PCR array, while protein ELISA showed all five genes were differentially and significantly expressed depending on the treatment used (δ- and/or γ-tocotrienols and β-carotene) compared to untreated K562 cells at a protein level. This study showed that β-carotene and δ- and γ-tocotrienols exert potent cytotoxicity towards K562 cells and regulated a number of genes annotated in the apoptotic and leukemic qPCR array.