DNA METHYLATION PATTERNS INDUCED BY GAMMA-TOCOTRIENOL IN THE CD4+ TLYMPHOCYTES AND TUMOURS ISOLATED FROM A MOUSE MODEL OF BREAST CANCER

Abstract

Lymphocytes, the key players in adaptive immune system have the ability to rearrange their antigen receptors to increase their capability to respond against wide range of immune challenges including tumour establishment. As these cells develop and respond to various immune challenges, they may undergo epigenetic changes such as DNA methylation and histone modification which may be integral to generation of immunological memory. Recent studies showed that some nutritional compounds resulted in epigenetic changes as well as reversing the abnormal gene activation and silencing mainly in tumour. Tocotrienols (T3), which are found abundantly in palm oil are micronutrients from the vitamin E family, had been reported to exhibit anticancer, anti-angiogenesis as well as immunomodulatory effects on breast cancer in murine mouse. However, the epigenetic changes exerted by gamma-tocotrienol (γ-T3) on tumour and host immune system have not been studied. Hence, this study targets on the DNA methylation patterns induced by tocotrienols in the CD4+ T-lymphocytes and tumours isolated from a mouse model of breast cancer. The anticancer effect of γ-T3 was initially investigated using a cellbased system (4T1 mouse mammary cancer cell line) using cell proliferation assay. This was followed by a syngeneic mouse model of breast cancer. In the in vivo study, the 5-week old BALB/c female mice were supplemented daily with T3 or vehicle (Soy oil) for two-weeks before these mice were inoculated with 4T1 cells. The supplementation continued for 35 days after tumour inoculation. The changes in tumour growth were measured every seven days using digital callipers. On dissection days, blood was withdrawn for CD4+ T-lymphocytes isolation followed by DNA extraction and DNA methylation studies. Tumour excised was subjected for DNA and RNA extraction followed by DNA methylation as well as gene expression studies. Organs such as lung, liver and kidney were used for histopathological analysis. Cell proliferation assay using the MTT and WST-1 showed that γ-T3 has significant dose- and time-dependant inhibition on 4T1 mouse breast cancer cell line. The IC50 values obtained following 72 hours of exposure using the MTT assay was 8.41 μg/ml whereas with the WST-1 assay, it was 8.04 μg/ml. There is a significant (p < 0.05) reduction in tumour volume in the γ-T3 supplemented mice as compared to vehicle-fed mice. Results from DNA methylation studies of CD4+ T-lymphocytes showed that, γ-T3 supplementation resulted in differential regulation of methylation levels in certain genes (HOXA10, IRF4 and RORα) reported to be involved in differentiation and clonal expansion of CD4+ T-cells which eventually could enhance immune response against cancer. Besides that, γ-T3 has also deferentially regulated the genes (GATA3, NR4A3 and TGIF1) that are responsible for the T-lymphocytes differentiation and proliferation in order to keep the immune system activated. Moreover, DNA methylation studies of tumour excised during autopsy showed that γ-T3 can differentially regulate genes (CCND2, SFN and TWIST1) that are reported to play vital roles in breast cancer progression. Besides that, breast cancer qPCR array showed γ-T3 differentially regulated genes that are involved in breast cancer progression. This study showed that γ-T3 exerts potent anti-cancer activity towards 4T1 cell lines as well as mouse model of breast cancer. Moreover, γ-T3 also exerts immunomodulatory effects in the mouse model of breast cancer via regulations of DNA methylation patterns in CD4+ T-lymphocytes. The results obtained from this study will be useful in designing and developing newer therapies for breast cancer.