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days, blood was withdrawn for CD4+ T-lymphocytes isolation followed by DNA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.