Molecular Mechanisms Of Breast Cancer Suppression With Tocotrienol Supplementation

Abstract

Tocotrienols, isoforms of vitamin E, are not only known for their antioxidant, lipid-lowering properties and as anti-proliferating agents but also for their inhibitory effects on the growth of human breast cancer cells in vitro and in vivo. In this study, the effects of tocotrienol-rich fraction (TRF) from palm oil and its isomers (α, δ and γ-tocotrienol) were examined in 4T1 mouse mammary cancer cells. The 4T1 cells were cultured and grown in RPMI medium supplemented with different concentrations of tocotrienols. Cell numbers were determined at the end of twelve days of culture. Results showed that TRF and the individual fractions of palm tocotrienols inhibited the growth of 4T1 cells in vitro at lower concentrations (6-20 μg/ml) compared to tocopherols (> 20 μg/ml). Deltatocotrienol was found to be most inhibitory followed by γ-tocotrienol. Complete inhibition of 4T1 proliferation was achieved with 6 μg/ml δ-tocotrienol and 10 μg/ml γ-tocotrienol. The effects of TRF and tocotrienol isomers were investigated for proapoptotic activity by expression of FasL gene. This study showed that γ- and δ- tocotrienol (8 μg/ml) treated 4T1 cells expressed significantly (P < 0.01) higher levels of FasL compared to untreated control cells. The TRF showed pro-apoptotic effects at higher concentrations from 20 – 80 μg/ml). Tumourigenesis was examined and compared against control in both nude and BALB/c mice models. The mice were injected with MDA-MB-231 and 4T1 cells respectively for the different models, and were fed with TRF by oral gavage. Tumour incidence was reduced by 33% and 57% in nude mice and BALB/c mice respectively. In addition, the tumour load was significantly (P < 0.05) reduced by 72% and 93.6% respectively in tocotrienol-supplemented nude and BALB/c mice, when compared to the vehicle-fed control group. This study shows that palm tocotrienols have strong inhibitory effects on the growth of both MDA-MB-231 and 4T1 cells both in vitro and in vivo. The immune modulatory effects of tocotrienols were investigated and it was found that TRF enhanced production of NK cells (P < 0.05) as well as IFN-γ (P < 0.05), which in turn regulate the immune protection against cancer cells. These observations were recorded in both mice models.

In this study, adipose tissue from control (vehicle-fed) and experimental (TRF-fed) animals were analysed for the presence of tocopherols and tocotrienols using HPLC. All the tocotrienol isomers increased significantly (P < 0.05) in the TRF supplemented mice compared to control whilst only α-T and γ-T showed significant increase in the supplemented nude mice and BALB/c mice. The anti-angiogenic activity of TRF and two tocotrienol isomers (γ and δ) were investigated using the human umbilical vein endothelial cells (HUVEC). Results showed that the level of Interleukin-8 (IL-8), an angiogenic activator was reduced in the HUVEC treated with TRF (P < 0.05) and the two tocotrienol isomers compared to α-tocopherol and control cells. The level of IL-8 was lowest in the δ-tocotrienol treated cells followed by γ-tocotrienol and TRF. In addition, TRF also down-regulated the expression of various angiogenic activators such as VEGF-C, KDR and FLT1 in tumours from mice fed with TRF as compared to control mice. Moreover, immunohistochemical expression of pro-angiogenic such as VEGF, VEGF-R2 and CD31 were found to be reduced in tumour and lung tissues of mice supplemented with TRF. This study shows that tocotrienols exhibited anti-angiogenic activity, which may play a key role in inhibiting the growth and metastasis of tumour. Microarray analysis was carried out on 4T1 cells treated with TRF, δ- tocotrienol, α-tocopherol and differential gene expression compared to untreated control cells were investigated. The outcome from this study revealed the identity of several interesting genes including Interleukin-24 (IL-24), death-associated protein 3 (Dap3), rat sarcoma oncogene (Rras) and platelet-derived growth factor- B (Pdgfb) that possess important functional role in cancer mechanisms. In this study, the microarray analysis was further subjected to investigate the effect of TRF on regulation of genes from tumour tissues of mice that were supplemented with TRF compared to that of control mice. This analysis again revealed the up-regulation of IL-24 in vivo with TRF-supplementation. In this study, we also found that tocotrienols up-regulated (P < 0.05) the expression of a novel-cytokine, IL-24 in the tumour and 4T1 cells. The IL-24 cytokine has been reported to play a significant role in tumour regression. In addition, while tocotrienols up-regulated the expression of IL-24, they also downregulated pro-angiogenic markers; VEGF and IL-8 at the same time. Studies have previously shown that IL-24 possess anti-angiogenic properties. Thus, the tumour protective mechanism exhibited by tocotrienols may be explained via the IL-24 mechanistic pathway.