Publication: Identification Of Putative Target And Ligand Molecules Of Tocotrienols In Mcf-7 Human Breast Cancer Cells
Date
2010-07
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Publisher
International Medical University
Abstract
Tocotrienol is a sub-class in the vitamin E family. Tocotrienols have been shown to play a key role in multiple cellular processes that are considered to be major anti-cancer mechanisms. These include antioxidant activity, inhibiting proliferation of cancer cells, inducing apoptosis in cancer cells and inhibiting metastasis. Tocotrienols have been shown to inhibit proliferation and growth of several types of cancer cells including breast and prostate cancer cells. The present work was on determining the dose and time-dependent effects of tocotrienol rich fractions (TRF), tocotrienol isomers (e.g. αT3, δT3 and γT3) and alpha tocopherol on the proliferation and growth of oestrogen-positive MCF-7 human breast cancer cell lines in vitro, gene expression profiling in MCF-7 cells co-cultured with the various vitamin E isomers at half maximal inhibitory concentration (IC50) using microarray human bead chips as well as use of in silico approach such as molecular modelling via computer simulation to understand the molecular mechanism(s) of tocotrienols on the functions of various important biological molecules such as NF-κB, ESR1, EGFR and MIG6 and TTK. The results from this study showed that tocotrienols significantly (p<0.05) inhibit the proliferation of MCF-7 cells when compared to control i.e. untreated cells. The inhibition observed was found to be dose and time dependent. In contrast, α-tocopherol can only significantly (p<0.05) inhibit proliferation of the MCF-7 cells when a higher concentration of this vitamin E isomer was used. The IC50 and the order of potency for the compounds tested for Day 3, 6 and 10 was found to be δT3 (Day 3: 7.6, Day 6: 5.0, Day 10: 3.0) < γT3 (Day 3: 7.71, Day 6: 3.94 , Day 10: 3.0) < TRF (Day 3: 10.86, Day 6: 5.2, Day 10: 3.2) < αT3 (Day 3: 12.4, Day 6: 7.2, Day 10: 6.06) < αT (Day 3: 50.2) and the trend maintained at all time interval studied. Our data suggest that tocotrienols act continuously over a period of time, with low doses being as effective as higher doses when the treatment period is inceased. Gene expression study of vitamin E (TRF, α-tocopherol and α-, δ-, and γ-tocotrienol) treated on MCF-7 cells revealed expression of genes that can serve as potential targets for the treatment of breast cancer. Expression levels of selected differentially expressed genes were verified by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that tocotrienols alter the expression of genes that encode for various immune responses, tumour and metastasis suppressors, apoptotic signalling proteins, transcription factors, protein biosynthesis regulators, and many others. In particular, our results showed that treatment with tocotrienols downregulated the expression of API5 and upregulated the expression MIG6 at higher fold-change difference compared with control and the same reconfirmed using qRT-PCR. These genes regulate the expression of other genes that are pertinent to breast cancer. A molecular modelling was performed using a docking program Autodock version 3.0.5, which employs Lamarkian Genetic Algorithm and Arguslab 4.0 to predict the binding affinities of four isomers of tocotrienol (α-T3, β-T3, δ-T3 and γ-T3), against the active sites of various proteins that have been described in the literature to play key roles in cancer (NF-κB, ESR1, EGFR and MIG6 and TTK). The approach helped to putatively identify the potential target(s) of the tocotrienol isomers. The surfaces of the proteins were explored using this approach to identify fields that are most favourable in energy wise for interaction with the tocotrienols. The efficacy of binding was quantified in terms of binding energy and co-efficient constant, Ki. The results indicated that isomers of tocotrienols have the high predicted binding affinities against NF-κB, ESR1, EGFR and MIG6 and TTK proteins, while δ-tocotrienol and γ-tocotrienol interacts the most with the essential amino acid residues of the proteins studied.
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Keywords
Tocotrienols, Breast Neoplasms, Antineoplastic Agents, Apoptosis