JOURNALS

Inflammation and oxidative damage contribute to the pathogenesis of asthma. Although corticosteroid is the first-line treatment for asthma, a subset of patients is steroid resistant, and chronic steroid use causes side effects. Because vitamin E isoform g-tocotrienol possesses both antioxidative and anti-inflammatory properties, we sought to determine protective effects of g-tocotrienol in a house dust mite (HDM) experimental asthma model. BALB/c mice were sensitized and challenged with HDM. Bronchoalveolar lavage (BAL) fluid was assessed for total and differential cell counts, oxidative damage biomarkers, and cytokine levels. Lungs were examined for cell infiltration and mucus hypersecretion, as well as the expression of antioxidants and proinflammatory bio-markers. Sera were assayed for IgE and g-tocotrienol levels. Airway hyperresponsiveness in response to methacholine was measured. g-Tocotrienol displayed better free radical–neutralizing activity in vitro and inhibition of BAL fluid total, eosinophil, and neutrophil counts in HDM mouse asthma in vivo, as compared with other vitamin E isoforms, including a-tocopherol. Besides, g-tocotrienol abated HDM-induced elevation of BAL fluid cytokine and chemokine levels, total reactive oxygen species and oxidative damage biomarker levels, and of serum IgE levels, but it promoted lung-endogenous antioxidant activities. Mech-anistically, g-tocotrienol was found to block nuclear NF-kB level and enhance nuclear Nrf2 levels in lung lysates to greater extents than did a-tocopherol and prednisolone. More importantly, g-tocotrienol markedly suppressed methacholine-induced airway hyperresponsiveness in experimental asthma. To our knowledge, we have shown for the first time the protective actions of vitamin E isoform g-tocotrienol in allergic asthma.

Angiogenesis is one of the key hallmarks of cancer. In this study, we investigated whether γ-tocotrienol can abrogate angiogenesis-mediated tumor growth in hepatocellular carcinoma (HCC) and if so, through what molecular mechanisms. We observed that γ-tocotrienol inhibited vascular endothelial growth factor (VEGF)-induced migration, invasion, tube formation and viability of HUVECs in vitro. Moreover, γ-tocotrienol reduced the number of capillary sprouts from matrigel embedded rat thoracic aortic ring in a dose-dependent manner. Also, in chick chorioallantoic membrane assay, γ-tocotrienol significantly reduced the blood vessels formation. We further noticed that γ-tocotrienol blocked angiogenesis in an in vivo matrigel plug assay. Furthermore, γ-tocotrienol inhibited VEGF-induced autophosphorylation of VEGFR2 in HUVECs and also suppressed the constitutive activation of AKT/mammalian target of rapamycin (mTOR) signal transduction cascades in HUVECs as well as in HCC cells. Interestingly, γ-tocotrienol was also found to significantly reduce the tumor growth in an orthotopic HCC mouse model and inhibit tumor-induced angiogenesis in HCC patient xenografts through the suppression of various biomarkers of proliferation and angiogenesis. Taken together, our findings strongly suggest that γ-tocotrienol might be a promising anti-angiogenic drug with significant antitumor activity in HCC.

Purpose: Because of poor prognosis and development of resistance against chemotherapeutic drugs, the existing treatment modalities for gastric cancer are ineffective. Hence, novel agents that are safe and effective are urgently needed. Whether g-tocotrienol can sensitize gastric cancer to capecitabine in vitro and in a xenograft mouse model was investigated.
Experimental Design: The effect of g-tocotrienol on proliferation of gastric cancer cell lines was examined by mitochondrial dye uptake assay, apoptosis by esterase staining, NF-kB activation by DNA-binding assay, and gene expression by Western blotting. The effect of g-tocotrienol on the growth and chemosensitization was also examined in subcutaneously implanted tumors in nude mice.
Results: g-Tocotrienol inhibited the proliferation of various gastric cancer cell lines, potentiated the apoptotic effects of capecitabine, inhibited the constitutive activation of NF-kB, and suppressed the NF-kB–regulated expression of COX-2, cyclin D1, Bcl-2, CXCR4, VEGF, and matrix metalloproteinase-9 (MMP-9). In a xenograft model of human gastric cancer in nude mice, we found that administration of g-tocotrienol alone (1 mg/kg body weight, intraperitoneally 3 times/wk) significantly suppressed the growth of the tumor and this effect was further enhanced by capecitabine. Both the markers of proliferation index Ki-67 and for microvessel density CD31 were downregulated in tumor tissue by the combination of capecitabine and g-tocotrienol. As compared with vehicle control, g-tocotrienol also suppressed the NF-kB activation and the expression of cyclin D1, COX-2, intercellular adhesion molecule-1 (ICAM-1), MMP-9, survivin, Bcl-xL, and XIAP.
Conclusions: Overall our results show that g-tocotrienol can potentiate the effects of capecitabine through suppression of NF-kB–regulated markers of proliferation, invasion, angiogenesis, and metastasis.

Emerging evidence supports that prostate cancer originates from a rare subpopulation of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to mainly target the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after treatment. Therefore, successful elimination of CSCs may be an effective strategy to achieve complete remission from this disease. Gamma-tocotrienols (c-T3) is one of the vitamin-E constituents, which have been shown to have anticancer effects against a wide range of human cancers. Recently,
we have reported that c-T3 treatment not only inhibits prostate cancer cell invasion but also sensitizes the cells to docetaxel-induced apoptosis, suggesting that c-T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that c-T3 can downregulate the expression of prostate CSC markers (CD133/CD44) in androgen-independent prostate cancer cell lines (PC-3 and DU145), as evident from Western blotting analysis. Meanwhile, the spheroid formation ability of the prostate cancer cells was significantly hampered by c-T3 treatment. In addition, pretreatment of PC-3 cells with c-T3 was found to suppress tumor initiation ability of the cells. More importantly, although CD133-enriched PC-3 cells were highly resistant to docetaxel treatment, these cells were as sensitive to c-T3 treatment as the CD133-depleted population. Our data suggest that c-T3 may be an effective agent in targeting prostate CSCs, which may account for its anticancer and chemosensitizing effects reported in previous studies.

γ-Tocotrienol (γT3) is known to selectively kill prostate cancer (PCa) cells and to sensitize the cells to docetaxel (DTX)induced apoptosis. In the present study, the pharmacokinetics of γT3 and the in vivo cytotoxic response of androgen-independent prostate cancer (AIPCa) tumor following γT3 treatment were investigated. Here, we investigated these antitumor effects for PCa tumors in vivo. The pharmacokinetic and tissue distribution of γT3 after exogenous γT3 supplementation were examined. Meanwhile, the response of the tumor to γT3 alone or in combination with DTX were studied by real-time in vivo bioluminescent imaging and by examination of biomarkers associated with cell proliferation and apoptosis. After intraperitoneal injection, γT3 rapidly disappeared from the serum and was selectively deposited in the AIPCa tumor cells. Administration of γT3 alone for 2 weeks resulted in a significant shrinkage of the AIPCa tumors. Meanwhile, further inhibition of the AIPCa tumor growth was achieved by combined treatment of γT3 and DTX (p<0.002). The in vivo cytotoxic antitumor effects induced by γT3 seem to be associated with a decrease in expression of cell proliferation markers (proliferating cell nuclear antigen, Ki-67 and Id1) and an increase in the rate of cancer cell apoptosis [cleaved caspase 3 and poly(ADP-ribose) polymerase]. Additionally, the combined agents may be more effective at suppressing the invasiveness of AIPCa. Overall, our results indicate that γT3, either alone or in combination with DTX, may provide a treatment strategy that can improve therapeutic efficacy against AIPCa while reducing the toxicity often seen in patients treated with DTX.

Gamma-tocotrienol has demonstrated anti-proliferative effect on breast cancer (BCa) cells, but mechanisms involved are largely unknown. This study aimed at deciphering the molecular pathways responsible for its activity. Our results showed that treatment of BCa cells with gamma-tocotrienol resulted in induction of apoptosis as evidenced by activation of pro-caspases, accumulation of sub-G1 cells and DNA fragmentations. Examination of the pro-survival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of Id1 and NF-jB through modulation of their upstream regulators (Src, Smad1/5/8, Fak and LOX). Meanwhile, gamma-tocotrienol treatment also resulted in the induction of JNK signaling pathway and inhibition of JNK activity by specific inhibitor partially blocked the effect of gamma-tocotrienol. Furthermore, synergistic effect was observed when cells were co-treated with gamma-tocotrienol and Docetaxel. Interestingly, in cells that treated with gamma-tocotrienol, alpha-tocopherol or b-aminoproprionitrile were found to partially restore Id1 expression. Meanwhile, this restoration of Id1 was found to protect the cells from gamma-tocotrienol induced apoptosis. Consistent outcome was observed in cells ectopically transfected with the Id-1 gene. Our results suggested that the anti-proliferative and chemosensitization effect of gamma-tocotrienol on BCa cells may be mediated through downregulation of Id1 protein.

To date, the most effective cure for metastatic melanoma re-mains the surgical resection of the primary tumor. Recently, tocotrienol-rich-fraction has shown antiproliferative effect on can-cer cells. To elucidate this anticancer property in malignant melanoma, this study aimed, first, to identify the most potent iso-mer for eliminating melanoma cells and second to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of gamma-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of pro-caspases and the accumulation of sub-G1 cell population. Examina-tion of the prosurvival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of NF-kappaB, EGF-R, and Id family proteins. Meanwhile, gamma-tocotrienol treatment also resulted in induction of JNK signaling pathway, and inhibition of JNK activity by selective inhibitor was able to par-tially block the effect of gamma-tocotrienol. Interestingly, gamma-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and gamma-catenin expression, which was associated with suppression of cell invasion capabil-ity. Furthermore, synergistic effect was observed when cells were cotreated with gamma-tocotrienol and chemotherapy drugs. To-gether, our results demonstrated for the first time the anti-invasion and chemonsensitization effect of gamma-tocotrienol against hu-man malignant melanoma cells.