Gamma-tocotrienol and hydroxy-chavicol synergistically inhibits growth and induces apoptosis of human glioma cells.

Abdul Rahman A, A Jamal AR, Harun R, Mohd Mokhtar N, Wan Ngah WZ.

Gamma-tocotrienol (GTT), an isomer of vitamin E and hydroxy-chavicol (HC), a major bioactive compound in Piper betle, has been reported to possess anti-carcinogenic properties by modulating different cellular signaling events. One possible strategy to overcome multi-drug resistance and high toxic doses of treatment is by applying combinational therapy especially using natural bioactives in cancer treatment.

METHODS:

In this study, we investigated the interaction of GTT and HC and its mode of cell death on glioma cell lines. GTT or HC alone and in combination were tested for cytotoxicity on glioma cell lines 1321N1 (Grade II), SW1783 (Grade III) and LN18 (Grade IV) by [3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)- 2H- tetrazolium, inner salt] MTS assay. The interactions of each combination were evaluated by using the combination index (CI) obtained from an isobologram.

RESULTS:

Individually, GTT or HC displayed mild growth inhibitory effects against glioma cancer cell lines at concentration values ranging from 42-100 mug/ml and 75-119 mug/ml respectively. However, the combination of sub-lethal doses of GTT + HC dramatically enhanced the inhibition of glioma cancer cell proliferation and exhibited a strong synergistic effect on 1321N1 with CI of 0.55, and CI = 0.54 for SW1783. While in LN18 cells, moderate synergistic interaction of GTT + HC was observed with CI value of 0.73. Exposure of grade II, III and IV cells to combined treatments for 24 hours led to increased apoptosis as determined by annexin-V FITC/PI staining and caspase-3 apoptosis assay, showing caspase-3 activation of 27%, 7.1% and 79% respectively.

CONCLUSION:

In conclusion, combined treatments with sub-effective doses of GTT and HC resulted in synergistic inhibition of cell proliferation through the induction of apoptosis of human glioma cells in vitro.

Haghighat N, Vafa M, Eghtesadi S, Heidari I, Hosseini A, Rostami A.

BACKGROUND:

Tocotrienols (T3) were neglected in the past; today, get attentions due to their antioxidant and none-antioxidant activity. The objective of this study was to evaluate the effects of the daily intake of 200 mg T3 added in canola oil over 8 weeks on microalbuminuria, inflammation, and nitrosative stress in type 2 diabetic patients.

METHODS:

This study was a double-blinded, placebo-controlled, randomized trial. A total of 50 patients with T2DM and FBS >126 mg/dl treated by non-insulin hypoglycemic drugs were randomly assigned to receive either 15 ml T3-enriched canola oil (200 mg/day T3) or pure canola oil for 8 weeks. Urine microalbumin, volume and creatinine levels, serum hs-CRP, and nitric oxide (NO) levels were measured before and after intervention.

RESULTS:

From 50 patients participated in this study, 44 completed the study. There were no significant differences in baseline characteristics, dietary intake, and physical activity between groups. Urine microalbumin and serum hs-CRP were declined significantly in T3-treated group. At the end of the study, patients who treated with T3 had lower urine microalbumin (11 (9, 25) vs. 22 (15, 39.75) nmol/dl, P = 0.003) and hs-CRP changes (-10.91 ± 15.5 vs. -9.88 ± 27.5 Pg/ml, P = 0.048) than control group. A non-significant decrease was also observed in serum NO level in T3-treated group with no changes in urine volume and creatinine levels.

CONCLUSIONS:

These findings indicate that T3 leads to ameliorate proteinuria and can protect the kidney against inflammation (hs-CRP) and nitrosative stress (NO).

Alqahtani S, Alayoubi A, Nazzal S, Sylvester PW, Kaddoumi A.

The aim of this study was to evaluate the in vitro and in vivo performance of γ-tocotrienol (γ-T3) incorporated in a self-emulsifying drug delivery system (SEDDS) and to compare its enhanced performance to a commercially available product, namely Tocovid Suprabio™ (hereafter Tocovid), containing tocotrienols. The solubilization of γ-T3 was tested in a dynamic in vitro lipolysis model followed by in vitro cellular uptake study for the lipolysis products. In addition, in vitro uptake studies using Caco2 cells were conducted at different concentrations of γ-T3 prepared as SEDDS, Tocovid, or mixed micelles. γ-T3 incorporated in SEDDS or Tocovid was orally administered to rats at different doses and absolute oral bioavailability from both formulations were determined. The dynamic in vitro lipolysis experiment showed about two fold increase in the solubilization of γ-T3 prepared as SEDDS compared to Tocovid, which correlated with higher cellular uptake in the subsequent uptake studies. In vitro cellular uptake and in vivo oral bioavailability studies have shown a twofold increase in the cellular uptake and oral bioavailability of γ-T3 incorporated in SEDDS compared to Tocovid as a result of improvement in its solubility and passive uptake as confirmed by in vitro studies. In conclusion, incorporation of γ-T3 in SEDDS formulation enhanced γ-T3 solubilization and passive permeability, thus its cellular uptake and oral bioavailability when compared to Tocovid.

Nasir NA, Agarwal R, Vasudevan S, Tripathy M, Alyautdin R, Ismail NM.

PURPOSE:

Oxidative and nitrosative stress underlies cataractogenesis, and therefore, various antioxidants have been investigated for anticataract properties. Several vitamin E analogs have also been studied for anticataract effects due to their antioxidant properties; however, the anticataract properties of tocotrienols have not been investigated. In this study, we investigated the effects of topically applied tocotrienol on the onset and progression of cataract and lenticular oxidative and nitrosative stress in galactosemic rats.

METHODS:

In the first part of this study, we investigated the effects of topically applied microemulsion formulation of tocotrienol (TTE) using six concentrations ranging from 0.01% to 0.2%. Eight groups of Sprague-Dawley rats (n = 9) received distilled water, vehicle, or one of the six TTE concentrations as pretreatment topically twice daily for 3 weeks while on a normal diet. After pretreatment, animals in groups 2-8 received a 25% galactose diet whereas group 1 continued on the normal diet for 4 weeks. During this 4-week period, topical treatment continued as for pretreatment. Weekly slit-lamp examination was conducted to assess cataract progression. At the end of the experimental period, the animals were euthanized, and the proteins and oxidative stress parameters were estimated in the lenses. In the second part of the study, we compared the anticataract efficacy of the TTE with the liposomal formulation of tocotrienol (TTL) using five groups of Sprague-Dawley rats (n = 15) that received distilled water, TTE, TTL, or corresponding vehicle. The mode of administration and dosing schedule were the same as in study 1. Weekly ophthalmic examination and lens protein and oxidative stress estimates were performed as in study 1. Lens nitrosative stress was also estimated.

RESULTS:

During the 4-week treatment period, the groups treated with 0.03% and 0.02% tocotrienol showed slower progression of cataract compared to the vehicle-treated group (p<0.05), whereas the group treated with 0.2% tocotrienol showed faster progression of cataract compared to the vehicle-treated group (p<0.05). The lenticular protein content, malondialdehyde, superoxide dismutase, and catalase levels were normalized in the groups that received 0.03% and 0.02% tocotrienol. The lenticular reduced glutathione also showed a trend toward normalization in these groups. In contrast, the group treated with 0.2% tocotrienol showed increased lenticular oxidative stress. When the microemulsion and liposomal formulations were compared, the effects on cataract progression, lens oxidative and nitrosative stress, and lens protein content did not show significant differences.

CONCLUSIONS:

Topically applied tocotrienol within the concentration range of less than 0.05% and more than 0.01% tends to delay the onset and progression of cataract in galactose-fed rats by reducing lenticular oxidative and nitrosative stress. However, topical tocotrienol at a concentration of 0.2% and higher aggravates cataractogenesis in galactose-fed rats by increasing lens oxidative stress. The anticataract efficacy of 0.03% microemulsion of tocotrienol did not differ from its liposomal formulations at the same concentration.

Radhakrishnan A, Tudawe D, Chakravarthi S, Chiew GS, Haleagrahara N.

Tocotrienols exhibit a significant anti-inflammatory and antioxidant effect in numerous human diseases. However, the anti-inflammatory and antioxidant effects of tocotrienols in arthritic conditions are not well documented. Therefore, the effect of γ-tocotrienol supplementation against oxidative stress and joint pathology in collagen-induced arthritis in rats was investigated in the present study. Adult female Dark Agouti rats were randomly divided into groups: Control, γ-tocotrienol alone, arthritis alone and arthritis with γ-tocotrienol. Arthritis was induced using 4 mg/kg body weight collagen in complete Freund’s adjuvant. The rats were treated orally with 5 mg/kg body weight of γ-tocotrienol between day 21 and day 45. After 45 days, serum C-reactive protein (CRP), tumor necrosis factor (TNF)-α, superoxide dismutase (SOD) and total glutathione (GSH) assays were conducted. γ-tocotrienol significantly reduced the arthritis-induced changes in body weight, CRP, TNF-α, SOD and the total GSH levels. There was a significant reduction in the arthritis-induced histopathological changes in the γ-tocotrienol treatment group. The data indicated that administration of γ-tocotrienol resulted in a significant antioxidant and anti-inflammatory effect on collagen-induced arthritis; therefore, γ-tocotrienol may have therapeutic potential as a long-term anti-arthritic agent in rheumatoid arthritis therapy.

Ran Cui., Zhu-Qing Liu., Qing Xu

Background

Epidemiological studies that have examined the association of blood α-tocopherol and γ-tocopherol (the principal bioactive form of vitamin E) levels with the risk of prostate cancer have yielded inconsistent results. In addition, a quantitative assessment of published studies is not available.

Methods and Findings

In this meta-analysis, relevant studies were sought by a search of the PubMed and Embase databases for articles published up to October 2013, with no restrictions. Bibliographies from retrieved articles also were scoured to find further eligible studies. Prospective studies that reported adjusted relative risk (RR) estimates with 95% confidence intervals (CIs) for the association between blood tocopherol levels and the risk of prostate cancer were included. Nine nested case–control studies involving approximately 370,000 participants from several countries were eligible. The pooled RRs of prostate cancer for the highest versus lowest category of blood α-tocopherol levels were 0.79 (95% CI: 0.68–0.91), and those for γ-tocopherol levels were 0.89 (95% CI: 0.71–1.12), respectively. Significant heterogeneity was present among the studies in terms of blood γ-tocopherol levels (p = 0.008) but not in terms of blood α-tocopherol levels (p = 0.33). The risk of prostate cancer decreased by 21% for every 25-mg/L increase in blood α-tocopherol levels (RR: 0.79; 95% CI: 0.69–0.91).

Conclusions

Blood α-tocopherol levels, but not γ-tocopherol levels, were inversely associated with the risk of prostate cancer in this meta-analysis.

Nasr M, Nafee N, Saad H, Kazem A.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide. Epirubicin (EPI), an anthracycline derivative, is one of the main line treatments for HCC. However, serious side effects including cardiomyopathy and congestive heart failure limit its long term administration. Our main goal is to develop a delivery strategy that ensures improved efficacy of the chemotherapeutic agent together with reduced cardiotoxicity. In this context, EPI was loaded in chitosan-PLGA nanoparticles linked with asialofetuin (EPI-NPs) selectively targeting hepatocytes. In an attempt to reduce cardiotoxicity, targeted EPI-NPs were coadministered with tocotrienols. EPI-NPs significantly enhanced the antiproliferative effect compared to free EPI as studied on Hep G2 cell line. Nanoencapsulated EPI injected in HCC mouse model revealed higher p53-mediated apoptosis and reduced angiogenesis in the tumor. Combined therapy of EPI-NPs with tocotrienols further enhanced apoptosis and reduced VEGF level in a dose dependent manner. Assessment of cardiotoxicity indicated that EPI-NPs diminished the high level of proinflammatory cytokine tumor necrosis factor-α (TNF-α) as well as oxidative stress-induced cardiotoxicity as manifested by reduced level of lipid peroxidation products (TBARS) and nitric oxide (NO). EPI-NPs additionally restored the diminished level of superoxide dismutase (SOD) and reduced glutathione (GSH) in the heart. Interestingly, tocotrienols provided both antitumor activity and higher protection against oxidative stress and inflammation induced by EPI in the heart. This hepatocyte-targeted biodegradable nanoparticle/tocotrienol combined therapy represents intriguing therapeutic strategy for EPI providing not only superior efficacy but also higher safety levels.

Tran AT, Ramalinga M, Kedir H, Clarke R, Kumar D.

Tocomin® represents commercially available mixture of naturally occurring tocotrienols (T3s) and tocopherols extracted from palm oil/palm fruits that possess powerful antioxidant, anticancer, neuro/cardioprotective and cholesterol-lowering properties. Cellular autophagy represents a defense mechanism against oxidative stress and several anticancer compounds. Recently, we reported that T3s induce apoptosis and endoplasmic reticulum stress in breast cancer cells.

METHODOLOGY:

We studied the effects of Tocomin® on MCF-7 and MDA-MB 231 breast cancer cells and non-tumor MCF-10A cells.

RESULTS:

Tocomin® inhibited cell proliferation and induced apoptosis in both MCF-7 and MDA-MB 231 breast cancer cell lines without affecting the viability of MCF-10A cells. We also showed that Tocomin® negatively modulates phosphoinositide 3-kinase and mTOR pathways and induces cytoprotective autophagic response in triple negative MDA-MB 231 cells. Lastly, we demonstrate that autophagy inhibitor 3-methyladenine (3-MA) potentiated the apoptosis induced by Tocomin® in MDA-MB 231 cells.

CONCLUSION:

Together, our data indicate anticancer effects of Tocomin® in breast cancer cells, which is potentiated by the autophagy inhibitor 3-MA.

Nakaso K, Tajima N, Horikoshi Y, Nakasone M, Hanaki T, Kamizaki K, Matsura T.

Tocotrienols (T3s) are members of the vitamin E family, have antioxidant properties, and are promising candidates for neuroprotection in the pathogenesis of neurodegenerative disorders such as Parkinson’s disease (PD). However, whether their antioxidant capacities are required for their cytoprotective activity remains unclear. In this regard, the antioxidant-independent cytoprotective activity of T3s has received considerable attention. Here, we investigated the signaling pathways that are induced during T3-dependent cytoprotection of human neuroblastoma SH-SY5Y cells, as these cells are used to model certain elements of PD. T3s were cytoprotective against 1-methyl-4-phenylpyridinium ion (MPP+) and other PD-related toxicities. γT3 and δT3 treatments led to marked activation of the PI3K/Akt signaling pathway. Furthermore, we identified estrogen receptor (ER) β as an upstream mediator of PI3K/Akt signaling following γT3/δT3 stimulation. Highly purified γT3/δT3 bound to ERβ directly in vitro, and knockdown of ERβ in SH-SY5Y cells abrogated both γT3/δT3-dependent cytoprotection and Akt phosphorylation. Since membrane-bound ERβ was important for the signal-related cytoprotective effects of γT3/δT3, we investigated receptor-mediated caveola formation as a candidate for the early events of signal transduction. Knockdown of caveolin-1 and/or caveolin-2 prevented the cytoprotective effects of γT3/δT3, but did not affect Akt phosphorylation. This finding suggests that T3s and, in particular, γT3/δT3, exhibit not only antioxidant effects but also a receptor signal-mediated protective action following ERβ/PI3K/Akt signaling. Furthermore, receptor-mediated caveola formation is an important event during the early steps following T3 treatment.

Sato A, Virgona N, Ando A, Ota M, Yano T.

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis and represents an attractive anticancer target. We have previously demonstrated that a redox-silent analogue of α-tocotrienol, 6-O-carboxypropyl-α-tocotrienol (T3E) exhibits potent anti-carcinogenic property in human malignant mesothelioma (MM) cells. However, inhibition of tumor growth by targeting VEGF pathway remains undetermined. In this study, we explored the inhibitory effect of T3E on the paracrine secretion of VEGF in MM cells under mimicked hypoxia by cobalt chloride (CoCl2). In this study we examine whether T3E can suppress the secretion of VEGF in MM cells exposed to mimic hypoxia by cobalt chloride (CoCl2). We found that CoCl2-induced hypoxia treatment leads to increased up-regulated hypoxia-inducible factor-2α (HIF-2α) and subsequently induced the secretion of VEGF in MM cells. This up-regulation activation mainly depended on the activation of Yes, a member of the Src family of kinases. Treatment of hypoxic MM cells with T3E effectively inhibited the secretion of VEGF, On the other hand, T3E inhibited CoCl2-induced gene expression of VEGF due to the inactivation of Yes/HIF-2α signaling. These data suggest that Yes/HIF2-α/VEGF could be a promising therapeutic target of T3E in MM cells.