Wen-Guang Sun

Inhibition of proliferation and induction of apoptosis by γ-tocotrienol in human colon carcinoma HT-29 cells

OBJECTIVE gamma-Tocotrienol is a major component of the tocotrienol-rich fraction of palm oil, but there is limited evidence that it has antitumor activity. In particular, the effects of gamma-tocotrienol on human colon carcinoma cells have not been reported. To investigate the chemopreventive effects of gamma-tocotrienol on colon cancer, we examined its capacity to inhibit proliferation and induce apoptosis in HT-29 cells and explored the mechanism underlying these effects. METHODS We cultured HT-29 cells in the presence of gamma-tocotrienol. The effect of gamma-tocotrienol on cell proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mitotic index, and colony formation. The cell-cycle distribution was investigated by flow cytometry. We measured apoptosis by nuclear staining, transmission electron microscopy, and DNA fragmentation. Apoptosis-related proteins and the nuclear factor-kappaB p65 protein were determined by western blotting and immunofluorescence. RESULTS gamma-Tocotrienol inhibited cell growth and arrested HT-29 cells in G(0)/G(1) phase. The 50% inhibitory concentration was 31.7 micromol/L (48 h). gamma-Tocotrienol-induced apoptosis in HT-29 cells was accompanied by downregulation of Bcl-2, upregulation of Bax, and activation of caspase-3. Furthermore, we found that gamma-tocotrienol reduced the expression level of total nuclear factor-kappaB p65 protein and inhibited its nuclear translocation. CONCLUSION The results indicated that gamma-tocotrienol inhibits cell proliferation and induces apoptosis in HT-29 cells in a time- and dose-dependent manner, and that this process is accompanied by cell-cycle arrest at G(0)/G(1), an increased Bax/Bcl-2 ratio, and activation of caspase-3. Our data also indicated that nuclear factor-kappaB p65 protein may be involved in these effects.

γ-Tocotrienol induces mitochondria-mediated apoptosis in human gastric adenocarcinoma SGC-7901 cells.

Tocotrienols are naturally occurring isoprenoid compounds highly enriched in palm oil, rice bran, oat, wheat germ, barley and rye. Tocotrienols have antioxidant properties as well as potent anticancer properties. In this study, the mechanisms underlying the apoptosis of gamma-tocotrienol on human gastric adenocarcinoma SGC-7901 cells were further studied, especially in correlation with the involvement of the apoptotic pathway. gamma-Tocotrienol inhibited SGC-7901 cell growth in a concentration- and time-dependent manner. The inhibitory effects of SGC-7901 cells were correlated with the DNA damage and arresting cell cycle at G(0)/G(1) phase in a time-dependent manner at 60 mumol/L concentration of gamma-tocotrienol. gamma-Tocotrienol induced activation of caspase-3 and increased the cleavage of the downstream substrate poly(ADP-ribose) polymerase. Furthermore, gamma-tocotrienol-induced apoptosis on SGC-7901 cells was mediated by activation of caspase-9. The data in this study suggested that gamma-tocotrienol could induce the apoptosis on human gastric cancer SGC-7901 cells via mitochondria-dependent apoptosis pathway. Thus, our findings revealed gamma-tocotrienol as a potential, new chemopreventive agent for human gastric cancer.

β‐Ionone suppresses mammary carcinogenesis, proliferative activity and induces apoptosis in the mammary gland of the Sprague‐Dawley rat

β‐Ionone demonstrates potent anticancer activity both in vitro and in vivo. We determined tumor incidence and the number of rats bearing tumors as well as cell proliferation and apoptosis in a rat mammary cancer model induced by 7, 12‐dimethylbenz[a]anthracene (DMBA). Rats were fed an AIN‐76A diet containing β‐ionone (0, 9, 18 or 36 mmol/kg), starting 2 weeks before DMBA administration and continuing for 24 weeks. A dose‐dependent inhibition of mammary carcinogenesis by dietary β‐ionone was observed. Corresponding tumor incidence values were 82.1, 53.3, 25.9 and 10.0% (p < 0.01 or 0.05). Time to tumor appearance increased and tumor multiplicity decreased with increasing dietary β‐ionone. Histopathological and immunohistochemical evaluations of tumors were performed on the 64, 31, 15 and 3 tumors, respectively, identified in rats from the respective groups of 30. The proportions of adenocarcinomas, adenomas and benign masses were equally distributed in the latter group. In proportions within the other groups, the proportions of adenocarcinomas and benign masses decreased and increased with increasing dietary β‐ionone. Proliferating cell nuclear antigen (PCNA), cyclin D1 and Bcl‐2 expression decreased, and Bax expression and nuclear fragmentation increased with increasing dietary β‐ionone. These results demonstrate the potent capacity of dietary β‐ionone to suppress DMBA‐initiated mammary cancer in rats.

γ-Tocotrienol-induced apoptosis in human gastric cancer SGC-7901 cells is associated with a suppression in mitogen-activated protein kinase signalling

Tocotrienols have been shown to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in tocotrienol-induced apoptosis are still unclear. In the present study, gamma-tocotrienol induced apoptosis in human gastric adenocarcinoma SGC-7901 cell line through down regulation of the extracellular signal-regulated kinase (ERK) signalling pathway. Furthermore, gamma-tocotrienol-induced apoptosis was accompanied by down regulation of Bcl-2, up regulation of Bax, activation of caspase-3, and subsequent poly (ADP-ribose) polymerase cleavage. These results indicated that up or down regulation of Bcl-2 family proteins play a major role in the initiation of gamma-tocotrienol-induced apoptosis as an activator of caspase-3. Gamma-tocotrienol also down regulated the activation of the Raf-ERK signalling pathway, and down regulated c-Myc by decreasing the expressions of Raf-1 and p-ERK1/2 proteins. The results suggest that key regulators in tocotrienol-induced apoptosis may be Bcl-2 families and caspase-3 in SGC-7901 cells through down regulation of the Raf-ERK signalling pathway.

Inhibitory effects of γ-tocotrienol on invasion and metastasis of human gastric adenocarcinoma SGC-7901 cells☆☆☆

Natural vitamin E is a mixture of two classes of compounds, tocopherols and tocotrienols. Recent research has revealed that tocotrienols, especially gamma-tocotrienol, exhibit not only the same antioxidant ability as tocopherols, but also remarkable anticancer capacity in cancer cell lines. In this study, the invasion and metastatic capacities of gastric adenocarcinoma SGC-7901 cells and the correlation with antimetastasis mechanisms induced by gamma-tocotrienol were explored. The results showed the inhibitory effects of gamma-tocotrienol at doses of 15, 30, 45 and 60 mumol/L for 48 h on cell migration and cell matrigel invasion; activities of matrix metalloproteinase (MMPs) increased in SGC-7901 cells when compared to the control group (P<.05 or P<.01). An increasing trend in the chemotactic responses to fibronectin (FN) in SGC-7901 cells was found in the gamma-tocotrienol treatments. SGC-7901 cell attachment decreased in the gamma-tocotrienol-treated groups in comparison with the control group (P<.01). The mRNA expressions of MMP-2 and MMP-9 showed that gamma-tocotrienol significantly reduced the matrigel invasion capability through down-regulation of the mRNA expressions of MMP-2 and MMP-9 (P<.01), and up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in SGC-7901 cells by treatment with gamma-tocotrienol for 48 h (P<.05). gamma-Tocotrienol also significantly increased the mRNA expression of nm23-H1 in SGC-7901 cells (P<.01). These findings suggest a potential mechanism of gamma-tocotrienol-mediated antitumor metastasis activity and indicate the role of vitamin E as potential chemopreventative agents against gastric cancer.

γ-Tocotrienol modulates the paracrine secretion of VEGF induced by cobalt(II) chloride via ERK signaling pathway in gastric adenocarcinoma SGC-7901 cell line.

Hypoxia is a common characteristic feature of solid tumors, and carcinoma cells are known to secrete many growth factors. These growth factors, such as vascular endothelial growth factor (VEGF), play a major role in the regulation of tumor angiogenesis and metastasis. In this study, the effect of gamma-tocotrienol, a natural product commonly found in palm oil and rice bran, on the accumulation of HIF-1alpha protein and the paracrine secretion of VEGF in human gastric adenocarcinoma SGC-7901 cell line induced by cobalt(II) chloride (as a hypoxia mimic) was investigated. These results showed that cobalt(II) chloride induced the high expression of VEGF in SGC-7901 cells at dose of 150 micromol/L for 24h. Both basal level and cobalt(II) chloride-induced HIF-1alpha protein accumulation and VEGF paracrine secretion were inhibited in SGC-7901 cells treated with gamma-tocotrienol at 60 micromol/L treatment for 24 h. U0126, a MEK1/2 inhibitor, decreased the expression of HIF-1alpha protein and the paracrine secretion of VEGF under normoxic and hypoxic conditions. In this study, gamma-tocotrienol also significantly inhibited the hypoxia-stimulated expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2). The mechanism seems to involve in inhibiting hypoxia-mediated activation of p-ERK1/2, it leads to a marked decrease in hypoxia-induced HIF-1alpha protein accumulation and VEGF secretion. These data suggest that HIF-1alpha/VEGF could be a promising target for gamma-tocotrienol in an effective method of chemoprevention and chemotherapy in human gastric cancer.

Effects of β-ionone on mammary carcinogenesis and antioxidant status in rats treated with DMBA.

Recent chemopreventive studies from our group showed that dietary β -ionone inhibited 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis by the inhibition of cell proliferation and apoptosis initiation. In this study, we examined the chemopreventive effects of varied doses of dietary β -ionone on the development and growth of DMBA-induced rat mammary tumors as well as plasma antioxidant status. β -ionone treatment groups were given 9, 18, and 36 mmol/kg in the AIN76A diet starting 2 wk prior to DMBA administration and continuing for the 24 wk. Results showed that tumor incidence was dose dependently reduced by 35.4, 68.3, and 87.8%, respectively, compared to the positive control. Tumor sizes were dose dependently smaller, and tumor weight was less in each group, each rat, and each tumor compared to the positive control ( P < 0.05). A significant decrease in lipid peroxidation was observed in the tumor-induced rats treated with dietary β -ionone, whereas the plasma activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, superoxide dismutase, and the nonenzymatic antioxidant glutathione were increased in the β -ionone treated rats when compared to control. The levels of catalase and lactate dehydrogenase were remarkably decreased in the β -ionone treated groups compared to the positive control group. These results suggest that dietary β -ionone has biologically relevant antioxidant activity and plays a chemopreventive role against DMBA induced mammary gland tumors.

γ-Tocotrienol inhibits angiogenesis of human umbilical vein endothelial cell induced by cancer cell

Antiangiogenic therapy mediated by food components is an established strategy for cancer chemoprevention. Growth factors play critical roles in tumor angiogenesis. A conditioned medium containing growth factors from human gastric adenocarcinoma SGC-7901 cell conditioned medium was used as an angiogenic stimulus in this study. The purpose of this study was to evaluate the inhibitory effect and possible mechanism of γ-tocotrienol on tumor angiogenesis. The results showed that γ-tocotrienol (10-40 μmol/L) significantly suppressed proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) induced by SGC-7901 cell conditioned medium in a dose-dependent manner. γ-Tocotrienol (800-1200 μg/egg) also inhibited new blood vessel formation on the growing chick embryo chorioallantoic membrane in a dose-dependent manner. Moreover, the inhibitory effects of γ-tocotrienol on HUVECs were correlated with inducing the apoptosis and arresting cell cycle at the G(0)/G(1) phase at a dose of 40 μmol/L γ-tocotrienol. In addition, γ-tocotrienol inhibited angiogenesis in HUVECs by down-regulation of β-catenin, cyclin D1, CD44, phospho-VEGFR-2 and MMP-9. The antiangiogenic effects of γ-tocotrienol on HUVECs may be attributable to regulation of Wnt signaling by decreasing β-catenin expression. Thus, our results suggest that γ-tocotrienol has a potential chemopreventive agent via antiangiogenesis.

γ-Tocotrienol inhibits cell viability through suppression of β-catenin/Tcf signaling in human colon carcinoma HT-29 cells

γ-Tocotrienol, a major component of the tocotrienol-rich fraction of palm oil, has been suggested to have antioxidant and anticancer activity as well as potent chemopreventive effects on tumor cells. In this study, the mechanisms underlying γ-tocotrienol-mediated growth inhibition of human carcinoma HT-29 cells were further investigated, especially in correlation with the involvement of β-catenin/T-cell factor (Tcf) signaling pathway. We found that γ-tocotrienol could strongly suppress the transcriptional activity of β-catenin/Tcf signaling pathway in HT-29 cells. γ-Tocotrienol inhibited the expression level of total β-catenin protein but did not significantly affect the phosphorylated β-catenin level. Meanwhile, γ-tocotrienol down-regulated the protein level of nuclear β-catenin and induced its redistribution to cell membrane. Furthermore, γ-tocotrienol suppressed the expression of downstream target genes such as c-myc, cyclin D1 and survivin. The results demonstrated that γ-tocotrienol-inhibited growth and -induced apoptosis in HT-29 cells were accompanied by significant inhibition of β-catenin/Tcf signaling. Blocking the expression of β-catenin with small interfering RNA significantly suppressed the ability of γ-tocotrienol to reduce viability and induce apoptosis in HT-29 cells. Thus, our data suggested that γ-tocotrienol exerts its anticancer activity through β-catenin/Tcf signaling, and β-catenin is a target for γ-tocotrienol in the Wnt/β-catenin signaling pathway.

Growth inhibition effect of β-catenin small interfering RNA-mediated gene silencing on human colon carcinoma HT-29 cells.

The β-catenin gene is a critical component of Wnt signaling pathway. Aberrant activation of Wnt/β-catenin signaling and subsequent upregulation of β-catenin is related to enhancing cell proliferation and developing colon polyps and colon cancer. In the present study, the effect of β-catenin knockdown on the growth and survival of the human colon cancer cell line HT-29 was investigated in vitro. The effect of knockdown of β-catenin on cell proliferation was investigated by MTT assay and colony formation. The cell cycle distribution was investigated by flow cytometry. Apoptosis was measured by nuclear staining and flow cytometry. The change of β-catenin and related proteins were determined by western blotting and immunofluorescence. The results showed that small interfering RNA directed against β-catenin markedly inhibited the expression and nuclear translocation of β-catenin and decreased the expression of known target genes such as cyclin D1 and c-myc; HT-29 cell proliferation was inhibited as indicated by growth reduction, cell cycle arrest in G0/G1 phase, and induction of apoptosis; and the inhibition of cell growth may be associated with switching off cyclin D1 and c-myc expression by small interfering RNA targeted against β-catenin in colon cancer HT-29 cells.