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Nutrition and Disease

Tocotrienol Inhibits Secretion of Angiogenic Factors from Human Colorectal Adenocarcinoma Cells by Suppressing Hypoxia-Inducible Factor-1^1–3,

⁴ Food and Biodynamic Chemistry Laboratory and ⁵ Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan and ⁶ Department of Pharmacology, Institute of Health Biosciences, University of Tokushima, Tokushima 770-8503, Japan

^* To whom correspondence should be addressed. E-mail: nkgw{at}biochem.tohoku.ac.jp.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Tocotrienol (T3), unsaturated vitamin E, has recently gained considerable attention as a potent antiangiogenic agent minimizing tumor growth, the exact intracellular mechanisms of which remain poorly understood. Because hypoxia-inducible factor-1 (HIF-1), its downstream target vascular endothelial growth factor (VEGF), and other angiogenic factors such as interleukin-8 (IL-8) and cyclooxygenase 2 (COX-2) play critical roles in neovascularization, we tested the hypothesis that the inhibitory effect of T3 on tumor angiogenesis is via regulation of these angiogenic factors. We used 2 cancer cell lines, human colorectal adenocarcinoma cells (DLD-1) and human hepatoma cells (HepG2). T3 isomers (2 µmol/L) inhibited hypoxia-induced VEGF secretion from DLD-1, with -T3 showing potent inhibition. -T3 suppressed hypoxia-induced VEGF and IL-8 expression in DLD-1 at both mRNA and protein levels, and we found the inhibitory mechanism of -T3 by reducing HIF-1 protein expression or increasing HIF-1 degradation. Also, -T3 (2 µmol/L) did not affect hypoxia-induced COX-2 mRNA expression; however, -T3 tended to suppress (P = 0.044) hypoxia-induced COX-2 protein expression, implying a possible post-transcriptional mechanism by -T3. Overall, our results confirmed that T3 has an inhibitory effect on angiogenic factor secretion from cancer cells and revealed the possible mechanisms, providing new information about the antiangiogenic effects of T3.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

The major physiological activity of vitamin E is its well-defined antioxidative action (4), with -Toc having the most activity. However, T3 has recently attracted increasing scientific interest as a result of its superior antioxidative (5), antihypercholesterolemic (6), and neuroprotective (7) activities, which differ somewhat from those of Toc. Moreover, several lines of evidence support the beneficial effect of T3 on inhibiting tumor development (8,9). Various factors have been associated with anticancer properties, including inhibition of free radical reaction (5), downregulation of telomerase (10), activation of caspases (11), and a decrease in protein kinase C activity (12) and phosphoinositide 3-kinase signal cascades (13). Nevertheless, the in vivo potency and exact intracellular mechanisms of the anticancer properties of T3 remain poorly understood.

Generally, the anticancer effect of some drugs and nutrients is explained by direct inhibition of tumor cell growth and by indirect mechanisms (14). Results from our previous studies showed that T3 inhibited angiogenesis (15,16) and implied that T3 may inhibit tumor growth indirectly by inhibiting proliferation, migration, and tube formation of endothelial cells via suppression of growth factor signaling, thereby reducing neovascularization and the supply of nutrients and oxygen into cancer cells. Normally, angioprevention is achieved by suppressing the expressions and effects of tumor-derived angiogenic factors. Among angiogenic factors, vascular endothelial growth factor (VEGF) is an important endothelial cell-selective mitogen (17). The VEGF induces angiogenesis and produces increased vascular permeability. Interleukin-8 (IL-8) is also a potent angiogenic inducer that contributes to cancer progression as a mitogenic, angiogenic, and motogenic factor (18). These angiogenic factors (VEGF and IL-8) are elevated in human tumors and are correlated with tumor progression (19). In addition to these factors, cyclooxygenase 2 (COX-2) is upregulated in a variety of malignancies and induces malignant cell growth by stimulating proliferation and angiogenesis (20). Therefore, whether T3 suppresses tumor growth through its suppressive effect on secretion of angiogenic factors (i.e. VEGF, IL-8, and COX-2) from cancer cells is of great interest.

Hypoxia is the major pathophysiological condition regulating angiogenesis (21). Indeed, several types of tumors (e.g. mammary carcinomas and gliomas) secrete angiogenic factors (e.g. VEGF and IL-8), which are promoted by hypoxic conditions (22). Increased angiogenesis in response to hypoxia is part of an adaptive response mediated by the key transcription factor, hypoxia-inducible factor (HIF)-1 (21). Under hypoxic conditions, HIF-1 is stabilized and heterodimerized with HIF-1β/Aryl hydrocarbon receptor nuclear translocator (Arnt) to bind an enhancer element called the hypoxia response element (HRE) in target genes (23). HIF-1 induces the transcription of >70 genes, including VEGF (24). HIF-1 expression is elevated in many human cancers (25), and its overexpression is related to more aggressive tumor phenotypes (26), thus supporting the notion that the hypoxia/HIF-1 system is a potential molecular target in cancer therapeutics (27).

In this study, we tested the hypothesis that the inhibitory effect of T3 on tumor angiogenesis is attributable to reduced expression of VEGF and other angiogenic factors, which are regulated by HIF-1 under hypoxic conditions. Moreover, we evaluated the antiangiogenic mechanism by measuring transcriptional factor activity and mRNA and protein expression in cell culture studies.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Materials. T3 isomers were purchased from Chromadex. We prepared the human PGL3 luciferase (Luc) reporter vector construct containing a tandem repeat of HRE (5'-RCGTG-3'). The PGL3 control Luc reporter vector and PGL4-renilla Luc vector were purchased from Promega. Activating protein-1 (AP-1), nuclear factor-B (NF-B), and control Luc reporter constructs (pAP-1-TA-Luc, pNF-B-TA-Luc, and pTAL-Luc, respectively) were obtained from Clontech. All other reagents were of analytical grade.

    Cell cultures. Human colorectal adenocarcinoma cells (DLD-1) were obtained from the Cell Resource Center for Biochemical Research at Tohoku University School of Medicine (Sendai, Japan). Human hepatoma cells (HepG2) were obtained from the RIKEN cell bank (Tsukuba, Japan). DLD-1 or HepG2 cells were cultured in RPMI-1640 medium (containing 0.3 g/L L-glutamine and 2.0 g/L sodium bicarbonate; Sigma) supplemented with 10% fetal bovine serum (FBS) (Dainippon Pharmaceutical), 100 kU/L penicillin (Gibco), and 100 mg/L streptomycin (Gibco). Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Hypoxia (<1% O₂) was achieved using an anaerobic jar (AnaeroPack Series, Mitsubishi Gas Chemical) equipped with an AnaeroPack disposable O₂-absorbing and CO₂-generating agent.

    Preparation of T3 for cell cultures. -, β-, -, or -T3 was dissolved in ethanol at a concentration of 20 µmol/L. The solution was diluted with RPMI-1640 medium (containing 1% FBS) to achieve the desired final concentration (0–2 µmol/L). The final concentration of ethanol in the medium was 0.1% (v:v), which did not affect cell viability. Similarly, T3-free medium was prepared as a control for the study.

    ELISA. DLD-1 or HepG2 cells were seeded onto a 96-well culture plate (1 x 10⁴ cells per well) and preincubated in RPMI-1640 medium containing 10% FBS for 48 h. The culture medium was then changed to a test medium (RPMI-1640 containing 1% FBS supplemented with or without T3) and incubated under normoxic or hypoxic conditions for 24 h. An aliquot (100 µL) of the conditioned medium was then used to measure the levels of VEGF and IL-8 using a commercial ELISA kit (R&D Systems), according to the manufacturer's instructions. The results were normalized to the number of cells per well.

    Total RNA isolation and mRNA analysis. After treating DLD-1 with -T3 under normoxic or hypoxic conditions up to 24 h, total RNA were isolated from the cells (4–8 x 10⁶) with the RNeasy plus Mini kit (Qiagen) for real-time quantitative RT-PCR using the DNA Engine Opticon 2 system (MJ Research). The amount of total RNA was spectrophotometrically determined at 260 and 280 nm. RNA integrity was confirmed by visualizing intact 28S and 18S ribosomal RNA on formaldehyde-denaturing agarose gel. cDNA was synthesized from the RNA using a Ready-To-Go T-Primed First-Strand kit (Amersham Pharmacia Biotech). The cDNA was subjected to PCR amplification using a SYBR Premix Ex Taq (Takara Bio) and gene-specific primers for VEGF, IL-8, COX-2, HIF-1, and β-actin (Supplemental Table 1). PCR conditions for VEGF, IL-8, COX-2, and β-actin were 95°C for 30 s, 95°C for 5 s, 60°C for 30 s, and 75°C for 15 s for 40 cycles. PCR conditions for HIF-1 were 95°C for 1 min, 95°C for 5 s, and 60°C for 20 s for 40 cycles. After each reaction, we performed melt curve analysis to confirm the presence of only a single reaction product. In addition, a representative PCR product was electrophoresed on 2.0% agarose gel to verify that only a single band was present. The ratio between the β-actin content in the control and test samples was defined as the normalization factor.

    Western blot analysis. DLD-1 cells were incubated with -T3 under normoxic or hypoxic conditions for up to 24 h. Thereafter, cellular proteins (40 µg/well) were prepared from the cells as previously described (15) and were separated by SDS-PAGE (4–20% e-PAGEL; Atto). The protein bands were then transferred to polyvinylidene fluoride membranes (Amersham Pharmacia Biotech). The membrane was blocked to nonspecific sites and probed with primary antibodies, followed by a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). Antibody reactions were detected using ECL Plus western blotting reagents (Amersham Pharmacia Biotech). The antibodies used were anti-HIF-1 (BD Transduction Laboratories), anti-COX-2 (IBL), and anti-β-actin (Cell Signaling).

    Transient transfection and Luc assay. DLD-1 cells were seeded onto a 6-well culture plate (2 x 10⁵ cells per well) and preincubated in RPMI-1640 containing 10% FBS for 12 h. Then, the cells were transiently transfected with HIF-1, AP-1, NF-B, and control reporter plasmid using Lipofectamine LTX (Invitrogen). The PGL4-renilla Luc plasmid was cotransfected into the cells as an internal control for transfection efficiency. We used an empty plasmid vector as a mock transfection control. After transfection, the cells were cultured for 24 h and incubated with -T3 under normoxic or hypoxic conditions for 24 h. Cells were then washed with PBS, lysed with Passive Lysis Buffer (Promega), and Luc activity was measured using the Luc assay system (Promega). The relative Luc activity (defined as each transcriptional factor reporter activity) was calculated as the ratio of firefly:renilla Luc activity and normalized to the control.

    Statistical analysis. Values are expressed as means ± SD. We performed statistical analysis using 2-way ANOVA with a Bonferroni/Dunn post hoc test when the interaction was significant. For statistical evaluation of effects on VEGF secretion, we conducted 1-way ANOVA followed by a Bonferroni/Dunn test. Differences were considered significant at P < 0.05.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
We examined the effect of T3 on VEGF secretion from DLD-1 or HepG2 cells under normoxic and hypoxic conditions (Fig. 1). Cells that were incubated in hypoxic conditions for 24 h secreted 1-fold more VEGF than those in normoxic cultures. All T3 isomers suppressed VEGF secretion in a dose-dependent manner, especially under hypoxic conditions, and the inhibitory effect was ranked as - > β- > - > -T3. No apparent cytotoxic effects, e.g. obvious cell death, were observed (data not shown). These results showed that T3 inhibits VEGF secretion from cancer cells. Because -T3 had a greater inhibitory effect on DLD-1 than HepG2 under hypoxic conditions, we decided to use -T3 and DLD-1 for later experiments. -T3 suppressed secretion of IL-8 (an important angiogenic cytokine) from DLD-1, especially under hypoxic conditions (Fig. 2).

View larger version (41K): [in this window] [in a new window]   FIGURE 1  T3 inhibited VEGF secretion from DLD-1 and HepG2 cells treated with 1% FBS RPMI-1640 medium containing different concentrations of each T3 isomer under normoxic (A,C) and hypoxic (B,D) conditions. After cultivation up to 24 h, VEGF protein in the conditioned medium was measured by ELISA. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

View larger version (9K): [in this window] [in a new window]   FIGURE 2  -T3 inhibited IL-8 secretion from DLD-1 cells treated with 1% FBS RPMI-1640 medium containing -T3 (02 µmol/L) under normoxic and hypoxic conditions. After incubation for 24 h, IL-8 protein in the conditioned medium was measured by ELISA. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

View larger version (45K): [in this window] [in a new window]   FIGURE 3  Effect of -T3 on the hypoxia-induced expression of VEGF, IL-8, COX-2, and HIF-1 mRNA in DLD-1 cells treated with 1% FBS RPMI-1640 medium containing -T3 (0 or 2 µmol/L) under normoxic or hypoxic conditions (<1% O2). After incubation for 1224 h, mRNA levels were measured by real-time quantitative RT-PCR and compared with a vehicle control in normoxia. Values are means ± SD, n = 6. Means without a common letter differ, P < 0.05.

View larger version (43K): [in this window] [in a new window]   FIGURE 4  Effect of -T3 on expression of HIF-1 and COX-2 protein in DLD-1 cells incubated with 1% FBS RPMI-1640 medium containing -T3 (0 or 2 µmol/L) under normoxic or hypoxic conditions (<1% O2) for 12 and 24 h, followed by western blot analysis. Each western blot is a representative example of data from 3 replicate experiments. Band intensities were evaluated by densitometric analysis and their values were compared with a vehicle control in normoxia. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

View larger version (28K): [in this window] [in a new window]   FIGURE 5  Effect of -T3 on the hypoxia-induced HIF-1, AP-1, and NF-B transcriptional activity in DLD-1 cells transfected with the PGL3 reporter vector containing a tandem repeat of HIF-1 binding site (HRE) and SV40 promoter (A), pAP-1-TA-Luc vector (B), or pNF-B-TA-Luc vector (C). DLD-1 cells were also transfected with PGL-3 control (D) or pTAL-Luc (E) reporter vector (as a negative control). As an internal reference vector, the pGL4.74 [hRluc/TK] vector, which expresses renilla Luc, was cotransfected. After transfection for 24 h, cells were treated with or without -T3 (0 or 2 µmol/L) under normoxic or hypoxic conditions (<1% O2) for 24 h. Reporter activity was calculated as the ratio of firefly:renilla Luc activity and normalized to the control. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

T3 suppressed VEGF secretion from DLD-1 or HepG2, especially under hypoxic conditions (Fig. 1). The inhibitory potency of the isomers varied in the order of - > β- > - > -T3. The same tendency was reported for the cellular uptake of T3 and for the inhibition of telomerase, DNA polymerase , or angiogenesis by T3 (10,15,33). Structurally, -T3 lacks the 5- and 7-methyl groups attached to its chroman ring, so it is possible that -T3 could pass easily through cell membranes. The better incorporation of -T3 into DLD-1 or HepG2 could explain its greater inhibitory effect on VEGF secretion. In our study, -T3 inhibited IL-8 secretion from DLD-1 under hypoxic conditions (Fig. 2) and suppressed the hypoxia-induced expression of VEGF and IL-8 mRNA (Fig. 3). Although under normoxic conditions, IL-8 secretion was slightly higher, with 2 µmol/L -T3 compared with 1 µmol/L, the differences were not so great. IL-8 is a potent lymphocyte chemoattractant that contributes to cancer progression by acting as a mitogenic, angiogenic, and motogenic factor (18). Because treating cancer cells with antibody for IL-8 resulted in attenuation of tumor growth and reduced tumor-associated angiogenesis (22), our findings (the suppressive effect of T3 on IL-8) seem to be pertinent to cancer prevention.

Hypoxia induces many stress response genes, such as HIF-1, AP-1, and NF-B, but it is still unclear which genes are specifically involved in hypoxia-induced changes in cancers (34). HIF-1 protein is constitutively expressed under normoxic conditions but is rapidly degraded by the ubiquitin-proteasome pathway (35), which is mediated by the specific binding of the von Hippel-Lindau tumor suppressor to HIF-1 (36). The prolyl hydroxylation of HIF-1 is required for the HIF-1-von Hippel-Lindau interaction, which is critical for degrading HIF-1 (35). Under hypoxic conditions, due to the inhibition of the prolyl hydroxylation, HIF-1 escapes from degradation, accumulates, translocates into the nucleus, and forms a heterodimer with HIF-1β. HIF-1 then activates expression of the VEGF gene by binding to HRE in the VEGF promoter region (29). Increased levels of VEGF expression and microvessel density in cancer directly correlate with a poor prognosis (37). In this study, -T3 not only inhibited hypoxia-induced HIF-1 protein accumulation (Fig. 4), but it also suppressed hypoxia-induced HIF-1–dependent transcriptional activity (Fig. 5). These findings indicate that suppression of the hypoxia-induced VEGF expression by T3 (Fig. 3) is due at least in part to its inhibitory effects on HIF-1 transactivation of the VEGF gene. In addition, although hypoxia had no obvious effects on HIF-1 mRNA expression, the level was reduced by -T3 treatment for 24 h (Fig. 3). Therefore, -T3 may inhibit HIF-1 protein expression through both transcriptional and post-transcriptional mechanisms.

In addition to the HIF-1 pathway, VEGF and IL-8 transcription are also highly responsive to NF-B and AP-1 activation (24). A recent study showed that NF-B– and AP-1–binding sites are indispensable for VEGF and IL-8 gene expression in hypoxic conditions (28). In this respect, VEGF and IL-8 expression are directly correlated with NF-B and AP-1 activity in melanoma and pancreatic cancer cell lines (38). These transcriptional factors are also implicated in the control of VEGF transcription, and inhibition of these activities reduces VEGF and IL-8 expression, as well as tumorigenicity and angiogenesis of prostate and ovarian cancer cells (39). In this study, -T3 inhibited the hypoxia-induced transcriptional factor activity (Fig. 5). It is likely that -T3 inhibits angiogenic factor secretion by regulating these transcriptional factors or upstream protein. Further studies are needed to clarify the mechanism. On the other hand, hypoxia caused a slight upregulation of pGL3-control vector (negative control) Luc activity. This may be due to some transcriptional factor binding elements in the backbone and promoter regions of this vector.

COX-2, the isoform responsible for prostanoid production, is upregulated in a variety of malignancies (40,41) and induces malignant cell growth by stimulating proliferation and angiogenesis (20). Its overexpression is closely related to tumor metastasis (42). Although the exact mechanisms responsible for the effects of COX-2 are still unknown, its products (prostaglandins) seem to be involved (43). In support of this notion, prostaglandin E2, a major COX-2 product, promotes tumor growth, angiogenesis, and cell motility as well as preventing apoptosis (44). In this study, -T3 did not inhibit hypoxia-induced COX-2 mRNA expression (Fig. 3) but slightly suppressed hypoxia-induced COX-2 protein expression (Fig. 4). -T3 reportedly suppresses tumor necrosis factor-–induced COX-2 expression in human chronic myeloid leukemia cells at the protein level (45). This knowledge (45), together with our findings (Figs. 3 and 4), indicate the possibility that T3 inhibits external stimulation-induced COX-2 expression by a post-transcriptional mechanism (i.e., by affecting COX-2 protein synthesis and/or degradation).

We have demonstrated that T3 inhibits angiogenesis in vitro and in vivo (15). The inhibitory effect was attributable to the regulation of growth factor-dependent phosphatidylinositol-3 kinase/phosphoinositide-dependent protein kinase/Akt signaling, as well as to apoptosis induction in endothelial cells. In the present study, T3 suppressed angiogenic factor secretion from cancer sells via the inhibition of a hypoxia-induced transcriptional factor. Thus, the inhibitory effects of T3 on angiogenic factor secretion are involved directly or indirectly in its antiangiogenic property. Recently, Kashiwagi et al. (46) reported that the survival and invasion capacity of tumor cells under hypoxic conditions were suppressed by 6-O-carboxypropyl--T3, T3 ether analogue, via the inactivation of c-Src. 6-O-carboxypropyl--T3 potently inhibited tumor hypoxic adaptation, but -T3 did not. In our study, -T3 was the most potent inhibitor of hypoxia-induced VEGF and IL-8 expressions from cancer cells, whereas -T3 had the least inhibition effect among T3 isomers; therefore it is likely that -T3 as well as its ether analogue may suppress tumor hypoxic adaptation via the inactivation of c-Src. Further elucidation of the detailed mechanisms for angioprevention by T3 is the subject of ongoing investigation. On the other hand, some antiangiogenic agents are available from natural sources (47–52). For instance, tea catechin (epigallocatechin gallate) and red wine polyphenol (resveratrol) have antiangiogenic effects via inhibition of gene expression or signal transduction of VEGF (48,49). Although these compounds, as well as T3, are natural products, questions regarding their safety and toxicity must be addressed. In the case of T3, there was no critical weight loss or adverse events in animals in preclinical studies (53). T3 is absorbed through the intestine (54) and is distributed into the bloodstream of humans, suggesting that T3 is bioavailable. T3 reached a concentration of 1.6 µmol/L in human plasma after T3 oral administration (700 mg/d) (55). In this study, the concentrations of -T3 (0.5–2 µmol/L) were high enough to inhibit in vitro angiogenic factor secretion. It is thus tempting to speculate that the inclusion of T3 in diets may have anticancer effects through angiogenesis inhibition.

In conclusion, we demonstrated that T3 inhibits hypoxia-induced VEGF and IL-8 expression in tumor cells via reduction of HIF-1 protein accumulation or suppression of transcriptional factors. These findings provide a mechanistic rationale for the development of T3 as an angiopreventive agent, which warrants testing of T3 in other cell or animal models of cancer, with a realistic prospect of its use in human therapy.

	FOOTNOTES

 
¹ Supported by a Grant-in-Aid from the Bio-oriented Technology Research Advancement Center of the National Agricultural and Biological Research Organization, Japan and by Project M Co. Ltd (Sendai, Japan).

² Author disclosures: A. Shibata, K. Nakagawa, P. Sookwong, T. Tsuzuki, S. Tomita, H. Shirakawa, M. Komai, and T. Miyazawa, no conflicts of interest.

³ Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org.

⁷ Abbreviations used: AP-1, activating protein-1; COX-2, cyclooxygenase 2; DLD-1, human colorectal adenocarcinoma cells; FBS, fetal bovine serum; HepG2, human hepatoma cells; HIF, hypoxia-inducible factor; HRE, hypoxia response element; IL-8, interleukin-8; Luc, luciferase; NF-B, nuclear factor-B; Toc, tocopherol; T3, tocotrienol; VEGF, vascular endothelial growth factor.

Manuscript received 21 May 2008. Initial review completed 20 June 2008. Revision accepted 26 August 2008.

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