Id1, inhibitor of differentiation, is a key protein mediating anti-tumor responses of gamma-tocotrienol in breast cancer cells

Cancer Letters 291 (2010) 187–199

Contents lists available at ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Id1, inhibitor of differentiation, is a key protein mediating anti-tumor responses of gamma-tocotrienol in breast cancer cells Wei Ney Yap a,1, Norazean Zaiden a,1, Yee Ling Tan b, Chang Piek Ngoh c, Xue Wu Zhang d, Y.C. Wong e, M.T. Ling f,*, Yee Leng Yap a,* a

Davos Life Science Pte. Ltd., Cancer Research Laboratory, 16 Tuas South Street 5, Singapore Singapore Bioimaging Consortium (SBIC), Biomedical Sciences Institutes, 11 Biopolis Way #02-02 Helios, Singapore 138667, Singapore c Duke-NUS Graduate Medical School Singapore, 8 College Road, Singapore 169857, Singapore d College of Light Industry and Food Sciences, South China University of Technology, Guang Zhou, China e Department of Anatomy, Cancer Biology Lab, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 1/F, Laboratory Block, 21 Sassoon Road, Hong Kong SAR, China f Australian Prostate Cancer Research Centre-Queensland Institute of Health and Biomedical Innovation (IHBI), QUT, Australia b

a r t i c l e

i n f o

Article history: Received 3 August 2009 Received in revised form 9 October 2009 Accepted 15 October 2009

Keywords: Tocotrienol Tocopherol Vitamin E Breast cancer Id-1 Docetaxel Chemosensatizing

a b s t r a c t 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. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Breast cancer (BCa) is the most common type of cancer in female population worldwide [1]. Despite advances in

* Corresponding authors. E-mail addresses: [email protected] (W.N. Yap), [email protected] (N. Zaiden), [email protected] (Y.L. Tan), [email protected] (C.P. Ngoh), [email protected] (X.W. Zhang), [email protected] (Y.C. Wong), [email protected] (M.T. Ling), [email protected] (Y.L. Yap). 1 Both authors contribute equally to this study. 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.10.012

early detection and the understanding of the molecular bases of breast cancer biology, 30% of women with early-stage breast cancer develop recurrent disease attributed to resistance to systemic therapies (cytotoxic, hormonal, and immunotherapeutic agents) [2,3]. In general, systemic agents are active at the beginning of therapy in majority of primary and metastatic breast cancers. However, after initial treatment, disease progression occurs frequently. For example, the overall response to Docetaxel therapy in patients developing recurrence is about 50% [4]. At advanced stage, tumor is usually resistance to most of the available therapy [2,3]. Prevailing chemotherapy

188

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

failure describes three resistant phenotypes: (1) cells with molecular alterations in trans-membrane drug transport; (2) increased detoxification and repair pathways; and (3) molecular alterations leading to failure of pro-apoptotic response [2,3]. Therefore, an alternative treatment which enhances the pro-apoptotic response with low toxicity is urgently needed for the treatment of BCa. Historically, natural products have been a rich sources of biologically active compounds for drug discovery [5]. Tocotrienols (T3) are important plant vitamin-E constituents found in palm. Together with tocopherols (TP), they provide a significant source of anti-oxidant activity to all living cells [6,7]. This common anti-oxidant attribute reflects the similarity in chemical structures between T3 and TP, which differ only in their structural side chain (contains farnesyl for T3 or saturated phytyl side chain for TP). The common hydrogen atom from the hydroxyl group on the chromanol ring acts to scavenge the chainpropagating peroxyl free radicals. Depending on the locations of methyl groups on their chromanol ring, T3 can be distinguished into four isomeric forms: alpha (a), beta (b), gamma (c), and delta (d). Apart from T3’s anti-oxidant, anti-inflammatory, antiangiogenic, anti-neurodegeneration, anti-hypercholesterolemic and anti-microbial properties, emerging in vitro and in vivo evidences have manifested the anti-cancer activity of T3 on numerous human cancers including prostate, colon, skin and gastric [8–13] (reviewed in [14]). Recently, we showed that gamma-T3 was the most potent isomer in inducing prostate cancer and melanoma cell apoptosis, as evidenced by the activation of pro-caspases and the presence of sub-G1 cell population after the treatment [15,16]. The apoptosis response was found to strongly associate with Id1 expression. Id1 is a member of the Helix–Loop–Helix protein family which plays important roles in cell proliferation, differentiation, and tumorigenesis. Id1 has been shown to be upregulated in a wide-range of cancers and its expression has been demonstrated to correlate with disease stages and poor prognosis of human cancers. Previously, examination of the pro-survival genes in prostate cancer and melanoma cells revealed that the gamma-T3-induced cell death was associated with suppression of NF-jB and EGF-R [15,16]. Meanwhile, gamma-T3 treatment also resulted in the induction of JNK signaling pathway and inhibition of JNK activity by specific inhibitor (SP600125) was able to partially block the effect of gamma-T3. Interestingly, gamma-T3 treatment was found to suppress mesenchymal markers expression and restore E-cadherin and gammacatenin expression [15], which was associated with suppression of cell invasion capability. Furthermore, synergistic effect was observed when cells were co-treated with gamma-T3 and Docetaxel. In a separate study, T3 treatment was also demonstrated to induce apoptosis through a p53 dependent mechanism [8]. Using p53+ human colon carcinoma RKO cells, researchers demonstrated that tocotrienol rich fraction (TRF) induced activation of the Bax gene through upregulation of p53. This was associated with the release of cytochrome c from nucleus to cytosol, induction of Apaf1 oligomerization and activation of caspase 9. Mean-

while, the tocotrienol-induced p53 expression was also found to result in the downregulation of Bcl-2 level, which eventually triggers apoptotic signaling cascade by increasing the bax/bcl-2 ratio. In addition to p53 pathway, phosphatidylinositol-3-kinase dependent kinase (PI3K)/ PI3K-dependent (PDK1) mitogenic signaling cascade [17] and NF-jB [18] pathways have both been suggested to contribute to T3-induced apoptosis. For example, in gamma-T3 treated cancer cells, inactivation of PDK1 and Akt as well as downregulation of their downstream effectors such as FLICE-inhibitory protein (FLIP) and p-NF-jB were observed [18]. FLIP reduction was shown to promote the cleavage of caspases 3 and 8, which results in growth arrest and apoptosis in T3-treated cells. These findings support that the anti-cancer effect of gamma-T3 may involve multiple signaling pathways. Here, we demonstrated that Id1, an inhibitor of differentiation, may be an important target of gamma-T3 in BCa cells. We found that gamma-T3 significantly downregulated Id1 through regulation of its upstream regulators. This suppression of Id1 level by gamma-T3 was associated with induction of apoptosis and promotion of chemosensitivity in BCa cells. Finally, partial restoration of Id1 levels was found to protect the cells from gamma-T3-induced apoptosis, suggesting that the anti-cancer effect of gamma-T3 is mediated through inactivation of Id1. 2. Materials and methods 2.1. Cancer cell lines, cell culture conditions and chemicals The human estrogen-dependent BCa cells (MCF-7), human estrogen-independent BCa cells (MDA-MB-231), androgen-independent prostate cancer cells (PC-3) (ATCC, Rockville, MD) were maintained in their respective medium recommended by ATCC (Invitrogen, Carlsbad, CA) supplemented with 2 mmol/l L-glutamine, 10% fetal calf serum (FCS) and 2% penicillin streptomycin at 37 °C in 5% CO2. The immortalized human non-tumorigenic breast epithelial cell line (MCF-10A) (ATCC, Rockville, MD) was maintained in MEBM, which is supplied as part of the MEGM Bullet Kit available from Clonetics Corporation. To make the complete growth medium, the following components were added into the base medium: all MEGM SingleQuot additives that are supplied with the kit except the GA-1000 (BPE 13 mg/ml, 2 ml; hydrocortisone 0.5 mg/ml, 0.5 ml; hEGF 10 ug/ml, 0.5 ml; insulin 5 mg/ml, 0.5 ml); 100 ng/ml cholera toxin. The stable Si-Id1 PC-3 cell line (Id1 knockdown model) was contributed by Prof. Y.C. Wong (HKU) based on previous protocol [19]. Docetaxel (Calbiochem, Darmstadt, Germany), JNK inhibitor SP600125, Erk inhibitor U0126 (Sigma–Aldrich, St. Louis, USA) and b-aminopropionitrile (APN) (TCI, Japan) were dissolved in dimethylsulfoxide (DMSO). The treatment solutions were diluted in culture medium to obtain the desired concentrations. 2.2. Generation of Id1 transfectants MDA-MB-231 cells (1  105 cells/well) were plated into 12-well culture plates and allowed to grow for 24 h. pc-Id1

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

or pcDNA (a gift from Prof. M.T. Ling, IHBI) was transfected into the cells using Fugene 6 reagent for 24 h before gamma-T3 treatment. 24 h later, the cells were either assayed for MTT cell viability or lysed for Western blotting. 2.3. Tocotrienol (T3) and tocopherol (TP) isomers T3 and TP isomers were extracted and purified from palm oil using Davos distillation technology [20]. The extraction facility is located at Tuas Singapore. Crude palm oil (CPO) feed was purchased from Kuala Lumpur Kepong Berhad. Using the corresponding T3 isomers as the reference standard, the purity of T3 and TP isomers was verified to be P97% by high performance liquid chromatography (HPLC) percentage area (%area). 2.4. Cell viability study and time course experiment For cell viability study, 5  103 cells re-suspended in 100 ll medium were plated into each well of a 96-well plate. The cells were then treated with different concentrations of the vitamin-E isomers for 24 h. After the treatment, 20 ll of MTT solution (1 mg/ml in PBS) (Sigma–Aldrich, St. Louis, USA) was added into each well and the cells were incubated at 37 °C for 2 h. The formazan crystals were then re-suspended in 200 ll of DMSO and the intensity at 595 nm were measured. For inhibitors study, cells were pre-treated with inhibitors (U0126, PD98059, APN and alpha-TP) at targeted dosage for 8 h prior to the addition of vitamin-E isomers. Each experiment was repeated three times in triplicate wells and the growth curves showed the means and standard deviations. To test the effect of gamma-T3 on the cytotoxicity of Docetaxel, cells were pre-incubated with gamma-T3 for 3 h before addition of Docetaxel. After 24 h, cells were subjected to Western blotting and MTT assays respectively. 2.5. DNA fragmentation assay After 24 h incubation with gamma-T3, 3  106 MDAMB-231 cells were harvested and suspended in lysis buffer (5 mM Tris–HCl (pH 8.0), 20 mM EDTA, and 0.5% (v/v) Triton X-100) for 60 min on ice. Samples were centrifuged, the supernatants were removed and incubated with 5 ll RnaseA (10 lg/ml) at 37 °C for 40 min, and 1 ml of anhydrous ethanol was added. Tubes were placed at 20 °C for 20 min and then centrifuged to pellet the DNA. DNA samples were analyzed by electrophoresis at 80 V for 3 h on a 2% agarose gel containing ethidium bromide (0.2 lg/ml) and visualized under UV illumination. 2.6. Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay DNA strand breaks during apoptosis was examined using in situ cell death detection reagent (Roche Applied Science). Briefly, 1  106 cells were pre-treated with gamma-T3 for 24 h. Thereafter, cells were incubated with reaction mixture for 60 min at 37 °C. Stained cells were analyzed and captured by fluorescence microscope on glass slide.

189

2.7. Matrigel-invasion assay Matrigel-invasion assay was performed according to a previously published method with modifications [21]. Briefly, the invasive estrogen-independent BCa cells (MDA-MB-231) were pre-incubated in a serum-free medium with or without gamma-T3 isomers for 24 h. The MDA-MB-231 cells, (2.5  105) re-suspended in 500 ll of serum-free medium containing 0.1% bovine serum albumin (BSA), were then added to the upper chamber of a 8 lm pore size insert (Millipore, Bedford, MA) manually coated with Matrigel (0.5 mg/ml) (BD Bioscience, Bedford, MA). Five hundred lliter of invasion buffer containing fibronectin (10 lg/ml) (Sigma–Aldrich, St. Louis, USA) and RPMI 1640 supplemented with 10% FCS were added in the lower chamber as a chemo-attractant. The MDA-MB231 cells were incubated at 37 °C for 24 h in 5% CO2 humidified conditions. At the end of incubation period, inserts were stained with Diff-Quick staining solution (Fischer Scientific). Non-invaded MDA-MB-231 cells on the inside of the insert were scraped off with a cotton swab. The invaded cells were examined by a phase-contrast microscope and were then extracted using extraction buffer (Millipore, Bedford, MA) and the cell number was estimated based on the absorbance at 595 nm. 2.8. Western blotting Detailed protocols have been described previously [22]. Briefly, cell lysates were prepared by suspending cell pellets in lysis buffer (50 mmol/l Tris–HCl (pH 8.0), 150 mmol/l NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 1 mg/ml aprotinin, 1 lg/ml leupeptin and 1 mmol/l phenylmethylsulfonyl fluoride). For nuclear protein extraction, NucBusterTM protein extraction kit (Novagen, Darmstadt, Germany) was used. Protein concentration was measured using the DC Protein Assay kit (Bio-Rad, Hercules, CA). Equal amount of protein (30 lg) was loaded onto a 10% SDS polyacrylamide gel for electrophoresis and then transferred onto a polyvinylidene difluoride membrane (Amersham, Piscataway, NJ). The membrane was then incubated with primary antibodies for 1 h at room temperature against E-cadherin (BD Biosciences, Bedford, MA), a-catenin, b-catenin, c-catenin, Id-1, Id-3, EGFR, p-c-jun, p-ATF2, cleaved PARP, vimentin, a-smooth muscle actin, twist, p-Smad1, Smad1/5/8 (Santa Cruz Biotechnology, CA), p-IkB-alpha (Ser32/36), p-SAPK/JNK (Thr183/Tyr185) G9, SAPK/JNK, NF-kB p65 (5A5), Erk1/2, p-Erk1/2, Src, LOX (Cell Signaling Technology Inc., Beverly, MA). After incubation with appropriate secondary antibodies, signals were visualized by ECL Western blotting system (Amersham, Piscataway, NJ). Expression of b-actin and histone H1 were assessed as an internal loading control for total cell lysate and nuclear protein lysate respectively. 2.9. Id-1 RT-PCR Total RNA was isolated using Trizol reagent according to the manufacturer’s protocol (Invitrogen). cDNA was synthesized using the SuperScript First Strand Synthesis System (Invitrogen) and was then amplified by PCR with

190

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

Id-1 specific primers (forward primer, Id1-S, 50 -CTC CAG CAC GTC ATC GAC TA-30 and reverse primer, Id1-AS,50 AAC GCA TGC CGC CTC-30 ). PCR cycling protocol was as follows: 30 cycles of 10 min at 95 °C, 30 s at 95 °C, 30 s at 55 °C, 1 min at 72 °C and 10 min at 72 °C. Glyceraldehyde 3-phosphate dehydrogenase was amplified as an internal control. The PCR products were electrophoresed on a 2% agarose gel and analyzed using a gel documentation system. 3. Results 3.1. Anti-proliferation effect of vitamin-E isomers on BCa cells BCa cells were treated with vitamin-E isomers for 24-h at increasing dosage (low: 20, medium: 40 lM and high: 80 lM). Our results showed that vitamin-E isomers did not affect the proliferation rate of normal breast epithelial cells (MCF-10A), but significantly suppressed the proliferation of MCF-7 and MDA-MB-231 (Fig. 1A). Surprisingly, MDA-MB-231 cells were more sensitive to the growth inhibition of the vitamin-E isomers than MCF-7 cells. The inhibition of cell proliferation was stronger for T3 isomers in MDA-MB-231, particularly for gamma-T3, which showed a dose-dependent inhibition (Supplementary 1). Based on the IC50 values in MDA-MB-231 cells incubated with various isomers for

24-h, the order of inhibitory effect is gamma-T3 > beta-T3 > delta-T3. Since MDA-MB-231 cells are considered to be more invasive and resistant to chemotherapeutic agents when compared to MCF-7 cells [23], for the subsequent experiments, we chose to investigate the effect of gammaT3 on MDA-MB-231. To study the mechanism responsible for gamma-T3-induced growth inhibition, cell cycle distribution and genomic DNA fragmentation of the cells with or without gamma-T3 treatment for 24 h were analyzed by flow cytometry, gel electrophoresis and TUNEL assays. Consequently, treatment of cells with gamma-T3 (IC50–90) resulted in an induction of sub-G1 cell population (Fig. 1B) and DNA fragmentations (Fig. 1C and D), indicating the presence of apoptotic cells after the treatment. The proportion of apoptotic cells (sub-G1 fraction) increased in a dose-dependent manner. To study further the mechanism of gamma-T3-induced apoptosis, we first investigated if the programmed cell death in MDA-MB-231 cells is caspase-dependent. As shown in Fig. 2A activation of procaspase 3, 7, 8, 9 as well as PARP, as evidenced from the appearance of the cleaved products, were observed in MDA-MB-231 cells treated with different gammaT3 dosage for 24 h. Downregulation of bcl-2 was also detected after the treatment, together with upregulation of bax expression (Fig. 2A). Meanwhile, these gamma-T3-mediated activations of the pro-apoptotic proteins as well as the change of bcl-2/Bax ratio were in a dose-dependent manner (Fig. 2A). In addition, activation of these pro-apoptotic genes by gamma-T3 treatment (Fig. 2B) was only observed in MDA-MB-231 and MCF-7 cells, but not in MCF-10A cells, indicating that gamma-T3 specifically induced apoptosis in BCa cells.

Fig. 1. Induction of apoptosis by gamma-T3 treatment. (A) IC50 of different vitamin-E isomers was determined by examination of cell viability by MTT assay 24 h after the treatment. Note that vitamin-E isomers, particularly beta-, gamma- and delta-T3, selectively inhibit the viability of the BCa cells at different degree, but do not have significant effect on the non-tumorigenic breast epithelial cells. UD represents undetermined IC50 value. (B) Cell cycle analysis by flow cytometry. Control cells and treated cells incubated with gamma-T3 at IC50 dose level for 24 hr were subjected to flow cytometry analysis. Note that the sub-G1 population increases after gamma-T3 treatment. (C) Gamma-T3 induces DNA fragmentation in MDA-MB-231 cells. Briefly, the cells were harvested and fragmented DNA was extracted and analyzed by electrophoresis in 2% agarose gel containing ethidium bromide. (D) DNA fragmentation induced by gamma-T3 was also detection by terminal deoxynucleotidyl transferase (TUNEL assay).

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199 3.2. Gamma-T3 downregulated the pro-survival signaling pathways in BCa cells Because NF-jB was reported to be constitutively activated in MDAMB-231 cells [24], the possibility that gamma-T3-induced cell apoptosis attributable to the suppression of NF-jB activation was considered. The NF-jB activities of MDA-MB-231 treated with gamma-T3 at different dos-

191

ages were measured by examining the nuclear translocation of NF-jB subunit p65. As illustrated in Fig. 3A, gamma-T3 treatment suppressed nuclear level of NF-jB p65 in a dose-dependent manner. The effect of gamma-T3 on NF-jB signaling was further explored by examining the expression of other upstream regulators, such as p-IjBa/b and IjBa/b. In gamma-T3 treated MDA-MB-231 cells, a dose-dependent decrease in the level of the phosphorylated IjBa/b were observed (Fig. 3A). This is

Fig. 2. Activation of pro-apoptosis molecules by gamma-T3 treatment. (A) gamma-T3 treatment induces activation of the critical apoptotic molecules (cleaved caspase 3, 7, 8, 9, PARP) and modulates the ratio between the amounts of bcl-2 and bax in a cell dose-dependent fashion. (B) gamma-T3 activates pro-apoptotic genes on MCF7 and MDA-MB-231 cells but not on the non-tumorigenic breast epithelial cells (MCF-10A).

Fig. 3. Inactivation of pro-survival pathways by gamma-T3. (A) Effect of gamma-T3 on the activity of NF-jB pathway was examined by Western blotting. The phosphorylation of IjB was inhibited by gamma-T3 treatment in total cell lysate. Similarly, the nuclear translocated NF-jB p65 was inhibited in nuclear protein extract. (B) Treatment of gamma-T3 resulted in downregulation of the expression of EGFR and Id family proteins in MDA-MB-231 cells. (C) Treatment of gamma-T3 also resulted in downregulation of the upstream regulators of Id1 in MDA-MB-231 cells (Src, Smad1/5/8 and LOX). The focal adhesion kinase activity (FAK) is strongly correlated with LOX activation. (D) MDA-MB-231 cells treated with gamma-T3 were lysed and the lysate was used for immunoprecipitation assay using the anti-Src antibody. Results indicated that physical interaction between Src and Smad1/5/8 was affected by gammaT3 treatment.

192

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

associated with the increase in the level of IjBa/b, as well as an inhibition of NF-jB p65 nuclear translocation. These results indicate that c-T3 suppressed NF-jB activity through the dephosphorylation and accumulation of IjBa/b. 3.3. Gamma-T3 downregulated the Id1 signaling pathway and its upstream regulator proteins in BCa cells Surprisingly, we found that gamma-T3 treatment also downregulated a number of the key proteins that are involved in the development and progression of BCa. As shown in Fig. 3B, Id1 and Id3 expressions were significantly suppressed to almost undetectable level by treatment with increasing dosages of gamma-T3. Similar effect on EGF-R protein level was observed. Since EGF-R and Id protein family are essential for BCa cell growth and survival [25], their downregulation may be associated with the gamma-T3-induced growth arrest and apoptosis. Because Id1 transcript and protein levels were previously shown to be regulated directly or indirectly by the Src, Smad1/5, LOX and Fak signaling pathways in BCa cells [26,27], we next examined the effect of gamma-T3 on the upstream regulators of Id1 in BCa cells. Our results showed that the Src phosphorylation, as well as the protein level of Smad1/5/8, LOX and activated Fak were repressed in a dose-dependent manner by gamma-T3 treatment (Fig. 3C). Meanwhile, immunoprecipitation assay revealed using anti-Src antibody revealed a decrease of interaction between Src and Smad1/5/8, which is likely due to the suppression of Smad1/5/8 protein level by gamma-T3 (Fig. 3D). This possibly led to decreased binding of Src–Smad complex to Src-responsive region of the Id-1 promoter, resulting in the observed suppression of Id1 protein expression by gamma-T3 [27]. 3.4. Gamma-T3 activated the pro-apoptotic signaling pathways in BCa cells The c-Jun N-terminal kinase (JNK) is an evolutionarily conserved serine/threonine protein kinase that is activated by stress and genotoxic agents [28]. JNK phosphorylates the amino terminal of all three Jun transcription factors and ATF-2 members of the AP-1 family. The activated

transcription factors modulate gene expression to generate appropriate biological responses, including cell migration and cell death. When MDA-MB-231 cells were treated with varies dosages of gamma-T3, a dose-dependent increase in JNK phosphorylation activities were detected (Fig. 4A). Meanwhile, phosphorylation of the JNK downstream effectors such as ATF-2 or c-jun were all upregulated by gamma-T3, supporting that JNK signaling pathway was activated by gamma-T3. To study the importance of JNK activation in gamma-T3-induced apoptosis in BCa cells, we investigated whether inactivation of JNK with a specific inhibitor, SP600125, could protect cells from gamma-T3. As shown in Fig. 4B, co-treatment of gamma-T3 together with 20 lM of SP600125 was found to increase the percentage of viable cells when compared to that treated with gamma-T3 alone, confirming that JNK activation may be required for gamma-T3-induced apoptosis. 3.5. Activation of MAPK/ERK pathway was not associated with gamma-T3induced apoptosis in BCa cells The MAPK/ERK kinase is one of the intracellular signaling pathway which is activated by different stimuli, including growth factors, cytokines and carcinogens [29–31]. Although mitogen-activated protein kinase (MAPK/ERK) pathway was found to be activated by gamma-T3 in MDA-MB-231, as evident by phosphorylation of Erk1/2, Mek1/2 and Elk1 (Fig. 4C), their activation may not be directly required for gammaT3-induced apoptosis because inactivation of MAPK by specific inhibitors, U0126/PD98059, were not able to restore cancer cell viability after gamma-T3 treatment (Fig. 4D). 3.6. Effect of gamma-T3 on inhibition of BCa cell invasion Although gamma-T3 has been shown to have anti-proliferation effect on many cancers, it is not clear if it affects BCa metastasis. Therefore, we examined whether gamma-T3 could suppress the invasive ability of the BCa cells. As shown in Fig. 5A, using matrigel-invasion assay, we found that gamma-T3 treated MDA-MB-231 cells for 24 h showed an at least 2-time lower invasion capability compared to the untreated control, as

Fig. 4. Jun N-terminal Kinase (JNK) and MAPK/ERK activation during gamma-T3-induced apoptosis. (A) JNK activity was examined by measuring the phosphorylation levels of SAPK/JNK, c-jun and ATF-2 after 24 h of gamma-T3 treatment. Note that phosphorylation levels of all the proteins were induced by gamma-T3, suggesting that JNK was activated by gamma-T3 treatment. (B) Cell viability, after incubation with gamma-T3 and JNK inhibitor (SP600125) for 24 h, was examined by MTT assay. Note that the addition of JNK inhibitor alleviates the cytotoxicity of gamma-T3 on MDA-MB-231 cells, suggesting that JNK mediates the anti-proliferation effect of gamma-T3. (C) MAPK/ERK activity, as examined by measuring the phosphorylation levels of Mek1/2, Erk1/2 and Elk1, was found to be elevated after 24 h of gamma-T3 treatment. (D) Cell viability, after incubation with gamma-T3 and MAPK/ERK inhibitor (U0126/ PD98059) for 24 h, was examined by MTT assay. Note that the addition of MAPK/ERK inhibitors had no impact on the cytotoxicity of gamma-T3 on MDAMB-231 cells.

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199 evidenced by the decreased in the number of cells invaded through the matrigel layer. This inhibitory effect on cell invasion was not the result of cell growth inhibition induced by gamma-T3 as the number of viable cells added into the invasion chamber was the same. These results indicate that gamma-T3 is able to inhibit the invasion ability of BCa cells, independent to their cytotoxic effects. Downregulation of E-cadherin expression is one of the most frequently reported characteristics of metastatic cancers [32,33]. Restoration of E-cadherin expression in cancer cells leads to suppression of metastatic ability [22,34]. In BCa, downregulation of E-cadherin expression is correlated with high-grade tumors and poor prognosis [33,35], indicating their roles in BCa progression. We were unable to detect MDA-MB-231 as it is an E-cadherin-negative human BCa cell line [36]. Meanwhile, gamma-T3 treatment failed to affect a- and b-catenin protein expression but enhanced the c-catenin expression. The expression of Snail and Twist, the two E-cadherin repressors [37,38]), were both downregulated after treatment with c-T3 (Fig. 5B). In addition, the mesenchymal markers a-SMA was downregulated after treatment with gamma-T3 for 24 h (Fig. 5B), suggesting that gamma-T3 may suppress BCa invasion through inhibition of epithelial to mesenchyme transition (EMT).

3.7. Effect of gamma-T3 treatment on Docetaxel-induced apoptosis Many of the natural products, such as aged garlic extract [39] or resveratrol [40] which are extracted from fruit or plant have been shown to have anti-cancer effect. Previous studies have shown that many of these

193

natural products increased the sensitivity of cancer cells to chemotherapy and enhanced the effectiveness of radiation treatment against prostate tumor [10,15]. To test if gamma-T3 can act synergistically with chemotherapeutic agent, we have compared the effect of gamma-T3 alone or in combination with Docetaxel. As shown in Fig. 6A, the percentage of apoptotic cells in MDA-MB-231 cell line following co-treatment of Docetaxel with gamma-T3 for 24 h was significantly higher than that treated with gamma-T3 or Docetaxel alone. Using Western blotting, we further demonstrated that gamma-T3 co-treatment with Docetaxel enhances cell apoptosis through activation of pro-apoptotic proteins (cleaved PARP, caspases 3, 7, 8, 9) and downregulation of pro-survival proteins (Id-1, EGFR) (Fig. 6B). Similar effect was also observed in MCF-7 cells (Fig. 6C), suggesting that gamma-T3 and Docetaxel may have synergistic effect against BCa cells.

3.8. Alpha-tocopherol (a-TP) and b-aminopropionitrile (APN) attenuated gamma-T3-induced apoptosis Because a-TP was previously reported to negate the cholesterol lowering capability of tocotrienol [41], we therefore evaluated a-TP’s ability to block gamma-T3-induced BCa cell apoptosis. Our results indicated that 50uM a-TP partially inhibits the effect of gamma-T3 treatment on BCa cell viability (Fig. 7A). Interestingly, a-TP not only suppressed the expression of pro-apoptosis genes (cleaved caspase 3, 7, 8 and PARP) in response to gamma-T3 treatment, but also partially restored the expression of Id1 protein (Fig. 7B). In contrast to the partial restoration of Id1 by a-TP,

Fig. 5. Inhibition of cell invasion by gamma-T3 treatment. (A) MDA-MB-231 cells treated with the indicated dosage of gamma-T3 was harvested and then plated into the matrigel-coated (0.5 mg/ml) insert. Cells invaded through the membrane were stained with crystal violet and the images were photographed under microscope. After lysed with extraction buffer, intensity at 595 nm was measured and presented with the means and standard deviations (Right panel). (B) 24 h dose-dependent gamma-T3 treatment had no impact on the expression of epithelial markers (a-, b-, c-catenin), but suppresses the expression of mesenchymal markers (Twist and a-SMA) and E-cadherin’s repressor (Snail, Twist). PC-3 represents the androgenindependent prostate cancer cell line expressing wild type E-cadherin. Consistent with previous report [15] indicated that gamma-T3-induced E-cadherin expression.

194

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

Fig. 6. Synergistic effect of gamma-T3 on Docetaxel-induced apoptosis. (A) Effect of Docetaxel and gamma-T3 co-treatment for 24 h. Cells were incubated with 50 nM of Docetaxel together with different dosages of gamma-T3 for 24 h. Cell viability was examined by MTT assay. The viable MDA-MB-231 cells following co-treatment of Docetaxel and gamma-T3 was significantly lower than that treated with either agent alone. (B–C) Using Western blotting, we further demonstrated that gamma-T3 co-treatment with Docetaxel for 24 h promote apoptosis of MDA-MB-231/MCF7 cell through activation of proapoptotic molecules (cleaved PARP, caspases 3, 7, 8, 9). Suppression of Id-1 and EGFR expressions were also confirmed by Western blotting analysis. Gamma-TP represents gamma tocopherol.

co-treatment of gamma-T3 with b-aminopropionitrile (APN; a non-specific inhibitor of LOX [42]) almost completely restored the expression of Id1 and at the same time inhibited the gamma-T3-induced caspasedependent apoptosis, as evident from the cell proliferation and Western blotting analysis (Fig. 7C–E). However, the marginal decrease in the levels of PARP cleavage as seen with gamma-T3 and gamma-T3-APN co-treatment suggested an induction of caspase-independent apoptosis. These findings are unexpected, and thus suggesting involvement of other mechanisms leading to Id1 induction during gamma-T3 and APN co-treatment (see Fig. 8).

4. Discussion Tocotrienol isomers have been previously shown to inhibit cancer cell proliferation, promote cell cycle arrest, and decrease angiogenesis (reviewed in [14]). We report here that gamma-T3, one of the eight vitamin-E isomers, inhibited BCa cell survival through modulation of pro-survival (EGFR and NF-jB) and pro-apoptotic (JNK, cleaved caspases and PARP) genes. Meanwhile, we also demonstrated that gamma-T3 inhibited BCa cell invasion by suppressing the expression of mesenchymal marker. In addition, we demonstrated that gamma-T3 suppressed Id1 expression, possibly through inhibition of its upstream regulators (Src, Smad1/5/8, LOX). Interestingly, alpha-TP was found to partially restore Id1 expression and attenuate the effect of gamma-T3 on cell viability. Together with the finding that gamma-T3 enhanced the anti-cancer effect of

Docetaxel, our study provide strong evidences that gamma-T3 may be used as a safe and effective anti-cancer agent for the treatment of BCa. In this report, we investigated our hypothesis that the anti-cancer effect of gamma-T3 may act by suppressing Id1 expression through inactivation of its upstream regulators (Src, Smad1/5/8, LOX). Id1 is a dominant negative regulator of basic helix–loop–helix transcription factors, and plays a key role in the control of breast epithelial cell growth, invasion and differentiation. Previous investigations have shown that Id1 mRNA was constitutively expressed in highly aggressive and invasive human BCa cells but not in non-transformed or non-aggressive cancerous cells [43,44]. Over-expression of Id1 was also found to be associated with poor survival rate of the BCa patients [45]. Due to its importance in breast carcinogenesis, Id1 has been suggested as a novel therapeutic target, and inactivation of Id1 has been shown to reduce BCa metastasis in vivo [25]. In this study, treatment of MDA-MB-231 cells led to suppression of activated lysyl oxidase encoded by human LOX gene. Previously, activation of LOX has been suggested to promote cancer cell invasion through induction of focal adhesion kinase (FAK) activity and cell-to-matrix adhesion. It was suggested that LOX may be required to create a niche permissive for metastatic cell growth and thus may be required for hypoxia-induced cancer metastasis [46]. Consistent with inactivation of LOX and

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

195

Fig. 7. Alpha-tocopherol antagonizes gamma-T3-induced cancer cell apoptosis. (A) Cell viability, after incubation with gamma-T3 and a-tocopherol for 24 h, was examined by MTT assay. Note that the addition of a-tocopherol alleviates the cytotoxicity of gamma-T3 on MDA-MB-231 cells. (B) Using Western blotting analysis, we further demonstrated that gamma-T3 co-treatment with a-tocopherol for 24 h suppresses MDA-MB-231 cell apoptosis through inactivation of pro-apoptotic molecules (cleaved PARP, caspases 3) and re-expression of Id1. (C) Cell viability, after incubation with gamma-T3 and APN for 24 h, was examined by MTT assay. Note that the addition of APN alleviates the cytotoxicity of gamma-T3 on MDA-MB-231 cells, suggesting that LOX mediates the anti-proliferation effect of gamma-T3. (D) Gamma T3 co-treatment with APN reversed the activation of pro-apoptosis genes (caspases 3, 7, 8, 9 and PARP) and partially restored the constitutive activation of Id1. (E) Id1 mRNA was determined to be repressed following gamma-T3 treatment. However, Id1 mRNA was restored partially following gamma-T3 co-treatment with either alpha-TP or APN. Amount of GAPDH was measured as loading control.

FAK proteins following gamma-T3 pre-treatment, we found that the invasiveness of BCa cells was also decreased significantly by gamma-T3 (Fig. 5A). Previously, Src was reported to regulate Id1 expression by binding directly to a Src-responsive region within the Id1 promoter [27]. This Src-responsive region was found to contain a Smad-binding element. In MDA-MB-231 cells treated with gamma-T3, both Src phosphorylation and Smad1/5/8 expression was downregulated in a dosedependent fashion (Fig. 3C), and inhibition of Src–Smad interaction by gamma-T3 was confirmed via immunoprecipitation (Fig. 3D). Thus, it seems plausible from our results that gamma-T3-induced BCa cell apoptosis involved cooperative interactions between LOX, Src and Smad-Id1 signaling pathways. Worth noting, published reports [47–55] indicated that gamma-T3 isomer suppressed BCa cell growth. In these reports, gamma-T3’s IC50 value for MDA-MB-231, MDA-

MB-435 and MCF7 cells varied between 4.5 and 85 lM. The difference between our results and those reported previously could be due to several reasons. Firstly, the IC50 values reported previously were for cells supplemented with gamma-T3 for 48–72 h incubation period. Secondly, the gamma-T3 isomer used in those studies may have different purity than that used in this study (P97%). Thirdly, the difference in the proliferation assays used ([3H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) resulted in data variation. Although gamma-T3 treatment caused significant apoptosis in BCa cells [54], gastric cancer cells [56] and human myeloid cells [18], several published reports have also indicated that d-T3 is equally potent for inducing apoptosis in other types of cancer [57–59]. For example, HepG2 and B16 melanoma cells treated with d-T3 showed significant reduction in cell viability with an IC50 9.6 lM and 10 lM respectively. In this study, d-T3 was found to be equally

196

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

Fig. 8. Id1 over-expression and Id1 knockdown affect gamma-T3’s anti-tumor effect. (A) MDA-MB-231 cell line over-expressing Id1 transiently (MDA-MB231 pcId1) showed increased cell viability compared to MDA-MB-231 pcDNA after 50 M gamma-T3 treatment for 24 h. In contrast, stable Si-Id1 transfectants PC3 showed decreased cell viability compared to wild type PC3. (B) Id1 over-expressing cell lines (MDA-MB-231 pcId1 and wild type PC3) showed lower level of caspase 3 and PARP activation. (C) The proposed pathway of gamma-T3 in BCa cells.

potent in inhibiting BCa cell proliferation. Taken together, it seems likely that c- and d-T3 may possess tumor suppressing activities across different types of cancer cell. In this study, we demonstrated that gamma-T3 suppressed constitutive NF-jB activity [24] through inhibition of IjB kinase activation, leading to apoptosis in BCa cells. This is in agreement with the previous study which showed that gamma-T3 can interfere with the TNF-induced NF-jB activation pathway in human myeloid KMB-5 cells and several other cancer cell lines [18]. In addition to their findings, we have demonstrated that gamma-T3-induced NF-jB inactivation also downregulates the level of bcl-2 in a dosage-dependent fashion. Consequently, this leads to induction of apoptosis via activation of caspases 3, 7, 8, 9 and PARP. Although our results is in agreement with majority of previous reports [13,49– 51,60], our results on activation of caspases 3, 7, 8, 9 and PARP were in stark contrast with one report [48] which used the same cell line and T3 isomer as our present study. They concluded that expression of Bax and Bcl-2 remained unchanged while poly-(ADP-ribose)-polymerase and caspases cleavage were not detected. Although the exact reason for the discrepancy is unknown, it is possibly caused by the difference in T3 purity between our and their studies [15]. It is worth noting that gamma-T3 was previously demonstrated to abolish NF-jB activation induced by epidermal growth factor (EGF) and other pro-inflammatory

cytokines [18], although the molecular mechanism involved was not clear. Our result revealed that downregulation of EGF receptor (EGF-R) was correlated to gammaT3-induced NF-jB inactivation (Fig. 3B). This finding may explain why gamma-T3 was able to suppress NF-jB activation by EGF treatment in KBM-5 cells [18]. Interestingly, the estrogen-independent BCa cell line MDA-MB-231 was found to be more sensitive to gamma-T3 treatment than the estrogen-dependent MCF-7 cells. MDA-MB-231 cells were found to have constitutive NF-jB activation and are in general more resistant to chemotherapeutic drugs-induced apoptosis than the MCF-7 cells. Although the exact reason for this observation is unclear, but based on the fact that non-tumorigenic breast epithelial cells are highly resistant to gamma-T3 as well, it is possible that gammaT3 may preferentially target the more aggressive BCa cells. In this study, we also showed that c-Jun N-terminal kinase participates in gamma-T3-induced apoptosis. When BCa cells were treated with gamma-T3, a series of molecules associated with JNK pathway, such as c-Jun and ATF-2 (Fig. 4A), were activated simultaneously. Meanwhile, we demonstrated that treatment of JNK inhibitor (SP600125) protects the BCa cells from gamma-T3-induced apoptosis (Fig. 4B). This further confirms the involvement of JNK pathway in gamma-T3-induced apoptosis in BCa cells. Worth noting is that, the JNK pathway is also known to be involved in cell apoptosis induced by the chemother-

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

apeutic drug, Docetaxel [61]. Taking these findings into consideration, we therefore question whether gamma-T3 possesses synergistic interaction with Docetaxel as a result of activation of JNK pathway. To this end, we compared the anti-proliferation capability of Docetaxel treatment alone with Docetaxel plus gamma-T3 co-treatment. Remarkably, we found that combination of Docetaxel with gamma-T3, but not with c-TP, resulted in higher proportion of apoptotic cells (Fig. 6B). This finding confirmed the synergistic role of gamma-T3 with the chemotherapeutic agent. Upregulation of mesenchymal markers (a-SMA and Twist) is one of the most frequently reported phenomena in metastatic cancers [34]. In this study, we demonstrated that the inhibition of BCa cell invasion by gamma-T3 was correlated with the expression of a-SMA Twist and LOX. Meanwhile, we found that the gamma-T3 treated BCa cells showed decreased Snail and Twist expressions, which was associated with reduced invasion ability. It is suggested that loss of E-cadherin expression is able to promote epithelial–mesenchymal transition (EMT), which plays a key role in the progression of cancer cells to metastatic stage. Although the precise mechanism responsible for E-cadherin inactivation in cancer cells is not clear, alterations at transcriptional level due to its repressors Snail and Twist seem to be one of the mechanisms responsible for its decreased expression in several cancer types. Although gamma-T3 treatment suppresses the expression of both Twist and Snail, we were unable to detect re-expression of E-cadherin in the BCa cells, suggesting that the inactivation of E-cadherin gene may involve a mechanism other than upregulation of Twist and Snail activities. Independent from the mesenchymal markers, the suppression of BCa cell invasion capability may also be resulted from the inhibition of LOX activation through collagen IV/laminin/gelatin matrix [26,62]. As shown in Fig. 7A and B, gamma-T3 co-treatment with a-TP led to partial restoration of Id1 expression, which was associated with an increase in the viability of MDA-MB-231 cells, suggesting that a-TP attenuates the anti-proliferation potency of gamma-T3. Although the mechanism involved is currently unknown, one possible explanation is the competition between gamma-T3 and a-TP for tocopherol-transport protein (TTP) that primarily transports a-TP and has low affinity for gamma-T3 [63]. Interestingly, a similar antagonistic interaction between gamma-T3 and a-TP was also observed for the cholesterol lowering property of tocotrienols [41], suggesting that highly purified gamma-T3, which separates from tocopherols, should be a better source for testing the therapeutic value of the vitamin E derivatives. In addition, gamma-T3 co-treatment with APN also led to an almost complete restoration of Id1 expression (Fig. 7D) and increase in the viability of MDA-MB-231 cells than gamma-T3 treatment alone (Fig. 7C), suggesting that APN may also act as an antagonist to gamma-T3. Although APN is known as a non-specific LOX inhibitor [42], the fact that LOX expression was suppressed by gamma-T3 in a dose-dependent manner (Fig. 3C) suggests that the unexpected antagonistic effect of APN on gamma-T3 is not likely due to its effect on LOX activity. Therefore, more research will be required to elucidate how APN could reverse gamma-T3-induced cell

197

apoptosis. Furthermore, the marginal decrease of PARP cleavage as seen with gamma-T3 and gamma-T3-APN cotreatment suggested an induction of caspase-independent apoptosis by gamma-T3. As summarized in Fig. 7C, our results demonstrated that gamma-T3 is a potent and specific inhibitor of BCa cell proliferation and invasion which acts through the Id1 pathway. Since no side effect can be observed after long term intake of natural T3 extract [11,64], our results support that gamma-T3 may be an ideal therapeutic agent to be used alone or in combination with chemotherapy for treating advanced stage BCa. Conflict of interest Y.C. Wong, M.T. Ling, and X.W. Zhang are researchers in The University of Hong Kong, Australian prostate cancer research centre and South China University of Technology respectively and have no conflict of interest. Wei Ney Yap, Norazean Zaiden and Yee Leng Yap are employee by Davos Life Science Pte. Ltd., a manufacturer of Tocotrienol based in Singapore. Y.L. Tan and C.P. Chang are researcher in Singapore Bioimaging Consortium (SBIC) and Duke-NUS Graduate Medical School Singapore respectively and have no conflict of interest. None declared. Acknowledgements This work was supported by research Grant from Kuala Lumpur Kepong Berhad to Davos Life Science Pte. Ltd. and RGC Grants to Y.C.W (HKU 7314/01 M, HKU7490/03 M and 7470/04 M). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2009. 10.012. References [1] L.A.G. Ries et al., SEER Cancer Statistics Review, 1975–2004, National Cancer Institute, Bethesda, MD, 2006. [2] C.W. Yde et al., The antipsychotic drug chlorpromazine enhances the cytotoxic effect of tamoxifen in tamoxifen-sensitive and tamoxifenresistant human breast cancer cells, Anticancer Drugs 20 (2009) 723–735. [3] F. Leonessa et al., Effect of tamoxifen on the multidrug-resistant phenotype in human breast cancer cells: isobologram, drug accumulation, and M(r) 170,000 glycoprotein (gp170) binding studies, Cancer Res. 54 (1994) 441–447. [4] I.C. Smith et al., Neoadjuvant chemotherapy in breast cancer: significantly enhanced response with docetaxel, J. Clin. Oncol. 20 (2002) 1456–1466. [5] P.B. Schiff, J. Fant, S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol, Nature 277 (1979) 665–667. [6] N.S. Ahmad et al., Tocotrienol offers better protection than tocopherol from free radical-induced damage of rat bone, Clin. Exp. Pharmacol. Physiol. 32 (2005) 761–770. [7] M. Mazlan et al., Comparative effects of alpha-tocopherol and gamma-tocotrienol against hydrogen peroxide induced apoptosis on primary-cultured astrocytes, J. Neurol. Sci. 243 (2006) 5–12. [8] M.K. Agarwal et al., Tocotrienol-rich fraction of palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis independent of cell cycle association, Cell Cycle 3 (2004) 205–211.

198

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199

[9] T. Eitsuka, K. Nakagawa, T. Miyazawa, Down-regulation of telomerase activity in DLD-1 human colorectal adenocarcinoma cells by tocotrienol, Biochem. Biophys. Res. Commun. 348 (2006) 170–175. [10] K.S. Kumar et al., Preferential radiation sensitization of prostate cancer in nude mice by nutraceutical antioxidant gammatocotrienol, Life Sci. 78 (2006) 2099–2104. [11] J.A. McAnally et al., Tocotrienols potentiate lovastatin-mediated growth suppression in vitro and in vivo, Exp. Biol. Med. (Maywood) 232 (2007) 523–531. [12] K. Nesaretnam et al., Tocotrienol-rich fraction from palm oil affects gene expression in tumors resulting from MCF-7 cell inoculation in athymic mice, Lipids 39 (2004) 459–467. [13] P.W. Sylvester, S. Shah, Intracellular mechanisms mediating tocotrienol-induced apoptosis in neoplastic mammary epithelial cells, Asia Pac J. Clin. Nutr. 14 (2005) 366–373. [14] C.K. Sen et al., Tocotrienols: the emerging face of natural vitamin E, Vitam. Horm. 76 (2007) 203–261. [15] W.N. Yap et al., Gamma-tocotrienol suppresses prostate cancer cell proliferation and invasion through multiple-signalling pathways, Br. J. Cancer 99 (2008) 1832–1841. [16] P.N. Chang et al., Evidence of gamma-tocotrienol as an apoptosisinducing, invasion-suppressing, and chemotherapy drug-sensitizing agent in human melanoma cells, Nutr. Cancer 61 (2009) 357–366. [17] G.V. Samant, P.W. Sylvester, gamma-Tocotrienol inhibits ErbB3dependent PI3K/Akt mitogenic signalling in neoplastic mammary epithelial cells, Cell Proliferat. 39 (2006) 563–574. [18] K.S. Ahn et al., Gamma-tocotrienol inhibits nuclear factor-kappaB signaling pathway through inhibition of receptor-interacting protein and TAK1 leading to suppression of antiapoptotic gene products and potentiation of apoptosis, J. Biol. Chem. 282 (2007) 809–820. [19] M.T. Ling et al., Overexpression of Id-1 in prostate cancer cells promotes angiogenesis through the activation of vascular endothelial growth factor (VEGF), Carcinogenesis 26 (2005) 1668– 1676. [20] L. Jacobs, Process for the Production of Tocotrienols, Archer– Daniels–Midland Company, USA, 2002. [21] A. Albini et al., A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res. 47 (1987) 3239–3245. [22] Q. Chu et al., A novel anticancer effect of garlic derivatives: inhibition of cancer cell invasion through restoration of E-cadherin expression, Carcinogenesis 27 (2006) 2180–2189. [23] G.M. Nagaraja et al., Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics, Oncogene 25 (2006) 2328–2338. [24] N.M. Patel et al., Paclitaxel sensitivity of breast cancer cells with constitutively active NF-kappaB is enhanced by IkappaBalpha superrepressor and parthenolide, Oncogene 19 (2000) 4159–4169. [25] S. Fong et al., Id-1 as a molecular target in therapy for breast cancer cell invasion and metastasis, Proc. Natl. Acad. Sci. USA 100 (2003) 13543–13548. [26] S.L. Payne et al., Lysyl oxidase regulates breast cancer cell migration and adhesion through a hydrogen peroxide-mediated mechanism, Cancer Res. 65 (2005) 11429–11436. [27] O. Gautschi et al., Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer, Cancer Res. 68 (2008) 2250–2258. [28] Y. Liu et al., Role of mitogen-activated protein kinase phosphatase during the cellular response to genotoxic stress. Inhibition of c-Jun N-terminal kinase activity and AP-1-dependent gene activation, J. Biol. Chem. 270 (1995) 8377–8380. [29] A. Miglietta et al., Conjugated linoleic acid induces apoptosis in MDA-MB-231 breast cancer cells through ERK/MAPK signalling and mitochondrial pathway, Cancer Lett. 234 (2006) 149–157. [30] D.G. Azzam et al., ERK/MAPK regulation of the androgen responsiveness of breast cancer cells, Adv. Exp. Med. Biol. 617 (2008) 429–435. [31] O.K. Mirzoeva et al., Basal subtype and MAPK/ERK kinase (MEK)phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition, Cancer Res. 69 (2009) 565–572. [32] B. Saha et al., Overexpression of E-cadherin protein in metastatic breast cancer cells in bone, Anticancer Res. 27 (2007) 3903–3908. [33] U. Jeschke et al., Expression of E-cadherin in human ductal breast cancer carcinoma in situ, invasive carcinomas, their lymph node metastases, their distant metastases, carcinomas with recurrence and in recurrence, Anticancer Res. 27 (2007) 1969–1974.

[34] R.A. Morton et al., Reduction of E-cadherin levels and deletion of the alpha-catenin gene in human prostate cancer cells, Cancer Res. 53 (1993) 3585–3590. [35] G. Turashvili et al., Expression of E-cadherin and c-erbB-2/HER-2/ neu oncoprotein in high-grade breast cancer, Cesk. Patol. 43 (2007) 87–92. [36] M. van de Wetering et al., Mutant E-cadherin breast cancer cells do not display constitutive Wnt signaling, Cancer Res. 61 (2001) 278– 284. [37] A. Cano et al., The transcription factor snail controls epithelial– mesenchymal transitions by repressing E-cadherin expression, Nat. Cell Biol. 2 (2000) 76–83. [38] F. Vesuna et al., Twist is a transcriptional repressor of E-cadherin gene expression in breast cancer, Biochem. Biophys. Res. Commun. 367 (2008) 235–241. [39] E.W. Howard et al., Garlic-derived S-allylmercaptocysteine is a novel in vivo antimetastatic agent for androgen-independent prostate cancer, Clin. Cancer Res. 13 (2007) 1847–1856. [40] M.T. Laux et al., Identification of a p53-dependent pathway in the induction of apoptosis of human breast cancer cells by the natural product, resveratrol, J. Altern. Complement Med. 10 (2004) 235–239. [41] A.A. Qureshi et al., Dietary alpha-tocopherol attenuates the impact of gamma-tocotrienol on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in chickens, J. Nutr. 126 (1996) 389–394. [42] D.A. Dawson, A.C. Rinaldi, G. Poch, Biochemical and toxicological evaluation of agent–cofactor reactivity as a mechanism of action for osteolathyrism, Toxicology 177 (2002) 267–284. [43] J. Singh et al., Constitutive expression of the Id-1 promoter in human metastatic breast cancer cells is linked with the loss of NF-1/Rb/ HDAC-1 transcription repressor complex, Oncogene 21 (2002) 1812–1822. [44] C.Q. Lin et al., A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells, Cancer Res. 60 (2000) 1332–1340. [45] S.F. Schoppmann et al., Overexpression of Id-1 is associated with poor clinical outcome in node negative breast cancer, Int. J. Cancer 104 (2003) 677–682. [46] J.T. Erler et al., Lysyl oxidase is essential for hypoxia-induced metastasis, Nature 440 (2006) 1222–1226. [47] W. Yu et al., Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols, Nutr. Cancer 33 (1999) 26–32. [48] K. Takahashi, G. Loo, Disruption of mitochondria during tocotrienolinduced apoptosis in MDA-MB-231 human breast cancer cells, Biochem. Pharmacol. 67 (2004) 315–324. [49] P.W. Sylvester, S.J. Shah, Mechanisms mediating the antiproliferative and apoptotic effects of vitamin E in mammary cancer cells, Front. Biosci. 10 (2005) 699–709. [50] K. Nesaretnam et al., Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status, Lipids 33 (1998) 461–469. [51] K. Nesaretnam et al., Effect of tocotrienols on the growth of a human breast cancer cell line in culture, Lipids 30 (1995) 1139– 1143. [52] K. Nesaretnam, S. Dorasamy, P.D. Darbre, Tocotrienols inhibit growth of ZR-75-1 breast cancer cells, Int. J. Food Sci. Nutr. 51 (Suppl) (2000) S95–S103. [53] K. Nesaretnam et al., Tocotrienol-rich fraction from palm oil and gene expression in human breast cancer cells, Ann. NY Acad. Sci. 1031 (2004) 143–157. [54] N. Guthrie et al., Inhibition of proliferation of estrogen receptornegative MDA-MB-435 and -positive MCF-7 human breast cancer cells by palm oil tocotrienols and tamoxifen, alone and in combination, J. Nutr. 127 (1997) 544S–548S. [55] S. Elangovan, T.C. Hsieh, J.M. Wu, Growth inhibition of human MDAmB-231 breast cancer cells by delta-tocotrienol is associated with loss of cyclin D1/CDK4 expression and accompanying changes in the state of phosphorylation of the retinoblastoma tumor suppressor gene product, Anticancer Res. 28 (2008) 2641–2647. [56] W. Sun et al., Gamma-tocotrienol-induced apoptosis in human gastric cancer SGC-7901 cells is associated with a suppression in mitogen-activated protein kinase signalling, Br. J. Nutr. 99 (2008) 1247–1254. [57] L. He et al., Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo, J. Nutr. 127 (1997) 668–674. [58] S. Wada et al., Tumor suppressive effects of tocotrienol in vivo and in vitro, Cancer Lett. 229 (2005) 181–191.

W.N. Yap et al. / Cancer Letters 291 (2010) 187–199 [59] A.A. Qureshi et al., Isolation and identification of novel tocotrienols from rice bran with hypocholesterolemic, antioxidant, and antitumor properties, J. Agric. Food Chem. 48 (2000) 3130– 3140. [60] B.S. McIntyre et al., Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells, Proc. Soc. Exp. Biol. Med. 224 (2000) 292–301. [61] X. Zhang et al., Inactivation of Id-1 in prostate cancer cells: a potential therapeutic target in inducing chemosensitization to taxol

199

through activation of JNK pathway, Int. J. Cancer 118 (2006) 2072– 2081. [62] D.A. Kirschmann et al., A molecular role for lysyl oxidase in breast cancer invasion, Cancer Res. 62 (2002) 4478–4483. [63] S. Khanna et al., Delivery of orally supplemented alpha-tocotrienol to vital organs of rats and tocopherol-transport protein deficient mice, Free Radic. Biol. Med. 39 (2005) 1310–1319. [64] V. Patel et al., Natural vitamin E alpha-tocotrienol: retention in vital organs in response to long-term oral supplementation and withdrawal, Free Radic. Res. 40 (2006) 763–771.