γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents*

Although γ-tocotrienol (T3), a vitamin E isolated primarily from palm and rice bran oil, has been linked with anticancer activities, the mechanism of this action is poorly understood. In this study, we investigated whether γ-T3 can modulate the STAT3 cell signaling pathway, closely linked to inflammation and tumorigenesis. We found that γ-T3 but not γ-tocopherol, the most common saturated form of vitamin E, inhibited constitutive activation of STAT3 in a dose- and time-dependent manner, and this inhibition was not cell type-specific. γ-T3 also inhibited STAT3 DNA binding. This correlated with inhibition of Src kinase and JAK1 and JAK2 kinases. Pervanadate reversed the γ-T3-induced down-regulation of STAT3 activation, suggesting the involvement of a protein-tyrosine phosphatase. When examined further, we found that γ-T3 induced the expression of the tyrosine phosphatase SHP-1, and gene silencing of the SHP-1 by small interfering RNA abolished the ability of γ-T3 to inhibit STAT3 activation, suggesting a vital role for SHP-1 in the action of γ-T3. Also γ-T3 down-modulated activation of STAT3 and induced SHP-1 in vivo. Eventually, γ-T3 down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) gene products; and this correlated with suppression of proliferation, the accumulation of cells in sub-G(1) phase of the cell cycle, and induction of apoptosis. This vitamin also sensitized the tumor cells to the apoptotic effects of thalidomide and bortezomib. Overall, our results suggest that γ-T3 is a novel blocker of STAT3 activation pathway both in vitro and in vivo and thus may have potential in prevention and treatment of cancers.

Although ␥-tocotrienol (T3), a vitamin E isolated primarily from palm and rice bran oil, has been linked with anticancer activities, the mechanism of this action is poorly understood. In this study, we investigated whether ␥-T3 can modulate the STAT3 cell signaling pathway, closely linked to inflammation and tumorigenesis. We found that ␥-T3 but not ␥tocopherol, the most common saturated form of vitamin E, inhibited constitutive activation of STAT3 in a dose-and timedependent manner, and this inhibition was not cell type-specific. ␥-T3 also inhibited STAT3 DNA binding. This correlated with inhibition of Src kinase and JAK1 and JAK2 kinases. Pervanadate reversed the ␥-T3-induced down-regulation of STAT3 activation, suggesting the involvement of a protein-tyrosine phosphatase. When examined further, we found that ␥-T3 induced the expression of the tyrosine phosphatase SHP-1, and gene silencing of the SHP-1 by small interfering RNA abolished the ability of ␥-T3 to inhibit STAT3 activation, suggesting a vital role for SHP-1 in the action of ␥-T3. Also ␥-T3 down-modulated activation of STAT3 and induced SHP-1 in vivo. Eventually, ␥-T3 down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) gene products; and this correlated with suppression of proliferation, the accumulation of cells in sub-G(1) phase of the cell cycle, and induction of apoptosis. This vitamin also sensitized the tumor cells to the apoptotic effects of thalidomide and bortezomib. Overall, our results suggest that ␥-T3 is a novel blocker of STAT3 activation pathway both in vitro and in vivo and thus may have potential in prevention and treatment of cancers.
Vitamin E includes a group of eight naturally occurring compounds that is further subdivided into two structurally associated subgroups called tocopherols (TP) 2 and tocotrienols (T3), each containing ␣-, ␤-, ␥-, and ␦-forms (1,2). Although both TPs and T3s demonstrate antioxidant properties (3), only T3s display potent anti-cancer activity at treatment doses that have little or no effect on normal cell proliferation (4 -6). T3 displays activity against a variety of chronic diseases, including cardiovascular diseases, neurological diseases, and diabetes, as well as cancer (7)(8)(9)(10). Although there is a lot known about TP, as indicated by over 30,000 citations, very little is known about T3. In particular, how T3 mediates its effects against cancers and other chronic diseases is not fully understood. The studies from our laboratory (11) established for the first time that ␥-T3 is a potent inhibitor of the NF-B activation pathway, which has been closely linked to tumorigenesis.
Another potential intriguing target of ␥-T3 is signal transducers and activators of transcription (STATs), which constitutes a family of six different transcription factors and has been linked to tumor development (12). Upon ligand-induced activation of cytokine receptors, STATs directly relay receptorgenerated signals into the nucleus. Activated STATs dimerize and translocate to the nucleus, where they adhere to specific DNA-response elements and induce expression of STATregulated gene expression. One of the members of the STAT family, STAT3, mediates IL-6 signaling through interaction with the IL-6 receptor, and studies using dominant-negative STAT3 proteins have demonstrated the role of STAT3 signaling in malignant transformation (13,14). Numerous reports suggest that activation of STAT3 by various growth factors can suppress apoptosis and promote proliferation, angiogenesis, chemoresistance, and inflammation (15)(16)(17)(18). Bhattacharya et al. (18) showed that activated STAT3 precluded apoptosis in polyamine-depleted cells through the transcription of the antiapoptotic proteins Bcl-2, Mcl-1, and c-IAP2. Both chemically induced and constitutively active STAT3 protect fibroblasts from ultraviolet-induced apoptosis and antagonize the proapoptotic effects of activated STAT1 (16). Thus, STAT3 can contribute to oncogenesis by defending cancer cells from apoptosis (12).
Based on these published results (19,20), we hypothesized that ␥-T3 may modulate the STAT3 cell signaling pathway and sensitize tumor cells to apoptosis. We tested this hypothesis in a series of tumor cell lines. We found that ␥-T3 suppressed the activation of the STAT3 pathway by activating a protein-tyrosine phosphatase, down-regulated STAT3-regulated proteins, inhibited cell proliferation, and sensitized cancer cells to chemotherapeutic agents.
Animal Protocol-The MIA PaCa-2 cells were orthotopically implanted as described previously (21). One week after implantation, the mice were randomized into the following treatment groups (n ϭ 6/group): (a) untreated control (olive oil, 100 l daily), and (b) tocotrienol (400 mg/kg once daily orally (per oral)). Therapy was continued for 4 weeks, and the animals were euthanized 1 week later. Primary tumors in the pancreas were excised, snap-frozen in liquid nitrogen, and stored at Ϫ80°C. Our experimental protocol (ACEF 10-05-11032) was reviewed and approved by the Institutional Animal Care and Use Committee at M. D. Anderson Cancer Center.
Electrophoretic Mobility Shift Assay-STAT3-DNA binding was analyzed by electrophoretic mobility shift assay using a 32 P-labeled high affinity cis-inducible element probe as described previously (22). Briefly, nuclear extracts were prepared from ␥-T3-treated cells and incubated with a high affinity cis-inducible element probe (5-CTTCATTTCCCG-TAAATCCCTAAAGCT-3 and 5-AGCTTTAGGGATTTA-CGGGAAATGA-3). The DNA-protein complex that formed was separated from free oligonucleotide on 5% native polyacrylamide gels. The dried gels were visualized, and the radioactive bands were quantitated with a Storm 820 and ImageQuant software (Amersham Biosciences).
Immunocytochemistry for STAT3 Localization-␥-T3-treated MM cells were plated on a glass slide by centrifugation using a Cytospin 4 (Thermoshendon), air-dried for 1 h at room temperature, and fixed with cold acetone. After a brief washing in PBS, slides were blocked with 5% normal goat serum for 1 h and then incubated with rabbit polyclonal anti-human STAT3 antibody (dilution, 1:100). After overnight incubation, the slides were washed and then incubated with goat anti-rabbit IgG-Alexa 594 (1:100) for 1 h and counterstained for nuclei with Hoechst (50 ng/ml) for 5 min. Stained slides were mounted with mounting medium (Sigma) and analyzed under an epifluorescence microscope (Labophot-2, Nikon). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon) and MetaMorph version 4.6.5 software (Universal Imaging).
Transfection with SHP-1 siRNA-Human squamous cell carcinoma (SCC4) cells were plated in 6-well plates and allowed to adhere for 24 h. On the day of transfection, 12 l of HiPerFect transfection reagent (Qiagen) were added to 50 nM SHP-1 siRNA (sense-GCAGGAGUCCGAGGAUACAtt, antisense-UGUACCUCGGACUCCUGCtt) (Ambion) in a final volume of 100 l of culture medium. After 48 h of transfection, cells were treated with ␥-T3 for 6 h, and whole-cell extracts were prepared for SHP-1, STAT3, and phospho-STAT3 analysis by Western blot.
Flow Cytometric Analysis-To determine the effect of ␥-T3 on the cell cycle, U266 cells were first synchronized by serum starvation and then exposed to ␥-T3 for the indicated time intervals. Thereafter, cells were washed, fixed with 70% ethanol, and incubated for 30 min at 37°C with 0.1% RNase A in PBS. Cells were then washed again, resuspended, and stained in PBS containing 25 ng/ml propidium iodide for 30 min at room temperature. Cell distribution across the cell cycle was analyzed with a FACS Calibur (Franklin Lakes, NJ).
Live/Dead Assay-Apoptosis of cells was also determined by the Live/Dead assay (Molecular Probes), which measures intracellular esterase activity and plasma membrane integrity, as described previously (23). Briefly, 1 ϫ 10 6 cells were incubated with ␥-T3/Velcade/thalidomide alone or in combination for 24 h at 37°C. Cells were stained with the Live/Dead reagent (5 M ethidium homodimer, 5 M calcein) and then incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2).

RESULTS
The goal of this study was to determine whether ␥-T3 affects the STAT3 activation pathway and, if so, through what mechanism. Among all isoforms of T3s, we used ␥-T3 because it exhibits maximum anticancer activity against most types of tumor cells (24). The structure of ␥-T3 is shown in Fig. 1A. We evaluated the effect of ␥-T3 on both constitutive and IL-6-induced STAT3 activation. For most studies we used MM cells, because STAT3 activation is better understood in these cells. We also investigated the effect of ␥-T3 on various mediators of cellular proliferation, cell survival, and apoptosis. Changes in the treatment dose (up to 80 M) and duration (up to 6 h) of ␥-T3 used for STAT3 experiments had no effect on cell viability (data not shown).
␥-T3 Inhibits Constitutive STAT3 Phosphorylation in a Doseand Time-dependent Manner-Whether ␥-T3 can modulate constitutive STAT3 activation in MM cells was examined. U266 cells were incubated with different concentrations of ␥-T3 for 6 h, and whole-cell extracts were prepared and examined for phosphorylated STAT3 by Western blot analysis using an antibody that recognizes STAT3 phosphorylated at tyrosine 705. As shown in Fig. 1B, ␥-T3 inhibited the constitutive activation of STAT3 in U266 cells, with maximum inhibition at 80 M. Under these conditions, ␥-T3 had no effect on the expression levels of STAT3 protein (Fig. 1B, bottom panel). We also determined the incubation time required for ␥-T3 to suppress STAT3 activation in U266 cells. As shown in Fig. 1C, ␥-T3 inhibited activation of STAT3 in a time-dependent manner, with maximum inhibition occurring at 6 h, again with no effect on the expression of STAT3 protein (Fig. 1C, bottom panel) or on cell viability (data not shown).
␥-T3 Inhibits DNA Binding Activity of STAT3-Because tyrosine phosphorylation causes dimerization of STATs, nuclear translocation, and DNA binding, thus resulting in gene transcription (22), we determined whether ␥-T3 suppresses DNA binding activity of STAT3. EMSA (electrophoretic mobility gel shift assay) analysis of nuclear extracts prepared from U266 cells showed that ␥-T3 decreased DNA binding activity of STAT3 in a dose-dependent (Fig. 1D) and time-dependent manner (Fig. 1E). Supershift analysis indicated that the binding of STAT3 to the DNA was blocked by anti-Stat3 antibody and by cold competitor oligonucleotide, thus confirming that the protein-DNA complex observed actually contained STAT3. No constitutive activation of STAT3 could be detected in human myeloid KBM-5 cell (Fig. 1F). Thus, ␥-T3 abrogated the DNAbinding ability of STAT3.
␥-T3 Depletes Nuclear Pool of STAT3 in MM Cells-Because nuclear translocation of STAT3 is central to the function of it (and other transcription factors), we determined whether ␥-T3 suppresses nuclear translocation of STAT3. Fig. 1G, as analyzed by Western blot and immuno cytochemistry (Fig. 1H), clearly shows that ␥-T3 inhibited the translocation of STAT3 to the nucleus in U266 cells.
STAT3 Inhibition by ␥-T3 Is Not Cell Type-specific-Next, we determined the effect of ␥-T3 on the constitutive activation of STAT3 in DU-145, PC3, and MiaPaCa-2 cell lines. ␥-T3 inhibited STAT3 activation in all these cell lines (Fig. 2A). These results clearly indicate that the STAT3 inhibition by ␥-T3 is not cell type-specific.
␥-T3 but Not ␥-TP Inhibits STAT3 Activation-T3s differ from TPs in that T3 is an unsaturated TP that contains three double bonds in the side chain. Whether ␥-TP can also inhibit activation of STAT3 in MM cells was examined. As shown in Fig. 2B, ␥-T3 inhibited activation of STAT3, but ␥-TP did not even at higher doses (Fig. 2C). Thus ␥-T3 differs in its biological effects from that of ␥-TP.
␥-T3 Inhibits Inducible STAT3 Phosphorylation in Human Cancer Cells-Because IL-6 is a growth factor for MM cells and it mediates its effects through induction of STAT3 phosphorylation (25, 26), we determined whether ␥-T3 could inhibit IL-6-induced STAT3 phosphorylation in MM1.S cells, which lack constitutively active STAT3. IL-6 induced phosphorylation of STAT3 as early as 5 min, and maximum activation could be seen at 30 min (Fig. 2D). IL-6-induced STAT3 phosphorylation was suppressed by ␥-T3 in a time-dependent manner (Fig. 2E). Exposure of cells to ␥-T3 for 4 h was sufficient to suppress IL-6-induced STAT3 phosphorylation.
␥-T3 Suppresses Constitutive Activation of c-Src, JAK1, and JAK2-Because STAT3 is also activated by soluble tyrosine kinases of the Src kinase families (27), we determined the effect of ␥-T3 on constitutive activation of c-Src kinase in U266 cells. We found that ␥-T3 suppressed the constitutive phosphorylation of c-Src kinase (Fig. 3A). The total levels c-Src kinase protein remained unchanged under these conditions (Fig. 3A, bottom panel). STAT3 has been reported to be activated by soluble tyrosine kinases of the JAK family (28); thus, we determined whether ␥-T3 affects constitutive activation of JAK1 in U266 cells. We found that ␥-T3 suppressed the constitutive phosphorylation of JAK1 (Fig. 3B). The levels of nonphosphorylated JAK1 remained unchanged under the same conditions (Fig. 3B, bottom panel).
To determine the effect of ␥-T3 on JAK2 phosphorylation, untreated and ␥-T3-treated whole-cell lysates were analyzed by Western blot with the anti-phospho-JAK2 antibody. As shown in Fig. 3C, JAK2 was constitutively active in U266 cells, and treatment with ␥-T3 inhibited this phosphorylation in a dose-dependent manner.
␥-T3-induced Inhibition of STAT3 Activation Is Reversed by Tyrosine Phosphatase Inhibitor-Because protein-tyrosine phosphatase (PTP) has been implicated in STAT3 inhibition (29), we determined whether ␥-T3-induced inhibition of STAT3 tyrosine phosphorylation could be due to activation of a PTP. Treatment of U266 cells with the broadacting PTP inhibitor sodium pervanadate reversed the ␥-T3-induced inhibition of STAT3 activation (Fig.  4A). This suggests that PTPs are involved in ␥-T3-induced inhibition of STAT3 activation.
␥-T3 Induces the Expression of SHP-1-SHP-1 is a nontransmembrane PTP that has been linked with regulation of STAT3 activation (30). Whether inhibition of STAT3 phosphorylation by ␥-T3 is due to induction of the expression of SHP-1 was examined. As shown in Fig. 4B, ␥-T3 indeed induced the expression of SHP-1 in a dose-dependent manner. During this treatment, ␤-actin levels remained unchanged. suppresses phospho-STAT3 levels in a dose-dependent manner. U266 cells (2 ϫ 10 6 /ml) were treated with the indicated concentrations of ␥-T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of protein were resolved on 7.5% SDS-polyacrylamide gel, electrotransferred onto nitrocellulose membrane, and probed for phospho-STAT3. C, ␥-T3 suppresses phospho-STAT3 levels in a time-dependent manner. U266 cells (2 ϫ 10 6 /ml) were treated with the 60 M ␥-T3 for the indicated time points, after which Western blotting was done as described above. D, ␥-T3 suppresses STAT3 DNA binding in a dose-dependent manner. U266 cells (2 ϫ 10 6 /ml) were treated with the indicated concentrations of ␥-T3 for 6 h and analyzed for nuclear STAT3 levels by EMSA. E, U266 cells (2 ϫ 10 6 /ml) were treated with 60 M ␥-T3 for the indicated durations and analyzed for nuclear STAT3 levels by EMSA. F, nuclear extracts from U266 cells were incubated with STAT3 antibody and an unlabeled STAT3 oligonucleotide probe. Nuclear extracts from myeloid leukemia (KBM-5) cells were taken alone. They were then assayed for STAT3 DNA binding by electrophoretic mobility shift assay. G, ␥-T3 inhibits translocation of STAT3 to the nucleus. U266 cells (1 ϫ 10 5 /ml) were incubated with or without 60 M ␥-T3 for 6 h and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry. The same slides were counterstained for nuclei with Hoechst (50 ng/ml) for 5 min. H, U266 cells (2 ϫ 10 6 /ml) were treated with the indicated concentrations of ␥-T3 for 6 h and analyzed for nuclear STAT3 levels by Western blot.

␥-T3-induced Inhibition of STAT3 Activation Is Reversed by Gene Silencing of SHP-1-We determined whether the suppression of SHP-1 expression by siRNA (small interfering RNA) would abrogate the induction of SHP-1 by ␥-T3.
Western blotting showed that ␥-T3-induced SHP-1 expression and that SHP-1 siRNA but not the control scRNA abolished the expression of SHP-1 induction by ␥-T3. We also found that ␥-T3 failed to suppress STAT3 activation in cells treated with SHP-1 siRNA (Fig. 4C). These results further corroborate our earlier evidence for the critical role of SHP-1 in suppression of STAT3 phosphorylation by ␥-T3. SHP-1 gene silencing with siRNA did not suppress constitutive activation of JAK1, JAK2 and Src in SCC4 cells (Fig. 4D).
SHP-1 siRNA Attenuates Cell Death by ␥-T3-We determined whether the silencing of SHP-1 expression by siRNA would abrogate the ␥-T3-induced cell death. We found that silencing of SHP-1 significantly reduced the ␥-T3-induced cell death (Fig. 4E). These results suggest the critical role of SHP-1 in ␥-T3-induced cell death.
␥-T3 Inhibits STAT3 Phosphorylation in Pancreatic Tumors in Vivo-Whether ␥-T3 inhibits STAT3 activation in vivo was examined in human pancreatic tissue from animals treated with ␥-T3. The Western blot analysis of tumor tissues showed that tissues from vehicle-treated animals express STAT3 phosphorylation, whereas that from ␥-T3-treated animals did not (Fig. 4F). This result clearly indicates that ␥-T3 inhibits STAT3 activation not only in vitro but also in vivo.
␥-T3 Down-regulates the Expression of Cell Survival, Proliferative, and Angiogenic Gene Products-We found that expression of antiapoptotic proteins (Mcl-1, Bcl-xL, Bcl-2, and survivin), cell cycle regulator proteins (cyclin D1), and angiogenic protein (VEGF), all reported to be regulated by STAT3 (12,31), were modulated by ␥-T3 treatment. ␥-T3 treatment down-regulated expression of these proteins in a time-dependent manner, with maximum suppression observed at 18 h after the beginning of treatment (Fig. 5A). (1 ϫ 10 6 /ml) were treated with 60 M ␥-T3 for 6 h, after which whole-cell extracts were prepared, and protein was resolved on 7.5% SDS-polyacrylamide gel, electrotransferred onto nitrocellulose membrane, and probed for phospho-STAT3. B and C, ␥-TP has no effect on activation of STAT3. U266 cells (2 ϫ 10 6 /ml) were treated with the indicated concentrations of either ␥-TP or ␥-T3 for 6 h, after which the whole-cell extracts were prepared, and phospho-STAT3 levels were detected by Western blot. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. D, ␥-T3 suppresses IL-6-induced phospho-STAT3 levels in a dose-dependent manner. MM.1s cells (2 ϫ 10 6 ) were treated with IL-6 (10 ng/ml) for the indicated times. Whole-cell extracts were prepared, and phospho-STAT3 level was detected by Western blot. E, ␥-T3 suppresses IL-6-induced phospho-STAT3 levels in a time-dependent manner. MM.1s cells (2 ϫ 10 6 ) were treated with 60 M ␥-T3 for the indicated times and then stimulated with IL-6 (10 ng/ml) for 30 min. Whole-cell extracts were then prepared and analyzed for phospho-STAT3 by Western blotting. A, ␥-T3 suppresses phospho-Src levels in a dose-dependent manner. U266 cells (2 ϫ 10 6 /ml) were treated with indicated doses of ␥-T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-Src antibody. The same blots were stripped and reprobed with Src antibody to verify equal protein loading. B, ␥-T3 suppresses phospho-JAK1 expression in a dose-dependent manner. U266 cells (2 ϫ 10 6 /ml) were treated with indicated doses of ␥-T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of those extracts were resolved on 7.5% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-JAK1. C, ␥-T3 suppresses phospho-JAK2 expression in a dose-dependent manner. U266 cells (2 ϫ 10 6 /ml) were treated with indicated doses of ␥-T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of those extracts were resolved on 7.5% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-JAK2. The same blots were stripped and reprobed with respective nonphosphorylated protein antibodies to verify equal protein loading.

␥-T3 Causes Accumulation of Cells in the Sub-G 1 Cell Cycle
Phase-Because D-type cyclins are required for cell progression from the G 1 to the S phase of the cell cycle (32) and because we observed a rapid decline in cyclin D1 levels in ␥-T3-treated cells, we sought to determine the effect of ␥-T3 on cell cycle phase distribution. We found that ␥-T3 caused significant accumulation of cells in the sub-G 1 phase, an indicator of apoptosis (Fig. 5C).
␥-T3 Potentiates the Apoptotic Effect of Bortezomib and Thalidomide in MM Cells-Recently, the drugs bortezomib, an inhibitor of proteasome, and thalidomide, an inhibitor of TNF expression, have been approved for the treatment of MM (33). We investigated whether ␥-T3 can sensitize the cancer cells to these drugs. U266 cells were treated with ␥-T3 together with either thalidomide or bortezomib. The cells were then examined for apoptosis by using the Live/Dead assay. ␥-T3 treatment significantly enhanced the apoptotic effects of thalidomide from 18 to 56% and of bortezomib from 18 to 77% (Fig. 5E).

DISCUSSION
Because STAT3 has been linked with inflammation, survival, proliferation, chemoresistance, and angiogenesis of tumor cells, its inhibitors have potential for the prevention and treatment of cancer. In this study, we describe a novel role of ␥-⌻3, i.e. inhibition of STAT3 activation. To our knowledge this is the first study ever to suggest that ␥-T3 can inhibit STAT3 signaling in MM cells. We observed that ␥-⌻3 inhibited both constitutive and IL-6-induced STAT3 activation. We found that inhibition of STAT3 activation is unique to ␥-tocotrienol as ␥-tocopherol did not. There are a number of mechanisms that could account for this difference. Because of structural differences, tocotrienols may be more uniformly distributed in the lipid bilayer, so the chromanol ring of tocotrienols may interact more efficiently with the lipid bilayer than that of tocopherols. Also tocotrienols have a 70 times higher cellular uptake than that of tocopherols (34) and have a higher recycling efficiency (35). All of these factors may contribute to the greater efficacy of tocotrienol. These results are similar to our recent report in which we showed that ␥-T3 can induce death receptors for tumor necrosis factor-related apoptosis-inducing ligand, but ␥-TP had no effect (36).
When examined for the molecular mechanisms of STAT3 inhibition by ␥-T3, we found that the mechanism involves inhibition of activation of JAK2, JAK1, and c-Src and the induction of SHP-1. This correlated with the inhibition of various STAT3-regulated gene products and induction of apoptosis of MM cells. Based on our experiments involving STAT3 phosphorylation at tyrosine 705, nuclear translocation, and DNA binding, we conclude that ␥-⌻3 inhibits STAT3 activation, and this inhibition was found to be not cell type-specific. We also found that ␥-T3 suppressed STAT3 activation induced FIGURE 4. ␥-T3 regulates STAT3 through induction of SHP-1. A, pervanadate reverses the phospho-STAT3 inhibitory effect of ␥-T3. U266 cells (2 ϫ 10 6 /ml) were treated with pervanadate (50 M) and 60 M ␥-T3 for 4 h, after which Western blotting was done as described above. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. B, ␥-T3 induces the expression of SHP-1 protein in U266 cells. U266 cells (2 ϫ 10 6 /ml) were treated with indicated concentrations of ␥-T3 for 6 h, after which whole-cell extracts were prepared, and 40-g portions of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with SHP-1 antibody. The same blots were stripped and reprobed with ␤-actin antibody to verify equal protein loading. C, effect of SHP-1 knockdown on ␥-T3-induced expression of SHP-1. SCC4 cells (1 ϫ 10 5 /ml) were transfected with either scrambled or SHP-1-specific siRNA (50 nM). After 48 h, cells were treated with 60 M ␥-T3 for 6 h, and whole-cell extracts were subjected to Western blot analysis for SHP-1, p-STAT3, and STAT3. D, effect of SHP-1 knockdown on the expression levels of constitutively active JAK1, JAK2, and Src. SCC4 cells (1 ϫ 10 5 /ml) were transfected with either scrambled or SHP-1-specific siRNA (50 nM). After 48 h, cells were treated with 60 M ␥-T3 for 6 h, and whole-cell extracts were subjected to Western blot analysis. E, knockdown of SHP-1 inhibits the apoptotic effect of ␥-T3. SCC4 cells (1 ϫ 10 5 /ml) were transfected with either scrambled or SHP-1-specific siRNA (50 nM). After 48 h, cells were treated with 25 M ␥-T3 for 24 h, and the percentage of apoptosis was analyzed by the Live/Dead assay. F, human pancreatic tissues from animals treated with vehicle and ␥-T3 (400 mg/kg body weight) for 4 weeks were homogenized and analyzed by Western blot for p-STAT3, STAT3, and SHP-1 expression levels as indicated under "Experimental Procedures." The blots were stripped nd reprobed with ␤-actin antibody to verify equal protein loading. *, p Ͻ 0.01. by IL-6, one of the major tumor cell growth factors that activate STAT3. The mechanism by which ␥-T3 inhibits STAT3 activation is summarized in Fig. 6.
Our results demonstrate that ␥-⌻3 can inhibit constitutively active c-Src activity in MM cells. Src-transformed cell lines have persistently activated STAT3, and dominantnegative STAT3 blocks transformation (37,38). Although ␥-T3 alone was inefficient, a combination of ␥-⌻3 plus erlotinib/gefitinib has been reported to inhibit STAT3 activation in murine mammary tumor cells (20). How ␥-⌻3 inhibits activation of STAT3 was not investigated in detail. Another study showed suppression of STAT3 activation in mesothelioma cells by redox-silent analog of ␣-tocotrienol, 6-O-carboxypropyl-␣-tocotrienol (19). Again, the mechanism by which this analog suppresses STAT3 activation was not examined. Thus, our studies are the first to investigate the mechanism by which ␥-T3 inhibits STAT3 activation. Previously, ␣-T3 has been shown to inhibit glutamate-induced pp60 (c-Src) kinase activation in neuronal cells (6), but it had no effect on the activity of recombinant c-Src kinase, suggesting that its mechanism of action may include regulation of SH domains. Besides c-Src, we also found for the first time that ␥-T3 can inhibit the activation of JAK1 and JAK2 that has been closely linked with STAT3 activation.
From numerous lines of evidence, we also found that PTP is involved in the down-regulation of STAT3 by ␥-T3. First, the broad-acting PTP inhibitor, pervanadate, inhibited the effect of ␥-T3 on STAT3 activation. What type of PTP is involved was further investigated. Several PTPs have been reported to regulate STAT3 signaling, including SHP-1 (39), SHP-2 (40), TC-PTP (41), PTEN (42), PTP-1D (43), CD45 (44), and PTP-⑀ (45). Also, it has been shown that loss of SHP-1 will enhance JAK3/STAT3 signaling in an ALK-positive anaplastic large cell lymphoma (29). In our FIGURE 5. ␥-T3 suppresses STAT3-regulated antiapoptotic gene products, induces apoptosis, and potentiates chemotherapeutic agents. A, ␥-T3 suppresses STAT3-regulated antiapoptotic gene products. U266 cells (2 ϫ 10 6 /ml) were treated with 25 M ␥-T3 for the indicated time intervals, after which whole-cell extracts were prepared, and 40-g portions of those extracts were resolved on 10% SDS-PAGE; the membrane was sliced according to molecular weight, and the gel was probed using antibodies against cyclin D1, Bcl-2, Bcl-xL, survivin, and VEGF. The same blots were stripped and reprobed with ␤-actin antibody to verify equal protein loading. B, ␥-T3 induces caspase-3-dependent PARP cleavage. U266 cells were treated with 60 M ␥-T3 for the indicated times, and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blotting against caspase-3 antibody and PARP antibody. The same blots were stripped and reprobed with ␤-actin antibody to show equal protein loading. C, ␥-T3 causes significant accumulation of cells in the sub-G 1 phase. U266 cells (2 ϫ 10 6 /ml) were synchronized by incubation overnight in the absence of serum and then treated with 25 M ␥-T3 for the indicated times, after which the cells were washed, fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. D, effects of ␥-T3 on the proliferation of multiple myeloma cells. MM cells were plated in triplicate, treated with the indicated concentrations of ␥-T3 for the indicated days, and then subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. E, ␥-T3 potentiates the apoptotic effect of thalidomide (Thal) and Velcade (Val). U266 cells (1 ϫ 10 6 /ml) were treated with 25 M ␥-T3 and 10 ng/ml thalidomide or 20 nM bortezomib alone or in combination for 24 h at 37°C. Cells were stained with a Live/Dead assay reagent for 30 min, then analyzed under a fluorescence microscope, and 20 random fields were counted. C, Control; T, Thalidomide; V, Velcade; *, p Ͻ 0.01; **; p Ͻ 0.05 versus control. studies ␥-⌻3 induced SHP-1 protein expression. Hence, it is possible that the induction of SHP-1 could have led to inhibition of STAT3 activation. We found that knockdown of SHP-1 by siRNA reversed the inhibitory effect of ␥-T3 on STAT3.
Whether our in vitro results have any relevance to that in vivo was also investigated. We found that ␥-T3 inhibited STAT3 activation in tumor tissue from animals treated with the agent in vivo. ␥-T3 also induced SHP-1 expression in human tumors from the animals. This indicates that downmodulation of STAT3 activation by ␥-T3 may be through upregulation of SHP-1 in vivo.
Consistent with suppression of STAT3 activation, ␥-T3 downregulated the expression of STAT3-regulated genes that are involved in proliferation (cyclin D1) and survival (Bcl-2, Bcl-xL, and Mcl-1) of cancer cells. Although several of these protein have been reported to be down-regulated by ␥-T3, the mechanism of this down-regulation is not understood. It is possible that abrogation of NF-B reported previously (11) and of STAT3 by ␥-T3 reported here is responsible for the suppression of proteins involved in survival and proliferation of tumor cells. Suppression of cell survival proteins led to activation of caspases and increased apoptosis.
We also found that down-regulation of cyclin D1 by ␥-T3 in MM cells that express constitutively active STAT3 caused suppression of proliferation and accumulation of cells in sub-G 1 . In addition, we found that the apoptotic effects of thalidomide and bortezomib were potentiated by ␥-T3.
We also found that ␥-T3 down-regulated the expression of VEGF, which is needed for angiogenesis of tumor cells. Previously, we have reported that ␥-T3 can inhibit NF-B activation through inhibition of IB kinase (IKK) activation (11). Whether inhibition of STAT3 by ␥-T3 is connected with suppression of IKK activation is not clear at this point. Although the p65 subunit of NF-B has been shown to communicate with STAT3 (46), different cytokines and different kinases activate them. Although tumor necrosis factor is the major activator of NF-kB, IL-6 that is regulated by NF-B, is the most potent inducer of STAT3. Collectively, our results show that ␥-T3 inhibits both inducible and constitutive STAT3 activation through the induction of tyrosine kinase phosphatase, which makes it a potentially effective suppressor of tumor cell survival, proliferation, and angiogenesis. Further clinical studies may provide important leads for using ␥-T3 as treatment for cancer and other inflammatory diseases through the suppression of STAT3.