# Synergistic Antiproliferative Effects of Combinedγ-Tocotrienol and PPARγ Antagonist Treatment Are Mediated through PPARγ-Independent Mechanisms in Breast Cancer Cells

Authors: Abhita Malaviya; Paul W. Sylvester
Journal: PPAR Research
 (2014)

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## Abstract

Previous findings showed that the anticancer effects of combinedγ-tocotrienol and peroxisome proliferator activated receptor γ (PPARγ) antagonist treatment caused a large reduction in PPARγ expression. However, other studies suggest that the antiproliferative effects of γ-tocotrienol and/or PPARγ antagonists are mediated, at least in part, through PPARγ-independent mechanism(s). Studies were conducted to characterize the role of PPARγ in mediating the effects of combined treatment of γ-tocotrienol with PPARγ agonists or antagonists on the growth of PPARγ negative +SA mammary cells and PPARγ-positive and PPARγ-silenced MCF-7 and MDA-MB-231 breast cancer cells. Combined treatment of γ-tocotrienol with PPARγ antagonist decreased, while combined treatment of γ-tocotrienol with PPARγ agonist increased, growth of all cancer cells. However, treatment with high doses of 15d-PGJ2, an endogenous natural ligand for PPARγ, had no effect on cancer cell growth. Western blot and qRT-PCR studies showed that the growth inhibitory effects of combined γ-tocotrienol and PPARγ antagonist treatment decreased cyclooxygenase (COX-2), prostaglandin synthase (PGDS), and prostaglandin D2 (PGD2) synthesis. In conclusion, the anticancer effects of combined γ-tocotrienol and PPARγ antagonists treatment in PPARγ negative/silenced breast cancer cells are mediated through PPARγ-independent mechanisms that are associated with a downregulation in COX-2, PGDS, and PGD2 synthesis.


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## Body

## 1. Introduction

γ-Tocotrienol is a member of the vitamin E family that displays potent anticancer activity at treatment doses that have little or no effect on normal cell function and viability [1–4]. The mechanism(s) involved in mediating the anticancer effects of γ-tocotrienol result from both direct and indirect actions on multiple intracellular targets [5, 6]. Recently, studies have shown that γ-tocotrienol stimulates endogenous PPARγ activity [7] and increases the production of a PPARγ ligand, 15-S-hydroxyeicosatetraenoic acid in human prostate cancer cells [8]. Furthermore, combined treatment of γ-tocotrienol with the PPARγ antagonists, GW9662 and T0070907, significantly inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and this effect was associated with a corresponding decrease in PPARγ activity and expression. In contrast, combined treatment of γ-tocotrienol with the PPARγ agonists, rosiglitazone and troglitazone, was found to stimulate tumor cell growth, and this effect was associated with an increase in PPARγ activity and expression [9]. While these findings suggest that treatments that reduce PPARγ activity suppress, whereas treatments that increase PPARγ activity, enhance breast cancer cell growth, the possibility exists that these effects are mediated, at least in part, through PPARγ-independent mechanism(s).Peroxisome proliferator activated receptorγ (PPARγ) is a ligand activated transcription factor that belongs to the nuclear receptor superfamily [10, 11]. Ligands for PPARγ include 15-deoxy-Δ12, 14-PGJ2 (15d-PGJ2), an endogenous prostaglandin, and synthetic agents such as the PPARγ agonist rosiglitazone and troglitazone that increase 15d-PGJ2 levels in adipocytes [12, 13]. 15d-PGJ2 is a nonenzymatically derived product of prostaglandin D2 [14], and its production is associated with elevated cyclooxygenase-2 (COX-2) and prostaglandin synthase (PGDS) activity [15]. Several reports have also suggested that endogenous PPARγ ligand production may be related to COX-2 expression in various forms of cancer [16–20]. Studies have also shown that treatment with mixed tocopherols and tocotrienols, reduced COX-2 expression [21], and combined treatment of γ-tocotrienol with the specific COX-2 inhibitor, celecoxib, resulted in a synergistic inhibition in mammary tumor cell growth [22, 23]. These anticancer effects were found to be associated with reduction in COX-2, but not COX-1 levels, and a corresponding suppression in PGE2 synthesis, and decrease in Akt and NFκB activation [22, 23]. Furthermore, treatment with high doses of γ-tocotrienol, PPARγ agonists, or PPARγ antagonist alone, inhibits mammary tumor cell growth [9]. Although the exact mechanism(s) has/have not yet been determined, it is very possible that some or all of these anticancer effects are mediated through PPARγ-independent mechanisms. Previous studies have shown that high dose treatment with PPARγ agonists and antagonists results in varying degrees of nonspecific effects in different types of cancer cells [24, 25].Therefore, studies were conducted to characterize the role of PPARγ in mediating the effects of combined treatment of γ-tocotrienol with PPARγ agonists (rosiglitazone and troglitazone) or antagonists (GW9662 and T007907) on the growth of PPARγ negative +SA mouse mammary epithelial cells and PPARγ-positive and PPARγ-silenced (siRNA transfected) MCF-7 and MDA-MB-231 human breast cancer cells. Additional studies evaluated the effects of these treatments alone and in combination on the levels and activity of COX-2, PGDS, PGD2synthesis, and various proteins involved in cell cycle progression in these same breast cancer cells.


## 2. Materials and Methods

### 2.1. Reagents and Antibodies

All reagents were purchased from Sigma Chemical Company unless otherwise stated. Purifiedγ-tocotrienol was generously provided as a gift by First Tech International Ltd (Hong Kong). PPARγ agonists, rosiglitazone (Cayman Chemical 71740) and troglitazone (Cayman Chemical 71750), and 15d-PGJ2(Cayman Chemical 18500) and the PPARγ antagonists GW9662 (Cayman Chemical 70785) and T0070907 (Cayman Chemical 10026) were used in this study. Antibodies, β-actin (Cell Signaling 4970), PPARγ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPARγ siRNAs (Santa Cruz sc-29455) were used in the present study. Secondary antibodies goat anti-rabbit (PerkinElmer Biosciences NEF812001EA) and anti-mouse (PerkinElmer Biosciences NEF822001EA) were used in this study.


### 2.2. Cell Lines and Culture Conditions

The neoplastic +SA cell line was derived from a mammary adenocarcinoma that developed spontaneously in a BALB/c female mouse. The +SA cell line is characterized as being highly malignant, estrogen-independent, and displays anchorage-independent growth when cultured in soft agarose gels [26, 27]. Cell culture and experimental details have been described previously in detail [1, 2]. Briefly, +SA cells were grown and maintained in serum-free modified Dulbecco’s modified Eagle Medium (DMEM/F12) media containing 5 mg/mL bovine serum albumin (BSA), 10 μg/mL insulin, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 ng/mL epidermal growth factor (EGF) as a mitogen at 37°C in an environment of 95% air and 5% CO2 in a humidified incubator. The estrogen-receptor negative MDA-MB-231 (ATCC HTB-26) and the estrogen-receptor positive MCF-7 (ATCC HTB-22) breast carcinoma cell lines were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (ATCC 30-2020), 10 μg/mL insulin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37°C in an environment of 95% air and 5% CO2 in a humidified incubator. For subculturing, +SA, MCF-7, and MDA-MB-231 cells were rinsed twice with sterile Ca2+- and Mg2+-free phosphate-buffered saline (PBS) and incubated in 0.05% trypsin containing 0.025% EDTA in PBS for 5 min at 37°C. The released cells were centrifuged, resuspended in serum containing media, and counted using a hemocytometer.


### 2.3. Experimental Treatments

The highly lipophilicγ-tocotrienol was suspended in a solution of sterile 10% BSA as described previously [1, 2]. Briefly, an appropriate amount of γ-tocotrienol was first dissolved in 100 μL of 100% ethanol, then added to a small volume of sterile 10% BSA in water, and incubated overnight at 37°C with continuous shaking. This stock solution was then used to prepare various concentrations of treatment media. Stock solutions of rosiglitazone, troglitazone, GW9662, T0070907, and 15d-PGJ2 were prepared in dimethyl sulfoxide (DMSO). Ethanol and/or DMSO was added to all treatment media such that the final concentration was the same in all treatment groups within any given experiment and was always less than 0.1%.


### 2.4.  Growth Studies

+SA cells were plated at a density of5
×
104 cells/well (6 replicates/group) in 24-well culture plates and allowed to adhere overnight. The next day, cells were divided into different treatment groups, and culture media were removed, washed with sterile PBS, then fed fresh media containing their respective treatments, and then returned to the incubator. Cells were treated with media containing 0–20 μM rosiglitazone, troglitazone, GW9662, T0070907, 0–50 μM 15d-PGJ2, or 0–4 μM γ-tocotrienol alone or in combination for a 4-day culture period. Cells in each treatment group were fed fresh media every other day throughout the experimental period. For apoptosis experiments, +SA cells were plated as described above. Cells were allowed to grow in control media for 3 days, after which they were exposed to the various treatments for a 24 h period. Treatment with 20 μM γ-tocotrienol has been previously shown to induce apoptosis in breast cancer cells [1] and was used as a positive control in this study.


### 2.5. Measurement of Viable Cell Number

+SA viable cell number was determined using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay as described previously [1, 2]. At the end of the treatment period, treatment media were removed and all cells were exposed for 4 h (24 well/plates) to fresh control media containing 0.41 mg/mL MTT at 37°C. Afterwards, media were removed and MTT crystals were dissolved in 1 mL of isopropanol. The optical density of each sample was measured at 570 nm at a SpectraCount microplate reader (Packard Bioscience Company) zeroed against a blank prepared from cell-free medium. The number of cells per well was calculated against a standard curve prepared by plating known cell densities, as determined by hemocytometer, in triplicate at the start of each experiment.


### 2.6. Western Blot Analysis

+SA, MCF-7, and MDA-MB-231 cells were plated at a density of1
×
106 cells/100 mm culture dish and exposed to control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Afterwards, cells were washed with PBS and isolated with trypsin, and whole cell lysates were prepared in Laemmli buffer [28] as described previously [29]. To study treatment effect on cell cycle progression, +SA cells in various groups were synchronized to prevent phase variation between different groups before mitogen treatment (EGF). The method of mitogen starvation was employed by using mitogen-free media to synchronize all cells in G1 phase of cell cycle. Control defined media were removed, and cells were washed with PBS to remove any traces of mitogen, followed by 48 h exposure to mitogen-free control and treatment media to allow cells to synchronize in G0/G1 phase of the cell cycle. Afterwards, media were removed and replaced with fresh control and treatment media containing EGF to initiate simultaneous cell cycle progression in all groups. Cells were then isolated with trypsin and whole cell lysates were prepared. The protein concentration in each sample was determined using Bio-Rad protein assay kit (Bio-Rad 500-0006). Equal amounts of protein from each sample in a given experiment were loaded onto SDS-polyacrylamide Minigels and electrophoresed through 5%–15% resolving gel. Proteins separated on each gel were transblotted at 30 V for 12–16 h at 4°C onto a polyvinylidene fluoride (PVDF) membrane (PerkinElmer Lifesciences NEF1000) in a Trans-Blot Cell (Bio-Rad) according to the method of Towbin et al. [30]. The membranes were then blocked with 2% BSA in 10 mM Tris HCl containing 50 mM NaCl and 0.1% Tween 20 pH 7.4 (TBST) and then incubated with specific primary antibodies against PPARγ, COX-2, PGDS, Cyclin D1, CDK4, CDK6, p16, phospho-Rb (ser780), and phospho-Rb (ser807/811), cleaved caspase-3, cleaved PARP or β-actin, diluted 1 : 500 to 1 : 5000 in TBST/2% BSA for 2 h. Membranes are washed 5 times with TBST followed by incubation with the respective horseradish peroxide-conjugated secondary antibodies diluted 1 : 3000 to 1 : 5000 in TBST/2% BSA for 1 h followed by rinsing with TBST. Protein bands bound to the antibody were visualized by chemiluminescence (Thermo Scientific 34078) according to the manufacturer’s instructions and images were obtained using a Kodak Gel Logic 1500 Imaging System (Carestream Health Inc.). The visualization of β-actin was performed to confirm equal sample loading in each lane. Images of protein bands on the film were acquired and the densitometric analysis was performed with Kodak molecular imaging software version 4.5. All experiments were repeated at least 3 times and a representative Western blot image from each experiment is shown in the figures.


### 2.7. Quantitative Real-Time PCR

+SA cells were plated at a density of2
×
103 cells/well (3 replicates per group) in 6-well plates and allowed to adhere overnight. In the next day, cells were divided into different treatment groups, and culture media were removed, washed with sterile PBS, then fed fresh media containing their respective treatments, and then returned to the incubator. Cells were treated with media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Total RNA was extracted using RNA kit (Applied Biosystems 4305895) according to the manufacturer’s instructions. First-strand cDNA was generated from total RNA for each sample using the cDNA kit (Applied Biosystems 18080200) according to the manufacturer’s instructions. Taqman PCR probes and gene-specific primer pairs were generated for COX-2, PGDS, and GAPDH using Integrated DNA technologies. qRT-PCR was performed on an Applied Biosystems Prism 7900 Sequence Detection System. Reactions were prepared in triplicate for each gene using Taqman gene expression assays. During thermal cycling, the threshold cycle (Ct) is defined as the cycle number when amplification of a specific PCR product is detected. The average Ct value of GAPDH was subtracted from average Ct value of target genes (COX-2, PGDS) to normalize the amount of sample RNA added to the reaction. Relative quantification describes the fold change in expression of a gene of interest in a test sample relative to a calibrator sample. With the comparative Ct (ΔΔCt) method, the level of the target gene mRNA in treatment samples relative to control samples was determined.


### 2.8. Transient Transfection

MCF-7 and MDA-MB-231 cells were plated at a density of2
×
105 cells/well (3 replicates per group) in 6-well plate in 2 mL antibiotic free media and allowed to adhere overnight. Transfections were performed using 5 μL lipofectamine 2000 (Invitrogen 11668027) according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs were diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to 2 mL of control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or a combination for a 4-day culture period. Afterwards, cells were harvested for Western blot analysis. To study the treatment effect of 15d-PGJ2 in MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. Transfections were performed using 0.25 μL lipofectamine 2000. For each well of cells to be transfected, 5 pmol of the scrambled or PPARγ siRNAs was diluted with 100 μL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% fetal bovine serum and cells were cultured for 18 h. Cells were then exposed to 100 μL of treatment media containing 10 μM and 50 μM of 15d-PGJ2 for a 4-day culture period. After this cell viability was determined by MTT assay.


### 2.9. Luciferase Reporter Assay

+SA, MCF-7, and MDA-MB-231 cells were plated at a density of2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. After this cells were transfected with 32 ng of PPRE X3-TK-luc (Addgene plasmid no.1015) [31] and 3.2 ng of Renilla luciferase plasmid per well (Promega E2261) and then cotransfected with scrambled or PPARγ siRNAs using 0.8 μL of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μL of treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Afterwards, cells were lysed with 75 μL of passive lysis buffer and treated according to manufacturer’s instructions using dual-glo luciferase assay system (Promega E2920). Luciferase activity of each sample was normalized by the level of Renilla activity. Data is represented as mean fold changes in treated cells as compared to control cells.


### 2.10. PGD2 Synthesis

+SA cells were plated at a density of1
×
106 cells/100 mm culture dish and exposed to control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Afterwards, cells were washed with PBS and isolated with trypsin, and whole cell lysates were prepared as described previously [29]. MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
105 cells/well in 6-well plates in 2 mL antibiotic-free media and allowed to adhere overnight. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to 2 mL of control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. The cell lysates were collected as described previously [29]. Cell lysates collected from +SA, MCF-7, and MDA-MB-231 cells were assayed for PGD2 according to the methods described in the Enzyme Immunoassay kit (Cayman Chemical 512031). Optical density was measured at 420 nm on a Synergy-2 Multimode Microplate Reader (BioTek Instruments Inc.). Data is represented as amount of PGD2synthesized (pg/mL) in treated cells as compared to vehicle-treated control cells.


### 2.11. Statistical Analysis

The level of interaction between PPARγ ligands and γ-tocotrienol was evaluated by isobologram method [32]. A straight line was formed by plotting IC50 doses of γ-tocotrienol and individual PPARγ ligands on the x-axes and y-axes, respectively, as determined by nonlinear regression curve fit analysis using GraphPad Prism 4. The data point in the isobologram corresponds to the actual IC50 dose of combined γ-tocotrienol and PPARγ ligands treatment. If a data point is on or near the line, this represents an additive treatment effect, whereas a data point that lies below or above the line indicates synergism or antagonism, respectively. Differences among the various treatment groups in growth studies and western blot studies were determined by analysis of variance followed by Dunnett’s multiple range tests. Differences were considered statistically significant at a value of P
<
0.05.


## 2.1. Reagents and Antibodies

All reagents were purchased from Sigma Chemical Company unless otherwise stated. Purifiedγ-tocotrienol was generously provided as a gift by First Tech International Ltd (Hong Kong). PPARγ agonists, rosiglitazone (Cayman Chemical 71740) and troglitazone (Cayman Chemical 71750), and 15d-PGJ2(Cayman Chemical 18500) and the PPARγ antagonists GW9662 (Cayman Chemical 70785) and T0070907 (Cayman Chemical 10026) were used in this study. Antibodies, β-actin (Cell Signaling 4970), PPARγ (Cell Signaling 2443), COX-2 (Cell Signaling 12282), Cyclin D1 (Cell Signaling 2922), CDK4 (Cell Signaling 2906), CDK6 (Cell Signaling 3136), phospho-Rb (ser780) (Cell Signaling 9307), phospho-Rb (Ser807/811) (Cell Signaling 9308), cleaved caspase-3 (Cell Signaling 9661), cleaved-PARP (Cell Signaling 9544), p16 (Santa Cruz sc-1661), PGDS (Santa Cruz sc-14816), and PPARγ siRNAs (Santa Cruz sc-29455) were used in the present study. Secondary antibodies goat anti-rabbit (PerkinElmer Biosciences NEF812001EA) and anti-mouse (PerkinElmer Biosciences NEF822001EA) were used in this study.


## 2.2. Cell Lines and Culture Conditions

The neoplastic +SA cell line was derived from a mammary adenocarcinoma that developed spontaneously in a BALB/c female mouse. The +SA cell line is characterized as being highly malignant, estrogen-independent, and displays anchorage-independent growth when cultured in soft agarose gels [26, 27]. Cell culture and experimental details have been described previously in detail [1, 2]. Briefly, +SA cells were grown and maintained in serum-free modified Dulbecco’s modified Eagle Medium (DMEM/F12) media containing 5 mg/mL bovine serum albumin (BSA), 10 μg/mL insulin, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 ng/mL epidermal growth factor (EGF) as a mitogen at 37°C in an environment of 95% air and 5% CO2 in a humidified incubator. The estrogen-receptor negative MDA-MB-231 (ATCC HTB-26) and the estrogen-receptor positive MCF-7 (ATCC HTB-22) breast carcinoma cell lines were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (ATCC 30-2020), 10 μg/mL insulin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37°C in an environment of 95% air and 5% CO2 in a humidified incubator. For subculturing, +SA, MCF-7, and MDA-MB-231 cells were rinsed twice with sterile Ca2+- and Mg2+-free phosphate-buffered saline (PBS) and incubated in 0.05% trypsin containing 0.025% EDTA in PBS for 5 min at 37°C. The released cells were centrifuged, resuspended in serum containing media, and counted using a hemocytometer.


## 2.3. Experimental Treatments

The highly lipophilicγ-tocotrienol was suspended in a solution of sterile 10% BSA as described previously [1, 2]. Briefly, an appropriate amount of γ-tocotrienol was first dissolved in 100 μL of 100% ethanol, then added to a small volume of sterile 10% BSA in water, and incubated overnight at 37°C with continuous shaking. This stock solution was then used to prepare various concentrations of treatment media. Stock solutions of rosiglitazone, troglitazone, GW9662, T0070907, and 15d-PGJ2 were prepared in dimethyl sulfoxide (DMSO). Ethanol and/or DMSO was added to all treatment media such that the final concentration was the same in all treatment groups within any given experiment and was always less than 0.1%.


## 2.4.  Growth Studies

+SA cells were plated at a density of5
×
104 cells/well (6 replicates/group) in 24-well culture plates and allowed to adhere overnight. The next day, cells were divided into different treatment groups, and culture media were removed, washed with sterile PBS, then fed fresh media containing their respective treatments, and then returned to the incubator. Cells were treated with media containing 0–20 μM rosiglitazone, troglitazone, GW9662, T0070907, 0–50 μM 15d-PGJ2, or 0–4 μM γ-tocotrienol alone or in combination for a 4-day culture period. Cells in each treatment group were fed fresh media every other day throughout the experimental period. For apoptosis experiments, +SA cells were plated as described above. Cells were allowed to grow in control media for 3 days, after which they were exposed to the various treatments for a 24 h period. Treatment with 20 μM γ-tocotrienol has been previously shown to induce apoptosis in breast cancer cells [1] and was used as a positive control in this study.


## 2.5. Measurement of Viable Cell Number

+SA viable cell number was determined using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay as described previously [1, 2]. At the end of the treatment period, treatment media were removed and all cells were exposed for 4 h (24 well/plates) to fresh control media containing 0.41 mg/mL MTT at 37°C. Afterwards, media were removed and MTT crystals were dissolved in 1 mL of isopropanol. The optical density of each sample was measured at 570 nm at a SpectraCount microplate reader (Packard Bioscience Company) zeroed against a blank prepared from cell-free medium. The number of cells per well was calculated against a standard curve prepared by plating known cell densities, as determined by hemocytometer, in triplicate at the start of each experiment.


## 2.6. Western Blot Analysis

+SA, MCF-7, and MDA-MB-231 cells were plated at a density of1
×
106 cells/100 mm culture dish and exposed to control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Afterwards, cells were washed with PBS and isolated with trypsin, and whole cell lysates were prepared in Laemmli buffer [28] as described previously [29]. To study treatment effect on cell cycle progression, +SA cells in various groups were synchronized to prevent phase variation between different groups before mitogen treatment (EGF). The method of mitogen starvation was employed by using mitogen-free media to synchronize all cells in G1 phase of cell cycle. Control defined media were removed, and cells were washed with PBS to remove any traces of mitogen, followed by 48 h exposure to mitogen-free control and treatment media to allow cells to synchronize in G0/G1 phase of the cell cycle. Afterwards, media were removed and replaced with fresh control and treatment media containing EGF to initiate simultaneous cell cycle progression in all groups. Cells were then isolated with trypsin and whole cell lysates were prepared. The protein concentration in each sample was determined using Bio-Rad protein assay kit (Bio-Rad 500-0006). Equal amounts of protein from each sample in a given experiment were loaded onto SDS-polyacrylamide Minigels and electrophoresed through 5%–15% resolving gel. Proteins separated on each gel were transblotted at 30 V for 12–16 h at 4°C onto a polyvinylidene fluoride (PVDF) membrane (PerkinElmer Lifesciences NEF1000) in a Trans-Blot Cell (Bio-Rad) according to the method of Towbin et al. [30]. The membranes were then blocked with 2% BSA in 10 mM Tris HCl containing 50 mM NaCl and 0.1% Tween 20 pH 7.4 (TBST) and then incubated with specific primary antibodies against PPARγ, COX-2, PGDS, Cyclin D1, CDK4, CDK6, p16, phospho-Rb (ser780), and phospho-Rb (ser807/811), cleaved caspase-3, cleaved PARP or β-actin, diluted 1 : 500 to 1 : 5000 in TBST/2% BSA for 2 h. Membranes are washed 5 times with TBST followed by incubation with the respective horseradish peroxide-conjugated secondary antibodies diluted 1 : 3000 to 1 : 5000 in TBST/2% BSA for 1 h followed by rinsing with TBST. Protein bands bound to the antibody were visualized by chemiluminescence (Thermo Scientific 34078) according to the manufacturer’s instructions and images were obtained using a Kodak Gel Logic 1500 Imaging System (Carestream Health Inc.). The visualization of β-actin was performed to confirm equal sample loading in each lane. Images of protein bands on the film were acquired and the densitometric analysis was performed with Kodak molecular imaging software version 4.5. All experiments were repeated at least 3 times and a representative Western blot image from each experiment is shown in the figures.


## 2.7. Quantitative Real-Time PCR

+SA cells were plated at a density of2
×
103 cells/well (3 replicates per group) in 6-well plates and allowed to adhere overnight. In the next day, cells were divided into different treatment groups, and culture media were removed, washed with sterile PBS, then fed fresh media containing their respective treatments, and then returned to the incubator. Cells were treated with media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Total RNA was extracted using RNA kit (Applied Biosystems 4305895) according to the manufacturer’s instructions. First-strand cDNA was generated from total RNA for each sample using the cDNA kit (Applied Biosystems 18080200) according to the manufacturer’s instructions. Taqman PCR probes and gene-specific primer pairs were generated for COX-2, PGDS, and GAPDH using Integrated DNA technologies. qRT-PCR was performed on an Applied Biosystems Prism 7900 Sequence Detection System. Reactions were prepared in triplicate for each gene using Taqman gene expression assays. During thermal cycling, the threshold cycle (Ct) is defined as the cycle number when amplification of a specific PCR product is detected. The average Ct value of GAPDH was subtracted from average Ct value of target genes (COX-2, PGDS) to normalize the amount of sample RNA added to the reaction. Relative quantification describes the fold change in expression of a gene of interest in a test sample relative to a calibrator sample. With the comparative Ct (ΔΔCt) method, the level of the target gene mRNA in treatment samples relative to control samples was determined.


## 2.8. Transient Transfection

MCF-7 and MDA-MB-231 cells were plated at a density of2
×
105 cells/well (3 replicates per group) in 6-well plate in 2 mL antibiotic free media and allowed to adhere overnight. Transfections were performed using 5 μL lipofectamine 2000 (Invitrogen 11668027) according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs were diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to 2 mL of control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or a combination for a 4-day culture period. Afterwards, cells were harvested for Western blot analysis. To study the treatment effect of 15d-PGJ2 in MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. Transfections were performed using 0.25 μL lipofectamine 2000. For each well of cells to be transfected, 5 pmol of the scrambled or PPARγ siRNAs was diluted with 100 μL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% fetal bovine serum and cells were cultured for 18 h. Cells were then exposed to 100 μL of treatment media containing 10 μM and 50 μM of 15d-PGJ2 for a 4-day culture period. After this cell viability was determined by MTT assay.


## 2.9. Luciferase Reporter Assay

+SA, MCF-7, and MDA-MB-231 cells were plated at a density of2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. After this cells were transfected with 32 ng of PPRE X3-TK-luc (Addgene plasmid no.1015) [31] and 3.2 ng of Renilla luciferase plasmid per well (Promega E2261) and then cotransfected with scrambled or PPARγ siRNAs using 0.8 μL of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μL of treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Afterwards, cells were lysed with 75 μL of passive lysis buffer and treated according to manufacturer’s instructions using dual-glo luciferase assay system (Promega E2920). Luciferase activity of each sample was normalized by the level of Renilla activity. Data is represented as mean fold changes in treated cells as compared to control cells.


## 2.10. PGD2 Synthesis

+SA cells were plated at a density of1
×
106 cells/100 mm culture dish and exposed to control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. Afterwards, cells were washed with PBS and isolated with trypsin, and whole cell lysates were prepared as described previously [29]. MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
105 cells/well in 6-well plates in 2 mL antibiotic-free media and allowed to adhere overnight. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to 2 mL of control or treatment media containing 3.2 μM rosiglitazone and GW9662 or 2 μM γ-tocotrienol alone or in combination for a 4-day culture period. The cell lysates were collected as described previously [29]. Cell lysates collected from +SA, MCF-7, and MDA-MB-231 cells were assayed for PGD2 according to the methods described in the Enzyme Immunoassay kit (Cayman Chemical 512031). Optical density was measured at 420 nm on a Synergy-2 Multimode Microplate Reader (BioTek Instruments Inc.). Data is represented as amount of PGD2synthesized (pg/mL) in treated cells as compared to vehicle-treated control cells.


## 2.11. Statistical Analysis

The level of interaction between PPARγ ligands and γ-tocotrienol was evaluated by isobologram method [32]. A straight line was formed by plotting IC50 doses of γ-tocotrienol and individual PPARγ ligands on the x-axes and y-axes, respectively, as determined by nonlinear regression curve fit analysis using GraphPad Prism 4. The data point in the isobologram corresponds to the actual IC50 dose of combined γ-tocotrienol and PPARγ ligands treatment. If a data point is on or near the line, this represents an additive treatment effect, whereas a data point that lies below or above the line indicates synergism or antagonism, respectively. Differences among the various treatment groups in growth studies and western blot studies were determined by analysis of variance followed by Dunnett’s multiple range tests. Differences were considered statistically significant at a value of P
<
0.05.


## 3. Results

### 3.1. Antiproliferative Effects ofγ-Tocotrienol, PPARγ Agonists (Rosiglitazone and Troglitazone), and PPARγ Antagonists (GW9662 and T0070907) on the Highly Malignant Mouse +SA Mammary Tumor Cells

Treatment with 3-4μM γ-tocotrienol was found to significantly inhibit growth of +SA cells in a dose-responsive manner as compared to cells in the vehicle-treated control group. Treatment with 0–20 μM PPARγ agonists, rosiglitazone and troglitazone, or 0–20 μM of PPARγ antagonists, GW9662 and T0070907, inhibited growth of +SA cells in a dose-dependent manner compared to vehicle-treated control cells (Figure 1).Treatment effects ofγ-tocotrienol, PPARγ agonists, and PPARγ antagonists on +SA cells. +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well culture plates and exposed to treatment media for a 4-day period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)
(c)
(d)
(e)


### 3.2. Effects of Combined Treatment ofγ-Tocotrienol with PPARγ Agonists (Rosiglitazone and Troglitazone) or PPARγ Antagonists (GW9662 and T0070907) on +SA Mammary Tumor Cell Growth

Treatment with 1–4μM γ-tocotrienol alone significantly inhibited growth of +SA cells after a 4-day treatment period. However, the growth inhibitory effects of γ-tocotrienol on +SA cells were reversed when given in combination with 3.2 μM of PPARγ agonist rosiglitazone or troglitazone (Figure 2(a)). Conversely, the growth inhibitory effects of 0.5–2 μM γ-tocotrienol were significantly enhanced when given in combination with 3.2 μM of PPARγ antagonist GW9662 or T0070907 (Figure 2(b)).Treatment effects ofγ-tocotrienol, (a) PPARγ agonists and (b) PPARγ antagonists in combination in +SA cells. +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well plates and exposed to treatment media for a 4-day period. Afterwards, viable cell number was determined using MTT colorimetric assay. Vertical bars indicate the mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls and #
P
<
0.05 as compared to their corresponding control treated with γ-tocotrienol alone.
(a)
(b)


### 3.3. Isobologram Analysis of Combined Treatment Effects ofγ-Tocotrienol with PPARγ Agonists (Rosiglitazone and Troglitazone) and Antagonists (GW9662 and T0070907) on +SA Mammary Tumor Cells

The combined treatment ofγ-tocotrienol with the PPARγ agonist rosiglitazone or troglitazone was found to be statistically antagonistic, as evidenced by the location of the data point in the isobologram being well above the line defining additive effect (Figure 3(a)). In contrast, the growth inhibitory effect of combined treatment of γ-tocotrienol with PPARγ antagonists, GW9662 and T0070907, was found to be statistically synergistic, as evidenced by the location of the data point in the isobologram being well below the line defining additive effect (Figure 3(b)).Isobologram analysis of treatment ofγ-tocotrienol with PPARγ agonists and antagonists in +SA cells. Individual IC50 doses for γ-tocotrienol, (a) PPARγ agonists (rosiglitazone and troglitazone) and (b) PPARγ antagonists (GW9662 and T0070907) were calculated and then plotted on the x-axes and y-axes, respectively. The data point on the isobologram represents the actual doses of combined γ-tocotrienol and PPARγ agonists and antagonists. Combined treatment of γ-tocotrienol with PPARγ agonists rosiglitazone and troglitazone was found to be antagonistic, as evidenced by the location of the data point in the isobologram being well above the line defining additive effect. In contrast, the growth inhibitory effect of combined treatment of γ-tocotrienol with PPARγ antagonists GW9662 and T0070907 was found to be synergistic, as evidenced by the location of the data point in the isobologram being well below the line defining additive effect for both cell lines.
(a)
(b)


### 3.4. Effects ofγ-Tocotrienol, Rosiglitazone, or GW9662 Treatment Alone or in Combination on PPARγ Protein Expression in Mouse (+SA) and Human (MCF-7 and MDA-MB-231) Mammary Tumor Cells

Western blot analysis showed that PPARγ levels were abundant in untreated control human MCF-7 and MDA-MB-231 breast cancer cells, whereas PPARγ levels were undetectable in untreated control +SA cells following a 4-day culture period (Figure 4(a)). Additional studies showed that treatment with γ-tocotrienol, rosiglitazone, or GW9662 alone or in combination had no effect of PPARγ expression in +SA mouse mammary tumor cells as compared to the vehicle-treated control group (Figure 4(b)). Since +SA cells were found to be PPARγ negative, treatment with PPARγ agonists and antagonists was found to induce significant growth effects on these cells when given alone or in combination with γ-tocotrienol; these findings suggested that treatment effects may be mediated through PPARγ-independent mechanisms. Additional studies were conducted to investigate this hypothesis.Western blot analysis to determine expression of PPARγ in mammary cancer cells. (a) PPARγ levels were determined in untreated +SA, MCF-7, and MDA-MB-231 cells and (b) PPARγ levels were determined after treatment with γ-tocotrienol, rosiglitazone, and GW9662 alone or in combination in +SA cells. +SA, MCF-7, and MDA-MB-231 cells were initially plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit).
(a)
(b)


### 3.5. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on COX-2 and PGDS Protein Expression and mRNA Levels in PPARγ Negative +SA Mammary Tumor Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), rosiglitazone (3.2 μM), or GW9662 (3.2 μM) alone had little or no effect on the expression of COX-2 and PGDS as compared to the vehicle-treated control group in +SA cells (Figure 5(a)). However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a significant increase in COX-2 and PGDS levels as compared to vehicle-treated +SA cells. In contrast, combined treatment with 2 μM γ-tocotrienol and 3.2 μM GW9662 resulted in a significant decrease in the expression of COX-2 and PGDS as compared to the vehicle-treated control group in +SA cells (Figure 5(a)). Additional qRT-PCR experiments showed that similar treatments with subeffective doses of γ-tocotrienol, rosiglitazone, or GW9662 showed no significant effect on COX-2 and PGDS mRNA levels as compared to the vehicle-treated control group in +SA cells (Figure 5(b)). However, combined treatment with similar doses of γ-tocotrienol and the PPARγ agonist, rosiglitazone, resulted in a slight increase in COX-2 and PGDS mRNA levels but these differences were not found to be significant (Figure 5(b)). Furthermore, combined treatment with 2 μM γ-tocotrienol and 3.2 μM GW9662 results in a significant decrease in COX-2 and PGDS mRNA levels as compared to the vehicle-treated control group in +SA cells (Figure 5(b)). These results demonstrate that combined treatment of γ-tocotrienol with PPARγ agonists or antagonists caused significant changes in prostaglandin signaling in PPARγ negative +SA cells, indicating that the actions of these agents are mediated through a PPARγ-independent mechanism.(a) Western blot and (b) qRT-PCR analysis to determine effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on levels of COX-2 and PGDS in +SA cells. For Western blot analysis, +SA cells were plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For qRT-PCR analysis, +SA cells were plated at a density of 2
×
103 cells/well (3 replicates per group) in 6-well plates and treated with control or treatment media for a 4-day culture period. Total RNA was extracted and first-strand cDNA was generated from total RNA for each sample according to the manufacturer’s instructions. COX-2, PGDS, and GAPDH were measured using Taqman technology. Changes in mRNA levels of COX-2 and PGDS were normalized to mRNA level of GAPDH and represented as bar graph. Vertical bars indicate the normalized Ct value ± SEM (Arbitrary Unit) in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


### 3.6. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on PGD2 Synthesis and the Effect of 15d-PGJ2 Treatment on the Growth of PPARγ Negative +SA Mammary Tumor Cells

Treatment withγ-tocotrienol (2 μM), the PPARγ agonist, rosiglitazone (3.2 μM), and the PPARγ antagonist, GW9662 (3.2 μM) alone had no significant effect on PGD2 synthesis in +SA breast cancer cells compared to vehicle-treated control cells. However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a slight increase in PGD2 synthesis in +SA cells compared to vehicle-treated control cells. On the other hand, combined treatment with similar doses of γ-tocotrienol and GW9662 resulted in a significant decrease in PGD2 synthesis in +SA cells compared to vehicle-treated control cells (Figure 6(a)). Previous studies have shown that 15d-PGJ2 induces antiproliferative effects between 10 and 50 μM doses in cancer cells [33]. However, treatment with 10 μM or 50 μM 15d-PGJ2 had no effect on +SA mammary tumor cell growth following a 4-day culture period (Figure 6(b)). These results demonstrate that antiproliferative effects of combined treatment of γ-tocotrienol with PPARγ antagonists are due to decrease in prostaglandin synthesis, mediated through a PPARγ-independent mechanism. This finding was further confirmed by performing similar experiments in PPARγ silenced PPARγ positive MCF-7 and MDA-MB-231 breast cancer cells.(a) Effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PGD2 synthesis and (b) treatment effect of 15d-PGJ2 in +SA cells. +SA cells were plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were assayed for PGD2 according to the manufacturer’s protocol. Vertical bars indicate the amount of PGD2 synthesized (pg/mL) ± SEM in each treatment group. To study effect of 15d-PGJ2, +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well culture plates and exposed to treatment media for a 4-day period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


### 3.7. Effects of PPARγ siRNA Transfection on PPARγ Expression and PPRE Mediated Reporter Activity in PPARγ Positive Human MCF-7 and MDA-MB-231 Breast Cancer Cells

Western blot analysis shows that PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells displayed decreased expression in PPARγ levels as compared to vehicle-treated or scrambled RNA transfected MCF-7 and MDA-MB-231 breast cancer cells (Figure 7(a)). Luciferase assay shows that PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells significantly decreased PPRE-mediated reporter activity as compared to vehicle-treated or scrambled RNA transfected MCF-7 and MDA-MB-231 breast cancer cells (Figure 7(b)). These results confirm that PPARγ siRNA successfully downregulated expression and activation of PPARγ in both MCF-7 and MDA-MB-231 breast cancer cells.(a) Western blot analysis and (b) luciferase assay to determine expression and activity of PPARγ in PPARγ siRNA transfected MCF-7 and MDA-MB-231 cells. For Western blot, cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic free media. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. For western blot analysis, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane). Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For luciferase assay, cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates. After this cells were transfected with 32 ng of PPRE X3-TK-luc and 3.2 ng of Renilla luciferase plasmid per well and then cotransfected with scrambled or PPARγ siRNAs using 0.8 μL of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μL of control media. Afterwards, cells were lysed with 75 μL of passive lysis buffer and treated according to manufacturer’s instructions using dual-glo luciferase assay system. Results were calculated as raw luciferase units divided by raw Renilla units. Vertical bars indicate PPRE mediated reporter activity ± SEM (arbitrary unit). *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


### 3.8. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on PPARγ Expression and PPRE Mediated Reporter Activity in PPARγ siRNA Transfected PPARγ Positive MCF-7 and MDA-MB-231 Human Breast Cancer Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), rosiglitazone (3.2 μM), or GW9662 (3.2 μM) alone or in combination had no significant effect on PPARγ levels in vehicle-treated or scrambled RNA transfected MCF-7 (Figure 8(a)) and MDA-MB-231 (Figure 8(b)) breast cancer cells. Treatment with these agents was also found to have no significant effect on downregulated PPARγ levels in PPARγ siRNA transfected MCF-7 (Figure 8(a)) and MDA-MB-231 (Figure 8(b)) breast cancer cells. Similarly, treatment with 2 μM γ-tocotrienol, 3.2 μM rosiglitazone, or 3.2 μM GW9662 alone or in combination had little or no effect on PPRE mediated activity in vehicle-treated or scrambled RNA transfected PPARγ positive MCF-7 (Figure 8(c)) and MDA-MB-231 (Figure 8(d)) breast cancer cells and no significant effect on downregulated PPARγ levels in PPARγ siRNA transfected MCF-7 (Figure 8(c)) and MDA-MB-231 (Figure 8(d)) breast cancer cells.Western blot analysis and luciferase assay to determine effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PPARγ expression and activity in PPARγ siRNA transfected MCF-7 and MDA-MB-231 cells. For Western blot analysis, (a) MCF-7 and (b) MDA-MB-231 cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media and allowed to adhere overnight. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Afterwards whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For luciferase assay, (c) MCF-7 and (d) MDA-MB-231 cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. After this cells were transfected with 32 ng of PPRE X3-TK-luc and 3.2 ng of Renilla luciferase plasmid per well and then cotransfected with scrambled or PPARγ siRNAs using 0.8 μL of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μL of control or treatment media. Afterwards, cells were lysed with 75 μL of passive lysis buffer and treated according to manufacturer’s instructions using dual-glo luciferase assay system. Results were calculated as raw luciferase units divided by raw Renilla units. Vertical bars indicate PPRE mediated reporter activity ± SEM (arbitrary unit) in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)
(c)
(d)


### 3.9. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on COX-2 and PGDS in PPARγ siRNA Transfected PPARγ Positive MCF-7 and MDA-MB-231 Human Breast Cancer Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), the PPARγ agonist, rosiglitazone (3.2 μM), alone had no significant effect on COX-2 and PGDS levels in vehicle-treated and scrambled RNA transfected MCF-7 (Figure 9(a)) and MDA-MB-231 (Figure 9(b)) breast cancer cells. However, combined treatment with these agents resulted in a slight but in significant increase in COX-2 and PGDS levels in PPARγ siRNA transfected MCF-7 (Figure 9(a)) and MDA-MB-231 (Figure 9(b)) breast cancer cells. Treatment with subeffective doses of γ-tocotrienol (2 μM), the PPARγ antagonist, GW9662 (3.2 μM), alone had no significant effect on COX-2 and PGDS levels in vehicle-treated and scrambled RNA transfected MCF-7 (Figure 9(c)) and MDA-MB-231 (Figure 9(d)) breast cancer cells. However, combined treatment with these agents resulted in a significant decrease in COX-2 and PGDS levels in PPARγ siRNA transfected MCF-7 (Figure 9(c)) and MDA-MB-231 (Figure 9(d)) breast cancer cells.Western blot was performed to determine effect of treatment ofγ-tocotrienol and rosiglitazone alone or in combination in PPARγ siRNA transfected (a) MCF-7 and (b) MDA-MB-231 cells. In addition, Western blot was performed to determine effect of treatment of γ-tocotrienol and GW9662 alone or in combination in PPARγ siRNA transfected (c) MCF-7 and (d) MDA-MB-231 cells. Cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to control or treatment media for a 4-day culture period. Afterwards whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)
(c)
(d)


### 3.10. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on PGD2 Synthesis and Effects of 15d-PGJ2 Treatment on the Growth of PPARγ siRNA Transfected PPARγ Positive MCF-7 and MDA-MB-231 Breast Cancer Cells

Treatment withγ-tocotrienol (2 μM), the PPARγ agonist, rosiglitazone (3.2 μM), and the PPARγ antagonist, GW9662 (3.2 μM), alone had no significant effect on PGD2 synthesis in PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells compared to vehicle-treated or scrambled RNA transfected cells. However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a significant increase in PGD2 synthesis in PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells compared to vehicle-treated or scrambled RNA transfected cells. On the other hand, combined treatment with similar doses of γ-tocotrienol and GW9662 resulted in a significant decrease in PGD2 synthesis in PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells compared to vehicle-treated or scrambled RNA transfected cells (Figure 10(a)). Treatment with 10 μM or 50 μM 15d-PGJ2 had no effect on the growth of vehicle-treated, scrambled RNA transfected or PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells after a 4-day treatment period (Figure 10(b)).(a) Effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PGD2 synthesis and (b) treatment effect of 15d-PGJ2 in PPARγ positive MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to control or treatment media for a 4-day culture period. Afterwards whole cell lysates were assayed for PGD2 according to the manufacturer’s protocol. Vertical bars indicate the amount of PGD2 synthesized (pg/mL) ± SEM in each treatment group. For 15d-PGJ2 effect, MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. Transfections were performed using 0.25 μL lipofectamine 2000. For each well of cells to be transfected, 5 pmol of the scrambled or PPARγ siRNAs was diluted with 100 μL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% fetal bovine serum and cells were cultured for 18 h. Cells were then exposed to 100 μL of control or treatment media for a 4-day culture period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


### 3.11. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on Cell Cycle Progression in PPARγ Negative +SA Mammary Tumor Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), rosiglitazone (3.2 μM), or GW9662 (3.2 μM) alone had no effect on cyclin D1, CDK4, and CDK6 levels as compared to PPARγ negative +SA cells in the vehicle-treated control group (Figure 11(a)). However, combined treatment with similar doses of γ-tocotrienol and the PPARγ agonist, rosiglitazone, resulted in a slight but insignificant increase in the levels of cyclin D1, CDK4, and CDK6 24 h after exposure to EGF (Figure 11(a)). In contrast, combined treatment with γ-tocotrienol and the PPARγ antagonist, GW9662, resulted in a significant decrease in cyclin D1, CDK4, and CDK6 in PPARγ negative +SA cells (Figure 11(a)). Other studies demonstrated that treatment with 2 μM γ-tocotrienol, 3.2 μM rosiglitazone, or GW9662 alone had no effect on the CKI protein, p16, or phosphorylated (inactive) retinoblastoma (Rb) levels (Figure 11(b)). However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a slight decrease in p16, and a slight but insignificant increase in phosphorylated Rb levels in PPARγ negative +SA cells (Figure 11(b)). In contrast, combined treatment of 2 μM γ-tocotrienol and 3.2 μM of the PPARγ antagonist, GW9662, caused a slight but insignificant increase in p16 and corresponding significant decrease in phosphorylated-Rb levels in PPARγ negative +SA cells as compared to cells in the vehicle-treated control group (Figure 11(b)).Western blot analysis of effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on levels of (a) cyclin and CDKs and (b) cyclin kinase inhibitors and phosphorylated retinoblastoma in +SA cells. +SA cells were plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


### 3.12. Apoptotic Effects ofγ-Tocotrienol, GW9662, and T0070907 Treatment Alone or in Combination on +SA Mammary Tumor Cells

In order to determine if the growth inhibitory effects resulting from combined treatment with subeffective doses ofγ-tocotrienol and PPARγ antagonists might result from a reduction in viable cell number, studies were conducted to determine the acute effects (24 h) and chronic effects (96 h) of these treatment on the initiation of apoptosis and cell viability. Western blot analysis shows that treatment with 2 μM γ-tocotrienol or 3.2 μM GW9662 or 3.2 μM T0070907 alone or in combination had no effect on the expression of cleaved PARP, cleaved caspase-3, or viable cell number after a 24 h and 96 h treatment exposure (Figures 12(a) and 12(b)). However, treatment with 20 μM γ-tocotrienol, a dose previously shown to induce apoptosis in mammary cancer cells [2] and used as a positive control in this study, was found to induce a large increase in cleaved PARP and cleaved caspase-3 levels and decrease viable cell number in +SA cells (Figures 12(a) and 12(b)).Apoptotic effects ofγ-tocotrienol and PPARγ antagonists alone or in combination on (a) cleaved caspase-3, cleaved PARP levels and (b) viable cell number on +SA cells. For Western blot studies, +SA cells were initially plated at 1
×
106 cells/100 mm culture dish and maintained on control media for a 3-day culture period. Afterwards, cells were divided into the various treatment groups, media were removed, and cells were exposed to their respective treatment media for a 24 h treatment period. In addition, cells were exposed to their respective treatment media for a 96 h treatment period, where fresh media were added every other day. +SA cells were exposed to treatment media containing 2 μM γ-tocotrienol and 3.2 μM GW9662 or T0070907 alone or in combination. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by western blot analysis. In parallel studies, (b) +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well culture plates and exposed to the same treatments as described above. After a 24 h treatment exposure, viable cell number in all treatment groups was determined using MTT assay. Vertical barsindicate the mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


## 3.1. Antiproliferative Effects ofγ-Tocotrienol, PPARγ Agonists (Rosiglitazone and Troglitazone), and PPARγ Antagonists (GW9662 and T0070907) on the Highly Malignant Mouse +SA Mammary Tumor Cells

Treatment with 3-4μM γ-tocotrienol was found to significantly inhibit growth of +SA cells in a dose-responsive manner as compared to cells in the vehicle-treated control group. Treatment with 0–20 μM PPARγ agonists, rosiglitazone and troglitazone, or 0–20 μM of PPARγ antagonists, GW9662 and T0070907, inhibited growth of +SA cells in a dose-dependent manner compared to vehicle-treated control cells (Figure 1).Treatment effects ofγ-tocotrienol, PPARγ agonists, and PPARγ antagonists on +SA cells. +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well culture plates and exposed to treatment media for a 4-day period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)
(c)
(d)
(e)


## 3.2. Effects of Combined Treatment ofγ-Tocotrienol with PPARγ Agonists (Rosiglitazone and Troglitazone) or PPARγ Antagonists (GW9662 and T0070907) on +SA Mammary Tumor Cell Growth

Treatment with 1–4μM γ-tocotrienol alone significantly inhibited growth of +SA cells after a 4-day treatment period. However, the growth inhibitory effects of γ-tocotrienol on +SA cells were reversed when given in combination with 3.2 μM of PPARγ agonist rosiglitazone or troglitazone (Figure 2(a)). Conversely, the growth inhibitory effects of 0.5–2 μM γ-tocotrienol were significantly enhanced when given in combination with 3.2 μM of PPARγ antagonist GW9662 or T0070907 (Figure 2(b)).Treatment effects ofγ-tocotrienol, (a) PPARγ agonists and (b) PPARγ antagonists in combination in +SA cells. +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well plates and exposed to treatment media for a 4-day period. Afterwards, viable cell number was determined using MTT colorimetric assay. Vertical bars indicate the mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls and #
P
<
0.05 as compared to their corresponding control treated with γ-tocotrienol alone.
(a)
(b)


## 3.3. Isobologram Analysis of Combined Treatment Effects ofγ-Tocotrienol with PPARγ Agonists (Rosiglitazone and Troglitazone) and Antagonists (GW9662 and T0070907) on +SA Mammary Tumor Cells

The combined treatment ofγ-tocotrienol with the PPARγ agonist rosiglitazone or troglitazone was found to be statistically antagonistic, as evidenced by the location of the data point in the isobologram being well above the line defining additive effect (Figure 3(a)). In contrast, the growth inhibitory effect of combined treatment of γ-tocotrienol with PPARγ antagonists, GW9662 and T0070907, was found to be statistically synergistic, as evidenced by the location of the data point in the isobologram being well below the line defining additive effect (Figure 3(b)).Isobologram analysis of treatment ofγ-tocotrienol with PPARγ agonists and antagonists in +SA cells. Individual IC50 doses for γ-tocotrienol, (a) PPARγ agonists (rosiglitazone and troglitazone) and (b) PPARγ antagonists (GW9662 and T0070907) were calculated and then plotted on the x-axes and y-axes, respectively. The data point on the isobologram represents the actual doses of combined γ-tocotrienol and PPARγ agonists and antagonists. Combined treatment of γ-tocotrienol with PPARγ agonists rosiglitazone and troglitazone was found to be antagonistic, as evidenced by the location of the data point in the isobologram being well above the line defining additive effect. In contrast, the growth inhibitory effect of combined treatment of γ-tocotrienol with PPARγ antagonists GW9662 and T0070907 was found to be synergistic, as evidenced by the location of the data point in the isobologram being well below the line defining additive effect for both cell lines.
(a)
(b)


## 3.4. Effects ofγ-Tocotrienol, Rosiglitazone, or GW9662 Treatment Alone or in Combination on PPARγ Protein Expression in Mouse (+SA) and Human (MCF-7 and MDA-MB-231) Mammary Tumor Cells

Western blot analysis showed that PPARγ levels were abundant in untreated control human MCF-7 and MDA-MB-231 breast cancer cells, whereas PPARγ levels were undetectable in untreated control +SA cells following a 4-day culture period (Figure 4(a)). Additional studies showed that treatment with γ-tocotrienol, rosiglitazone, or GW9662 alone or in combination had no effect of PPARγ expression in +SA mouse mammary tumor cells as compared to the vehicle-treated control group (Figure 4(b)). Since +SA cells were found to be PPARγ negative, treatment with PPARγ agonists and antagonists was found to induce significant growth effects on these cells when given alone or in combination with γ-tocotrienol; these findings suggested that treatment effects may be mediated through PPARγ-independent mechanisms. Additional studies were conducted to investigate this hypothesis.Western blot analysis to determine expression of PPARγ in mammary cancer cells. (a) PPARγ levels were determined in untreated +SA, MCF-7, and MDA-MB-231 cells and (b) PPARγ levels were determined after treatment with γ-tocotrienol, rosiglitazone, and GW9662 alone or in combination in +SA cells. +SA, MCF-7, and MDA-MB-231 cells were initially plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit).
(a)
(b)


## 3.5. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on COX-2 and PGDS Protein Expression and mRNA Levels in PPARγ Negative +SA Mammary Tumor Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), rosiglitazone (3.2 μM), or GW9662 (3.2 μM) alone had little or no effect on the expression of COX-2 and PGDS as compared to the vehicle-treated control group in +SA cells (Figure 5(a)). However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a significant increase in COX-2 and PGDS levels as compared to vehicle-treated +SA cells. In contrast, combined treatment with 2 μM γ-tocotrienol and 3.2 μM GW9662 resulted in a significant decrease in the expression of COX-2 and PGDS as compared to the vehicle-treated control group in +SA cells (Figure 5(a)). Additional qRT-PCR experiments showed that similar treatments with subeffective doses of γ-tocotrienol, rosiglitazone, or GW9662 showed no significant effect on COX-2 and PGDS mRNA levels as compared to the vehicle-treated control group in +SA cells (Figure 5(b)). However, combined treatment with similar doses of γ-tocotrienol and the PPARγ agonist, rosiglitazone, resulted in a slight increase in COX-2 and PGDS mRNA levels but these differences were not found to be significant (Figure 5(b)). Furthermore, combined treatment with 2 μM γ-tocotrienol and 3.2 μM GW9662 results in a significant decrease in COX-2 and PGDS mRNA levels as compared to the vehicle-treated control group in +SA cells (Figure 5(b)). These results demonstrate that combined treatment of γ-tocotrienol with PPARγ agonists or antagonists caused significant changes in prostaglandin signaling in PPARγ negative +SA cells, indicating that the actions of these agents are mediated through a PPARγ-independent mechanism.(a) Western blot and (b) qRT-PCR analysis to determine effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on levels of COX-2 and PGDS in +SA cells. For Western blot analysis, +SA cells were plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For qRT-PCR analysis, +SA cells were plated at a density of 2
×
103 cells/well (3 replicates per group) in 6-well plates and treated with control or treatment media for a 4-day culture period. Total RNA was extracted and first-strand cDNA was generated from total RNA for each sample according to the manufacturer’s instructions. COX-2, PGDS, and GAPDH were measured using Taqman technology. Changes in mRNA levels of COX-2 and PGDS were normalized to mRNA level of GAPDH and represented as bar graph. Vertical bars indicate the normalized Ct value ± SEM (Arbitrary Unit) in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


## 3.6. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on PGD2 Synthesis and the Effect of 15d-PGJ2 Treatment on the Growth of PPARγ Negative +SA Mammary Tumor Cells

Treatment withγ-tocotrienol (2 μM), the PPARγ agonist, rosiglitazone (3.2 μM), and the PPARγ antagonist, GW9662 (3.2 μM) alone had no significant effect on PGD2 synthesis in +SA breast cancer cells compared to vehicle-treated control cells. However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a slight increase in PGD2 synthesis in +SA cells compared to vehicle-treated control cells. On the other hand, combined treatment with similar doses of γ-tocotrienol and GW9662 resulted in a significant decrease in PGD2 synthesis in +SA cells compared to vehicle-treated control cells (Figure 6(a)). Previous studies have shown that 15d-PGJ2 induces antiproliferative effects between 10 and 50 μM doses in cancer cells [33]. However, treatment with 10 μM or 50 μM 15d-PGJ2 had no effect on +SA mammary tumor cell growth following a 4-day culture period (Figure 6(b)). These results demonstrate that antiproliferative effects of combined treatment of γ-tocotrienol with PPARγ antagonists are due to decrease in prostaglandin synthesis, mediated through a PPARγ-independent mechanism. This finding was further confirmed by performing similar experiments in PPARγ silenced PPARγ positive MCF-7 and MDA-MB-231 breast cancer cells.(a) Effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PGD2 synthesis and (b) treatment effect of 15d-PGJ2 in +SA cells. +SA cells were plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were assayed for PGD2 according to the manufacturer’s protocol. Vertical bars indicate the amount of PGD2 synthesized (pg/mL) ± SEM in each treatment group. To study effect of 15d-PGJ2, +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well culture plates and exposed to treatment media for a 4-day period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


## 3.7. Effects of PPARγ siRNA Transfection on PPARγ Expression and PPRE Mediated Reporter Activity in PPARγ Positive Human MCF-7 and MDA-MB-231 Breast Cancer Cells

Western blot analysis shows that PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells displayed decreased expression in PPARγ levels as compared to vehicle-treated or scrambled RNA transfected MCF-7 and MDA-MB-231 breast cancer cells (Figure 7(a)). Luciferase assay shows that PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells significantly decreased PPRE-mediated reporter activity as compared to vehicle-treated or scrambled RNA transfected MCF-7 and MDA-MB-231 breast cancer cells (Figure 7(b)). These results confirm that PPARγ siRNA successfully downregulated expression and activation of PPARγ in both MCF-7 and MDA-MB-231 breast cancer cells.(a) Western blot analysis and (b) luciferase assay to determine expression and activity of PPARγ in PPARγ siRNA transfected MCF-7 and MDA-MB-231 cells. For Western blot, cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic free media. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. For western blot analysis, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane). Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For luciferase assay, cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates. After this cells were transfected with 32 ng of PPRE X3-TK-luc and 3.2 ng of Renilla luciferase plasmid per well and then cotransfected with scrambled or PPARγ siRNAs using 0.8 μL of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μL of control media. Afterwards, cells were lysed with 75 μL of passive lysis buffer and treated according to manufacturer’s instructions using dual-glo luciferase assay system. Results were calculated as raw luciferase units divided by raw Renilla units. Vertical bars indicate PPRE mediated reporter activity ± SEM (arbitrary unit). *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


## 3.8. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on PPARγ Expression and PPRE Mediated Reporter Activity in PPARγ siRNA Transfected PPARγ Positive MCF-7 and MDA-MB-231 Human Breast Cancer Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), rosiglitazone (3.2 μM), or GW9662 (3.2 μM) alone or in combination had no significant effect on PPARγ levels in vehicle-treated or scrambled RNA transfected MCF-7 (Figure 8(a)) and MDA-MB-231 (Figure 8(b)) breast cancer cells. Treatment with these agents was also found to have no significant effect on downregulated PPARγ levels in PPARγ siRNA transfected MCF-7 (Figure 8(a)) and MDA-MB-231 (Figure 8(b)) breast cancer cells. Similarly, treatment with 2 μM γ-tocotrienol, 3.2 μM rosiglitazone, or 3.2 μM GW9662 alone or in combination had little or no effect on PPRE mediated activity in vehicle-treated or scrambled RNA transfected PPARγ positive MCF-7 (Figure 8(c)) and MDA-MB-231 (Figure 8(d)) breast cancer cells and no significant effect on downregulated PPARγ levels in PPARγ siRNA transfected MCF-7 (Figure 8(c)) and MDA-MB-231 (Figure 8(d)) breast cancer cells.Western blot analysis and luciferase assay to determine effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PPARγ expression and activity in PPARγ siRNA transfected MCF-7 and MDA-MB-231 cells. For Western blot analysis, (a) MCF-7 and (b) MDA-MB-231 cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media and allowed to adhere overnight. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Afterwards whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For luciferase assay, (c) MCF-7 and (d) MDA-MB-231 cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. After this cells were transfected with 32 ng of PPRE X3-TK-luc and 3.2 ng of Renilla luciferase plasmid per well and then cotransfected with scrambled or PPARγ siRNAs using 0.8 μL of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μL of control or treatment media. Afterwards, cells were lysed with 75 μL of passive lysis buffer and treated according to manufacturer’s instructions using dual-glo luciferase assay system. Results were calculated as raw luciferase units divided by raw Renilla units. Vertical bars indicate PPRE mediated reporter activity ± SEM (arbitrary unit) in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)
(c)
(d)


## 3.9. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on COX-2 and PGDS in PPARγ siRNA Transfected PPARγ Positive MCF-7 and MDA-MB-231 Human Breast Cancer Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), the PPARγ agonist, rosiglitazone (3.2 μM), alone had no significant effect on COX-2 and PGDS levels in vehicle-treated and scrambled RNA transfected MCF-7 (Figure 9(a)) and MDA-MB-231 (Figure 9(b)) breast cancer cells. However, combined treatment with these agents resulted in a slight but in significant increase in COX-2 and PGDS levels in PPARγ siRNA transfected MCF-7 (Figure 9(a)) and MDA-MB-231 (Figure 9(b)) breast cancer cells. Treatment with subeffective doses of γ-tocotrienol (2 μM), the PPARγ antagonist, GW9662 (3.2 μM), alone had no significant effect on COX-2 and PGDS levels in vehicle-treated and scrambled RNA transfected MCF-7 (Figure 9(c)) and MDA-MB-231 (Figure 9(d)) breast cancer cells. However, combined treatment with these agents resulted in a significant decrease in COX-2 and PGDS levels in PPARγ siRNA transfected MCF-7 (Figure 9(c)) and MDA-MB-231 (Figure 9(d)) breast cancer cells.Western blot was performed to determine effect of treatment ofγ-tocotrienol and rosiglitazone alone or in combination in PPARγ siRNA transfected (a) MCF-7 and (b) MDA-MB-231 cells. In addition, Western blot was performed to determine effect of treatment of γ-tocotrienol and GW9662 alone or in combination in PPARγ siRNA transfected (c) MCF-7 and (d) MDA-MB-231 cells. Cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to control or treatment media for a 4-day culture period. Afterwards whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)
(c)
(d)


## 3.10. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on PGD2 Synthesis and Effects of 15d-PGJ2 Treatment on the Growth of PPARγ siRNA Transfected PPARγ Positive MCF-7 and MDA-MB-231 Breast Cancer Cells

Treatment withγ-tocotrienol (2 μM), the PPARγ agonist, rosiglitazone (3.2 μM), and the PPARγ antagonist, GW9662 (3.2 μM), alone had no significant effect on PGD2 synthesis in PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells compared to vehicle-treated or scrambled RNA transfected cells. However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a significant increase in PGD2 synthesis in PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells compared to vehicle-treated or scrambled RNA transfected cells. On the other hand, combined treatment with similar doses of γ-tocotrienol and GW9662 resulted in a significant decrease in PGD2 synthesis in PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells compared to vehicle-treated or scrambled RNA transfected cells (Figure 10(a)). Treatment with 10 μM or 50 μM 15d-PGJ2 had no effect on the growth of vehicle-treated, scrambled RNA transfected or PPARγ siRNA transfected MCF-7 and MDA-MB-231 breast cancer cells after a 4-day treatment period (Figure 10(b)).(a) Effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PGD2 synthesis and (b) treatment effect of 15d-PGJ2 in PPARγ positive MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
105 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to control or treatment media for a 4-day culture period. Afterwards whole cell lysates were assayed for PGD2 according to the manufacturer’s protocol. Vertical bars indicate the amount of PGD2 synthesized (pg/mL) ± SEM in each treatment group. For 15d-PGJ2 effect, MCF-7 and MDA-MB-231 cells were plated at a density of 2
×
104 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. Transfections were performed using 0.25 μL lipofectamine 2000. For each well of cells to be transfected, 5 pmol of the scrambled or PPARγ siRNAs was diluted with 100 μL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% fetal bovine serum and cells were cultured for 18 h. Cells were then exposed to 100 μL of control or treatment media for a 4-day culture period. Afterwards viable cell number was determined using MTT colorimetric assay. Vertical bars indicate mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


## 3.11. Effects ofγ-Tocotrienol, Rosiglitazone, and GW9662 Treatment Alone or in Combination on Cell Cycle Progression in PPARγ Negative +SA Mammary Tumor Cells

Western blot analysis shows that treatment with subeffective doses ofγ-tocotrienol (2 μM), rosiglitazone (3.2 μM), or GW9662 (3.2 μM) alone had no effect on cyclin D1, CDK4, and CDK6 levels as compared to PPARγ negative +SA cells in the vehicle-treated control group (Figure 11(a)). However, combined treatment with similar doses of γ-tocotrienol and the PPARγ agonist, rosiglitazone, resulted in a slight but insignificant increase in the levels of cyclin D1, CDK4, and CDK6 24 h after exposure to EGF (Figure 11(a)). In contrast, combined treatment with γ-tocotrienol and the PPARγ antagonist, GW9662, resulted in a significant decrease in cyclin D1, CDK4, and CDK6 in PPARγ negative +SA cells (Figure 11(a)). Other studies demonstrated that treatment with 2 μM γ-tocotrienol, 3.2 μM rosiglitazone, or GW9662 alone had no effect on the CKI protein, p16, or phosphorylated (inactive) retinoblastoma (Rb) levels (Figure 11(b)). However, combined treatment with similar doses of γ-tocotrienol and rosiglitazone resulted in a slight decrease in p16, and a slight but insignificant increase in phosphorylated Rb levels in PPARγ negative +SA cells (Figure 11(b)). In contrast, combined treatment of 2 μM γ-tocotrienol and 3.2 μM of the PPARγ antagonist, GW9662, caused a slight but insignificant increase in p16 and corresponding significant decrease in phosphorylated-Rb levels in PPARγ negative +SA cells as compared to cells in the vehicle-treated control group (Figure 11(b)).Western blot analysis of effect ofγ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on levels of (a) cyclin and CDKs and (b) cyclin kinase inhibitors and phosphorylated retinoblastoma in +SA cells. +SA cells were plated at 1
×
106 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


## 3.12. Apoptotic Effects ofγ-Tocotrienol, GW9662, and T0070907 Treatment Alone or in Combination on +SA Mammary Tumor Cells

In order to determine if the growth inhibitory effects resulting from combined treatment with subeffective doses ofγ-tocotrienol and PPARγ antagonists might result from a reduction in viable cell number, studies were conducted to determine the acute effects (24 h) and chronic effects (96 h) of these treatment on the initiation of apoptosis and cell viability. Western blot analysis shows that treatment with 2 μM γ-tocotrienol or 3.2 μM GW9662 or 3.2 μM T0070907 alone or in combination had no effect on the expression of cleaved PARP, cleaved caspase-3, or viable cell number after a 24 h and 96 h treatment exposure (Figures 12(a) and 12(b)). However, treatment with 20 μM γ-tocotrienol, a dose previously shown to induce apoptosis in mammary cancer cells [2] and used as a positive control in this study, was found to induce a large increase in cleaved PARP and cleaved caspase-3 levels and decrease viable cell number in +SA cells (Figures 12(a) and 12(b)).Apoptotic effects ofγ-tocotrienol and PPARγ antagonists alone or in combination on (a) cleaved caspase-3, cleaved PARP levels and (b) viable cell number on +SA cells. For Western blot studies, +SA cells were initially plated at 1
×
106 cells/100 mm culture dish and maintained on control media for a 3-day culture period. Afterwards, cells were divided into the various treatment groups, media were removed, and cells were exposed to their respective treatment media for a 24 h treatment period. In addition, cells were exposed to their respective treatment media for a 96 h treatment period, where fresh media were added every other day. +SA cells were exposed to treatment media containing 2 μM γ-tocotrienol and 3.2 μM GW9662 or T0070907 alone or in combination. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by western blot analysis. In parallel studies, (b) +SA cells were plated at a density of 5
×
104 (6 wells per group) in 24-well culture plates and exposed to the same treatments as described above. After a 24 h treatment exposure, viable cell number in all treatment groups was determined using MTT assay. Vertical barsindicate the mean cell count ± SEM in each treatment group. *P
<
0.05 as compared with vehicle-treated controls.
(a)
(b)


## 4. Discussion

Results in these studies demonstrate that, when given alone, treatment withγ-tocotrienol, PPARγ agonists (rosiglitazone and troglitazone), or PPARγ antagonists (GW9662 and T0070907) induced a significant dose-responsive inhibition in the viability of +SA mammary tumor cells in culture. However, when used in combination, treatment with low doses of PPARγ agonists was found to reverse, whereas treatment with low doses of PPARγ antagonists synergistically enhanced the antiproliferative effects of γ-tocotrienol. Additional studies determined that the synergistic inhibition of +SA tumor cell growth resulting from combined low dose treatment of γ-tocotrienol with PPARγ antagonists was associated with a reduction in COX-2, PGDS, PGD2 synthesis, and a corresponding inhibition of cell cycle progression. Conversely, enhancement in +SA tumor cell growth resulting from combined low dose treatment of γ-tocotrienol with PPARγ agonists was associated with an increase in COX-2, PGDS, and PGD2 synthesis. Western blot data showed that +SA cells did not contain detectable levels of PPARγ when compared with MCF-7 and MDA-MB-231 human breast cancer cells suggesting that the antiproliferative effects of combined treatment of γ-tocotrienol with PPARγ antagonist may be mediated through PPARγ-independent mechanisms. This hypothesis was confirmed by treating PPARγ siRNA transfected PPARγ positive MCF-7 and MDA-MB-231 human breast cancer cells with similar doses of γ-tocotrienol, PPARγ agonists, and antagonists alone and in combination. Combined treatment of γ-tocotrienol with PPARγ agonists increased COX-2, PGDS, and PGD2synthesis, while combined treatment of γ-tocotrienol with PPARγ antagonists decreased COX-2, PGDS, and PGD2synthesis in PPARγ siRNA transfected PPARγ positive MCF-7 and MDA-MB-231 breast cancer cells. Interestingly, 15d-PGJ2 did not affect the viability of PPARγ negative +SA or PPARγ positive PPARγ silenced (siRNA transfected) MCF-7 and MDA-MB-231 breast cancer cells. Taken together, these findings demonstrate that combined treatment of γ-tocotrienol with PPARγ antagonists displays synergistic anticancer activity by PPARγ-independent mechanisms primarily by decreasing expression of COX-2 and prostaglandin synthesis in breast cancer cells.Numerous investigations have established thatγ-tocotrienol acts as a potent anticancer agent that inhibits the growth of mouse [5, 34] and human [35, 36] breast cancer cells. Furthermore, studies have shown that combined treatment of γ-tocotrienol with other traditional chemotherapies results in synergistic or additive inhibition in cancer cell growth [34]. PPARγ is often found to be overexpressed in breast cancer cells [24, 37–39]. However, the exact role of PPARγ in breast cancer cell proliferation and survival is not clearly understood. Previously, it has been shown that combined treatment of γ-tocotrienol with PPARγ antagonists inhibits growth of human breast cancer cells by decreasing the expression and activity of PPARγ [9]. In addition, γ-tocotrienol inhibits growth of human prostate cancer cells through a partially PPARγ-dependent pathway and downregulation of TGFβ2 receptor [8]. Furthermore, studies have shown that treatment with PPARγ agonists, rosiglitazone and troglitazone, or conversely with PPARγ antagonists, GW9662 and T0070907, were both found to significantly inhibit the growth of a wide variety of cancer cell lines [40, 41]. An explanation for these conflicting findings is not clearly evident. Results in the present study show that treatment with high doses of PPARγ agonists or antagonists alone decreased viability of PPARγ negative +SA mammary tumor cells. However, when combined with γ-tocotrienol, PPARγ agonists increased, while PPARγ antagonists decreased +SA cell viability. These results confirm and extend previous findings observed in PPARγ positive MCF-7 and MDA-MB-231 breast cancer cells [9]. Western blot analysis shows that PPARγ is undetectable in untreated +SA cells as compared to untreated MCF-7 and MDA-MB-231 cells. In addition, PPARγ was not detected after treatment with γ-tocotrienol, PPARγ agonist, and antagonist when used alone or in combination at the end of 4-day incubation period in +SA cells. These results show that +SA cells are PPARγ negative, and the anticancer effects induced by combination of γ-tocotrienol with PPARγ antagonists, therefore, must be mediated through PPARγ-independent mechanisms. However, it is also possible that these PPARγ-independent effects may be mediated through other PPAR isoforms such as PPARα and PPARβ/δ. Previous studies have shown that PPARγ agonists and antagonists can modulate PPARα and PPARβ/δ receptor activation and signaling [42]. Additional studies are required to determine if this hypothesis is correct.COX-2 expression and activity are elevated in a wide range of cancer cell types and are associated with enhanced resistance to apoptosis, metastatic phenotype appearance and behavior, and angiogenesis [43–46]. Previous studies showed that the antiproliferative effects of γ-tocotrienol in combination with celecoxib, a COX-2 inhibitor, was associated with decreased expression of COX-2 and PGE2 synthesis [22, 23]. Furthermore, COX-2 inhibition has been associated with decreased PGDS expression and PGD2 synthesis [47]. Results in the present study show that combined treatment of γ-tocotrienol with PPARγ antagonist decreased the protein and mRNA levels of COX-2 and PGDS, as well as decreased PGD2 synthesis in +SA mammary tumor cells. In contrast, combined treatment of γ-tocotrienol with PPARγ agonist produced the opposite effects in PPARγ negative +SA breast cancer cells. Cyclin D1 is commonly overexpressed in a variety of cancers and is associated with enhanced tumor progression and metastasis, and loss of cyclin D1 can cause G0-G1 arrest [48]. Studies have shown that γ-tocotrienol significantly reduced cyclin D1, cyclin-dependent kinases CDK4 and CDK6 levels between 4 and 24 h after EGF exposure in mammary cancer cells [49]. In addition, previous studies have shown that PPARγ ligands show PPARγ-independent effects by inducing cell cycle arrest in cancer cells [50]. Results shown in the present study are consistent with these previous findings demonstrating that increased PGD2 synthesis is associated with increased cancer cell proliferation [51], whereas a decrease in PGD2 synthesis is associated with inhibition in cancer growth and G0-G1 cell cycle arrest [52].Many of the anticancer effects of PPARγ and PPARγ agonists are mediated indirectly from the actions of 15d-PGJ2, a biologically active cyclopentenone that induces cell cycle arrest and apoptosis in cancer cells [53]. Studies show that 15d-PGJ2 is formed through the action of COX-2 on arachidonic acid with the help of cell-specific synthases. One such synthase is PGDS, which catalyzes the isomerization of PGH2 to PGD2, which subsequently undergoes spontaneous dehydration to form 15d-PGJ2 [12, 13]. Previous studies have shown that treatment with 15d-PGJ2 significantly inhibited cancer cell growth [33]. However, in the present study, treatment with high doses of 15d-PGJ2 had no effect on +SA growth or viability. These findings provide further evidence that the anticancer effects of combined treatment of γ-tocotrienol with PPARγ antagonist are mediated through PPARγ-independent mechanisms in +SA PPARγ negative mammary tumor cells. However, other studies showed that the antiproliferative effects of γ-tocotrienol in prostate cancer cells result from PPARγ-dependent mechanisms associated with increased synthesis of the PPARγ endogenous ligand, 15-HETE [8]. Differences between these results and those in the present study can be attributed to differences in the cancer cell types investigated.


## 5. Conclusion

Results from the present study demonstrate that the effects resulting from combined treatment ofγ-tocotrienol with PPARγ agonist or antagonist are mediated through PPARγ-independent mechanism(s). This suggestion is supported by the findings that the anticancer effects of combined treatment of γ-tocotrienol with PPARγ antagonists are observed in PPARγ-negative +SA mammary tumor cells, as well as PPARγ silenced (siRNA transfected) PPARγ positive MCF-7 and MDA-MB-231 breast cancer cells. Similarly, the anticancer effects of γ-tocotrienol treatment were reversed when combined with PPARγ agonists in these same PPARγ negative (+SA) or PPARγ silenced (siRNA transfected MCF-7 and MDA-MB-231) mammary tumor cells. Furthermore, the anticancer effects of combined γ-tocotrienol and PPARγ antagonist treatments were found to be associated with a corresponding reduction in COX-2 and PGDS expression and corresponding decrease in PGD2 synthesis. Conversely, the stimulatory effects of combined γ-tocotrienol and PPARγ agonist treatment in PPARγ negative or PPARγ silenced mammary tumor cells were associated with an increase in COX-2 and PGDS expression and corresponding increase in PGD2 synthesis. Finally, these findings suggest that combined treatment of γ-tocotrienol with PPARγ antagonists might provide some benefit in the treatment of breast cancer in women.



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