Cancer Epidemiology 38 (2014) 765–772

Contents lists available at ScienceDirect

Cancer Epidemiology The International Journal of Cancer Epidemiology, Detection, and Prevention journal homepage: www.cancerepidemiology.net

Resveratrol suppresses the proliferation of breast cancer cells by inhibiting fatty acid synthase signaling pathway Arif Khan a,*, Ahmad N. Aljarbou a, Yousef H. Aldebasi a, Syed M. Faisal b,1, Masood A. Khan a a b

College of Applied Medical Sciences, Qassim University, Buraidah, Al-Qassim, Saudi Arabia College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 January 2014 Received in revised form 2 September 2014 Accepted 13 September 2014 Available online 25 October 2014

In breast cancer cells, overexpression of human epidermal growth factor receptor 2 (HER2) increases the translation of fatty acid synthase (FASN) by altering the activity of PI3K/Akt signaling pathways. Cancer chemotherapy causes major side effects and is not effective enough in slowing down the progression of the disease. Earlier studies showed a role for resveratrol in the inhibition of FASN, but the molecular mechanisms of resveratrol-induced inhibition are not known. In the present study, we examined the novel mechanism of resveratrol on Her2-overexpressed breast cancer cells. The effect of resveratrol on the growth of breast cancer cells was assessed as percent cell viability by cytotoxicity-based MTT assay and the induction of apoptosis was determined by cell-death detection ELISA and flow cytometric analysis of Annexin-V–PI binding. Western immunobloting was used to detect signaling events in human breast cancer (SKBR-3) cells. Data showed that resveratrol-mediated down-regulation of FASN and HER2 genes synergistically induced apoptotic death in SKBR-3 cells. This concurrently caused a prominent up-regulation of PEA3, leads to down-regulation of HER2 genes. Resveratrol also alleviated the PI3K/Akt/mTOR signaling by down-regulation of Akt phosphorylation and up-regulation of PTEN expression. These findings suggest that resveratrol alters the cell cycle progression and induce cell death via FASN inhibition in HER2 positive breast cancer. ß 2014 Elsevier Ltd. All rights reserved.

Keywords: Akt FASN Her2 PI3K Resveratrol SKBR-3

1. Introduction In recent years, the potential of dietary constituents to restrain carcinogenesis has attracted the extensive attention in the chemoprevention of cancer [1–3]. The naturally occurring agent resveratrol (3,40 ,5-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other foods, has been shown to possess many biological activities relevant to human cancer prevention and treatment [4–6]. As substantiated from several studies that resveratrol, a promising anti-cancer therapeutic agent, affects all three discrete stages of carcinogenesis (initiation, promotion, and progression). It affects multiple signaling pathways that control cell division and growth, apoptosis, inflammation, angiogenesis, and metastasis [6–8]. The clinical efficiency and safety of

* Corresponding author at: Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraidah 51452, Saudi Arabia. Tel.: +966 63800050x4166. E-mail addresses: [email protected], [email protected] (A. Khan). 1 Current address: National Institute of Animal Biotechnology, Hyderabad, India. http://dx.doi.org/10.1016/j.canep.2014.09.006 1877-7821/ß 2014 Elsevier Ltd. All rights reserved.

resveratrol is also under investigation for the treatment of cancer in its early stages [9,10]. Many studies enlighten the anti-proliferative/apoptotic effects of resveratrol as the possible molecular targets. It has been seen to be associated in the induction of cell cycle arrest with the decreased level of cyclin D1 and up-regulation of both the tumor suppressor p53 and the cdk inhibitor p21 [11–13]. Resveratrol was also shown to inhibit other known key regulator of cell proliferation such as protein kinase C (PKC) [14]. The induction of cell death was shown to be associated with Bcl-2 phosphorylation [15], BAX mitochondrial translocation [16], inhibition of ribonucleotide reductase thereby interfering with DNA synthesis [17] and autophagocytosis in ovarian cancer cells [18]. Nevertheless, the effects of resveratrol in human breast cancer cells are more complicated because it has been shown to exert both proliferative [19] and anti-proliferative [20,21] effects depending on cell types and cell culture conditions. Therefore, the exact mechanism by which resveratrol acts in human breast cancer is not well understood. Recently, several studies have shown fatty acid synthase (FASN) as a potential therapeutic target for breast, prostrate,

766

A. Khan et al. / Cancer Epidemiology 38 (2014) 765–772

endometrium, ovary and colon carcinomas [22–24]. The biosynthetic enzyme FASN is the major enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids, and it functions normally in cells with high lipid metabolism. A number of environmental, hormonal, and nutritional signals tightly regulate FASN activity under normal physiological conditions [25]. However, as evident from human studies that infiltrating carcinomas of the breast constitutively express high levels of FASN compared to non-transformed human epithelial tissue [24]. Moreover, there is a potential link between increased risk of breast cancer development and higher levels of FASN [26]. Astonishingly, FASN overexpression and hyperactivity is associated with more persistent breast cancers [27,28]. It is well-known that increased FASN activity plays an active role in cancer evolution by regulating oncogenic proteins closely related to malignant transformation. Recent studies have shown that FASN-dependent signaling regulates the expression, activity, and cellular localization of HER2 (c-erbB-2) oncogene in breast cancer cells [29–31]. Amplification of the HER2 gene and/or over expression of its protein product has been found in up to 25–30% of human breast cancers [32]. Considerable interest has been developed in identifying novel inhibitors of this enzyme. The natural occurring active dietary constituents such as the green tea polyphenol epigallocatechin-3gallate (EGCG) and other flavonoids have been shown to induce anti-cancer effects by suppressing FASN activity and/or expression, which may account for the epidemiologically observed inverse correlation between green-tea drinking and cancer risk in oriental populations [33–35]. Several studies have shown the role of resveratrol as a potent inhibitor of FASN [36,37]. Thus, we sought to elucidate the ultimate molecular mechanism of resveratrol in human breast cancer system. The present study explored the correlation of resveratrol-induced cell growth inhibition with the status of FASN and Her2 expression in breast cancer cells. 2. Materials and methods 2.1. Materials Rapamycin and resveratrol were purchased from Santa Cruz Biotechnology Inc. (California, USA). Antibodies (Abs) against p185Her2/Neu, FASN, cyclin D1, b-actin, PEA3, rabbit anti mouse horseradish peroxidase or goat anti rabbit horseradish peroxidaseconjugated secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (California, USA). Akt, phospho-Akt (Ser473), p85-PI3K, PTEN antibodies were purchased from Cell Signaling Technology (Beverly, USA). The polyvinylidene fluoride (PVDF) membrane was obtained from Santa Cruz Biotechnology. The rest of the chemicals were of analytical grade of purity and were procured locally.

medium to obtain the desired final concentration used for the treatment of cells. The cells (80% confluent) were treated with varying concentrations of resveratrol for different time intervals in complete medium for dose- and time-dependent studies. In the controls, the cells were incubated with the vehicle (DMSO) alone. 2.4. MTT assay The cytotoxicity-based MTT assay was performed to select the resveratrol treatment dose according to published method [38]. Briefly, 1  104 cells (200 ml) in complete culture medium were plated 96-well microtitre plates, treated with a dose range from 0 to 150 mM of resveratrol and incubated at 37 8C in humidified incubator before assessing the cell proliferation. Following 24, 48 and 72 h of incubation with resveratrol, MTT was added and samples were read at 530 nm on a scanning multiwell spectrophotometer. The effect of resveratrol on growth inhibition was assessed as percent cell viability; control cells treated with the vehicle were considered 100% viable. 2.5. Measurement of apoptosis with enzyme linked immunosorbent assay (ELISA) based cell-death detection kit The cells were grown at a density of 1  106 cells in 100-mm culture dishes and then treated with various concentration of resveratrol (0, 20, 40 and 60 mM) for 24 h. Apoptotic cell death was determined with the cell-death detection ELISAplus (Roche Diagnostics, Heidelberg, Germany). 2.6. Flow-cytometric analysis of cell cycle The cells were grown at a density of 1  106 cells in 100-mm culture dishes and then treated with various concentration of resveratrol (0, 20, 40 and 60 mM) for 24 h. Cells were trypsinized, washed twice with Phosphate-buffered saline (PBS) and centrifuged at 200 g. The cell pellet was re-suspended in 50 ml PBS and fixed in 2 ml of 70% ice-cold ethanol. Cells were centrifuged and treated with 0.1% Triton X-100 for 5 min. After incubation, cells were centrifuged and re-suspended in 1 ml of PBS. Ribonuclease (100 mg/ml) was then added and the cells were incubated at 37 8C for 30 min. Cells were centrifuged at 200 g and resuspended in 1 ml of PBS containing 50 mg/ml propidium iodide (PI) and further incubated for 30 min at 4 8C. The cells were centrifuged, suspended in 500 ml of PBS and acquired on flow cytometer (Beckton Dickinson, San Jose, USA), followed data analysis through Cell Quest 3.3 software [39]. 2.7. Flow-cytometric analysis of Annexin-V–PI binding

SKBR-3 breast cancer was purchased from American Type Culture Collection (Manassas, USA) and grown in cell culture medium (McCoy’s 5A medium; Sigma–Aldrich, St. Louis, USA) containing 10% heat-inactivated fetal bovine serum (FBS), 1% Lglutamine, 1% sodium pyruvate, 50 U/ml penicillin and 50 mg/ml streptomycin. Cells were maintained at 37 8C in a humidified atmosphere of 95% air and 5% CO2. Cells were screened periodically for Mycoplasma contamination. Cells reaching to 70–80% confluency were subjected to different treatments.

Apoptosis was measured using flow cytometry to quantify the levels of detectable phosphatidylserine on the outer membranes of apoptotic cells [40]. Annexin-V FITC detection Kit (Beckton Dickinson, San Jose, USA) was used for the differentiation of apoptotic and necrotic cells following manufacturer’s protocol. Briefly, 5  105 cells were grown to about 60% confluence and treated with various concentrations of resveratrol (0, 20, 40 and 60 mM) for 24 h. Annexin-V/PI fluorescence was analyzed; for each sample, fluorescence of 10,000 cells was gated and counted using CellQuest 3.3 software. The percentages of cells in the upper-right (late apoptotic cells), upper-left (necrotic cells), lower-right (earlyapoptotic cells), and lower-left (viable cells) panels of the resulting histogram were calculated for comparison.

2.3. Treatment of cells

2.8. Western-blot analysis

The stock solution of resveratrol of 1 mM concentration was prepared in dimethyl sulphoxide (DMSO) and mixed with fresh

Western blotting was carried out as described previously [41]. Briefly, protein concentrations of cell lysates were measured

2.2. Cell culture

A. Khan et al. / Cancer Epidemiology 38 (2014) 765–772

using the BCA protein assay kit (Thermo Scientific, Rockford, USA) using BSA as a standard. Proteins (40 mg) were resolved on 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels and electroblotted on PVDF membranes. The blots were blocked for 1 h with 5% skimmed milk and probed with various antibodies at dilutions recommended by the suppliers. Immunoblots were detected through chemiluminescence using enhanced chemiluminescence reagents obtained from Millipore (Billerica, USA). To quantify equal loading, membranes were reprobed with b-actin antibody. Data is presented as the relative density of protein bands normalized to bactin. The intensities of the bands were quantitated using Image Analysis software on an Image Gel Documentation System. 2.9. Reverse transcriptase-polymerase chain reaction (RT-PCR) assay Total RNA was extracted by using the Trizol reagent (Invitrogen). RT-PCR for Her2 transcript was done on Bio-Rad C1000 Touch Thermal Cycler by using a GeneAmp RNA RT-PCR kit (Perkin-Elmer, Roche Molecular systems Inc., Branchburg, USA) as per the manufacturer’s instructions. The primers sequences to amplify HER2 transcript were 50 -GACCCGCTGAACAATACCAC-30 (forward) and 50 -TGCCGTCGTCTTCTAGGCCTTCAT-30 (reverse). Amplification of GAPDH cDNAs was carried out as an internal control in each reaction. Reaction mixture was first denatured at 95 8C for 10 min. PCR conditions were 95 8C for 1 min, 60 8C for 2 min, and 72 8C for 2 min, for 30 cycles, followed by 72 8C for 10 min. 2.10. Statistical analysis Mean values and standard deviation (SD) were calculated for all treated samples and vehicle controls. The Student’s t-test for the paired samples and one-way ANOVA Holm–Sidak for different treated sample were used to evaluate the statistical significance of differences between parameters (Sigma Stat 3.5, Systate Software Inc., San Jose, USA). Synergistic effects of the combination of resveratrol and rapamycin on cell proliferation were assessed using the Chou–Talalay method [42] and Calcusyn software (Biosoft, Ferguson, USA). Briefly, the dose–effect curve for each drug was determined based on the experimental observations using the median-effect principle; the combination index (CI) for each experimental combination was then calculated according to the following equation:

CI ¼

ðDÞ1 ðDÞ2 ðDÞ1 ðDÞ2 þ þ ðDx Þ1 ðDx Þ2 ðDx Þ1 ðDx Þ2

where (D)1 and (D)2 are the doses of resveratrol and rapamycin, respectively, that have x effect when used in combination and (Dx)1 and (Dx)2 are the doses of resveratrol and rapamycin that have the same x effect when used alone. CI values equaling 1 indicate additive effects; CI values less than 1 indicate a greater than expected additive effect (synergism).

767

resveratrol (5–150 mM). The IC50 value for growth inhibition was obtained at 80 mM of Resveratrol after 24 h, 48 and 72 h of treatment. Based on these observations, we selected various concentrations of resveratrol below IC50 i.e. 20, 40 and 60 mM for further mechanistic studies over a 24 h period. 3.2. Resveratrol altered the cell cycle progression in Her-2 overexpressed SKBR-3 breast cancer cells The potential of resveratrol in cell growth inhibition was investigated by analyzing cell cycle arrest through flow-cytometry. The treatment of cells with resveratrol showed that cells accumulated in sub G1 phase in dose dependent manner. Up to 34% cells accumulated in sub G1 phase upon treatment with 60 mM Resveratrol as compared to 2.4% in vehicle treated (0 mM) cells (Fig. 1B). The result also showed a concomitant decrease in G0/G1 and S-phase with increase in concentration of resveratrol treatment. 3.3. Resveratrol induced cell death in dose-dependent manner Apoptosis is one of the most potent defense mechanism against cancer [43]. To characterize the resveratrol-induced cell death, we quantified the extent of apoptosis by flow-cytometric analysis of cells labeled with Annexin-V-PI staining. Exposure of Her2overexpressed SKBR-3 breast cancer cells to varying concentration (20–60 mM) of resveratrol for 24 h resulted in a gradual increase in the apoptotic cell population. Our result shows clear increase in the number of both early (lower-right quadrant) and late (upper-right quadrant) apoptotic cells with increase in concentration of resveratrol treatment after 24 h (Fig. 1C). Almost 35% cells advanced to late apoptotic stage upon treatment with 60 mM resveratrol as compared to 0.1% in untreated controls. The number of early apoptotic cells also increased with increasing dose of resveratrol (Fig. 1C). 3.4. Resveratrol-induced inhibition of Akt phosphorylation alters Akt/ PI3K/mTOR pathway PI3K kinase regulates the important signaling pathways required for biological processes like cell survival, proliferation, cell growth and cell motility [44]. The activation of AKT by phosphorylation at serine 473 plays a pivotal role in fundamental cellular functions such as cell proliferation and cell survival by phosphorylating a variety of downstream substrates. Several studies have reported the involvement of PI3K/AKT signaling pathway in resveratrol-induced inhibition of growth of different cell types [45,46]. As expected, the result of Western blot analysis revealed a gradual inhibition in Akt phosphorylation and increased the level of its inhibitor PTEN (p < 0.001 against vehicle control) in dose-dependent manner (Fig. 2). 3.5. Resveratrol augments the effect of mTOR inhibitor rapamycin in growth inhibition and apoptotic induction synergistically

3. Results 3.1. Resveratrol mediated growth inhibition of HER2 over-expressed Sk-Br3 breast cancer cells To explore the molecular mechanism of resveratrol on Her2 over-expressed breast cancer proliferation, SKBr-3 breast cancer cell lines were cultured with various concentrations of Resveratrol. Preliminary screening was performed to assess the effect of resveratrol on the cellular proliferation and cell viability at different time intervals using the MTT assay. As depicted in Fig. 1A, the continuous and time dependent reductions in the viability of Sk-Br3 cells were observed with increasing doses of

Further, we investigated the inhibitory effect of resveratrol in combination with rapamycin as we know that mammalian target of rapamycin (mTOR) is an important downstream regulator of the phosphatidyl-inositol 3-kinase (PI3K)-Akt pathway. The mTOR inhibitors have been shown to have higher antitumor activity in breast cancer system with reduced toxicity [47]. Keeping the view into consideration, we hypothesized that inhibition of mTOR may enhance the sensitivity of the breast cancer cells to resveratrol. It was clearly observed that mTOR inhibitor rapamycin increased the effect of resveratrol (p < 0.001) in induction of apoptosis (Fig. 3A) and growth inhibition (Fig. 3B) when compared with corresponding doses (20, 40, 60 mM).

768

A. Khan et al. / Cancer Epidemiology 38 (2014) 765–772

Fig. 1. (A) Effect of resveratrol on Her2-overexpressed SKBR-3 breast cancer cells. 1  104 cells (200 ml) were treated with resveratrol (0–150 mM) for 24, 48 and 72 h. The percentage of cell growth inhibition was measured by MTT assay as described in Section 2. Values are represented as the percentage of viable cells; vehicle-treated cells were considered as 100% viable. Data represent mean percentages of viable cells  SD of three independent experiments. (B) Effect of resveratrol on cell cycle progression in HER2overexpressed SKBR-3 breast cancer cells. Cells were treated with resveratrol (20, 40 and 60 mM) for 24 h, and DNA cell-cycle analysis was performed as described in Section 2. Vehicle control cells showed no sub-G1 peak, whereas resveratrol-treated cells contained some cells in sub-G1, as determined by flow-cytometry analysis; PI fluorescence intensity was measured as an indicator of cellular DNA content; (C) Induction of apoptosis in HER2-overexpressed SKBR-3 breast cancer cells by resveratrol. Data shown are the results of three independent experiments. Annexin-V/PI dual staining of SKBR-3 cells was performed following exposure to resveratrol with 20, 40 and 60 mM for 24 h. Cells in the upper-left (UL) and upper-right (UR) portions are necrotic and late apoptotic cells and, whereas cells in the lower-left (LL) and lower-right (LR) portions are viable and earlyapoptotic cells, respectively.

Fig. 2. Resveratrol inhibits Akt phosphorylation and increases the expression of its inhibitor PTEN in dose-dependent manner. Cells were treated with increasing concentrations of resveratrol in serum free DMEM medium for 3 h and followed by incubation in medium containing 5% FBS for 20 min, the total protein was isolated and Western blot analysis was performed as described in Section 2 with antibodies specific for phosphorylated-S473-Akt, Akt and PTEN. Data shown are the results of three independent experiments and are represented as the relative densities of protein bands normalized to b-actin. *Significant difference compared with vehicle control (0 mM) for p-5473-Akt (p < 0.001). #Significant difference compared with vehicle control (0 mM) for PTEN (p < 0.001).

A. Khan et al. / Cancer Epidemiology 38 (2014) 765–772

769

activity that leads to the down-regulation of Her2 transcription and translation as well. 3.7. Discussions

Fig. 3. Resveratrol increased the effect of mTOR inhibitor rapamycin on cell apoptosis and proliferation. HER2-overexpressed breast cancer SKBR-3 cells (1  104) were seeded in 96-well-plate, incubated with various concentrations of resveratrol (20, 40 and 60 mM) in the absence or presence of mTOR inhibitor rapamycin (10 nM) for 24 h. Cells were also treated with various concentrations of rapamycin in the absence of resveratrol as indicated. (A) Cell apoptosis and (B) proliferation was measured by cell-death ELISA and MTT assay, respectively, as described in Section 2. Results are normalized to untreated controls, presented as means  SD of three assays. *Significant difference compared with vehicle control (0 mM). #Significant difference compared with corresponding doses (20, 40 and 60 mM).

3.6. Resveratrol down-regulates the expression of HER2 by inhibiting FASN activity To gain additional insight into the molecular mechanisms of the effect of resveratrol on Her2 over-expressed breast cancer cell lines, we determined the changes in the expression of FASN and Her2. Interestingly, we observed the continuous decreased in FASN and Her2 expression in dose dependent manner. These data demonstrate the uncharacterized molecular targets of resveratrol that follow the inhibition of FASN in Her2 over-expressing breast cancer cells. To characterize the specific mechanism through which resveratrol induced inhibition of FASN activity molecularly modulated Her2 expression, we examined the expression of HER2 regulators PEA3 and Cyclin D1. PEA3, a member of the Ets transcription factor family is a DNA-binding protein, downregulates Her2 promoter activity by targeting it specifically, thus inhibiting Her2 promoted tumorigenesis [48,49]. Remarkably, a significant upregulation of PEA3 was found following resveratrol exposure in dose-dependent manner (Fig. 4A). Noticeably, the decreased expression of cyclin D1 was observed as it is required for HER2 induced transformation and could reciprocally up-regulate HER2 expression [50,51]. Further, we studied whether the accumulation of PEA3 induced by resveratrol was associated with a transcriptional response of Her2 gene. As depicted in Fig. 4B that mRNA expression of Her2 was down-regulated upon treatment with resveratrol. These findings convincingly establish that decreased FASN activity by resveratrol upregulates PEA3 to bind effectively and inhibit Her2 promoter

Cancer chemoprevention by using non-toxic chemical substances is regarded as a promising alternative strategy to control human cancer. In recent years, many naturally occurring substances have been shown to protect against experimental carcinogenesis [8,13]. In this regard, resveratrol (3,5,40 -trihydroxy stilibene) has shown its immense potential to protect against experimental carcinogenesis [52–54]. Besides its chemoprevention role against cancer, the antioxidant activity of resveratrol helps in attenuating the development of atherosclerosis and other inflammatory diseases [55]. The chemopreventive efficacy of resveratrol is well documented against hepatocellular, lung, skin and prostate cancers through multiple regulatory mechanisms [56–58]. However, the exact mechanism(s) of action of resveratrol in cancer especially breast cancer remains largely unknown, thus limiting its therapeutic use. The lipogenic enzyme fatty acid synthase (FASN), also called oncogenic antigen-519, is selectively and highly expressed in breast cancers [27,28]. As evident from various studies, FASN is a critical enzyme involved in the anabolic conversion of dietary carbohydrates to fatty acids in mammals [59–61]. The upregulation of FASN activity and expression in many human cancers shows that FASN is involved in the development, maintenance, and enhancement of the malignant phenotype [27,62,63]. In this regard, pharmacological inhibitors of FASN are increasingly receiving more attention as the inhibition of FASN clearly reduces proliferation and at the same time enhances the apoptotic rates of human and mouse melanoma cells [64]. Recently, many polyphenols have been found to be effective as natural FASN inhibitors [33–35,37]. There are strong evidences that suggest the potential inhibitory effect of resveratrol on FASN expression and activity [36,37]. In the present study we have explored the molecular mechanism of resveratrol induced inhibition of FASN in Her2 overexpressed breast cancer cells. The data shows that resveratrol causes dose dependent inhibition of human breast cancer cells (Fig. 1A), alters cell cycle progression (by inducing S-phase arrest) and induces apoptotic cell death via FASN inhibition (Figs. 1 and 2). Our results not only reinforce the notion that FASN activity is a novel molecular target of resveratrol in breast cancer, but further demonstrate a close involvement of Her2/Neu oncogene, the overexpression and/or activation of which is known to play an important role in the etiology, aggressive progression and poor clinical outcome of breast carcinomas. The survival of HER2overexpressed breast cancer cells is heavily dependent on lipid metabolism [65] as HER2 increases translation of FASN [28,66]. The FASN over-expression markedly increases EGFR and HER2 signaling resulting in enhanced cell growth [30] that has been associated with poor prognosis in breast cancer patients [24]. The molecular connection between HER2 and FASN originally discovered by Kumar-Sinha et al. showed that HER2 mediates FASN induction by activating the FASN promoter via a PI3K-dependent pathway [67]. Later, it was suggested that HER2 over-expression is responsible in upregulating FASN activity [68] and showed that FASN inhibitors (for example, cerulenin and C75) and small interfering RNAs (siRNAs) targeting FASN specifically suppressed HER2 protein and mRNA expression by upregulating the HER2specific transcriptional repressor PEA3 [69]. Pharmacologically- or siRNA-induced inhibition of FASN in breast cancer cells results in major changes in the synthesis of phospholipids that, in turn, impair the proper localization of HER2 to the cell membrane [70]. In the current findings, we identified that resveratrol-mediated

770

A. Khan et al. / Cancer Epidemiology 38 (2014) 765–772

Fig. 4. Resveratrol inhibited the FASN activity and down-regulated Her-2/Neu promoter activity through binding of its transcriptional regulator PEA3. SKBR-3 cells were treated with increasing concentrations of resveratrol for 24 h. (A) The total protein was isolated and western blot analysis was performed as described in Section 2 with antibodies specific for p185Her2, Cyclin-D, FASN, PEA3. Equal loading was confirmed by re-probing the membrane with b-actin. (B) RT-PCR analyses for Her2 and GADPH transcripts and expression were performed as described in Section 2. Data shown are the results of three independent experiments and are represented as the relative densities of bands from western blot and RT-PCR analysis normalized to b-actin and GAPDH, respectively. a–dSignificant difference compared with vehicle control (0 mM) for Cyclin D, PEA3, FASN and Her2, respectively (p < 0.001). *Significant difference compared with vehicle control for Her-2/neu (p < 0.001).

inhibition of FASN down-regulated the expression of HER2 at the transcriptional level. As depicted in Fig. 4A, FASN inhibition concomitantly up-regulates the expression of Ets factor, PEA3, which specifically reverses the in vitro transformed phenotype of HER2-overexpressing cancer cells through the attenuation of the HER2 oncogene promoter activity [48,71]. Some recent findings have also shown that FASN expression is induced by HER2-overexpression, which activates the PI3K/AKT pathway and subsequently stimulates the mTOR leading to increased FASN translation [66,67,72]. Our current data demonstrates that resveratrol remarkably down-regulated the expression of phosphorylated Akt, whereas the PTEN, the antagonizer of the PI3k and Akt activity, was up-regulated (Fig. 2). Significantly, we also showed that the reduction of PI3K/Akt/mTOR signaling by inhibition of the downstream effector mTOR using rapamycin synergistically increased the resveratrol-induced cellular apoptosis (Fig. 3). This result not only sustains the involvement of PI3K/Akt/mTOR signaling in resveratrol-induced cellular events, but also highlights that resveratrol and mTOR inhibitor may possess clinical potential in treatment of various breast cancers. The molecular mechanisms of therapeutic agents that target HER2/Akt/PI3K/mTOR pathway have been shown to downregulate FASN expression [66,73]; conversely, FASN inhibitors block Her2 protein expression and its kinase activity [31,69]. These studies imply a potential role for FASN in apoptosis, cell proliferation and invasion in HER2-overexpressing breast cancer cells and indicate the possibility that agents targeting FASN could

be useful for treating breast cancers, especially those in which HER2 is over-expressed. Our results are compatible with the hypothesis that resveratrol inhibits FAS activity, which is necessary to integrate a number of signaling pathways important to regulate metabolism, proliferation, and survival in HER2 over-expressing cancer cells. Consistent with these findings, our data also suggest FASN as a novel therapeutic target of dietary constituents in HER2-overexpressing breast cancer. In terms of a clinical perspective, the present study provides a molecular rationale to design the novel therapies directed against FASN in HER2-overexpressing breast cancer. Conflict of interest statement The authors declare no competing financial interests. Authorship contribution AK who conceived the study, designed the experiments, supervised the work and wrote the manuscript. ANA performed the cell viability assay, discussed the experiments and participated in critically revising the manuscript. YHA participated in statistical analysis and the interpretation of the data. SMF performed cell cycle and apoptotic analysis by flow cytometry. MAK participated in experimental design and revised the manuscript critically for important and intellectual content. All authors read and approved the revised manuscript.

A. Khan et al. / Cancer Epidemiology 38 (2014) 765–772

Acknowledgements We acknowledge all the staff members of college of pharmacy for their co-operation to conduct this research. This study was supported in part by the Deanship Scientific Research, Grant No. 946, Qassim University, Kingdom of Saudi Arabia.

References [1] de Kok TM, de Waard P, Wilms LC, van Breda SG. Antioxidative and antigenotoxic properties of vegetables and dietary phytochemicals: the value of genomics biomarkers in molecular epidemiology. Mol Nutr Food Res 2010;54: 208–17. [2] Bickers DR, Athar M. Novel approaches to chemoprevention of skin cancer. J Dermatol 2000;27:691–5. [3] Keloff GJ, Sigman CC, Greenwald PP. Cancer chemoprevention: progress and promise. Eur J Cancer 1999;35:2031–8. [4] Bishayee A. Cancer prevention and treatment with resveratrol: from rodent studies to clinical trials. Cancer Prev Res 2009;2:409–18. [5] Kundu JK, Surh YJ. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives. Cancer Lett 2008;269:243–61. [6] Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997;275:218–20. [7] Sengottuvelan M, Viswanathan P, Nalini N. Chemopreventive effect of transresveratrol-a phytoalexin against colonic aberrant crypt foci and cell proliferation in 1,2-dimethylhydrazine induced colon carcinogenesis. Carcinogenesis 2006;27:1038–46. [8] Kapadia GJ, Azuine MA, Tokuda H, Takasaki M. Chemopreventive effect of resveratrol, sesamol, sesame oil and sunflower oil in the Epstein–Barr virus early antigen activation assay and the mouse skin two-stage carcinogenesis. Pharmacol Res 2002;45:499–505. [9] Brown VA, Patel KR, Viskaduraki M, et al. Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: safety, pharmacokinetics and effect on the insulin-like growth factor axis. Cancer Res 2010;70:9003–11. [10] Patel KR, Brown VA, Jones DJ, et al. Clinical pharmacology of resveratrol and its metabolites in colorectal cancer patients. Cancer Res 2010;70:7392–9. [11] Joe AK, Liu H, Suzui M, et al. Resveratrol induces growth inhibition, S-phase arrest, apoptosis, and changes in biomarker expression in several human cancer cell lines. Clin Cancer Res 2002;8:893–903. [12] Narayanan BA, Narayanan NK, Re GG, Nixon DW. Differential expression of genes induced by resveratrol in LNCaP cells: p53-mediated molecular targets. Int J Cancer 2003;104:204–12. [13] Ahmad N, Adhami VM, Afaq F, Feyes DK, Mukhtar H. Resveratrol causes WAF1/p21-mediated G1-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 cells. Clin Cancer Res 2001;7:1466–73. [14] Stewart JR, Ward NE, Ioannides CG, O’Brien CA. Resveratrol preferentially inhibits protein kinase C-catalyzed phosphorylation of a cofactor-independent, arginine-rich protein substrate by a novel mechanism. Biochemistry 1999;38:13244–51. [15] Tinhofer I, Bernhard D, Senfter M, et al. Resveratrol, a tumor-suppressive compound from grapes, induces apoptosis via a novel mitochondrial pathway controlled by Bcl-2. FASEB J 2001;15:1613–5. [16] Mahyar-Roemer M, Kohler H, Roemer K. Role of Bax in resveratrol induced apoptosis of colorectal carcinoma cells. BMC Cancer 2002;2:27–35. [17] Fontecave M, Lepoivre M, Elleingand E, Gerez C, Guittet D. Resveratrol, a remarkable inhibitor of ribonucleotide reductase. FEBS Lett 1998;421:277–9. [18] Opipari Jr AW, Tan L, Boitano AE, Sorenson DR, Aurora A, Liu JR. Resveratrolinduced autophagocytosis in ovarian cancer cells. Cancer Res 2004;64: 696–703. [19] Basly JP, Marre-Fournier F, Le-Bail JC, Habrioux G, Chulia AJ. Estrogenic/ antiestrogenic and scavenging properties of (E)- and (Z)-resveratrol. Life Sci 2000;66:769–77. [20] Zhang S, Cao HJ, Davis FB, et al. Oestrogen inhibits resveratrol-induced posttranslational modification of p53 and apoptosis in breast cancer cells. Br J Cancer 2004;91:178–85. [21] Nakagawa H, Kiyozuka Y, Uemura Y, et al. Resveratrol inhibits human breast cancer cell growth and may mitigate the effect of linoleic acid, a potent breast cancer cell stimulator. J Cancer Res Clin Oncol 2001;127:258–64. [22] Shimokawa T, Kumar MV, Lane MD. Effect of a fatty acid synthase inhibitor on food intake and expression of hypothalamic neuropeptides. Proc Natl Acad Sci USA 2002;99:66–71. [23] Loftus TM, Jaworsky DE, Frehywot GL, et al. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 2000;288:2379–81. [24] Kuhajda FP, Pizer ES, Li JN, et al. Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proc Natl Acad Sci USA 2000;97:3450–4. [25] Sul HS, Wang D. Nutritional and hormonal regulation of enzymes in fat synthesis: studies of fatty acid synthase and mitochondrial glycerol-3phosphate acyltransferase gene transcription. Annu Rev Nutr 1998;18: 331–51.

771

[26] Milgraum LZ, Witters LA, Pasternack GR, Kuhajda FP. Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma. Clin Cancer Res 1997;3:2115–20. [27] Alo PL, Visca P, Trombetta G, et al. Fatty acid synthase (FAS) predictive strength in poorly differentiated early breast carcinomas. Tumori 1999;85:35–40. [28] Kuhajda FP, Jenner K, Wood FD, et al. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci USA 1994;91:6379–83. [29] Jin Q, Yuan LX, Boulbes D, et al. Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells. Breast Cancer Res 2010;12:R96. [30] Vazquez-Martin A, Colomer R, Brunet J, Lupu R, Menendez JA. Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells. Cell Prolif 2008;41:59–85. [31] Menendez JA, Vellon L, Mehmi I, et al. Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells. Proc Natl Acad Sci USA 2004;101:10715–20. [32] Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–82. [33] Puig T, Va´zquez-Martı´n A, Relat J, et al. Fatty acid metabolism in breast cancer cells: differential inhibitory effects of epigallocatechin gallate (EGCG) and C75. Breast Cancer Res Treat 2008;109:471–9. [34] Pan MH, Lin CC, Lin JK, Chen WJ. Tea polyphenol ()-epigallocatechin 3-gallate suppresses heregulin-beta1-induced fatty acid synthase expression in human breast cancer cells by inhibiting phosphatidylinositol 3-kinase/Akt and mitogen activated protein kinase cascade signaling. J Agric Food Chem 2007;55:5030–7. [35] Tian WX. Inhibition of fatty acid synthase by polyphenols. Curr Med Chem 2006;13:967–77. [36] Gnoni GV, Paglialonga G. Resveratrol inhibits fatty acid and triacylglycerol synthesis in rat hepatocytes. Eur J Clin Invest 2009;39:211–8. [37] Li BH, Ma XF, Wang Y, Tian WX. Structure–activity relationship of polyphenols that inhibit fatty acid synthase. J Biochem 2005;138:679–85. [38] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. [39] Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139:271–9. [40] Evens AM, Prachand S, Shi B, et al. Imexon-induced apoptosis in multiple myeloma tumor cells is caspase-8 dependent. Clin Cancer Res 2004;10: 1481–91. [41] Khan A, Aijaz AK, Dwivedi V, et al. Tuftsin augments antitumor efficacy of liposomised etoposide against fibrosarcoma in Swiss albino mice. Mol Med 2007;13:266–76. [42] Chou TC, Talalay P. Quantitative analysis of dose–effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55. [43] Sun SY, Hail Jr N, Lotan R. Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 2004;96:662–72. [44] Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositideregulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 1999;68:965–1041. [45] Rachid O, Alkhalaf M. Resveratrol regulation of PI3KAKT signaling pathway genes in MDA-MB-231 breast cancer cells. Cancer Genomics Proteomics 2006;3:383–8. [46] Poolman TM, Ng LL, Farmer PB, Manson MM. Inhibition of the respiratory burst by resveratrol in human monocytes: correlation with inhibition of PI3K signalling. Free Radic Biol Med 2005;39:118–32. [47] Altomare DA, Testa JR. Perturbations of the AKT signaling pathway in human cancer. Oncogene 2005;24:7455–64. [48] Wang SC, Hung MC. Transcriptional targeting of the HER2/neu oncogene. Drugs Today (Barc) 2000;36:835–43. [49] Oliver FJ, de la Rubia G, Rolli V, et al. Importance of poly(ADPribose) polymerase and its cleavage in apoptosis: lesson from an uncleavable mutant. J Biol Chem 1998;273:33533–39. [50] Nahta R, Iglehart D, Kempkes B, Schmidt EV. Rate-limiting effects of Cyclin D1 in transformation by ErbB2 predicts synergy between herceptin and flavopiridol. Cancer Res 2002;62:2267–71. [51] Yu Q, Gen Y, Sicinski P. Specific protection against breast cancers by cyclin D1 ablation. Nature 2001;411:1017–21. [52] She QB, Bode AM, Ma WY, Chen V, Dong Z. Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer Res 2001;61:1604–10. [53] Surh YJ, Hurh YJ, Kang JY, et al. Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Cancer Lett 1999;140:1–10. [54] Clement MV, Hirpara JL, Pervaiz S. Chemopreventive agent resveratrol, a natural product derived from grapes triggers Cd95 signalling-dependent apoptosis in human tumor cells. Blood 1998;92:996–1002. [55] Soleas GJ, Grass L, Josephy PD, Goldberg DM, Diamandis EP. A comparison of the anticarcinogenic properties of four red wine polyphenols. Clin Biochem 2002;35:119–24. [56] Liu H, Zang C, Fenner MH, et al. Growth inhibition and apoptosis in human Philadelphia chromosome-positive lymphoblastic leukemia cell lines by treatment with the dual PPARalpha/gamma ligand TZD18. Blood 2006;107: 3683–92.

772

A. Khan et al. / Cancer Epidemiology 38 (2014) 765–772

[57] Roy P, Kalra N, Prasad S, George J, Shukla Y. Chemopreventive potential of resveratrol in mouse skin tumors through regulation of mitochondrial and PI3K/AKT signaling pathways. Pharm Res 2009;26:211–7. [58] Seeni A, Takahashi S, Takeshita K, et al. Suppression of prostate cancer growth by resveratrol in the transgenic rat for adenocarcinoma of prostate (TRAP) model. Asian Pac J Cancer Prev 2008;9:7–14. [59] Rangan VS, Joshi AK, Smith S. Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro. Biochemistry 2001;40:10792–99. [60] Chirala SS, Jayakumar A, Gu ZW, Wakil SJ. Human fatty acid synthase: role of interdomain in the formation of catalytically active synthase dimmer. Proc Natl Acad Sci USA 2001;98:3104–8. [61] Joshi AK, Witkowski A, Smith S. Mapping of functional interactions between domains of the animal fatty acid synthase by mutant complementation in vitro. Biochemistry 1997;36:2316–22. [62] Wang YY, Kuhajda FP, Li J, et al. Fatty acid synthase as a tumor marker: its extracellular expression in human breast cancer. J Exp Ther Oncol 2004;4:101–10. [63] Alo PL, Visca P, Botti C, et al. Immunohistochemical expression of human erythrocyte glucose transporter and fatty acid synthase in infiltrating breast carcinomas and adjacent typical/atypical hyperplastic or normal breast tissue. Am J Clin Pathol 2001;116:129–34. [64] Carvalho MA, Zecchin KG, Seguin F, et al. Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model. Int J Cancer 2008;123:2557–65.

[65] Kourtidis A, Srinivasaiah R, Carkner RD, Brosnan MJ, Conklin DS. Peroxisome proliferator-activated receptor-gamma protects ERBB2-positive breast cancer cells from palmitate toxicity. Breast Cancer Res 2009;11:R16. [66] Yoon S, Lee MY, Park SW, et al. Upregulation of acetyl-CoA carboxylase alpha and fatty acid synthase by human epidermal growth factor receptor 2 at the translational level in breast cancer cells. J Biol Chem 2007;282:26122–31. [67] Kumar-Sinha C, Ignatoski KW, Lippman ME, Ethier SP, Chinnaiyan AM. Transcriptome analysis of HER2 reveals a molecular connection to fatty acid synthesis. Cancer Res 2003;63:132–9. [68] Menendez JA. Fine-tuning the lipogenic/lipolytic balance to optimize the metabolic requirements of cancer cell growth: molecular mechanisms and therapeutic perspectives. Biochim Biophys Acta 2010;1801:381–91. [69] Menendez JA, Lupu R, Colomer R. Targeting fatty acid synthase: potential for therapeutic intervention in her-2/neu-overexpressing breast cancer. Drug News Perspect 2005;18:375–85. [70] Menendez JA, Vellon L, Lupu R. Targeting fatty acid synthase-driven lipid rafts: a novel strategy to overcome trastuzumab resistance in breast cancer cells. Med Hypotheses 2005;64:997–1001. [71] Xing X, Wang SC, Xia W, et al. The ets protein PEA3 suppresses HER-2/neu overexpression and inhibits tumorigenesis. Nat Med 2000;6:189–95. [72] Sabbisetti V, Di Napoli A, Seeley A, et al. p63 promotes cell survival through fatty acid synthase. PLoS ONE 2009;4:e5877. [73] Grunt TW, Wagner R, Grusch M, et al. Interaction between fatty acid synthaseand ErbB-systems in ovarian cancer cells. Biochem Biophys Res Commun 2009;385:454–9.