Journal of Surgical Research 161, 1822 (2010) doi:10.1016/j.jss.2009.06.

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ASSOCIATION FOR ACADEMIC SURGERY Pterostilbene Inhibits Lung Cancer Through Induction of Apoptosis1
John G. Schneider, M.D.,*, Julie A. Alosi, M.D.,*, Debbie E. McDonald, B.S.,* and David W. McFadden, M.D.*,,2
*Department of Surgery, University of Vermont, Burlington, Vermont; and Department of Surgery, Fletcher Allen Health Care, Burlington, Vermont Submitted for publication February 19, 2009

INTRODUCTION Background. Lung cancer remains the leading cause of cancer mortality in the United States. Resveratrol is a potent antioxidant found in grapes that inhibits several types of cancer, including lung cancer. Herein, we investigated the effects of pterostilbene, an analog of resveratrol found in blueberries, on lung cancer, in vitro. We hypothesized that pterostilbene would inhibit lung cancer cell growth in vitro by a pro-apoptotic mechanism. Methods. Two lung cancer cell lines (NCI-H460 and SK-MES-1) were cultured using standard techniques. Cells were treated with increasing doses of pterostilbene (10100mM). Cell viability was measured at 24, 48, and 72 h using a MTT assay. Apo-ONE Caspase-3/7 assay was used to evaluate caspase activity. T-test and two-way ANOVA were used for statistical analysis. Results. Pterostilbene signicantly decreased cell viability in lung cancer cells in a concentration- and time-dependent manner (P < 0.001). Concentrations greater than 20 mM of pterostilbene produced signicant growth inhibition by 72 h (P < 0.001). Apoptosis and caspase-3/7 activity were signicantly increased by pterostilbene treatment (P < 0.05). Conclusions. Pterostilbene inhibits growth via apoptosis induction in vitro. Further in vitro mechanistic studies and in vivo experiments are warranted to determine the potential role for pterostilbene in lung cancer treatment or prevention. 2010 Elsevier Inc. All rights
reserved.

Key Words: pterostilbene; lung cancer; apoptosis.

1 This work was presented at the 4th Annual Academic Surgical Congress and at the New England Surgical Society Resident and Fellow Research Day in 2009. 2 To whom correspondence and reprint requests should be addressed at Department of Surgery, University of Vermont, 111 Colchester Avenue, Fletcher House 301, Burlington, VT 05401. E-mail: David.McFadden@vtmednet.org.

Lung cancer continues to be the leading cause of cancer mortality in the United States [1]. The principal cause for the development of lung cancer is tobacco smoke. Despite nationwide campaigns for smoking cessation, 21.9% of adult men and 17.6% of adult women in the United States continue to smoke [2]. Patients continue to present with advanced disease and many are unresectable at the time of diagnosis. Even with advances in chemotherapy and radiation, overall 5-y survival is only 15% [1]. Thus, compounds that could be used for chemoprevention or adjuvant treatment have the potential to have a signicant impact in the treatment of lung cancer. Epidemiologic studies have linked diets rich in fruits and vegetables with lower risk for several common cancers. A postulated explanation for this observation is that these foods are rich in phytochemicals, naturallyoccurring, non-nutritive substances that have disease preventive or protective properties. One such phytonutrient is resveratrol, a polyphenol found in grapes and red wine, which has demonstrated signicant antitumor and cardioprotective effects. Studies have shown that resveratrol inhibits lung cancer growth in vitro [3, 4]. Pterostilbene is a naturally occurring analog of resveratrol found in blueberries. Previous investigations have found that pterostilbene has similar antioxidant, anti-inammatory, and anticancer properties to those of resveratrol [5, 6]. Melanoma, breast, gastric, and hepatocellular carcinomas are malignancies that have been shown to be pterostilbene sensitive [5]. However, investigations using pterostilbene have only begun recently, and relatively little is known about the mechanisms by which its effects are mediated and which tumors are most sensitive to it.

0022-4804/$36.00 2010 Elsevier Inc. All rights reserved.

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To date, pterostilbene has not been investigated in the treatment of lung cancer. The goal of this study is to examine the effects of pterostilbene treatment on lung cancer in vitro and identify intracellular mechanisms by which pterostilbene exerts its effects.
MATERIALS AND METHODS Reagents
Pterostilbene was purchased from Sigma-Aldrich (St. Louis, MO). It was dissolved in dimethyl sulfoxide (DMSO; Sigma) and further diluted in sterile culture medium immediately prior to use.

color change. Color development was proportional to the amount of nucleosomes captured in the antibody sandwich, and was measured spectrophotometrically at 405 nm.

Caspase Activity Assay

Cells were plated into 96-well plates with opaque sidewalls for this experiment. Cells were then treated with pterostilbene or DMSO control for 12, 24, and 36 h. The Apo-ONE homogeneous caspase 3/7 assay substrate (Promega, Madison, WI) was utilized to evaluate the activities of caspase-3 and -7, effector caspases that cleave intracellular protein substrates triggering the apoptotic process. The caspase-3/7 substrate rhodamine 110, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide; Z-DEVD-R110), is acted upon by caspase-3 and-7 resulting in a uorescent leaving group. The amount of uorescent product generated is proportional to the amount of caspase-3/7 cleavage activity present in the sample. Caspase-3/7 substrate was added to each well and incubated at room temperature for 12 h. A spectrouorometer was used to measure uorescence (excitation wavelength 485 6 20 nm, emission wavelength 528 6 20 nm).

Cell Lines
Two human lung carcinoma cell lines, NCI-H460 and SK-MES-1, were purchased from the American Type Culture Collection (ATCC, Manassas, VA). NCI-H460 cells represent a large cell lung carcinoma and SK-MES-1 cells represent a squamous cell lung carcinoma. Both originated from malignant pleural effusion. NCI-H460 and SK-MES -1 cells were cultured, according to the suppliers recommendations, in RPMI-1640 and Eagles minimal essential media. Cells were maintained at 37 C in a water-jacketed 5% CO2 incubator (Fischer Scientic, Houston, TX). For experiments, cells were harvested from culture monolayers at 80%90% conuence. Cells were plated with 104 cells per well in 96-well plates and allowed to attach overnight. Cells were then exposed to graduated doses of pterostilbene (10 100 mM) or DMSO control.

Statistical Analysis
Data were presented as mean values 6 standard error. Statistical comparisons among groups were performed by Students t-test or analysis of variance (ANOVA), followed by Bonferroni post-tests for multiple comparisons. These analyses were run using GraphPad Software (GraphPad Inc., San Diego, CA).

RESULTS Pterostilbene Anti-tumor Activity In Vitro

Cell Viability Assay

A MTT colorimetric assay was performed to detect cell viability after 24, 48, and 72 h of exposure to pterostilbene (10100 mM). Culture media was removed, and MTT, a tetrazolium dye (3-[4, 5dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; thiazolyl blue, Sigma) diluted in culture media was added to each well. Plates were incubated at 37 C in the CO2 incubator for 1 h. Mitochondrial dehydrogenase activity reduced the yellow MTT dye to a purple formazan, which was solubilized in DMSO (Sigma). Absorbance was read at 570 nm via an enzyme-linked immunosorbent assay (ELISA) plate reader.

Pterostilbene signicantly decreased cell viability in a concentration- and time-dependent (Fig. 1) manner in both cell lines.
Pterostilbene Induces Apoptosis in Lung Cancer Cells

Growth Inhibition
Cells were treated with pterostilbene at increasing doses for 24, 48, and 72 h. Cells were then harvested and counted by hemocytometer. The growth of treated cells was expressed as a percentage of untreated control cells. The concentration of pterostilbene that decreased cell count by 50% (IC50) was calculated by nonlinear least-squares curve tting of experimental data.

DNA Fragmentation Assay

The Cell Death Detection ELISA kit (Roche, Mannheim, Germany), a sandwich-enzyme-immunoassay-based method, was used to detect the occurrence of nuclear DNA fragmentation. This kit employs mouse monoclonal antibodies directed against DNA and histones to recognize released nucleosomes after DNA internucleosomal fragmentation. After the cells were exposed to various doses of pterostilbene for 18 h, supernatants were removed and stored at 4 for subsequent analysis of necrosis. Adherent cells were lysed and centrifuged to produce a nucleosome-containing supernatant. Samples were transferred to a streptavidin-coated microplate and incubated with antihistone and anti-DNA antibodies, followed by a peroxidase substrate resulting in

Programmed cell death is characterized by chromatin condensation, internucleosomal DNA degradation, and apoptotic body formation. An assay to examine released nucleosomes was performed to investigate whether the cytotoxic effects of pterostilbene resulted from necrosis or apoptosis. Increase in released nucleosomes was observed in both cell lines after treatment with pterostilbene by 18 h post-treatment. Specically, 1.5-fold (P < 0.01) and 2.4-fold (P < 0.05) increases were observed in NCI-H460 cells treated with 50 and 75 mM of pterostilbene, respectively. In SK-MES-1 cells, 1.4-fold (P < 0.05) and 2.3-fold (P < 0.001) increases were observed with the same pterostilbene concentrations (Fig. 2). This assay was performed 18 h posttreatment to optimize measurement of the initial cell death mechanism after pterostilbene treatment.
Pterostilbene Up-Regulates Effector Caspases

A caspase activity assay was performed to determine the nature of apoptosis induction by pterostilbene. Specically, the effector caspases 3 and 7 were targeted

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FIG. 2. Pterostilbene and nucleosome release.

FIG. 1. Pterostilbene inhibition of lung cancer growth.

with this test. Treatment with pterostilbene resulted in a signicant increase in caspase 3/7 activity as compared to controls in both NCI-H460 (5.1-fold increase at 36 h, P < 0.05) and SK-MES-1cells (1.5-fold increase at 24 h, P < 0.05) (Fig. 3.) Specic time points were chosen from the data as the greatest differences were observed at these intervals.
DISCUSSION

cancer cells [7]. Other related stilbenoid compounds have shown similar activity against lung cancer cells, although pterostilbene has not been evaluated previously [8, 9]. Pterostilbene is a stilbenoid compound that is similar to resveratrol in structure and function. Remsberg et al. demonstrated that this compound has signicant antiinammatory, antioxidant, anti-tumor and analgesic properties [5]. Rimando et al. found that this analog has similar antioxidant potency to resveratrol [6]. Additionally, these studies conrmed anti-neoplastic effects of pterostilbene on melanoma, gastric, hepatocelluar, and breast carcinomas in vitro. Our study demonstrates for the rst time that pterostilbene is an effective anti-proliferative agent against

Lung cancer continues to be the leading cause of cancer-related mortality in the United States, with over 160,000 new deaths estimated for 2008. Additionally, the American Cancer Society predicts that there will be over 200,000 new cases diagnosed this year [1]. Treatment for lung cancer can involve surgery, radiation and chemotherapy depending on the stage and type of cancer. Despite advances in radiation and chemotherapy, the 5-y overall survival for lung cancer remains poor. Resveratrol is a naturally occurring stilbene found in red wine and grapes. This compound has demonstrated direct anti-neoplastic effects against lung cancer cells in vitro [3, 4]. In addition, Liao et al. found that resveratrol treatment enhanced the radiosensitivity of lung

FIG. 3. Pterostilbene and caspase 3/7 activity.

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lung cancer. Our data suggest that the mechanism for growth inhibition may be through increased apoptosis as indicated by an increase in released nucleosomes. To test this hypothesis, we analyzed the activity of caspases 3 and 7. These are enzymes involved in the effector phase of apoptosis. Both cell lines demonstrated a signicant increase in activity of caspases 3 and 7, thus supporting that pterostilbene inhibits lung cancer growth via induction of apoptosis. Our results are similar to those of Pan et al. that showed an increase in caspase-dependent apoptosis in gastric cancer cells [10]. This may, in part, explain the anti-neoplastic effects of pterostilbene. However, Tolomeo et al. had conicting ndings when investigating pterostilbene in leukemia cells [11]. Their group could not demonstrate inhibition of pterostilbene-mediated apoptosis when leukemia cells were exposed to both pterostilbene and a pancaspase inhibitor. Hence, pterostilbenes mechanism of action could include both caspase-dependent and independent pathways. Indeed, Pan et al. found that gastric cancer cells underwent a G1 phase cell cycle arrest with disruption of the mitochondrial membrane. Our laboratory has also shown mitochondrial membrane disruption in pancreatic cancer cells treated with pterostilbene [12]. Consequently, alternative pathways that affect cell cycle progression and mitochondrial membrane stability could be inuenced by pterostilbene as well. Further investigation into the biochemical pathways of pterostilbenemediated apoptosis is warranted. Our study indicates that pterostilbene is effective in vitro against human large and squamous lung cancer cells. However, we did not investigate its effects on normal human lung cells. Previously, Tolomeo et al. demonstrated that pterostilbene was not cytotoxic to hematopoietic cells [11]. Similarly, Pan et al. have found pterostilbene to lack noxious effects on polymorphonucelar leukocytes [10]. Further evidence to support the idea that pterostilbene does not harm normal cells is the recent study by Ruiz et al. [13]. In this investigation, mice were fed pterostilbene enriched diets with supplement doses ranging from 0 to 3000 mg per kg body weight for 1 mo. There were no differences in biochemical markers, including blood counts and liver function tests, nor histopathologic examination of liver, heart, brain, kidneys, pancreas, and, importantly, lungs, at the time of necropsy. Accordingly, we believe that it is unlikely that pterostilbene would be cytotoxic to normal lung cells, but intend to conrm this with further analysis. In addition, we intend to study further histologic types of lung cancer, particularly adenocarcinoma, as it is one of the most prevalent [14]. Lastly, we would like to investigate the action of pterostilbene on in vivo lung tumors. With our in vitro results, we believe that pterostilbene would be effective

against lung tumors in spontaneous, carcinogeninduced, or orthotopic murine models. Suh et al. have previously demonstrated suppression of aberrant crypt foci in rats using an azoxymethane-induced colon carcinogenesis model, demonstrating that pterostilbene can suppress precancerous lesions in vivo [15]. Thus, investigation in a spontaneous murine lung cancer model could lead to the use of pterostilbene in a chemoprevention role for at-risk patients.
CONCLUSIONS

We have demonstrated for the rst time that pterostilbene is an effective anti-proliferative agent against lung cancer cell lines. We have shown that one mechanism for this effect is the induction of caspase-dependent apoptosis. The presence of this compound in safe, natural foods makes it an attractive therapy for the treatment or prevention of lung cancer. The results reported here encourage further investigation into determining the predominant apoptotic pathway, extrinsic or intrinsic, and specic biochemical signals by which pterostilbene produces increases in caspase-dependent apoptosis. Further understanding of these pathways will allow researchers to develop in vivo investigations, and bring us closer to clinical applications.
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JOURNAL OF SURGICAL RESEARCH: VOL. 161, NO. 1, JUNE 1, 2010 13. Ruiz MJ, Fernandez M, Pico Y, et al. Dietary administration of high doses of pterostilbene and quercetin to mice is not toxic. J Agric Food Chem 2009;57:3180. 14. Bryant A, Cerfolio RJ. Differences in the epidemiology, histology and survival between cigarette smokers and never smokers who develop non-small-cell lung cancer. Chest 2007;132:185. 15. Suh N, Paul S, Hao X, et al. Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats. Clin Cancer Res 2007;13:350.

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