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Pterostilbene induce autophagy on human oral cancer cells through modulation of Akt and mitogen-activated protein kinase pathway
Oral Oncology 51 (2015) 593–601
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Pterostilbene induce autophagy on human oral cancer cells through modulation of Akt and mitogen-activated protein kinase pathway Chung-Po Ko a,b, Chiao-Wen Lin c,d, Mu-Kuan Chen e, Shun-Fa Yang b,f, Hui-Ling Chiou g,h,⇑, Ming-Ju Hsieh b,i,j,⇑ a
Department of neurosurgery, Tungs’ Taichung Metro Harbor Hospital, Taichung, Taiwan Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan Institute of Oral Sciences, Chung Shan Medical University, Taichung 40201, Taiwan d Department of Dentistry, Chung Shan Medical University Hospital, Taichung 40201, Taiwan e Department of Otorhinolaryngology-Head and Neck Surgery, Changhua Christian Hospital, Changhua 500, Taiwan f Department of Medical Research, Chung Shan Medical University Hospital, Taichung 40201, Taiwan g School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan h Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan i Cancer Research Center, Changhua Christian Hospital, Changhua 500, Taiwan j School of Optometry, Chung Shan Medical University, Taichung 40201, Taiwan b c
a r t i c l e
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Article history: Received 10 November 2014 Received in revised form 28 January 2015 Accepted 19 March 2015 Available online 14 April 2015 Keywords: Pterostilbene Phytoalexin Oral cancer Apoptosis Autophagy MAPK
s u m m a r y Objectives: Extensive research supports the administration of herbal medicines or natural foods during cancer therapy. Pterostilbene, a naturally occurring phytoalexin, has various pharmacological activities, including antioxidant activity, cancer prevention activity, and cytotoxicity to many cancers. However, the effect of pterostilbene on the autophagy of tumor cells has not been clarified. Materials and methods: In this study, the unique effects of pterostilbene on the autophagy of human oral cancer cells were investigated. Results: The results of this study showed that pterostilbene effectively inhibited the growth of human oral cancer cells by inducing cell cycle arrest and apoptosis. In addition, the formation of acidic vesicular organelles and LC3-II production also demonstrated that pterostilbene induced autophagy. Administering 3-methylamphetamine (3-MA) and bafilomycin A1 (BafA1) exerted differing effects on the pterostilbeneinduced death of human oral cancer cells. Pterostilbene-induced autophagy was triggered by activation of JNK1/2 and inhibition of Akt, ERK1/2, and p38. Conclusion: In conclusion, this study demonstrated that pterostilbene caused autophagy and apoptosis in human oral cancer cells, suggesting that pterostilbene could serve as a new and promising agent for treating human oral cancer. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, and has a poor prognosis and low survival rate [1]. Chemotherapy is suggested to treat advanced-stage cancers. Many vegetables, fruits, and grains offer substantial protection against ⇑ Corresponding authors at: School of Medical Laboratory and Biotechnology, Chung Shan Medical University, 110, Section 1, Chien-Kuo N. Road, Taichung 402, Taiwan, ROC. Tel.: +886 4 24730022x12423; fax: +886 4 23248171 (H.-L. Chiou). Cancer Research Center, Changhua Christian Hospital, Changhua, 135 Nanhsiao St, Changhua, Taiwan ROC. Tel.: +886 4 7238595x4881; fax: +886 4 7232942 (M.-J. Hsieh). E-mail addresses: [email protected] (H.-L. Chiou), [email protected] (M.-J. Hsieh). http://dx.doi.org/10.1016/j.oraloncology.2015.03.007 1368-8375/Ó 2015 Elsevier Ltd. All rights reserved.
various cancers [2–4]. There is increasing focus on providing a scientific basis for using these agents as a preventive strategy for people with a high risk of cancer. Therefore, inducing the death of cancer cells using chemotherapeutic agents may help to achieve a successful chemotherapy in patients. Pterostilbene (trans-3,5-dimethoxy-40-hydroxystilbene) is a naturally occurring phytoalexin that has been identified in several plants of the genus Pterocarpus, in Vitis vinifera leaves, in certain berries (e.g. blueberries and cranberries), and in grapes [5]. Pterostilbene, an analog of the well-studied resveratrol, has diverse pharmacological activities including antioxidant activity, cancer prevention activity [5], and the ability to inhibit DNA synthesis [6]. It has also been demonstrated to be cytotoxic to several cancer cell lines in vitro, including breast, skin, colon, liver, and gastric
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cancer cells [5,7,8]. Although pterostilbene exerts antiproliferative and proapoptotic activities in various cancer cell types, the underlying mechanisms of autophagy remain unclear. Autophagy is a major intracellular degradation mechanism that operates under stress conditions to promote survival during starvation or to cause programmed type II cell death under specific conditions (e.g. apoptosis inhibition) [9–11]. Autophagy is initiated when large sections of cytoplasm are engulfed by a crescentshaped phagophore that elongates to form an autophagosome, which subsequently fuses with a lysosome, resulting in the degradation of cellular content by lysosomal hydrolases [12,13]. Because autophagy is vital for regulating growth and maintaining homeostasis, defective autophagy contributes to the pathogenesis of various diseases including certain cancer [14]. Recently, extensive attention has been paid to the role of autophagy in cancer development and therapy [15–17]. The relationship between apoptosis and autophagy is complex, and varies according to cell types and stressors. Certain agents kill cancer cells through non-apoptotic pathways, and may circumvent chemoresistance. Such agents may be promising for treating resistant cancers [18]. For this reason, tumor cells with apoptosis defects undergo autophagy, so inhibiting autophagy causes tumor cells to die through alternative mechanisms [13]. It has also been suggested that autophagy may provide a useful method to prevent cancer development, limit tumor progression, and enhance the efficacy of cancer treatments [12]. The aim of this study was to characterize the effects of pterostilbene on human lung oral cancer cells, and elucidate the underlying molecular mechanism responsible for autophagy and apoptosis in pterostilbene-induced cytotoxicity. Experimental details Cell lines The SAS and OECM-1 human oral cancer cell lines were purchased from ATCC (ATCC: American Type Culture Collection, Manassas, VA, USA). SAS cells were cultured in Dulbecco’s modified Eagle’s medium-F12 supplemented with 10% fetal bovine serum (FBS), 1% NEAA, 1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4), hydrocrostine (0.4 mg/L) and 1 mM sodium pyruvate (Sigma, St. Louis, Mo, USA). OECM-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4) and 1 mM sodium pyruvate (Sigma, St. Louis, Mo, USA). The cells culture were maintained at 37 °C in a humidified atmosphere of 5% CO2. Antibodies and other reagents Pterostilbene of P98% (HPLC) purity was purchased from Enzo Life Sciences. Stock solution of pterostilbene was made at 10, 20 and 40 mM concentration in dimethyl sulfoxide (DMSO) and stored at 20 °C. The final concentration of DMSO for all treatments was less than 0.1%. Other chemicals, were obtained from Sigma Chemical Co. Specific caspase inhibitors for caspase 3 (Z-DEVEFMK), caspase 8 (Z-IETD-FMK) or caspase 9 (Z-LEHO-FMK) were purchased from BioVision (Mountain View, CA). The FITC Annexin V Apoptosis Detection Kit I was obtained from BD Biosciences and Millipore, USA. Antibodies were obtained from Cell Signaling. Cell cytotoxicity As previously described [2]. Briefly, cells were cultured in 96well plates and stimulated with different concentrations of
pterostilbene. After 24 or 48 h, MTT was added to each well and incubated for further 4 h. The viable cell number was directly proportional to the production of formazan, reflected by the color intensity measured at 595 nm, following the solubilization with isopropanol. DAPI staining After being subjected to indicate treatment, cells were fixed with 4% paraformaldehyde for 20 min. Extensive PBS washing was conducted between each reaction to remove any residual solvent. Cells were subjected to DAPI staining for 15 min and then observed under fluorescence microscopy equipped with filters for UV. Cell cycle analysis Cells were first cultured in serum-free medium for starvation at 18 h and then exposed to pterostilbene for 24 h. After cells were washed, fixed with 70% ethanol, and incubated for 30 min in the dark at room temperature with propidium iodide (PI) buffer. The cell cycle distribution was analyzed by a FACS Vantage flow cytometer that uses the Cellquest acquisition and analysis program. Annexin V/PI double staining Briefly, cells were resuspended in 100 ll 1 binding buffer. With an addition of 5 ll of FITC Annexin V and 5 ll PI, the cell suspension was gently mixed and then incubated for 15 min at room temperature in the dark. Afterward, 400 ll of 1 binding buffer was added to each tube followed by flow cytometry analysis within 1 h. Cell transfection Cells were grown on 6-well cell culture dish overnight and then transfected with 2 lg of pEGFPC1-LC3 [12] for 6 h, followed by an indicated treatment. Afterward, cells were fixed with 4% paraformaldehyde for 30 min and then incubated with 0.1% Triton X-100 for 10 min. The dot formation of GFP-LC3 was detected under a fluorescence microscope after drug treatment. Western blot analysis Cell lysates were separated in a polyacrylamide gel and transferred onto a PVDF membrane. The blot was subsequently incubated with 3% non-fat milk in PBS for 1 h and probed with a corresponding antibody against a specific protein for overnight at 4 °C, and then with an appropriate peroxidase conjugated secondary antibody for 1 h. Signal was developed by ECL detection system and relative photographic density was quantitated by a gel documentation and analysis system. Detection and quantification of acidic vesicular organelle (AVO) formation Briefly, pterostilbene-treated cells were washed with PBS, followed by staining with 1 lg/ml acridine orange for 30 min. Afterward, cells were washed with PBS and then observed under fluorescence microscopy equipped with filters for 488 nm. For quantification of AVOs, acridine orange-stained cells were harvested, washed twice with PBS, resuspended in PBS containing 5% FBS and then analyzed by flow cytometry.
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Statistical analysis Statistically significant differences were calculated using the Student’s t-test (Sigma-Stat 2.0, Jandel Scientific, San Rafael, CA, USA). A P value <0.05 was considered to be statistically significant. Values represent the means ± standard deviation and the experiments were repeated three times (n = 3). Results Cytotoxic effects of pterostilbene on human oral cancer cell lines The chemical structure of pterostilbene is shown in Fig. 1A. To assess the effects of pterostilbene on cell viability, SAS and OECM-1 cells were treated with pterostilbene at various concentrations (0–40 lM) for 24 and 48 h, and then analyzed using the MTT assay. Pterostilbene substantially decreased the cell viability of SAS and OECM-1 cells, compared with untreated cells (Fig. 1B).
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Pterostilbene (40 lM) also decreased the growth rate of SAS and OECM-1 cells in a time-dependent manner, compared with untreated cells (Fig. 1C). Pterostilbene induced cell cycle arrest and apoptosis in human oral cancer cell lines Treatment with pterostilbene for 24 h induced an increase in the G0/G1 and S phase, and a decrease in the G2/M phase, compared with the controls (Fig. 2A). The results indicated that pterostilbene delayed cell cycle progression in both cell types. Therefore, pterostilbene-induced cell cycle arrest likely contributed to the overall growth suppression in SAS and OECM-1 cells. To determine whether the pterostilbene-induced cell death was related to apoptosis, DAPI staining was performed to analyze the changes in nuclear morphology. The results revealed the condensed and fragmented nuclei in both cell types after 24 h of pterostilbene (40 lM) treatment (Fig. 2B). Annexin V/PI double staining was also
Fig. 1. Pterostilbene inhibits cell proliferation in SAS and OECM-1 cell lines. (A) Structure of pterostilbene. (B) Cell viability of SAS and OECM-1 cells cultured in presence of pterostilbene (0–40 lM) for 24 and 48 h, as analyzed by MTT assay. (C) Cell viability of SAS and OECM-1 cells cultured in presence of pterostilbene (40 lM) for 6, 12 and 24 h, as analyzed by MTT assay. Results are shown as mean ± SD from 3 determinations per condition repeated 3 times. ⁄⁄⁄P < 0.001, compared with the control (0 lM or 0 h).
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Fig. 2. Pterostilbene inhibits cell cycle and induce apoptosis in SAS and OECM-1 cell lines. (A) SAS and OECM-1 cells were incubated for 18 h in the absence of serum and then treated with indicated concentrations of pterostilbene (40 lM), after which the cells were stained with PI, and analyzed for DNA content by flow cytometry. (B) Cells were treated with different concentration of pterostilbene (0–40 lM) for 24 h were and then subjected to DAPI staining for DNA (blue areas) by fluorescence microscopy. (C) SAS and OECM-1 cells treated with pterostilbene (40 lM) for 24 h. For quantitative analysis of apoptosis, pterostilbene-treated cells were harvested and then subjected to Annexin-V and PI double-stained flow cytometry. (D) Cells were treated with an indicated concentration of pterostilbene for 24 h and then analyzed by western blotting with an antibody against PARP, caspase-3, caspase-8 and caspase-9. b-actin as an internal control. (E) Furthermore, Cell were treated with 40 lM pterostilbene for 24 h in the presence or absence of 2 lM Z-DEVE-FMK, 20 lM Z-IETD-FMK, and 20 lM Z-LEHD-FMK and then subjected to MTT assay for cell viability. Results are shown as mean ± SD from 3 determinations per condition repeated 3 times. ⁄⁄⁄P < 0.001, compared with the untreated cells. ##P < 0.01, compared with the pterostilbene treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
determined using flow cytometry, and the results showed an increase in the percentage of cells displaying phosphatidylserine (PS) externalization in pterostilbene-treated SAS and OECM-1 cells (Fig. 2C). To investigate the underlying mechanism involved in pterostilbene-induced apoptosis. The results indicated that after a 24-h treatment with pterostilbene, the levels of cleaved PARP increased, whereas the cleaved-form caspase-3, -8, and -9 increased (Fig. 2D). To further confirm the involvement of caspase activation in pterostilbene-induced apoptosis, caspase-specific inhibitors were used. The results shown in Fig. 2E indicate that pretreatment with caspase-3, -8, and -9 specific inhibitors effectively attenuated pterostilbene-induced cell death. These data suggested that pterostilbene-induced apoptosis was dependent on the activation of caspase-3, -8, and -9.
Pterostilbene induced autophagy in human oral cancer cell lines Previous data has shown that pterostilbene causes autophagy in various cancer cells, including bladder cancer cells, breast cancer cells, and MOLT4 human leukemia cells [19]. In this study, various numbers of vacuoles were observed in the cytoplasm 24 h after pterostilbene (20 lM and 40 lM) treatment (Fig. 3A). Therefore, we hypothesized that other mechanisms mediated the response to pterostilbene. The formation of vacuoles in pterostilbene-treated cells was similar to that during cell autophagy, a general phenomenon that occurs when cells respond to stress [20]. We assessed whether pterostilbene induced autophagy in pterostilbene-induced human oral cancer cells. As shown in Fig. 3B, after transfection of pEGFPC1-LC3 and treatment with pterostilbene for 24 h, cytoplasmic LC3 formation was observed, indicating the
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Fig. 3. Pterostilbene induce autophagy in SAS and OECM-1 cells. (A) SAS and OECM-1 cells were treated with 40 lM pterostilbene for 24 h and microscopic observation revealed the formation vacuoles in cytoplasms of treated cells. (B) After a successful transfection with pEGFPC1-LC3, SAS and OECM-1 cells were treated with pterostilbene of 40 lM for 24 h, followed by an observation for LC-3 (green fluorescence) under fluorescence microscopy. (C) Quantitation of EGFP-LC3 puncta formation. Cells with EGFP-LC3 puncta formation were quantitated under a fluorescence confocal microscope. At least 200 cells on each slide were counted and the percentages of EGFP-LC3 puncta containing cells were calculated. The data shown are the mean ± SD of three individual experiments. (D) SAS and OECM-1 cells were treated with different concentration of pterostilbene (0–40 lM) for 24 h and stained by acridine orange to detect AVOs under fluorescence microscopy. (E) The percentage of cells with the formation of AVOs was detected by flow cytometry in a concentration and (F) time-dependent manner. Results are shown as mean ± SD. ⁄⁄⁄P < 0.001, compared with the SAS control (0 lM). ### P < 0.001, compared with the OECM-1 control (0 lM). (G) SAS and OECM-1 cells were treated with an indicated concentration of pterostilbene for 24 h and then analyzed by western blotting with an antibody against LC3-I/II and Beclin-1. b-actin as an internal control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
formation of autophagosomes in both cell lines treated with pterostilbene (40 lM). A substantial change in LC3 puncta formation was observed 24 h after treatment with 40 lM of pterostilbene (Fig. 3C). Pterostilbene caused induction of acidic vesicular organelles (AVOs) in SAS and OECM-1 cells in a concentration(Fig. 3D and E) and time-dependent manner (Fig. 3F). To further determine whether pterostilbene induced autophagy, the protein expression of LC3-II and Beclin-1 was analyzed. Compared with the control, the protein expression of LC3-II and Beclin-1 increased for both cells treated with pterostilbene (40 lM) for 24 h (Fig. 3G). These findings, combined with the previous results, showed that autophagy was induced by pterostilbene. Pterostilbene-induced cell death by treatment with autophagy inhibitors in human oral cancer cell lines To elucidate the interaction between pterostilbene-induced apoptosis and autophagy. SAS and OECM-1 cells were pre-treated
with 3-MA for 1 h before pterostilbene treatment; apoptosis-related molecules were examined using Western blotting. The results displayed in Fig. 4A indicated that adding 3-MA combined with pterostilbene to SAS and OECM-1 cells resulted in a slight increase in the levels of cleaved-form caspase-3, -8 and -9. To assess the effects of pterostilbene combined with autophagy inhibitors on cell viability, SAS and OECM-1 cells were treated with pterostilbene at various concentrations (0–40 lM), combined with 3-MA, for 24 h, and then analyzed using the MTT assay. As shown in Fig. 4B, pterostilbene combined with 3-MA substantially increased the cell viability of SAS and OECM-1 cells. Furthermore, after treatment with the pterostilbene/3-MA combination, the percentage of Annexin V-positive cells in the SAS and OECM-1 group were no significantly effected (Fig. 4C). In addition, similar results concerning cell viability and Annexin V-positive cells were observed after combined treatment with pterostilbene and BafA1 (Fig. 4D and E). Furthermore, a co-treatment with pterostilbene and Z-VAD-FMK (a broad-spectrum caspase inhibitor) was
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Fig. 4. Effects of autophagy inhibitors on pterostilbene-induced cell death. (A) SAS and OECM-1 cells were treated with pterostilbene of 40 lM for 24 h in the presence or absence of autophagy inhibitor 3-MA of 5 mM and then subjected to western blotting for caspase-3, caspase-8 and caspase-9 and b-actin. (B) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor 3-MA (5 mM), as analyzed by MTT assay. (C) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor 3-MA (5 mM), these treated cells were double-stained with Annexin-V and PI and then analyzed by flow cytometry. (D) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor BafA1 (10 nM), as analyzed by MTT assay. (E) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor BafA1 (10 nM), these treated cells were doublestained with Annexin-V and PI and then analyzed by flow cytometry. (F) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor 3-MA (5 mM) or Z-VAD-FMK (20 lM) and analyzed by MTT assay. (G) SAS and OECM-1 cells were treated with pterostilbene (40 lM) in the presence or absence of autophagy inducer rapamycin (100 nM), as analyzed by MTT assay. (H) SAS and OECM-1 cells were treated with pterostilbene (40 lM) in the presence or absence of autophagy inducer rapamycin (100 nM), these treated cells were double-stained with Annexin-V and PI and then analyzed by Muse Cell Analyzer flow cytometry and analysis data by the MuseÒ Cell Analyzer Assays (Millipore) (upper plane). Quantitative percentage of Annexin-V positive cells was calculated (under plane). Results are shown as mean ± SD. ⁄⁄⁄P < 0.001, compared with the SAS control (0 lM). $$$P < 0.001, compared with the OECM-1 control (0 lM). ###P < 0.001, compared with the SAS treated with pterostilbene (40 lM). &P < 0.05; &&&P < 0.001, compared with the OECM-1 treated with pterostilbene (40 lM).
conducted to show that the pterostilbene-induced reduction of the percentage of cell viability was prevented by Z-VAD-FMK. Nevertheless, cell viability in SAS and OECM-1 cells were increased when 3-MA was included (Fig. 4F). To assess the effects of pterostilbene combined with autophagy inhibitors on cell viability, SAS and OECM-1 cells were also treated with pterostilbene (40 lM), combined with rapamycin (mTOR inhibitor) for 24 h, and then analyzed for cell viability and the percentage of Annexin V-positive cells. As shown in Fig. 4G, pterostilbene combined with rapamycin substantially decreased the cell viability of SAS and OECM-1 cells. However, the percentage of Annexin V-positive cells was not significantly affected (Fig. 4H). Therefore, these results indicated that autophagy played differing roles in pterostilbene-induced SAS and OECM-1 cell death. Pterostilbene induced autophagy through inhibition of Akt, ERK1/2, p38 and upregulated JNK1/2 activation in human oral cancer cell lines To investigate the mechanism of pterostilbene-induced autophagy in human oral cancer cell lines. The results showed that pterostilbene treatment resulted in the activation of JNK1/2, which increased in a dose-dependent manner. By contrast, treatment with pterostilbene decreased the level of phosphorylated p38, Akt, and ERK1/2 in both cell lines (Fig. 5A and B). To elucidate whether Akt, p38 or ERK1/2 inhibition, or JNK1/2 activation, contributed to autophagy, LY294002, SB203580, U0126, and SP600125 were added. As shown in Fig. 5C, the AVOs resulting
from pterostilbene treatment and pterostilbene treatment with LY294002, SB203580, and U0126 increased considerably. The JNK1/2 inhibitor (SP600125) was used to further confirm the role of the JNK1/2 pathway in pterostilbene-induced autophagy. The expression levels of the AVOs for pterostilbene treatment with SP600125 decreased considerably compared with those of pterostilbene treatment alone. These results indicated that pterostilbeneinduced autophagy was mediated by inhibition of Akt p38, and ERK1/2, as well as activation of JNK1/2. We also investigated the role of p38, Akt, ERK1/2, and JNK1/2 in pterostilbene-induced cell viability. SAS and OECM-1 cells pretreated with LY294002, SB203580, or U0126 exhibited a decreased cell viability compared with those treated only with pterostilbene. The cell viability of SAS cells pretreated with SP600125 increased. However, pretreatment of OECM-1 cells with SP600125 effectively inhibited cell viability, compared with treatment using pterostilbene alone (Fig. 5D).
Pterostilbene induced the activation of AMPK, Raptor, ULK and downregulated mTOR activation in human oral cancer cell lines To further investigate the mechanism of pterostilbene-induced autophagy in human oral cancer cell lines, the expression levels of the phosphorylated forms of AMPK, Raptor, ULK and mTOR were examined using Western blotting. The results showed that pterostilbene treatment resulted in the activation of AMPK, Raptor and ULK at Ser555 in a dose-dependent manner. By contrast, the level
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Fig. 5. Pterostilbene induces autophagy through inhibition of the Akt, ERK1/2, p38 and activation of the JNK1/2 in SAS and OECM-1 cells. (A) Cells were treated with different concentrations of pterostilbene (0–40 lM) for 24 h and then subjected to western blotting. The levels of phosphorylation of Akt, ERK1/2, p38 and JNK1/2 were investigated by western blotting with b-actin being used as an internal control. (B) Quantitative results of Akt, ERK1/2 and JNK1/2 and p38 protein level which were adjusted with b-actin protein level. Results are shown as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001, compared with the control (0 lM). (C) SAS and OECM-1 cells were pretreated with LY294002 (20 lM), SP600125 (20 lM), SB203580 (20 lM) or U0126 (10 lM) for 1 h followed by treatment with pterostilbene (40 lM) for 24 h and stained by acridine orange to detect AVOs. Measurement of AVOs was detected by flow cytometry. (D) SAS and OECM-1 cells were pretreated with LY294002 (20 lM), SP600125 (20 lM), SB203580 (20 lM) or U0126 (10 lM) for 1 h followed by treatment with pterostilbene (40 lM) for 24 h and analyzed by MTT assay.
of phosphorylated ULK at Ser757 was decreased in both cell lines (Fig. 6A and B). Discussion Chemotherapy is suggested for treatment of advanced-stage cancers. However, the ability of cancer cells to become resistant to various drugs limits the efficiency of chemotherapy [21]. Natural products, which have been used traditionally in various ethnic societies worldwide, are crucial for the development of novel chemotherapeutics. Pterostilbene has been reported to inhibit herbicide-induced oxidative damage, inducing apoptosis in sensitive and chemoresistant lymphoma cell lines through the caspase-independent pathway, and is also known to act against B16 melanoma growth and metastatic activity [5,7,8]. The results of this study showed that pterostilbene induced the death of SAS and OECM-1 human oral cancer cells by arresting their cell cycle. Pterostilbene-induced apoptosis of oral cancer cells was dependent on the activation of caspase-3, -8, and -9 (Figs. 1 and 2). These results were consistent with other reports showing that pterostilbene induced a G0/G1 arrest in BCR-ABL-expressing leukemia cells [22]. Pan et al. showed that pterostilbene exerted growth inhibition by inducing apoptosis and arresting gastric cancer cells in the G0/G1 phase. Caspase-2, -3, -8, and -9 are all activated by pterostilbene [23]. In MOLT4 human leukemia cells, pterostilbene inhibited cell proliferation, resulting in a transient accumulation of cells in the G0/G1 cell cycle phase, followed by arrest in the S-phase, and induced apoptosis by caspase-3 activation [19]. Autophagy is a key cellular response for various environmental stimuli and diseases, including cancers [24–26]. This form of cell death was defined as autophagic programmed cell death [27]. Many anticancer agents, including sulforaphane, rapamycin, arsenic trioxide, and temozolomide were reported to induce autophagy [28,29]. Autophagy has attracted the interest of scientists in the cancer therapy field because drug-resistant cancer cells with antiapoptotic properties may be vulnerable to death by autophagy
[30,31]. Although pterostilbene exerts antiproliferative and proapoptotic activities in various cancer cell types, the underlying mechanisms of autophagy remain unclear. Furthermore, experiments conducted in other types of cancer cells may provide additional data to identify the signaling pathways involved in pterostilbene-induced autophagy. Thus, because of its capability to induce both the apoptotic and autophagic pathways, pterostilbene may be an effective multidrug resistance agent for antitumor treatment. Our results showed that pterostilbene resulted in autophagy after 24 h of administration. Pterostilbene was added to the culture medium, and the expression of LC3-II and AVOs indicated that the induction of autophagy was dose- and time-dependent (Fig. 3). The most substantial expression of the autophagy process was observed 24 h after pterostilbene (40 lM) treatment, whereas a slight expression of LC3-II was observed 24 h after pterostilbene (20 lM) treatment (Fig. 3G). However, treatment with pterostilbene (20 lM) for 24 h did not result in any activation of caspase-3, -8, or -9. These results suggested that the pterostilbene-induced autophagy process occurred as early as apoptosis. The autophagy inhibitor 3-MA can block autophagosome formation by inhibiting the type III PI-3K that prevents early-stage autophagy [25], as well as BafA1, an inhibitor of vacuolar H+-ATPase that prevents late-stage autophagy by inhibiting fusion of autophagosomes and lysosomes [26]. To further investigate the role of autophagy in pterostilbene-induced cell death, we combined pterostilbene treatment with an autophagy inhibitor, and analyzed cell viability and activation of caspases. The results indicated that the presence of 3-MA in SAS and OECM-1 cells resulted in a slight increase in the levels of cleaved-form caspase-3, -8 and -9 (Fig. 4A). In addition, inhibition of autophagy by 3-MA increased the cell viability of the SAS and OECM-1cell line (Fig. 4B and C). Similarly, accumulation of abnormal autophagosomes by BafA1 treatment of SAS and OECM-1 cells reduced cell death (Fig. 4D and E). Although pterostilbene could promote a cell arrest phenotype, but it is unclear whether the residual cells become dormant and more resistant to other common adjuvant therapeutic agents, such as mTOR inhibitors, HDAC inhibitors and cisplatin.
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Fig. 6. Pterostilbene induce activation of the AMPK, Raptor, ULK and inhibition of mTOR in SAS and OECM-1 cells. (A) Cells were treated with different concentrations of pterostilbene (0–40 lM) for 24 h. The phosphorylation levels of AMPK, Raptor, mTOR and ULK were investigated by western blotting with b-actin being used as an internal control. (B) Quantitative results of phospho-AMPK, phospho-Raptor, phospho-mTOR and phospho-ULK protein levels were adjusted with b-actin protein level. Results are shown as mean ± SD. ⁄P < 0.05, compared with the control (0 lM).
Especially considering it is also an autophagy-promoting agent, the potential impact on increasing cancer cell resistance needs to be tested. The results indicated that pterostilbene combined with rapamycin substantially decreased the cell viability of SAS and OECM-1 cells without significant change in the percentage of Annexin V-positive cells (Fig. 4G and H). Ellington et al. showed that soybean B-group triterpenoid saponins induced autophagy by inhibiting Akt and activating ERK activity [32]. The present study clearly demonstrated that pterostilbene inhibited Akt, ERK1/2, and p38, and activated the JNK1/2 pathway to promote autophagy. In both cell lines, blocking Akt, p38 or ERK1/2 with LY294002, SB203580 or U0126 enhanced the expression of AVOs by pterostilbene. These findings may highlight the common mechanisms (such as Akt inhibition and ERK1/2 activation) that regulate autophagy in cancer cells treated with anticancer agents. We suggested that inhibiting JNK1/2 activation using SP600125 could prevent the induction of AVOs by pterostilbene. For both cell lines, cell death increased in the LY294002, SB203580, and U0126 groups; however, inhibiting JNK1/2 activation with SP600125 caused different viability effects in both cell lines. This further confirmed that pterostilbene-induced autophagy had different mechanisms in SAS and OECM-1 cell lines (Fig. 5D). During low nutrient conditions, AMPK directly phosphorylates ULK1 at multiple sites including Ser317, Ser555, and Ser777 [33,34]. Conversely, mTOR, a regulator of cell growth and an inhibitor of autophagy, phosphorylates ULK1 at Ser757 and disrupts the interaction between ULK1 and AMPK [35]. Phosphorylation of
ULK1 by AMPK at Ser555 is critical for starvation-induced autophagy, cell survival under conditions of low nutrients and energy, and mitochondrial homeostasis [33]. AMPK phosphorylates raptor on Ser722/Ser792. This phosphorylation is essential for the inhibition of the raptor-containing mTOR complex 1 (mTORC1) and induces cell cycle arrest when cells are stressed for energy [36]. In this study, it was clearly demonstrated that pterostilbene induced phosphorylation of AMPK, Raptor and ULK at Ser555, and inhibited the phosphorylation of mTOR and ULK at Ser757 (Fig 6). Autophagy is suppressed by functional p53 and certain cytoplasmic p53 mutants. Tasdemir et al. reported that cytoplasmic p53 suppressed autophagy, and p53 inhibition induced autophagy [33]. Furthermore, nuclear expression of p53 may stimulate autophagy by DRAM upregulation and mTOR inhibition [34], suggesting that p53 exerts an opposite effect on autophagy regulation. Therefore, pterostilbene-induced autophagy and apoptosis in SAS and OECM-1 cells may be partially attributed to the functional p53, which could be verified by additional experiments exploring the role of p53 in pterostilbene-induced autophagy. Conclusion In conclusion, this was the first study to demonstrate that pterostilbene suppressed cell growth and induced autophagy in SAS and OECM-1 cell lines by inhibiting Akt, p38, ERK1/2, and activating the JNK1/2 pathway. Autophagy was induced in the
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early stage of pterostilbene-induced apoptosis. We also observed that pterostilbene-induced autophagy had a different mechanism in SAS and OECM-1 cell lines. Conflict of interest statement The authors declare that there are no conflicts of interest. Competing Interests No. Author Contributions Performed the revised experiments and analyzed the data. Acknowledgements This study was supported by grants from Ministry of Science and Technology, Taiwan (MOST 103-2314-B-371-007-MY2), Chung Shan Medical University Hospital, Taiwan (CSH-2013-C012) and Changhua Christian Hospital (103-CCH-IRP-071) (103CCH-ICO-006). The authors of the manuscript do not have a direct financial relation with the commercial identity mentioned in this paper. References [1] Spiro RH, Alfonso AE, Farr HW, Strong EW. Cervical node metastasis from epidermoid carcinoma of the oral cavity and oropharynx. A critical assessment of current staging. Am J Surg 1974;128:562–7. [2] Chen PN, Hsieh YS, Chiou HL, Chu SC. Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways. Chem Biol Interact 2005;156:141–50. [3] Huang HP, Shih YW, Chang YC, Hung CN, Wang CJ. Chemoinhibitory effect of mulberry anthocyanins on melanoma metastasis involved in the Ras/PI3K pathway. J Agric Food Chem 2008;56:9286–93. [4] Vieira JR, de Souza IA, do Nascimento SC, Leite SP. Indigofera suffruticosa: an alternative anticancer therapy. Evidence-based Complement Altern Med: eCAM 2007;4:355–9. [5] Rimando AM, Cuendet M, Desmarchelier C, Mehta RG, Pezzuto JM, Duke SO. Cancer chemopreventive and antioxidant activities of pterostilbene, a naturally occurring analogue of resveratrol. J Agric Food Chem 2002;50:3453–7. [6] Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, et al. Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. J Biol Chem 2001;276:22586–94. [7] Ferrer P, Asensi M, Segarra R, Ortega A, Benlloch M, Obrador E, et al. Association between pterostilbene and quercetin inhibits metastatic activity of B16 melanoma. Neoplasia 2005;7:37–47. [8] Remsberg CM, Yanez JA, Ohgami Y, Vega-Villa KR, Rimando AM, Davies NM. Pharmacometrics of pterostilbene: preclinical pharmacokinetics and metabolism, anticancer, antiinflammatory, antioxidant and analgesic activity. Phytother Res: PTR 2008;22:169–79. [9] Cohen D, Higman SM, Hsu SI, Horwitz SB. The involvement of a LINE-1 element in a DNA rearrangement upstream of the mdr1a gene in a taxol multidrugresistant murine cell line. J Biol Chem 1992;267:20248–54. [10] Davies AM, Lara Jr PN, Mack PC, Gandara DR. Docetaxel in non-small cell lung cancer: a review. Expert Opin Pharmacother 2003;4:553–65. [11] Green MR. The evolving role of gemcitabine and pemetrexed (Alimta) in the management of patients with malignant mesothelioma. Clin Lung Cancer 2002;3(Suppl 1):S26–9. [12] Hsieh MJ, Yang SF, Hsieh YS, Chen TY, Chiou HL. Autophagy inhibition enhances apoptosis induced by dioscin in huh7 cells. Evidence-based Complement Altern Med: eCAM 2012;2012:134512.
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Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Pterostilbene induce autophagy on human oral cancer cells through modulation of Akt and mitogen-activated protein kinase pathway Chung-Po Ko a,b, Chiao-Wen Lin c,d, Mu-Kuan Chen e, Shun-Fa Yang b,f, Hui-Ling Chiou g,h,⇑, Ming-Ju Hsieh b,i,j,⇑ a
Department of neurosurgery, Tungs’ Taichung Metro Harbor Hospital, Taichung, Taiwan Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan Institute of Oral Sciences, Chung Shan Medical University, Taichung 40201, Taiwan d Department of Dentistry, Chung Shan Medical University Hospital, Taichung 40201, Taiwan e Department of Otorhinolaryngology-Head and Neck Surgery, Changhua Christian Hospital, Changhua 500, Taiwan f Department of Medical Research, Chung Shan Medical University Hospital, Taichung 40201, Taiwan g School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan h Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan i Cancer Research Center, Changhua Christian Hospital, Changhua 500, Taiwan j School of Optometry, Chung Shan Medical University, Taichung 40201, Taiwan b c
a r t i c l e
i n f o
Article history: Received 10 November 2014 Received in revised form 28 January 2015 Accepted 19 March 2015 Available online 14 April 2015 Keywords: Pterostilbene Phytoalexin Oral cancer Apoptosis Autophagy MAPK
s u m m a r y Objectives: Extensive research supports the administration of herbal medicines or natural foods during cancer therapy. Pterostilbene, a naturally occurring phytoalexin, has various pharmacological activities, including antioxidant activity, cancer prevention activity, and cytotoxicity to many cancers. However, the effect of pterostilbene on the autophagy of tumor cells has not been clarified. Materials and methods: In this study, the unique effects of pterostilbene on the autophagy of human oral cancer cells were investigated. Results: The results of this study showed that pterostilbene effectively inhibited the growth of human oral cancer cells by inducing cell cycle arrest and apoptosis. In addition, the formation of acidic vesicular organelles and LC3-II production also demonstrated that pterostilbene induced autophagy. Administering 3-methylamphetamine (3-MA) and bafilomycin A1 (BafA1) exerted differing effects on the pterostilbeneinduced death of human oral cancer cells. Pterostilbene-induced autophagy was triggered by activation of JNK1/2 and inhibition of Akt, ERK1/2, and p38. Conclusion: In conclusion, this study demonstrated that pterostilbene caused autophagy and apoptosis in human oral cancer cells, suggesting that pterostilbene could serve as a new and promising agent for treating human oral cancer. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, and has a poor prognosis and low survival rate [1]. Chemotherapy is suggested to treat advanced-stage cancers. Many vegetables, fruits, and grains offer substantial protection against ⇑ Corresponding authors at: School of Medical Laboratory and Biotechnology, Chung Shan Medical University, 110, Section 1, Chien-Kuo N. Road, Taichung 402, Taiwan, ROC. Tel.: +886 4 24730022x12423; fax: +886 4 23248171 (H.-L. Chiou). Cancer Research Center, Changhua Christian Hospital, Changhua, 135 Nanhsiao St, Changhua, Taiwan ROC. Tel.: +886 4 7238595x4881; fax: +886 4 7232942 (M.-J. Hsieh). E-mail addresses: [email protected] (H.-L. Chiou), [email protected] (M.-J. Hsieh). http://dx.doi.org/10.1016/j.oraloncology.2015.03.007 1368-8375/Ó 2015 Elsevier Ltd. All rights reserved.
various cancers [2–4]. There is increasing focus on providing a scientific basis for using these agents as a preventive strategy for people with a high risk of cancer. Therefore, inducing the death of cancer cells using chemotherapeutic agents may help to achieve a successful chemotherapy in patients. Pterostilbene (trans-3,5-dimethoxy-40-hydroxystilbene) is a naturally occurring phytoalexin that has been identified in several plants of the genus Pterocarpus, in Vitis vinifera leaves, in certain berries (e.g. blueberries and cranberries), and in grapes [5]. Pterostilbene, an analog of the well-studied resveratrol, has diverse pharmacological activities including antioxidant activity, cancer prevention activity [5], and the ability to inhibit DNA synthesis [6]. It has also been demonstrated to be cytotoxic to several cancer cell lines in vitro, including breast, skin, colon, liver, and gastric
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cancer cells [5,7,8]. Although pterostilbene exerts antiproliferative and proapoptotic activities in various cancer cell types, the underlying mechanisms of autophagy remain unclear. Autophagy is a major intracellular degradation mechanism that operates under stress conditions to promote survival during starvation or to cause programmed type II cell death under specific conditions (e.g. apoptosis inhibition) [9–11]. Autophagy is initiated when large sections of cytoplasm are engulfed by a crescentshaped phagophore that elongates to form an autophagosome, which subsequently fuses with a lysosome, resulting in the degradation of cellular content by lysosomal hydrolases [12,13]. Because autophagy is vital for regulating growth and maintaining homeostasis, defective autophagy contributes to the pathogenesis of various diseases including certain cancer [14]. Recently, extensive attention has been paid to the role of autophagy in cancer development and therapy [15–17]. The relationship between apoptosis and autophagy is complex, and varies according to cell types and stressors. Certain agents kill cancer cells through non-apoptotic pathways, and may circumvent chemoresistance. Such agents may be promising for treating resistant cancers [18]. For this reason, tumor cells with apoptosis defects undergo autophagy, so inhibiting autophagy causes tumor cells to die through alternative mechanisms [13]. It has also been suggested that autophagy may provide a useful method to prevent cancer development, limit tumor progression, and enhance the efficacy of cancer treatments [12]. The aim of this study was to characterize the effects of pterostilbene on human lung oral cancer cells, and elucidate the underlying molecular mechanism responsible for autophagy and apoptosis in pterostilbene-induced cytotoxicity. Experimental details Cell lines The SAS and OECM-1 human oral cancer cell lines were purchased from ATCC (ATCC: American Type Culture Collection, Manassas, VA, USA). SAS cells were cultured in Dulbecco’s modified Eagle’s medium-F12 supplemented with 10% fetal bovine serum (FBS), 1% NEAA, 1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4), hydrocrostine (0.4 mg/L) and 1 mM sodium pyruvate (Sigma, St. Louis, Mo, USA). OECM-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4) and 1 mM sodium pyruvate (Sigma, St. Louis, Mo, USA). The cells culture were maintained at 37 °C in a humidified atmosphere of 5% CO2. Antibodies and other reagents Pterostilbene of P98% (HPLC) purity was purchased from Enzo Life Sciences. Stock solution of pterostilbene was made at 10, 20 and 40 mM concentration in dimethyl sulfoxide (DMSO) and stored at 20 °C. The final concentration of DMSO for all treatments was less than 0.1%. Other chemicals, were obtained from Sigma Chemical Co. Specific caspase inhibitors for caspase 3 (Z-DEVEFMK), caspase 8 (Z-IETD-FMK) or caspase 9 (Z-LEHO-FMK) were purchased from BioVision (Mountain View, CA). The FITC Annexin V Apoptosis Detection Kit I was obtained from BD Biosciences and Millipore, USA. Antibodies were obtained from Cell Signaling. Cell cytotoxicity As previously described [2]. Briefly, cells were cultured in 96well plates and stimulated with different concentrations of
pterostilbene. After 24 or 48 h, MTT was added to each well and incubated for further 4 h. The viable cell number was directly proportional to the production of formazan, reflected by the color intensity measured at 595 nm, following the solubilization with isopropanol. DAPI staining After being subjected to indicate treatment, cells were fixed with 4% paraformaldehyde for 20 min. Extensive PBS washing was conducted between each reaction to remove any residual solvent. Cells were subjected to DAPI staining for 15 min and then observed under fluorescence microscopy equipped with filters for UV. Cell cycle analysis Cells were first cultured in serum-free medium for starvation at 18 h and then exposed to pterostilbene for 24 h. After cells were washed, fixed with 70% ethanol, and incubated for 30 min in the dark at room temperature with propidium iodide (PI) buffer. The cell cycle distribution was analyzed by a FACS Vantage flow cytometer that uses the Cellquest acquisition and analysis program. Annexin V/PI double staining Briefly, cells were resuspended in 100 ll 1 binding buffer. With an addition of 5 ll of FITC Annexin V and 5 ll PI, the cell suspension was gently mixed and then incubated for 15 min at room temperature in the dark. Afterward, 400 ll of 1 binding buffer was added to each tube followed by flow cytometry analysis within 1 h. Cell transfection Cells were grown on 6-well cell culture dish overnight and then transfected with 2 lg of pEGFPC1-LC3 [12] for 6 h, followed by an indicated treatment. Afterward, cells were fixed with 4% paraformaldehyde for 30 min and then incubated with 0.1% Triton X-100 for 10 min. The dot formation of GFP-LC3 was detected under a fluorescence microscope after drug treatment. Western blot analysis Cell lysates were separated in a polyacrylamide gel and transferred onto a PVDF membrane. The blot was subsequently incubated with 3% non-fat milk in PBS for 1 h and probed with a corresponding antibody against a specific protein for overnight at 4 °C, and then with an appropriate peroxidase conjugated secondary antibody for 1 h. Signal was developed by ECL detection system and relative photographic density was quantitated by a gel documentation and analysis system. Detection and quantification of acidic vesicular organelle (AVO) formation Briefly, pterostilbene-treated cells were washed with PBS, followed by staining with 1 lg/ml acridine orange for 30 min. Afterward, cells were washed with PBS and then observed under fluorescence microscopy equipped with filters for 488 nm. For quantification of AVOs, acridine orange-stained cells were harvested, washed twice with PBS, resuspended in PBS containing 5% FBS and then analyzed by flow cytometry.
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Statistical analysis Statistically significant differences were calculated using the Student’s t-test (Sigma-Stat 2.0, Jandel Scientific, San Rafael, CA, USA). A P value <0.05 was considered to be statistically significant. Values represent the means ± standard deviation and the experiments were repeated three times (n = 3). Results Cytotoxic effects of pterostilbene on human oral cancer cell lines The chemical structure of pterostilbene is shown in Fig. 1A. To assess the effects of pterostilbene on cell viability, SAS and OECM-1 cells were treated with pterostilbene at various concentrations (0–40 lM) for 24 and 48 h, and then analyzed using the MTT assay. Pterostilbene substantially decreased the cell viability of SAS and OECM-1 cells, compared with untreated cells (Fig. 1B).
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Pterostilbene (40 lM) also decreased the growth rate of SAS and OECM-1 cells in a time-dependent manner, compared with untreated cells (Fig. 1C). Pterostilbene induced cell cycle arrest and apoptosis in human oral cancer cell lines Treatment with pterostilbene for 24 h induced an increase in the G0/G1 and S phase, and a decrease in the G2/M phase, compared with the controls (Fig. 2A). The results indicated that pterostilbene delayed cell cycle progression in both cell types. Therefore, pterostilbene-induced cell cycle arrest likely contributed to the overall growth suppression in SAS and OECM-1 cells. To determine whether the pterostilbene-induced cell death was related to apoptosis, DAPI staining was performed to analyze the changes in nuclear morphology. The results revealed the condensed and fragmented nuclei in both cell types after 24 h of pterostilbene (40 lM) treatment (Fig. 2B). Annexin V/PI double staining was also
Fig. 1. Pterostilbene inhibits cell proliferation in SAS and OECM-1 cell lines. (A) Structure of pterostilbene. (B) Cell viability of SAS and OECM-1 cells cultured in presence of pterostilbene (0–40 lM) for 24 and 48 h, as analyzed by MTT assay. (C) Cell viability of SAS and OECM-1 cells cultured in presence of pterostilbene (40 lM) for 6, 12 and 24 h, as analyzed by MTT assay. Results are shown as mean ± SD from 3 determinations per condition repeated 3 times. ⁄⁄⁄P < 0.001, compared with the control (0 lM or 0 h).
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Fig. 2. Pterostilbene inhibits cell cycle and induce apoptosis in SAS and OECM-1 cell lines. (A) SAS and OECM-1 cells were incubated for 18 h in the absence of serum and then treated with indicated concentrations of pterostilbene (40 lM), after which the cells were stained with PI, and analyzed for DNA content by flow cytometry. (B) Cells were treated with different concentration of pterostilbene (0–40 lM) for 24 h were and then subjected to DAPI staining for DNA (blue areas) by fluorescence microscopy. (C) SAS and OECM-1 cells treated with pterostilbene (40 lM) for 24 h. For quantitative analysis of apoptosis, pterostilbene-treated cells were harvested and then subjected to Annexin-V and PI double-stained flow cytometry. (D) Cells were treated with an indicated concentration of pterostilbene for 24 h and then analyzed by western blotting with an antibody against PARP, caspase-3, caspase-8 and caspase-9. b-actin as an internal control. (E) Furthermore, Cell were treated with 40 lM pterostilbene for 24 h in the presence or absence of 2 lM Z-DEVE-FMK, 20 lM Z-IETD-FMK, and 20 lM Z-LEHD-FMK and then subjected to MTT assay for cell viability. Results are shown as mean ± SD from 3 determinations per condition repeated 3 times. ⁄⁄⁄P < 0.001, compared with the untreated cells. ##P < 0.01, compared with the pterostilbene treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
determined using flow cytometry, and the results showed an increase in the percentage of cells displaying phosphatidylserine (PS) externalization in pterostilbene-treated SAS and OECM-1 cells (Fig. 2C). To investigate the underlying mechanism involved in pterostilbene-induced apoptosis. The results indicated that after a 24-h treatment with pterostilbene, the levels of cleaved PARP increased, whereas the cleaved-form caspase-3, -8, and -9 increased (Fig. 2D). To further confirm the involvement of caspase activation in pterostilbene-induced apoptosis, caspase-specific inhibitors were used. The results shown in Fig. 2E indicate that pretreatment with caspase-3, -8, and -9 specific inhibitors effectively attenuated pterostilbene-induced cell death. These data suggested that pterostilbene-induced apoptosis was dependent on the activation of caspase-3, -8, and -9.
Pterostilbene induced autophagy in human oral cancer cell lines Previous data has shown that pterostilbene causes autophagy in various cancer cells, including bladder cancer cells, breast cancer cells, and MOLT4 human leukemia cells [19]. In this study, various numbers of vacuoles were observed in the cytoplasm 24 h after pterostilbene (20 lM and 40 lM) treatment (Fig. 3A). Therefore, we hypothesized that other mechanisms mediated the response to pterostilbene. The formation of vacuoles in pterostilbene-treated cells was similar to that during cell autophagy, a general phenomenon that occurs when cells respond to stress [20]. We assessed whether pterostilbene induced autophagy in pterostilbene-induced human oral cancer cells. As shown in Fig. 3B, after transfection of pEGFPC1-LC3 and treatment with pterostilbene for 24 h, cytoplasmic LC3 formation was observed, indicating the
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Fig. 3. Pterostilbene induce autophagy in SAS and OECM-1 cells. (A) SAS and OECM-1 cells were treated with 40 lM pterostilbene for 24 h and microscopic observation revealed the formation vacuoles in cytoplasms of treated cells. (B) After a successful transfection with pEGFPC1-LC3, SAS and OECM-1 cells were treated with pterostilbene of 40 lM for 24 h, followed by an observation for LC-3 (green fluorescence) under fluorescence microscopy. (C) Quantitation of EGFP-LC3 puncta formation. Cells with EGFP-LC3 puncta formation were quantitated under a fluorescence confocal microscope. At least 200 cells on each slide were counted and the percentages of EGFP-LC3 puncta containing cells were calculated. The data shown are the mean ± SD of three individual experiments. (D) SAS and OECM-1 cells were treated with different concentration of pterostilbene (0–40 lM) for 24 h and stained by acridine orange to detect AVOs under fluorescence microscopy. (E) The percentage of cells with the formation of AVOs was detected by flow cytometry in a concentration and (F) time-dependent manner. Results are shown as mean ± SD. ⁄⁄⁄P < 0.001, compared with the SAS control (0 lM). ### P < 0.001, compared with the OECM-1 control (0 lM). (G) SAS and OECM-1 cells were treated with an indicated concentration of pterostilbene for 24 h and then analyzed by western blotting with an antibody against LC3-I/II and Beclin-1. b-actin as an internal control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
formation of autophagosomes in both cell lines treated with pterostilbene (40 lM). A substantial change in LC3 puncta formation was observed 24 h after treatment with 40 lM of pterostilbene (Fig. 3C). Pterostilbene caused induction of acidic vesicular organelles (AVOs) in SAS and OECM-1 cells in a concentration(Fig. 3D and E) and time-dependent manner (Fig. 3F). To further determine whether pterostilbene induced autophagy, the protein expression of LC3-II and Beclin-1 was analyzed. Compared with the control, the protein expression of LC3-II and Beclin-1 increased for both cells treated with pterostilbene (40 lM) for 24 h (Fig. 3G). These findings, combined with the previous results, showed that autophagy was induced by pterostilbene. Pterostilbene-induced cell death by treatment with autophagy inhibitors in human oral cancer cell lines To elucidate the interaction between pterostilbene-induced apoptosis and autophagy. SAS and OECM-1 cells were pre-treated
with 3-MA for 1 h before pterostilbene treatment; apoptosis-related molecules were examined using Western blotting. The results displayed in Fig. 4A indicated that adding 3-MA combined with pterostilbene to SAS and OECM-1 cells resulted in a slight increase in the levels of cleaved-form caspase-3, -8 and -9. To assess the effects of pterostilbene combined with autophagy inhibitors on cell viability, SAS and OECM-1 cells were treated with pterostilbene at various concentrations (0–40 lM), combined with 3-MA, for 24 h, and then analyzed using the MTT assay. As shown in Fig. 4B, pterostilbene combined with 3-MA substantially increased the cell viability of SAS and OECM-1 cells. Furthermore, after treatment with the pterostilbene/3-MA combination, the percentage of Annexin V-positive cells in the SAS and OECM-1 group were no significantly effected (Fig. 4C). In addition, similar results concerning cell viability and Annexin V-positive cells were observed after combined treatment with pterostilbene and BafA1 (Fig. 4D and E). Furthermore, a co-treatment with pterostilbene and Z-VAD-FMK (a broad-spectrum caspase inhibitor) was
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Fig. 4. Effects of autophagy inhibitors on pterostilbene-induced cell death. (A) SAS and OECM-1 cells were treated with pterostilbene of 40 lM for 24 h in the presence or absence of autophagy inhibitor 3-MA of 5 mM and then subjected to western blotting for caspase-3, caspase-8 and caspase-9 and b-actin. (B) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor 3-MA (5 mM), as analyzed by MTT assay. (C) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor 3-MA (5 mM), these treated cells were double-stained with Annexin-V and PI and then analyzed by flow cytometry. (D) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor BafA1 (10 nM), as analyzed by MTT assay. (E) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor BafA1 (10 nM), these treated cells were doublestained with Annexin-V and PI and then analyzed by flow cytometry. (F) SAS and OECM-1 cells were treated with pterostilbene (0–40 lM) in the presence or absence of autophagy inhibitor 3-MA (5 mM) or Z-VAD-FMK (20 lM) and analyzed by MTT assay. (G) SAS and OECM-1 cells were treated with pterostilbene (40 lM) in the presence or absence of autophagy inducer rapamycin (100 nM), as analyzed by MTT assay. (H) SAS and OECM-1 cells were treated with pterostilbene (40 lM) in the presence or absence of autophagy inducer rapamycin (100 nM), these treated cells were double-stained with Annexin-V and PI and then analyzed by Muse Cell Analyzer flow cytometry and analysis data by the MuseÒ Cell Analyzer Assays (Millipore) (upper plane). Quantitative percentage of Annexin-V positive cells was calculated (under plane). Results are shown as mean ± SD. ⁄⁄⁄P < 0.001, compared with the SAS control (0 lM). $$$P < 0.001, compared with the OECM-1 control (0 lM). ###P < 0.001, compared with the SAS treated with pterostilbene (40 lM). &P < 0.05; &&&P < 0.001, compared with the OECM-1 treated with pterostilbene (40 lM).
conducted to show that the pterostilbene-induced reduction of the percentage of cell viability was prevented by Z-VAD-FMK. Nevertheless, cell viability in SAS and OECM-1 cells were increased when 3-MA was included (Fig. 4F). To assess the effects of pterostilbene combined with autophagy inhibitors on cell viability, SAS and OECM-1 cells were also treated with pterostilbene (40 lM), combined with rapamycin (mTOR inhibitor) for 24 h, and then analyzed for cell viability and the percentage of Annexin V-positive cells. As shown in Fig. 4G, pterostilbene combined with rapamycin substantially decreased the cell viability of SAS and OECM-1 cells. However, the percentage of Annexin V-positive cells was not significantly affected (Fig. 4H). Therefore, these results indicated that autophagy played differing roles in pterostilbene-induced SAS and OECM-1 cell death. Pterostilbene induced autophagy through inhibition of Akt, ERK1/2, p38 and upregulated JNK1/2 activation in human oral cancer cell lines To investigate the mechanism of pterostilbene-induced autophagy in human oral cancer cell lines. The results showed that pterostilbene treatment resulted in the activation of JNK1/2, which increased in a dose-dependent manner. By contrast, treatment with pterostilbene decreased the level of phosphorylated p38, Akt, and ERK1/2 in both cell lines (Fig. 5A and B). To elucidate whether Akt, p38 or ERK1/2 inhibition, or JNK1/2 activation, contributed to autophagy, LY294002, SB203580, U0126, and SP600125 were added. As shown in Fig. 5C, the AVOs resulting
from pterostilbene treatment and pterostilbene treatment with LY294002, SB203580, and U0126 increased considerably. The JNK1/2 inhibitor (SP600125) was used to further confirm the role of the JNK1/2 pathway in pterostilbene-induced autophagy. The expression levels of the AVOs for pterostilbene treatment with SP600125 decreased considerably compared with those of pterostilbene treatment alone. These results indicated that pterostilbeneinduced autophagy was mediated by inhibition of Akt p38, and ERK1/2, as well as activation of JNK1/2. We also investigated the role of p38, Akt, ERK1/2, and JNK1/2 in pterostilbene-induced cell viability. SAS and OECM-1 cells pretreated with LY294002, SB203580, or U0126 exhibited a decreased cell viability compared with those treated only with pterostilbene. The cell viability of SAS cells pretreated with SP600125 increased. However, pretreatment of OECM-1 cells with SP600125 effectively inhibited cell viability, compared with treatment using pterostilbene alone (Fig. 5D).
Pterostilbene induced the activation of AMPK, Raptor, ULK and downregulated mTOR activation in human oral cancer cell lines To further investigate the mechanism of pterostilbene-induced autophagy in human oral cancer cell lines, the expression levels of the phosphorylated forms of AMPK, Raptor, ULK and mTOR were examined using Western blotting. The results showed that pterostilbene treatment resulted in the activation of AMPK, Raptor and ULK at Ser555 in a dose-dependent manner. By contrast, the level
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Fig. 5. Pterostilbene induces autophagy through inhibition of the Akt, ERK1/2, p38 and activation of the JNK1/2 in SAS and OECM-1 cells. (A) Cells were treated with different concentrations of pterostilbene (0–40 lM) for 24 h and then subjected to western blotting. The levels of phosphorylation of Akt, ERK1/2, p38 and JNK1/2 were investigated by western blotting with b-actin being used as an internal control. (B) Quantitative results of Akt, ERK1/2 and JNK1/2 and p38 protein level which were adjusted with b-actin protein level. Results are shown as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001, compared with the control (0 lM). (C) SAS and OECM-1 cells were pretreated with LY294002 (20 lM), SP600125 (20 lM), SB203580 (20 lM) or U0126 (10 lM) for 1 h followed by treatment with pterostilbene (40 lM) for 24 h and stained by acridine orange to detect AVOs. Measurement of AVOs was detected by flow cytometry. (D) SAS and OECM-1 cells were pretreated with LY294002 (20 lM), SP600125 (20 lM), SB203580 (20 lM) or U0126 (10 lM) for 1 h followed by treatment with pterostilbene (40 lM) for 24 h and analyzed by MTT assay.
of phosphorylated ULK at Ser757 was decreased in both cell lines (Fig. 6A and B). Discussion Chemotherapy is suggested for treatment of advanced-stage cancers. However, the ability of cancer cells to become resistant to various drugs limits the efficiency of chemotherapy [21]. Natural products, which have been used traditionally in various ethnic societies worldwide, are crucial for the development of novel chemotherapeutics. Pterostilbene has been reported to inhibit herbicide-induced oxidative damage, inducing apoptosis in sensitive and chemoresistant lymphoma cell lines through the caspase-independent pathway, and is also known to act against B16 melanoma growth and metastatic activity [5,7,8]. The results of this study showed that pterostilbene induced the death of SAS and OECM-1 human oral cancer cells by arresting their cell cycle. Pterostilbene-induced apoptosis of oral cancer cells was dependent on the activation of caspase-3, -8, and -9 (Figs. 1 and 2). These results were consistent with other reports showing that pterostilbene induced a G0/G1 arrest in BCR-ABL-expressing leukemia cells [22]. Pan et al. showed that pterostilbene exerted growth inhibition by inducing apoptosis and arresting gastric cancer cells in the G0/G1 phase. Caspase-2, -3, -8, and -9 are all activated by pterostilbene [23]. In MOLT4 human leukemia cells, pterostilbene inhibited cell proliferation, resulting in a transient accumulation of cells in the G0/G1 cell cycle phase, followed by arrest in the S-phase, and induced apoptosis by caspase-3 activation [19]. Autophagy is a key cellular response for various environmental stimuli and diseases, including cancers [24–26]. This form of cell death was defined as autophagic programmed cell death [27]. Many anticancer agents, including sulforaphane, rapamycin, arsenic trioxide, and temozolomide were reported to induce autophagy [28,29]. Autophagy has attracted the interest of scientists in the cancer therapy field because drug-resistant cancer cells with antiapoptotic properties may be vulnerable to death by autophagy
[30,31]. Although pterostilbene exerts antiproliferative and proapoptotic activities in various cancer cell types, the underlying mechanisms of autophagy remain unclear. Furthermore, experiments conducted in other types of cancer cells may provide additional data to identify the signaling pathways involved in pterostilbene-induced autophagy. Thus, because of its capability to induce both the apoptotic and autophagic pathways, pterostilbene may be an effective multidrug resistance agent for antitumor treatment. Our results showed that pterostilbene resulted in autophagy after 24 h of administration. Pterostilbene was added to the culture medium, and the expression of LC3-II and AVOs indicated that the induction of autophagy was dose- and time-dependent (Fig. 3). The most substantial expression of the autophagy process was observed 24 h after pterostilbene (40 lM) treatment, whereas a slight expression of LC3-II was observed 24 h after pterostilbene (20 lM) treatment (Fig. 3G). However, treatment with pterostilbene (20 lM) for 24 h did not result in any activation of caspase-3, -8, or -9. These results suggested that the pterostilbene-induced autophagy process occurred as early as apoptosis. The autophagy inhibitor 3-MA can block autophagosome formation by inhibiting the type III PI-3K that prevents early-stage autophagy [25], as well as BafA1, an inhibitor of vacuolar H+-ATPase that prevents late-stage autophagy by inhibiting fusion of autophagosomes and lysosomes [26]. To further investigate the role of autophagy in pterostilbene-induced cell death, we combined pterostilbene treatment with an autophagy inhibitor, and analyzed cell viability and activation of caspases. The results indicated that the presence of 3-MA in SAS and OECM-1 cells resulted in a slight increase in the levels of cleaved-form caspase-3, -8 and -9 (Fig. 4A). In addition, inhibition of autophagy by 3-MA increased the cell viability of the SAS and OECM-1cell line (Fig. 4B and C). Similarly, accumulation of abnormal autophagosomes by BafA1 treatment of SAS and OECM-1 cells reduced cell death (Fig. 4D and E). Although pterostilbene could promote a cell arrest phenotype, but it is unclear whether the residual cells become dormant and more resistant to other common adjuvant therapeutic agents, such as mTOR inhibitors, HDAC inhibitors and cisplatin.
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Fig. 6. Pterostilbene induce activation of the AMPK, Raptor, ULK and inhibition of mTOR in SAS and OECM-1 cells. (A) Cells were treated with different concentrations of pterostilbene (0–40 lM) for 24 h. The phosphorylation levels of AMPK, Raptor, mTOR and ULK were investigated by western blotting with b-actin being used as an internal control. (B) Quantitative results of phospho-AMPK, phospho-Raptor, phospho-mTOR and phospho-ULK protein levels were adjusted with b-actin protein level. Results are shown as mean ± SD. ⁄P < 0.05, compared with the control (0 lM).
Especially considering it is also an autophagy-promoting agent, the potential impact on increasing cancer cell resistance needs to be tested. The results indicated that pterostilbene combined with rapamycin substantially decreased the cell viability of SAS and OECM-1 cells without significant change in the percentage of Annexin V-positive cells (Fig. 4G and H). Ellington et al. showed that soybean B-group triterpenoid saponins induced autophagy by inhibiting Akt and activating ERK activity [32]. The present study clearly demonstrated that pterostilbene inhibited Akt, ERK1/2, and p38, and activated the JNK1/2 pathway to promote autophagy. In both cell lines, blocking Akt, p38 or ERK1/2 with LY294002, SB203580 or U0126 enhanced the expression of AVOs by pterostilbene. These findings may highlight the common mechanisms (such as Akt inhibition and ERK1/2 activation) that regulate autophagy in cancer cells treated with anticancer agents. We suggested that inhibiting JNK1/2 activation using SP600125 could prevent the induction of AVOs by pterostilbene. For both cell lines, cell death increased in the LY294002, SB203580, and U0126 groups; however, inhibiting JNK1/2 activation with SP600125 caused different viability effects in both cell lines. This further confirmed that pterostilbene-induced autophagy had different mechanisms in SAS and OECM-1 cell lines (Fig. 5D). During low nutrient conditions, AMPK directly phosphorylates ULK1 at multiple sites including Ser317, Ser555, and Ser777 [33,34]. Conversely, mTOR, a regulator of cell growth and an inhibitor of autophagy, phosphorylates ULK1 at Ser757 and disrupts the interaction between ULK1 and AMPK [35]. Phosphorylation of
ULK1 by AMPK at Ser555 is critical for starvation-induced autophagy, cell survival under conditions of low nutrients and energy, and mitochondrial homeostasis [33]. AMPK phosphorylates raptor on Ser722/Ser792. This phosphorylation is essential for the inhibition of the raptor-containing mTOR complex 1 (mTORC1) and induces cell cycle arrest when cells are stressed for energy [36]. In this study, it was clearly demonstrated that pterostilbene induced phosphorylation of AMPK, Raptor and ULK at Ser555, and inhibited the phosphorylation of mTOR and ULK at Ser757 (Fig 6). Autophagy is suppressed by functional p53 and certain cytoplasmic p53 mutants. Tasdemir et al. reported that cytoplasmic p53 suppressed autophagy, and p53 inhibition induced autophagy [33]. Furthermore, nuclear expression of p53 may stimulate autophagy by DRAM upregulation and mTOR inhibition [34], suggesting that p53 exerts an opposite effect on autophagy regulation. Therefore, pterostilbene-induced autophagy and apoptosis in SAS and OECM-1 cells may be partially attributed to the functional p53, which could be verified by additional experiments exploring the role of p53 in pterostilbene-induced autophagy. Conclusion In conclusion, this was the first study to demonstrate that pterostilbene suppressed cell growth and induced autophagy in SAS and OECM-1 cell lines by inhibiting Akt, p38, ERK1/2, and activating the JNK1/2 pathway. Autophagy was induced in the
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early stage of pterostilbene-induced apoptosis. We also observed that pterostilbene-induced autophagy had a different mechanism in SAS and OECM-1 cell lines. Conflict of interest statement The authors declare that there are no conflicts of interest. Competing Interests No. Author Contributions Performed the revised experiments and analyzed the data. Acknowledgements This study was supported by grants from Ministry of Science and Technology, Taiwan (MOST 103-2314-B-371-007-MY2), Chung Shan Medical University Hospital, Taiwan (CSH-2013-C012) and Changhua Christian Hospital (103-CCH-IRP-071) (103CCH-ICO-006). The authors of the manuscript do not have a direct financial relation with the commercial identity mentioned in this paper. References [1] Spiro RH, Alfonso AE, Farr HW, Strong EW. Cervical node metastasis from epidermoid carcinoma of the oral cavity and oropharynx. A critical assessment of current staging. Am J Surg 1974;128:562–7. [2] Chen PN, Hsieh YS, Chiou HL, Chu SC. Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways. Chem Biol Interact 2005;156:141–50. [3] Huang HP, Shih YW, Chang YC, Hung CN, Wang CJ. Chemoinhibitory effect of mulberry anthocyanins on melanoma metastasis involved in the Ras/PI3K pathway. J Agric Food Chem 2008;56:9286–93. [4] Vieira JR, de Souza IA, do Nascimento SC, Leite SP. Indigofera suffruticosa: an alternative anticancer therapy. Evidence-based Complement Altern Med: eCAM 2007;4:355–9. [5] Rimando AM, Cuendet M, Desmarchelier C, Mehta RG, Pezzuto JM, Duke SO. Cancer chemopreventive and antioxidant activities of pterostilbene, a naturally occurring analogue of resveratrol. J Agric Food Chem 2002;50:3453–7. [6] Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, et al. Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. J Biol Chem 2001;276:22586–94. [7] Ferrer P, Asensi M, Segarra R, Ortega A, Benlloch M, Obrador E, et al. Association between pterostilbene and quercetin inhibits metastatic activity of B16 melanoma. Neoplasia 2005;7:37–47. [8] Remsberg CM, Yanez JA, Ohgami Y, Vega-Villa KR, Rimando AM, Davies NM. Pharmacometrics of pterostilbene: preclinical pharmacokinetics and metabolism, anticancer, antiinflammatory, antioxidant and analgesic activity. Phytother Res: PTR 2008;22:169–79. [9] Cohen D, Higman SM, Hsu SI, Horwitz SB. The involvement of a LINE-1 element in a DNA rearrangement upstream of the mdr1a gene in a taxol multidrugresistant murine cell line. J Biol Chem 1992;267:20248–54. [10] Davies AM, Lara Jr PN, Mack PC, Gandara DR. Docetaxel in non-small cell lung cancer: a review. Expert Opin Pharmacother 2003;4:553–65. [11] Green MR. The evolving role of gemcitabine and pemetrexed (Alimta) in the management of patients with malignant mesothelioma. Clin Lung Cancer 2002;3(Suppl 1):S26–9. [12] Hsieh MJ, Yang SF, Hsieh YS, Chen TY, Chiou HL. Autophagy inhibition enhances apoptosis induced by dioscin in huh7 cells. Evidence-based Complement Altern Med: eCAM 2012;2012:134512.
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