Concentration effects of grape seed extracts in anti-oral cancer cells involving differential apoptosis, oxidative stress, and DNA damage
© Yen et al.; licensee BioMed Central. 2015
Received: 28 November 2014
Accepted: 21 February 2015
Published: 29 March 2015
Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer.
The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage.
High concentrations (50–400 μg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1–10 μg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations.
Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.
KeywordsGSE Apoptosis Oxidative stress DNA damage Oral cancer
Betel quid chewing is one of the main causes leading to oral cancer in Taiwan . Arecoline, one of main effective components in betel quid, was reported to lead to DNA damage and apoptosis through the formation of reactive oxygen species (ROS) and contribute to oral carcinogenesis [2-5]. Therefore, the modulation of ROS level may be helpful for oral cancer prevention and therapy.
Grape seed extract (GSE) is a common dietary health supplement due to its natural ROS modulating ability . Commercial preparations of GSE are marketed in the world as a dietary health supplements due to their natural free radical scavenging ability . The cancer chemoprevention and anticancer potential of GSE has been well reviewed previously  including skin, colorectal, prostate, breast, lung, and gastric cancers. However, the GSE effects with respect to oral cancer cells are less studied as yet.
ROS modulation effect has been well reviewed [8,9]. For example, cellular ROS may regulate apoptosis through the mitochondrial pathway [10-13]. Pro-oxidants induce ROS specifically targeting cancer cells, thereby activating signal transduction pathways that are responsible for cell cycle arrest and/or apoptosis . Similarly, GSE was reported to generate a strong superoxide radical-associated oxidative stress and result in the apoptosis of non-small-cell lung cancer cells  as well as in the induction of DNA damage .
Different concentrations of GSE were reported to generate diverse biological effects in several cancer studies [17-20]. For example, high concentrations (25–100 μg/ml) of GSE showed cytotoxicity or antiproliferation of human bladder , colorectal , and breast  cancer cell lines. In contrast, a low concentration (2.5 μg/ml) of GSE was reported to inhibit the micronuclei frequency and ROS generation in a lymphocyte culture, demonstrating that its antioxidant property has a protective effect during oxidative stress . However, more detailed mechanisms between cancer chemoprevention and anticancer effects of GSE in terms of concentration effects remain unclear.
Since GSE is a natural ROS scavenger, we hypothesize that GSE modulates ROS to further regulate proliferation, apoptosis, mitochondrial function, and DNA damage. Since concentration responses of GSE for these regulations may be relevant, in this study we aim to define the critical concentrations that may or may not be able to induce apoptosis in oral cancer cells.
The IH636 premium grade proanthocyanidin grape seed (Vitis vinifera) extract (GSE, commercially known as ActiVin®) was purchased from InterHealth Nutraceuticals Inc. (Benicia, CA, USA), which included 75–80% oligomeric proanthocyanidins and 3–5% monomeric proanthocyanidins as described previously .
Cell lines of human oral gingival cancer Ca9-22  and gingival fibroblast HGF-1  were routinely maintained in DMEM/F12 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum, 0.03% glutamine, 1 mM sodium pyruvate, and penicillin/streptomycin mixtures. Cells were kept at 37°C in a humidified incubator containing 5% CO2.
Determination of cell viability
Viability analysis was performed using Cell Titer 96™ Aqueous One solution cell proliferation (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) MTS) assay kit (Promega Madison, WI, USA) as described previously  with minor modification. In brief, cells were treated with various concentrations of GSE in fresh media in triplicates. The non-toxic concentration of DMSO (less than 1% v/v) was used to prepare test solutions in all assays. The plates were then incubated for 24 h under standard growth conditions. Subsequently, MTS reagent was loaded to each well (5 mg/ml in PBS) and cells were again incubated for another 2 h. Then, absorbance of each well was recorded directly at 490 nm by ELISA multi-Plate Reader (MTX Lab Systems, Inc., Vienna, VA, USA).
Determination of sub-G1 population
Measurement of DNA content for cell cycle analysis were carried out by flow cytometry, based on a previously described protocol . In brief, Ca9-22 cells were treated with either DMSO only or different GSE concentrations for 24 h. After incubation, cells were harvested for washing and fixing in 70% ethanol overnight. After harvest, cells were resuspended in 1 ml PBS containing 10 μg/ml PI (Sigma, St Louis, MO, USA) in the dark. Subsequently, cells were analyzed using a flow cytometer (FACScan; Becton-Dickinson, Mansfield, MA) at excitation and emission settings of 480 and 525 nm, respectively, and Win-MDI software (http://facs.scripps.edu/software.html).
Determination of apoptosis by annexin V/PI
The induction of apoptosis by GSE-treated Ca9-22 cells was analyzed by annexin V staining as previously described . Briefly, cells were treated with either vehicle or various GSE concentrations for 24 h. Subsequently, the cells were trypsinized, washed twice with PBS and stained with fluorescein isothiocyanate (FITC)-labelled annexin V. Then, the samples were measured with a flow cytometer (FACSCalibur; Becton-Dickinson) for the quantification of apoptotic cells at excitation and emission settings of 480 and 525 nm, respectively, and Win-MDI software.
Determination of apoptosis by pan-caspase activity
The induction of apoptosis by GSE-treated Ca9-22 cells was analyzed by activation of caspases (caspase-1, 3, 4, 5, 6, 7, 8, 9) by the generic caspase activity assay kit (Abcam, Cambridge, UK) as previously described . Briefly, cells were treated with either vehicle or various GSE concentrations for 24 h. After harvest, the cells were suspended and stained with 1 X fluorescent TF2-Val-Ala-Asp (VAD)-FMK at the cell incubator for 1 h. Then, the samples were measured with a flow cytometer (BD Accuri C6; Becton-Dickinson, Mansfield, MA, USA) and a BD Accuri C6 Software (version 1.0.264) for the quantification of pan-caspase positive populations at excitation and emission settings of 480 and 525 nm, respectively.
Determination of intracellular ROS
Intracellular redox state were determined by the ROS-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma Chemical Co., St. Louis, MO, USA) as previously described [25,28]. Ca9-22 cells were treated with various concentrations of GSE for 24 h. Subsequently, cells were harvested, thoroughly washed, resuspended in 10 μM DCFH-DA in PBS and then incubated at 37°C for 30 min in darkness. After incubation, cells were washed, resuspended in PBS, and analyzed with a FACSCalibur flow cytometer at excitation and emission settings of 480 and 525 nm, respectively, and Win-MDI software.
Determination of mitochondrial membrane potential
Mitochondrial membrane potential (MitoMP) was determined by flow cytometry using MitoProbe™ DiOC2(3) assay kit (Invitrogen, San Diego, CA, USA) as described previously . In brief, cells were incubated with various GSE concentrations at 37°C for 24 h. Subsequently, cells were incubated in culture medium (containing 50 μM of DiOC2(3)) at 37°C for 20 min in an incubator. After washing and resuspension in PBS, cells were subjected to flow cytometric analysis. The fluorescence intensity was measured using 488 and 525 nm filter settings for the excitation and emission wavelengths, respectively. The data were analyzed with Win-MDI software.
Determination of DNA double strand breaks (DSBs) by γH2AX/PI double staining
DSBs were measured by flow cytometry as described previously . Ca9-22 cells were incubated with various GSE concentrations for 24 h, followed by fixation with 70% ethanol overnight. After washing twice with BSA-T-PBS (1% bovine serum albumin and 0.2% Triton X-100 in PBS), cells were treated with 100 μl of BSA-T-PBS solution containing 0.2 μg monoclonal antibody against p-Histone H2A.X (Ser 139) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for overnight at 4°C. After washing, cells were resuspended in Alexa Fluor 488-tagged secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at a 1:100 dilution for 1 h at 4°C. After washing, cells were resuspended in 1 ml PBS containing 5 μg/ml PI and analyzed by a FACSCalibur flow cytometer and Win-MDI software.
Statistical analysis was performed with JMP 9 software. One-way ANOVA with Tukey’s HSD Post Hoc Test was used to analyze significant differences between treatments. Unless otherwise indicated, all experiments were repeated in triplication.
Cell cycle distribution by GSE treatments
Apoptotic cell death: annexin V/PI
Apoptotic cell death: caspase activity
DNA damages caused by GSE treatment
Accumulating evidence of the antiproliferative effect of GSE had been reported in several oral cancer cell lines. For example, high concentrations (50–600 μg/ml) of GSE of Vitis vinifera were found to inhibit cell proliferation and induce apoptosis of the KB cells but less harmful to non-cancerous human umbilical vein endothelial cells (HUVEC) by trypan blue assay at 24 h GSE treatment . Similarly, we found that the low and high concentrations of GSE to normal oral HGF-1 cells based on MTS analysis. The KB cells was used to be regarded as the oral cancer cell line, however, it was recently confirmed to be the contaminant cervical cancer HeLa cells . Moreover, the low concentrations of GSE were not investigated in this study. Recently, the differential concentration effect of GSE to differentially inhibit proliferation of oral cancer cells has been demonstrated. For example, low concentrations of GSE (10–20 μg/ml) did not displayed the antiproliferation of oral cancer CAL 27 cells but high concentrations of GSE (30–80 μg/ml) were able to inhibit its proliferation . Similarly, we found that low (1–10 μg/ml) and high (50–400 μg/ml) concentrations of GSE displayed the differential cytotoxic effects to cell viability of oral cancer Ca9-22 cells. Similar results also reported in other cancer cells. In the example of skin cancer HaCaT cells, high concentrations of GSE (IC50 = 76.44 μg GAE/ml) displayed the growth inhibitory effect, but low concentrations of GAE (10–20 μg GAE/ml) protected against UVB irradiation (50–100 mJ/cm2)-induced skin cancer . These findings suggested that different cancer cell lines may require different but high concentrations of GSE for antiproliferation purpose.
ROS induction by GSE was reported in non-small-cell lung cancer H1299 and A549 cells but it only tested at high concentrations (20–100 μg/ml) without detecting the mitochondrial function . ROS generation of high GSE (40 μg/ml) also reported to induce apoptosis in head and neck cancer Detroit 562 and FaDu cells . In oral cancer CAL 27 cells, GSE also reported to induce mRNA overexpression of apoptosis-associated signaling such as caspase-2 and caspase-8 . In head and neck cancer cells, GSE also reported to induce DNA damage . Our results further validated that GSE at high concentrations (50–400 μg/ml) have high oxidative stress and apoptosis in terms of ROS generation, mitochondrial depolarization, annexin V/PI staining, and caspase activation but not for low concentrations (<10 μg/ml) of GSE in oral cancer Ca9-22 cells.
Moreover, this differential concentration effect of GSE was also found in cancer cell migration. For example, GSE was reported to inhibit migration and invasion of breast cancer MDA-MB231 cell . High concentrations (50–100 μg/ml) of GSE inhibited cell proliferation and induced apoptosis. Conversely, low GSE (25 μg/ml) concentrations decreased cell migration and invasion. Therefore, the differential concentration effect of GSE in oral cancer cell migration is warranted for further investigation.
We demonstrated that GSE shows differential concentration effects in the antiproliferation of oral cancer cells through differential expressions of apoptosis, oxidative stress, and DNA damage. We showed that the antiproliferative effect of high GSE concentrations is associated with an overproduction of ROS causing DNA damage and apoptosis of cancer cells.
This work was partly supported by funds of the Ministry of Science and Technology (MOST 103-2320-B-037-008), the ChiMei-KMU Joint Project (103CM-KMU-09), the Kaohsiung Medical University “Aim for the Top Universities Grant, grant No. KMU-TP103A33”, the Kaohsiung Municipal Ta-Tung Hospital (kmtth-102-011), the National Sun Yat-sen University-KMU Joint Research Project (#NSYSU-KMU 104-p036), and the Health and welfare surcharge of tobacco products, the Ministry of Health and Welfare, Taiwan, Republic of China (MOHW104-TDU-B-212-124-003). We also thank for the help in English editing by Dr. Hans-Uwe Dahms and technical support with the flow cytometer by Mr. Yi-An Chung.
- Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med. 1995;24(10):450–3.View ArticlePubMedGoogle Scholar
- Nair UJ, Obe G, Friesen M, Goldberg MT, Bartsch H. Role of lime in the generation of reactive oxygen species from betel-quid ingredients. Environ Health Perspect. 1992;98:203–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Ji WT, Yang SR, Chen JY, Cheng YP, Lee YR, Chiang MK, et al. Arecoline downregulates levels of p21 and p27 through the reactive oxygen species/mTOR complex 1 pathway and may contribute to oral squamous cell carcinoma. Cancer Sci. 2012;103(7):1221–9.View ArticlePubMedGoogle Scholar
- Lee SS, Tsai CH, Yu CC, Chang YC. Elevated snail expression mediates tumor progression in areca quid chewing-associated oral squamous cell carcinoma via reactive oxygen species. PLoS One. 2013;8(7):e67985.View ArticlePubMedPubMed CentralGoogle Scholar
- Yen CY, Lin MH, Liu SY, Chiang WF, Hsieh WF, Cheng YC, et al. Arecoline-mediated inhibition of AMP-activated protein kinase through reactive oxygen species is required for apoptosis induction. Oral Oncol. 2011;47(5):345–51.View ArticlePubMedGoogle Scholar
- Bagchi D, Garg A, Krohn RL, Bagchi M, Tran MX, Stohs SJ. Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro. Res Commun Mol Pathol Pharmacol. 1997;95(2):179–89.PubMedGoogle Scholar
- Kaur M, Agarwal C, Agarwal R. Anticancer and cancer chemopreventive potential of grape seed extract and other grape-based products. J Nutr. 2009;139(9):1806S–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Chang HW. The fate of marine algal natural products-treated cells depend on it ROS modulating effects. J Rashid Latif Med College. 2013;2:8–10.Google Scholar
- Lee JC, Hou MF, Huang HW, Chang FR, Yeh CC, Tang JY, et al. Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int. 2013;13(1):55.View ArticlePubMedPubMed CentralGoogle Scholar
- Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med. 2010;48(6):749–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Song W, Hu P, Shan Y, Du M, Liu A, Ye R. Cartilage polysaccharide induces apoptosis in K562 cells through a reactive oxygen species-mediated caspase pathway. Food Funct. 2014;5(10):2486–93.View ArticlePubMedGoogle Scholar
- Cheng AC, Tsai ML, Liu CM, Lee MF, Nagabhushanam K, Ho CT, et al. Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through induction of ROS-dependent apoptosis. Food Funct. 2010;1(3):301–7.View ArticlePubMedGoogle Scholar
- Sun L, Luo C, Liu J. Hydroxytyrosol induces apoptosis in human colon cancer cells through ROS generation. Food Funct. 2014;5(8):1909–14.View ArticlePubMedGoogle Scholar
- Wondrak GT. Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal. 2009;11(12):3013–69.View ArticlePubMedPubMed CentralGoogle Scholar
- Tyagi A, Raina K, Gangar S, Kaur M, Agarwal R, Agarwal C. Differential effect of grape seed extract against human non-small-cell lung cancer cells: The role of reactive oxygen species and apoptosis induction. Nutr Cancer. 2013;65 Suppl 1:44–53.View ArticlePubMedGoogle Scholar
- Praphasawat R, Klungsupya P, Muangman T, Laovitthayanggoon S, Arunpairojana V, Himakoun L. Antimutagenicity and antioxidative DNA damage properties of oligomeric proanthocyanidins from Thai grape seeds in TK6 cells. Asian Pac J Cancer Prev. 2011;12(5):1317–21.PubMedGoogle Scholar
- Raina K, Tyagi A, Kumar D, Agarwal R, Agarwal C. Role of oxidative stress in cytotoxicity of grape seed extract in human bladder cancer cells. Food Chem Toxicol. 2013;61:187–95.View ArticlePubMedGoogle Scholar
- Dinicola S, Pasqualato A, Cucina A, Coluccia P, Ferranti F, Canipari R, et al. Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion. Eur J Nutr. 2014;53(2):421–31.View ArticlePubMedGoogle Scholar
- Stankovic M, Tesevic V, Vajs V, Todorovic N, Milosavljevic S, Godevac D. Antioxidant properties of grape seed extract on human lymphocyte oxidative defence. Planta Med. 2008;74(7):730–5.View ArticlePubMedGoogle Scholar
- Perde-Schrepler M, Chereches G, Brie I, Tatomir C, Postescu ID, Soran L, et al. Grape seed extract as photochemopreventive agent against UVB-induced skin cancer. J Photochem Photobiol B. 2013;118:16–21.View ArticlePubMedGoogle Scholar
- Kaur M, Singh RP, Gu M, Agarwal R, Agarwal C. Grape seed extract inhibits in vitro and in vivo growth of human colorectal carcinoma cells. Clin Cancer Res. 2006;12(20 Pt 1):6194–202.View ArticlePubMedGoogle Scholar
- Chen BH, Hung MH, Chen JYF, Chang HW, Yu ML, Wan L, et al. Anti-allergic activity of grapeseed extract (GSE) on RBL-2H3 mast cells. Food Chem. 2012;132(2):968–74.View ArticleGoogle Scholar
- Yeh CC, Yang JI, Lee JC, Tseng CN, Chan YC, Hseu YC, et al. Anti-proliferative effect of methanolic extract of Gracilaria tenuistipitata on oral cancer cells involves apoptosis, DNA damage, and oxidative stress. BMC Complement Altern Med. 2012;12(1):142.PubMedPubMed CentralGoogle Scholar
- Yen YH, Farooqi AA, Li KT, Butt G, Tang JY, Wu CY, et al. Methanolic extracts of Solieria robusta inhibits proliferation of oral cancer Ca9-22 cells via apoptosis and oxidative stress. Molecules. 2014;19:18721–32.View ArticlePubMedGoogle Scholar
- Chiu CC, Haung JW, Chang FR, Huang KJ, Huang HM, Huang HW, et al. Golden berry-derived 4beta-hydroxywithanolide E for selectively killing oral cancer cells by generating ROS, DNA damage, and apoptotic pathways. PLoS One. 2013;8(5):e64739.View ArticlePubMedPubMed CentralGoogle Scholar
- Yen CY, Chiu CC, Haung RW, Yeh CC, Huang KJ, Chang KF, et al. Antiproliferative effects of goniothalamin on Ca9-22 oral cancer cells through apoptosis; DNA damage and ROS induction. Mutat Res. 2012;747(2):253–8.View ArticlePubMedGoogle Scholar
- Yeh CC, Tseng CN, Yang JI, Huang HW, Fang Y, Tang JY, et al. Antiproliferation and induction of apoptosis in Ca9-22 oral cancer cells by ethanolic extract of Gracilaria tenuistipitata. Molecules. 2012;17(9):10916–27.View ArticlePubMedGoogle Scholar
- Chan WH, Wu HJ. Methylglyoxal and high glucose co-treatment induces apoptosis or necrosis in human umbilical vein endothelial cells. J Cell Biochem. 2008;103(4):1144–57.View ArticlePubMedGoogle Scholar
- Aghbali A, Hosseini SV, Delazar A, Gharavi NK, Shahneh FZ, Orangi M, et al. Induction of apoptosis by grape seed extract (Vitis vinifera) in oral squamous cell carcinoma. Bosn J Basic Med Sci. 2013;13(3):186–91.PubMedPubMed CentralGoogle Scholar
- Masters J. False cell lines. Carcinogenesis. 2002;23(2):371.View ArticlePubMedGoogle Scholar
- Chatelain K, Phippen S, McCabe J, Teeters CA, O’Malley S, Kingsley K. Cranberry and grape seed extracts inhibit the proliferative phenotype of oral squamous cell carcinomas. Evid Based Complement Alternat Med. 2011;2011:467691.View ArticlePubMedGoogle Scholar
- Shrotriya S, Deep G, Gu M, Kaur M, Jain AK, Inturi S, et al. Generation of reactive oxygen species by grape seed extract causes irreparable DNA damage leading to G2/M arrest and apoptosis selectively in head and neck squamous cell carcinoma cells. Carcinogenesis. 2012;33(4):848–58.View ArticlePubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.