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Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells
G Model BIONUT-184; No. of Pages 5
ARTICLE IN PRESS Biomedicine & Preventive Nutrition xxx (2014) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells V. Cijo George a , D.R. Naveen Kumar a , P.K. Suresh a , R. Ashok Kumar b,∗ a b
Cell culture lab (TT-114), School of Bio Sciences and Technology, VIT University, Vellore, India Department of Zoology, Government Arts College, Dharmapuri, India
a r t i c l e
i n f o
Article history: Received 21 August 2013 Accepted 16 September 2013 Keywords: Oleanolic acid Melanoma Apoptosis DNA fragmentation ELISA
a b s t r a c t Melanoma is a life threatening condition, which mostly effects cocassions despite the advancements in current chemotherapeutic techniques. The aim of present study is to investigate the apoptotic inducing potential of oleanolic acid (OA) in A375 human melanoma cells. The anti-proliferative effects of OA (12.5–200 M) were assessed by cell growth and XTT assay. The morphological and nuclear damage studies were carried out by Wright-Giemsa and DAPI staining, respectively. Further, the apoptotic inducing potential of OA in A375 cells were measured by DNA fragmentation ELISA. The results showed a doseresponsive effect of OA by inhibiting the cell growth significantly (P < 0.05) at 24 and 48 h with a decrease in cell viability (XTT data). The significant morphological changes included cellular annihilation, which was observed in A375 cells when compared to the control cells. Quantitative dose-dependent increase in apoptotic-DNA fragments in ELISA and nuclear fragments in DAPI results, further demonstrated the potential of this triterpenoid to induce apoptotic cell death at a concentration, particularly higher than 50 M. Thus, we conclude that OA has wielded both anti-proliferative and apoptotic inducing potentials against A375 melanoma cells and can be a better choice for its progression. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Skin cancer accounts for 5% of deaths worldwide, but the empirical data shows an exponential increase in the number of people suffering from different types of skin cancer [1]. Metastatic melanoma is the most deadly form of skin cancer, developed by the proliferation of transformed melanocytes from the basal region of the epidermis. Melanoma causes 75% of deaths when compared to non-melanoma skin cancers [2]. Hence, there is an urgent need for better therapeutic agents including ethno-based compounds. The induction of apoptosis has gained attention in cancer chemotherapy, which has prompted researchers to isolate plantbased derivatives that have the potential to induce cell death in cancer cells by activating various cell death signalling pathways. Plant compounds, like vincristine, vinblastine, vindesine, vinorelbine, etc. have already been used successfully in treating cancer either alone or in combinations [3]. Hence, the present research strategies continue to focus more to identify a potent template from natural resources that can be used to treat various cancers. In the present study, we investigated the anti-proliferative and apoptotic potentials of a pentacyclic triterpenoid (Fig. 1),
∗ Corresponding author. Department of Zoology, Government Arts College, Dharmapuri 636705, Tamil Nadu, India. Tel.: +91 99 943 336 88. E-mail address: ashoku [email protected] (R.A. Kumar).
oleanolic acid (OA) in A375 melanoma cells. OA has been reported for its promising anti-cancer activities in several cancer cell lines [4–6]. Though OA has been reported for its cytotoxic effects [7], to the best of our knowledge, its apoptotic activity against human melanoma A375 cells has not been investigated. In addition, our previous study [8] showed significant effects of OA on human keratinocyte (HaCaT) cells, which further prompted us to extend our investigation in A375 melanoma cells due to a close and important functional association between keratinocytes and melanocytes [9].
2. Materials and methods 2.1. Chemicals and reagents Dulbecco’s Modified Eagle Medium (DMEM) with (4.5 g/L of glucose and l-glutamine), Dulbecco’s phosphate buffered saline (PBS) (Ca2+ /Mg2+ free), Phenazine methosulfate (PMS) (also known as N-methylphenazonium methosulfate), Giemsa stain were purchased from Himedia Laboratories Pvt. Ltd. (India). XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]2H-tetrazolium hydroxide}, DAPI (4,6-diamidino-2-phenylindole dihydrochloride) and OA were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Apoptotic kit, Cellular DNA fragmentation ELISA (# 11 585 045 001) was obtained from Roche Diagnostics,
2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2013.09.003
Please cite this article in press as: Cijo George V, et al. Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2013.09.003
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2.6. Wright-Geimsa staining
Fig. 1. The structure of OA.
A375 cells were allowed to grow till 70% confluence and treated with different concentrations of OA for a period of 24 h. The cells were then washed in PBS and kept in PBS/methanol (1:1) for 2 min. Cell fixation was done by incubating cells in methanol for 10 min. After removing the methanol, the cells were stained with WrightGiemsa stain for 2 min and observed under inverted phase-contrast microscope. 2.7. DAPI staining
Germany. The remaining chemicals and solvents used were of standard analytical grade. 2.2. Drug preparation Stock solutions of OA were prepared at 25.77 mM in 100% dimethyl sulfoxide (DMSO) and the final concentration never exceeded 1% DMSO (v/v).
A375 cells were grown on cover slips to attain 70% confluence. Cells were treated by OA at 277.5 M (IC50 value from XTT assay) for 24 h followed by washing with PBS for two times. The cells were fixed with 4% paraformaldehyde for 15 min and then washed with PBS. DAPI (1 g/mL) staining was then performed as described previously [12] and observed for nuclear fragments under fluorescence microscopy at a magnification of 100×. 2.8. Apoptotic detection – cellular DNA fragmentation ELISA
2.3. Cell culture A375 (human melanoma) cells were obtained from National Centre for Cell Science (NCCS, Pune, India). The cells were propagated in DMEM media supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 ◦ C. The cells were maintained at the above-mentioned culture conditions for all the experiments and confluent cells between the second and the sixth passages were used for all the experiments. 2.4. Cell growth assay Cell growth assay was performed accordingly with minor modifications as described earlier [10]. Cells (1 × 105 ) were initially seeded in 6 well culture plate at time 0 h. Cells were then exposed to OA concentrations (12.5–200 M) for 24 and 48 h, respectively. Live cell quantification was achieved by trypsinization of the cultured cells and its consequent counting using a hemocytometer with trypan blue (0.4%) staining. The experiment was performed in triplicate. 2.5. Cytotoxicity analysis: XTT assay The effect of OA in A375 cells was tested by the method of XTT–formazan dye formation [11]. Then, 1 × 104 cells were seeded in a 96-well plate and 200 L of the culture medium was added to the cell suspension in the micro wells and incubated at 37 ◦ C for a period of 24 h. The media were then replaced with 200 L of the fresh media containing varying concentrations of OA, and subsequently, re-incubated for an additional 24 h. Drug medium was then substituted by 200 L of the fresh medium. About 50 L of XTT reagent, prepared in the medium (0.6 mg/mL) containing 25 M of PMS was then added to all the wells and the plate was incubated under humidified conditions in the dark at 37 ◦ C for 4 h. After incubation, the orange coloured complex formed was read at 450 nm using a Dynex Opsys MRTM Microplate Reader (Dynex Technologies, VA, USA) with a 630 nm reference filter. Wells containing cells without the OA served as the control and wells containing only culture medium and XTT reagent served as the blank. The percentage cytotoxicity of the extracts was calculated by using the formula: % Cytotoxicity =
(OD of control − OD of treated cells) × (100) OD of control
The potential of OA to induce apoptosis was studied using cellular DNA fragmentation ELISA kit as per the supplier’s instructions. Briefly, A375 cells were labelled with 10 M BrdU at 1 × 105 cells/mL density. Then, 100 L of these BrdU-labelled cells in culture medium were treated with varying concentrations of OA for a period of 4 h. The cells were then lysed and the apoptotic fragments were obtained after centrifugation at 1500 rpm for 10 min and subjected to ELISA. About 100 L of this obtained sample was transferred to an anti-DNA coated 96-well, flat-bottom microplates (MTPs). The plates were incubated for 90 min at 15–25 ◦ C. DNA was then denatured by microwave irradiation (500 W for 5 min) followed by the addition of 100 l anti-BrdU-POD conjugate solution. The plates were further incubated for 90 min and were washed 3 times with wash buffer (1×). Then, 100 L substrate (TMB) solution was then added for a blue colour development. The absorbance was read at 450 nm after the addition of 25 L of stop solution. 2.9. Statistical data analysis All the analytical experiments were carried out in triplicate (n = 3). Data was expressed as mean ± standard deviation (SD). Statistical analyses were performed by one-way ANOVA. MATLAB ver. 7.0 (Natick, MA, USA), GraphPad Prism 5.0 (San Diego, CA, USA) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations. Significant differences between groups were determined at P < 0.05. 3. Results 3.1. OA inhibited cell growth A375 cells were treated with OA for a period of 24 and 48 h at different concentrations and analysed by trypan blue assay to assess cell viability. OA exhibited significant reduction in cell numbers when compared to control cells in a concentration-dependent manner. Longer exposure time (48 h) of A375 cells to OA markedly inhibited cell growth when compared to the exposure time of 24 h (Fig. 2). 3.2. Cytotoxic effects of OA Cytotoxicity of OA on A375 cells were analyzed by XTT assay after 24 h treatment. XTT is metabolically reduced by mitochondrial dehydrogenase enzyme in viable cells to a water-soluble
Please cite this article in press as: Cijo George V, et al. Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2013.09.003
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Fig. 3. Time- and dose-dependent responses of OA in A375 cells. Data expressed as mean ± SD of n = 3 samples (* P < 0.05). D.C = DMSO control. Fig. 2. Effects of OA on A375 cell growth, which was markedly inhibited (* P < 0.05) at 24 and 48 h with increasing concentrations. Data expressed as mean ± SD of n = 3 independent experiments.
formazan product, which can be measured spectrophotometrically [13]. OA showed dose- and time-dependent cytotoxicity in A375 cells (Fig. 3). Maximum cytotoxicity observed for a concentration of 200 M was 36.10%.
cells showed changes in cell morphology, which included loss of cell–cell contacts, membrane damage, and membrane blebbing (Fig. 4). These characteristic changes were usually considered as the hallmarks for apoptosis [15].
3.4. DAPI staining 3.3. OA alters cellular morphologies Cytotoxicity of a chemotherapeutic drug often involves the annihilation of cellular morphologies, which may result in inducing cell death by activating various upstream/downstream elements (proteins) in apoptotic pathways [14]. A375 cells were exposed to various concentrations of OA for a time period of 24 h. Treated
Nuclear fragmentation was generally considered as an indication of apoptosis by observing the nuclei damage with fluorescent microscopy [16]. A375 cells were treated with OA for a period of 24 h. Treated cells showed the nuclear fragments stained in blue color (Fig. 5) when compared to the control A375 cells in which the nuclei remained intact.
Fig. 4. Morphological changes of A375 cells after treatment with OA, as showed by Giemsa staining. Cells showed changes in cell morphology, loss of cell–cell contacts, membrane damage (arrows) and membrane blebbing with increasing doses of OA when compared to the control cells.
Please cite this article in press as: Cijo George V, et al. Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2013.09.003
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Fig. 5. OA induces nuclear fragmentation in A375 cells. Cells were incubated with OA for period of 24 h. Nuclear fragments were stained by DAPI (B) in treated cells when compared to the control cells (A). Data are derived from three independent experiments.
3.5. Apoptotic potentials of OA Apoptosis is a physiological program that helps to maintain homeostasis in normal cells, in which cell death occurs naturally [17]. Apoptotic potentials of OA were assessed by cellular DNA fragmentation assay. BrdU-labelled DNA fragments formed after pre-treatment with OA, that were quantified at 450 nm. A concentration-dependent increase in DNA fragments was observed in A375 cells indicating dose-dependent apoptotic potentials of OA (Fig. 6). 4. Discussion and conclusion Plant derived anti-cancer compounds exert their effects on cancer cells by inducing various pathways of which apoptotic induction has been a new target for innovative mechanismsbased drug discovery [18]. Triterpenoids were previously reported for their apoptotic inductive potentials in various cell model systems, particularly at non-cytotoxic doses [19,20]. OA is one such common triterpenoid reported to possess varied therapeutic potentials, hitherto, has not been reported for apoptotic potential in melanoma cells. Hence, the present study scrutinized OA for its anti-proliferative and apoptotic potentials against melanoma. The cell viability and cytotoxic responses were studied to analyse the potential of OA on A375 cells. The amount of viable cells was significantly (P < 0.05) reduced with increasing concentrations of OA as showed by the trypan blue assay. The percentage cytotoxicity in A375 cells was found to be higher with increasing concentrations
Fig. 6. Apoptosis induction abilities of OA in A375 cells as demonstrated by cellular DNA fragmentation ELISA with increasing DNA fragments with increasing concentrations. Data expressed as mean ± SD of n = 3 samples.
of OA, and this possibly suggested that the cell viability was affected with a concentration and time-dependent manner and thus, substantiate its anti-proliferative effects. In our studies, the IC50 value (calculated from XTT assay) was found to be 277.5 M, which does not reveal any significant cytotoxic effects of OA on A375 cells when compared to its higher cytotoxic effects reported on other cancer model systems [21]. The observed cytotoxicity of a chemotherapeutic compound often coincides with the induction of apoptosis in cancer cells [22].
Please cite this article in press as: Cijo George V, et al. Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2013.09.003
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Cell death by apoptosis may result due to a genetically encoded suicide program that leads to cellular, morphological, and biochemical changes which include cell volume loss, mitochondrial depolarization, activation of caspase, chromatin condensation, and nuclear fragmentation [23]. In our study, morphological changes of A375 cells were observed after the treatment with OA for 24 h as recorded from Wright-Giemsa staining. Higher dosage of OA disrupts the cell wall and reduces the cell volume when compared to the control cells. Nuclear damage was shown by DAPI staining, and this suggested the possibility of cell death process through apoptotic induction, which was initiated by OA. Induction of apoptosis in tumor cells is a specific therapeutic approach towards developing cancer chemotherapeutic drugs [24]. Accordingly, DNA fragmentation was assessed by ELISA method, which showed dose-dependent response of OA in damaging DNA fragments in A375 cells. At a concentration < 50 M, there was only moderate DNA fragmentation and cytotoxicity when compared to those measured at higher doses (> 50 M). Significantly higher (almost two-fold) cell death occurring at elevated doses, possibly suggest that there is a differential sensitivity of A375 cells to OA. Similar results were reported before in human skin fibroblast cells [25]. Our results were also consistent with the results reported by other investigators in various primary cells, like HL60 cells (Human leukemia) and Panc-28 (Pancreatic) cells, which demonstrated the DNA fragmentation, caspase activation and cell cycle arresting potentials of OA [26,27]. However, the molecular elements/proteins involved in this pathway are yet to be elucidated. In conclusion, from this study we have observed that OA inhibited cell growth at 24 and 48 h with significant reduction in cell viability. Exposure to higher concentrations (> 50 M) of OA resulted in apoptotic-DNA fragmentation followed by cellular and nuclear destructions. Hence, we conclude that OA has the potential to induce apoptosis in A375 melanoma cells and these results warrant us to study further about the underlying molecular mechanism of this triterpenoid in A375 cells. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We are thankful to the management of VIT University, Vellore, Tamil Nadu, India, for providing the necessary infrastructure for the successful accomplishment of this research work. The first author, Mr Cijo George V, also thank the Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India for providing grants in the form of Senior Research Fellowship (SRF). References [1] Califano J, Nance M. Malignant melanoma. Facial Plast Surg Clin North Am 2009;17:337–48. [2] Boniol M, Autier P, Boyle P, Gandini S. Cutaneous melanoma attributable to sunbed use: systematic review and meta-analysis. BMJ 2012;345:e4757. [3] Lucas DM, Still PC, Perez LB, Grever MR, Kinghorn AD. Potential of plant-derived natural products in the treatment of leukemia and lymphoma. Curr Drug Targets 2010;11:812–22.
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[4] Liu J. Oleanolic acid and ursolic acid: research perspectives. J Ethnopharmacol 2005;100:92–4. [5] Chiang YM, Chang JY, Kuo CC, Chang CY, Kuo YH. Cytotoxic triterpenes from aerial roots of Ficus microcarpa. Phytochemistry 2005;66:495–501. [6] Wu M, Kodani I, Dickinson D, Huff F, Ogbureke KU, Qin H, et al. Exogenous expression of caspase-14 induces tumor suppression in human salivary cancer cells by inhibiting tumor vascularization. Anticancer Res 2009;29: 3811–8. [7] Wang D, Xia M, Cui Z. New triterpenoids isolated from the root bark of Ulmus pumila L. Chem Pharm Bull 2006;54:775–8. [8] George VC, Kumar DRN, Suresh PK, Kumar RA. Apoptosis-induced cell death due to oleanolic acid in HaCaT keratinocyte cells -a proof-of-principle approach for chemopreventive drug development. Asian Pac J Cancer Prev 2012;13:2015–20. [9] Chung H, Suh EK, Han IO, Oh ES. Keratinocyte-derived laminin-332 promotes adhesion and migration in melanocytes and melanoma. J Biol Chem 2011;15:13438–47. [10] Wu PK, Chi Shing Tai W, Liang ZT, Zhao ZZ, Hsiao WL. Oleanolic acid isolated from Oldenlandia diffusa exhibits a unique growth inhibitory effect against rastransformed fibroblasts. Life Sci 2009;85:113–21. [11] Weislow R, Kiser DL, Fine D, Bader J, Shoemaker RH, Boyd MR. New solubleformazan assay for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity. J Natl Cancer Inst 1989;81:577–86. [12] Lin CC, Kuo CL, Lee MH, Lai KC, Lin JP, Yang JS, et al. Wogonin triggers apoptosis in human osteosarcoma U-2 OS cells through the endoplasmic reticulum stress, mitochondrial dysfunction and caspase-3-dependent signaling pathways. Int J Oncol 2011;39:217–24. [13] Maioli E, Torricelli C, Fortino V, Carlucci F, Tommassini V, Pacini A. Critical appraisal of the MTT assay in the presence of rottlerin and uncouplers. Biol Proced Online 2009;11:227–40. [14] Ricci MS, Zong WX. Chemotherapeutic approaches for targeting cell death pathways. Oncologist 2006;11:342–57. [15] Tang HL, Tang HM, Mak KH, Hu S, Wang SS, Wong KM, et al. Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response. Mol Biol Cell 2012;23:2240–52. [16] Cifuentes MC, Castaneda DM, Uruena CP, Fiorentino S. A fraction from Petiveria alliacea induces apoptosis via a mitochondria-dependent pathway and regulates HSP70 expression. Universitas Scientiarum 2009;14:125–34. [17] Strasser A, Cory S, Adams JM. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J 2011;30: 3667–83. [18] Huang T, Gong WH, Li XC, Zou CP, Jiang GJ, Li XH, et al. Induction of apoptosis by a combination of Paclitaxel and Carboplatin in the presence of hyperthermia. Asian Pac J Cancer Prev 2012;13:81–5. [19] Fang ZZ, Nian Y, Li W, Wu JJ, Ge GB, Dong PP, et al. Cycloartane triterpenoids from Cimicifuga yunnanensis induce apoptosis of breast cancer cells (MCF7) via p53-dependent mitochondrial signaling pathway. Phytother Res 2011;25:17–24. [20] Zhang X, Zhao M, Chen L, Jiao H, Liu H, Wang L, et al. A triterpenoid from Thalictrum fortunei induces apoptosis in BEL-7402 cells through the P53-induced apoptosis pathway. Molecules 2011;16:9505–19. [21] Moulisha B, Kumar GA, Kanti HP. Anti-leishmanial and anti-cancer activities of a pentacyclic triterpenoid isolated from the leaves of Terminalia arjuna combretaceae. Trop J Pharm Res 2010;9:135–40. [22] Fecker LF, Geilen CC, Hossini AM, Schwarz C, Fechner H, Bartlett DL, et al. Selective induction of apoptosis in melanoma cells by tyrosinase promoter-controlled CD95 ligand overexpression. J Invest Dermatol 2005;124: 221–8. [23] Mgbonyebi OP, Russo J, Russo IH. Roscovitine induces cell death and morphological changes indicative of apoptosis in MDA-MB-231 breast cancer cells. Cancer Res 1999;59:1903–10. [24] Kumar DRN, George VC, Suresh PK, Kumar RA. Anticancer and anti-metastatic activities of rheum emodi rhizome chloroform extracts. Asian J Pharm Clin Res 2012;5:189–94. [25] Wojciak-Kosior M, Paduch R, Matysik-Wozniak A, Niedziela P, Donica H. The effect of ursolic and oleanolic acids on human skin fibroblast cells. Folia Histochem Cytobiol 2011;49:664–9. [26] Zhang P, Li H, Chen D, Ni J, Kang Y, Wang S. Oleanolic acid induces apoptosis in human leukemia cells through caspase activation and poly (ADP-ribose) polymerase cleavage. Acta Biochim Biophys Sin 2007;9:803–9. [27] Wei J, Liu M, Liu H, Wang H, Wang F, Zhang Y, et al. Oleanolic acid arrests cell cycle and induces apoptosis via ROS-mediated mitochondrial depolarization and lysosomal membrane permeabilization in human pancreatic cancer cells. J Appl Toxicol 2012, http://dx.doi.org/10.1002/jat.2725.
Please cite this article in press as: Cijo George V, et al. Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2013.09.003
ARTICLE IN PRESS Biomedicine & Preventive Nutrition xxx (2014) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells V. Cijo George a , D.R. Naveen Kumar a , P.K. Suresh a , R. Ashok Kumar b,∗ a b
Cell culture lab (TT-114), School of Bio Sciences and Technology, VIT University, Vellore, India Department of Zoology, Government Arts College, Dharmapuri, India
a r t i c l e
i n f o
Article history: Received 21 August 2013 Accepted 16 September 2013 Keywords: Oleanolic acid Melanoma Apoptosis DNA fragmentation ELISA
a b s t r a c t Melanoma is a life threatening condition, which mostly effects cocassions despite the advancements in current chemotherapeutic techniques. The aim of present study is to investigate the apoptotic inducing potential of oleanolic acid (OA) in A375 human melanoma cells. The anti-proliferative effects of OA (12.5–200 M) were assessed by cell growth and XTT assay. The morphological and nuclear damage studies were carried out by Wright-Giemsa and DAPI staining, respectively. Further, the apoptotic inducing potential of OA in A375 cells were measured by DNA fragmentation ELISA. The results showed a doseresponsive effect of OA by inhibiting the cell growth significantly (P < 0.05) at 24 and 48 h with a decrease in cell viability (XTT data). The significant morphological changes included cellular annihilation, which was observed in A375 cells when compared to the control cells. Quantitative dose-dependent increase in apoptotic-DNA fragments in ELISA and nuclear fragments in DAPI results, further demonstrated the potential of this triterpenoid to induce apoptotic cell death at a concentration, particularly higher than 50 M. Thus, we conclude that OA has wielded both anti-proliferative and apoptotic inducing potentials against A375 melanoma cells and can be a better choice for its progression. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Skin cancer accounts for 5% of deaths worldwide, but the empirical data shows an exponential increase in the number of people suffering from different types of skin cancer [1]. Metastatic melanoma is the most deadly form of skin cancer, developed by the proliferation of transformed melanocytes from the basal region of the epidermis. Melanoma causes 75% of deaths when compared to non-melanoma skin cancers [2]. Hence, there is an urgent need for better therapeutic agents including ethno-based compounds. The induction of apoptosis has gained attention in cancer chemotherapy, which has prompted researchers to isolate plantbased derivatives that have the potential to induce cell death in cancer cells by activating various cell death signalling pathways. Plant compounds, like vincristine, vinblastine, vindesine, vinorelbine, etc. have already been used successfully in treating cancer either alone or in combinations [3]. Hence, the present research strategies continue to focus more to identify a potent template from natural resources that can be used to treat various cancers. In the present study, we investigated the anti-proliferative and apoptotic potentials of a pentacyclic triterpenoid (Fig. 1),
∗ Corresponding author. Department of Zoology, Government Arts College, Dharmapuri 636705, Tamil Nadu, India. Tel.: +91 99 943 336 88. E-mail address: ashoku [email protected] (R.A. Kumar).
oleanolic acid (OA) in A375 melanoma cells. OA has been reported for its promising anti-cancer activities in several cancer cell lines [4–6]. Though OA has been reported for its cytotoxic effects [7], to the best of our knowledge, its apoptotic activity against human melanoma A375 cells has not been investigated. In addition, our previous study [8] showed significant effects of OA on human keratinocyte (HaCaT) cells, which further prompted us to extend our investigation in A375 melanoma cells due to a close and important functional association between keratinocytes and melanocytes [9].
2. Materials and methods 2.1. Chemicals and reagents Dulbecco’s Modified Eagle Medium (DMEM) with (4.5 g/L of glucose and l-glutamine), Dulbecco’s phosphate buffered saline (PBS) (Ca2+ /Mg2+ free), Phenazine methosulfate (PMS) (also known as N-methylphenazonium methosulfate), Giemsa stain were purchased from Himedia Laboratories Pvt. Ltd. (India). XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]2H-tetrazolium hydroxide}, DAPI (4,6-diamidino-2-phenylindole dihydrochloride) and OA were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Apoptotic kit, Cellular DNA fragmentation ELISA (# 11 585 045 001) was obtained from Roche Diagnostics,
2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2013.09.003
Please cite this article in press as: Cijo George V, et al. Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2013.09.003
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ARTICLE IN PRESS V. Cijo George et al. / Biomedicine & Preventive Nutrition xxx (2014) xxx–xxx
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2.6. Wright-Geimsa staining
Fig. 1. The structure of OA.
A375 cells were allowed to grow till 70% confluence and treated with different concentrations of OA for a period of 24 h. The cells were then washed in PBS and kept in PBS/methanol (1:1) for 2 min. Cell fixation was done by incubating cells in methanol for 10 min. After removing the methanol, the cells were stained with WrightGiemsa stain for 2 min and observed under inverted phase-contrast microscope. 2.7. DAPI staining
Germany. The remaining chemicals and solvents used were of standard analytical grade. 2.2. Drug preparation Stock solutions of OA were prepared at 25.77 mM in 100% dimethyl sulfoxide (DMSO) and the final concentration never exceeded 1% DMSO (v/v).
A375 cells were grown on cover slips to attain 70% confluence. Cells were treated by OA at 277.5 M (IC50 value from XTT assay) for 24 h followed by washing with PBS for two times. The cells were fixed with 4% paraformaldehyde for 15 min and then washed with PBS. DAPI (1 g/mL) staining was then performed as described previously [12] and observed for nuclear fragments under fluorescence microscopy at a magnification of 100×. 2.8. Apoptotic detection – cellular DNA fragmentation ELISA
2.3. Cell culture A375 (human melanoma) cells were obtained from National Centre for Cell Science (NCCS, Pune, India). The cells were propagated in DMEM media supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 ◦ C. The cells were maintained at the above-mentioned culture conditions for all the experiments and confluent cells between the second and the sixth passages were used for all the experiments. 2.4. Cell growth assay Cell growth assay was performed accordingly with minor modifications as described earlier [10]. Cells (1 × 105 ) were initially seeded in 6 well culture plate at time 0 h. Cells were then exposed to OA concentrations (12.5–200 M) for 24 and 48 h, respectively. Live cell quantification was achieved by trypsinization of the cultured cells and its consequent counting using a hemocytometer with trypan blue (0.4%) staining. The experiment was performed in triplicate. 2.5. Cytotoxicity analysis: XTT assay The effect of OA in A375 cells was tested by the method of XTT–formazan dye formation [11]. Then, 1 × 104 cells were seeded in a 96-well plate and 200 L of the culture medium was added to the cell suspension in the micro wells and incubated at 37 ◦ C for a period of 24 h. The media were then replaced with 200 L of the fresh media containing varying concentrations of OA, and subsequently, re-incubated for an additional 24 h. Drug medium was then substituted by 200 L of the fresh medium. About 50 L of XTT reagent, prepared in the medium (0.6 mg/mL) containing 25 M of PMS was then added to all the wells and the plate was incubated under humidified conditions in the dark at 37 ◦ C for 4 h. After incubation, the orange coloured complex formed was read at 450 nm using a Dynex Opsys MRTM Microplate Reader (Dynex Technologies, VA, USA) with a 630 nm reference filter. Wells containing cells without the OA served as the control and wells containing only culture medium and XTT reagent served as the blank. The percentage cytotoxicity of the extracts was calculated by using the formula: % Cytotoxicity =
(OD of control − OD of treated cells) × (100) OD of control
The potential of OA to induce apoptosis was studied using cellular DNA fragmentation ELISA kit as per the supplier’s instructions. Briefly, A375 cells were labelled with 10 M BrdU at 1 × 105 cells/mL density. Then, 100 L of these BrdU-labelled cells in culture medium were treated with varying concentrations of OA for a period of 4 h. The cells were then lysed and the apoptotic fragments were obtained after centrifugation at 1500 rpm for 10 min and subjected to ELISA. About 100 L of this obtained sample was transferred to an anti-DNA coated 96-well, flat-bottom microplates (MTPs). The plates were incubated for 90 min at 15–25 ◦ C. DNA was then denatured by microwave irradiation (500 W for 5 min) followed by the addition of 100 l anti-BrdU-POD conjugate solution. The plates were further incubated for 90 min and were washed 3 times with wash buffer (1×). Then, 100 L substrate (TMB) solution was then added for a blue colour development. The absorbance was read at 450 nm after the addition of 25 L of stop solution. 2.9. Statistical data analysis All the analytical experiments were carried out in triplicate (n = 3). Data was expressed as mean ± standard deviation (SD). Statistical analyses were performed by one-way ANOVA. MATLAB ver. 7.0 (Natick, MA, USA), GraphPad Prism 5.0 (San Diego, CA, USA) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations. Significant differences between groups were determined at P < 0.05. 3. Results 3.1. OA inhibited cell growth A375 cells were treated with OA for a period of 24 and 48 h at different concentrations and analysed by trypan blue assay to assess cell viability. OA exhibited significant reduction in cell numbers when compared to control cells in a concentration-dependent manner. Longer exposure time (48 h) of A375 cells to OA markedly inhibited cell growth when compared to the exposure time of 24 h (Fig. 2). 3.2. Cytotoxic effects of OA Cytotoxicity of OA on A375 cells were analyzed by XTT assay after 24 h treatment. XTT is metabolically reduced by mitochondrial dehydrogenase enzyme in viable cells to a water-soluble
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Fig. 3. Time- and dose-dependent responses of OA in A375 cells. Data expressed as mean ± SD of n = 3 samples (* P < 0.05). D.C = DMSO control. Fig. 2. Effects of OA on A375 cell growth, which was markedly inhibited (* P < 0.05) at 24 and 48 h with increasing concentrations. Data expressed as mean ± SD of n = 3 independent experiments.
formazan product, which can be measured spectrophotometrically [13]. OA showed dose- and time-dependent cytotoxicity in A375 cells (Fig. 3). Maximum cytotoxicity observed for a concentration of 200 M was 36.10%.
cells showed changes in cell morphology, which included loss of cell–cell contacts, membrane damage, and membrane blebbing (Fig. 4). These characteristic changes were usually considered as the hallmarks for apoptosis [15].
3.4. DAPI staining 3.3. OA alters cellular morphologies Cytotoxicity of a chemotherapeutic drug often involves the annihilation of cellular morphologies, which may result in inducing cell death by activating various upstream/downstream elements (proteins) in apoptotic pathways [14]. A375 cells were exposed to various concentrations of OA for a time period of 24 h. Treated
Nuclear fragmentation was generally considered as an indication of apoptosis by observing the nuclei damage with fluorescent microscopy [16]. A375 cells were treated with OA for a period of 24 h. Treated cells showed the nuclear fragments stained in blue color (Fig. 5) when compared to the control A375 cells in which the nuclei remained intact.
Fig. 4. Morphological changes of A375 cells after treatment with OA, as showed by Giemsa staining. Cells showed changes in cell morphology, loss of cell–cell contacts, membrane damage (arrows) and membrane blebbing with increasing doses of OA when compared to the control cells.
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Fig. 5. OA induces nuclear fragmentation in A375 cells. Cells were incubated with OA for period of 24 h. Nuclear fragments were stained by DAPI (B) in treated cells when compared to the control cells (A). Data are derived from three independent experiments.
3.5. Apoptotic potentials of OA Apoptosis is a physiological program that helps to maintain homeostasis in normal cells, in which cell death occurs naturally [17]. Apoptotic potentials of OA were assessed by cellular DNA fragmentation assay. BrdU-labelled DNA fragments formed after pre-treatment with OA, that were quantified at 450 nm. A concentration-dependent increase in DNA fragments was observed in A375 cells indicating dose-dependent apoptotic potentials of OA (Fig. 6). 4. Discussion and conclusion Plant derived anti-cancer compounds exert their effects on cancer cells by inducing various pathways of which apoptotic induction has been a new target for innovative mechanismsbased drug discovery [18]. Triterpenoids were previously reported for their apoptotic inductive potentials in various cell model systems, particularly at non-cytotoxic doses [19,20]. OA is one such common triterpenoid reported to possess varied therapeutic potentials, hitherto, has not been reported for apoptotic potential in melanoma cells. Hence, the present study scrutinized OA for its anti-proliferative and apoptotic potentials against melanoma. The cell viability and cytotoxic responses were studied to analyse the potential of OA on A375 cells. The amount of viable cells was significantly (P < 0.05) reduced with increasing concentrations of OA as showed by the trypan blue assay. The percentage cytotoxicity in A375 cells was found to be higher with increasing concentrations
Fig. 6. Apoptosis induction abilities of OA in A375 cells as demonstrated by cellular DNA fragmentation ELISA with increasing DNA fragments with increasing concentrations. Data expressed as mean ± SD of n = 3 samples.
of OA, and this possibly suggested that the cell viability was affected with a concentration and time-dependent manner and thus, substantiate its anti-proliferative effects. In our studies, the IC50 value (calculated from XTT assay) was found to be 277.5 M, which does not reveal any significant cytotoxic effects of OA on A375 cells when compared to its higher cytotoxic effects reported on other cancer model systems [21]. The observed cytotoxicity of a chemotherapeutic compound often coincides with the induction of apoptosis in cancer cells [22].
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Cell death by apoptosis may result due to a genetically encoded suicide program that leads to cellular, morphological, and biochemical changes which include cell volume loss, mitochondrial depolarization, activation of caspase, chromatin condensation, and nuclear fragmentation [23]. In our study, morphological changes of A375 cells were observed after the treatment with OA for 24 h as recorded from Wright-Giemsa staining. Higher dosage of OA disrupts the cell wall and reduces the cell volume when compared to the control cells. Nuclear damage was shown by DAPI staining, and this suggested the possibility of cell death process through apoptotic induction, which was initiated by OA. Induction of apoptosis in tumor cells is a specific therapeutic approach towards developing cancer chemotherapeutic drugs [24]. Accordingly, DNA fragmentation was assessed by ELISA method, which showed dose-dependent response of OA in damaging DNA fragments in A375 cells. At a concentration < 50 M, there was only moderate DNA fragmentation and cytotoxicity when compared to those measured at higher doses (> 50 M). Significantly higher (almost two-fold) cell death occurring at elevated doses, possibly suggest that there is a differential sensitivity of A375 cells to OA. Similar results were reported before in human skin fibroblast cells [25]. Our results were also consistent with the results reported by other investigators in various primary cells, like HL60 cells (Human leukemia) and Panc-28 (Pancreatic) cells, which demonstrated the DNA fragmentation, caspase activation and cell cycle arresting potentials of OA [26,27]. However, the molecular elements/proteins involved in this pathway are yet to be elucidated. In conclusion, from this study we have observed that OA inhibited cell growth at 24 and 48 h with significant reduction in cell viability. Exposure to higher concentrations (> 50 M) of OA resulted in apoptotic-DNA fragmentation followed by cellular and nuclear destructions. Hence, we conclude that OA has the potential to induce apoptosis in A375 melanoma cells and these results warrant us to study further about the underlying molecular mechanism of this triterpenoid in A375 cells. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We are thankful to the management of VIT University, Vellore, Tamil Nadu, India, for providing the necessary infrastructure for the successful accomplishment of this research work. The first author, Mr Cijo George V, also thank the Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India for providing grants in the form of Senior Research Fellowship (SRF). References [1] Califano J, Nance M. Malignant melanoma. Facial Plast Surg Clin North Am 2009;17:337–48. [2] Boniol M, Autier P, Boyle P, Gandini S. Cutaneous melanoma attributable to sunbed use: systematic review and meta-analysis. BMJ 2012;345:e4757. [3] Lucas DM, Still PC, Perez LB, Grever MR, Kinghorn AD. Potential of plant-derived natural products in the treatment of leukemia and lymphoma. Curr Drug Targets 2010;11:812–22.
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Please cite this article in press as: Cijo George V, et al. Oleanolic acid inhibits cell growth and induces apoptosis in A375 melanoma cells. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2013.09.003