To examine the role of carvacrol in modulating PI3K/AKT signaling involved in human breast cancer pathogenesis using in vitro experimental model MCF-7 cells.
MTT and lactate dehydrogenase assays were performed with cells treated with different doses of carvacrol (0-250 p mol/L) at different time points (24 and 48 h). The nuclear morphology was assessed in MCF-7 cells with propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) staining and analyzed by fluorescence microscopy. Events like cell cycle arrest, apoptosis was observed by flow cytometric analysis and expressions of p-Rb, cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6, Bax, Bcl-2, PI3K/p-AKT was analyzed by immunoblot.
Carvacrol significantly reduced cell viability with the half maximal inhibitory concentration value of 200 µmol/L at 24 and 48 h (P<0.05). importantly, there was a significant increase in the accumulation of the G0/G1 phase upon treatment with carvacrol in MCF-7 cells (P<0.05 or P<0.01). A remarkable decrease in protein expressions of p-Rb, cyclin D1, CDK4 and CDK6 denotes cell cycle arrest (P<0.05 or P<0.01). In addition, carvacrol treatment significantly inhibited PI3K/p-AKT protein expressions leading to induction of apoptosis mediated by decreased Bcl2 and increased Bax protein expressions. Further, Annexin V/PI staining by FACS analysis, dual staining by AO/EB and PI staining studies suggests induction of apoptosis by carvacrol through PI3K/Akt signaling pathway in MCF-7 cells.
Carvacrol significantly inhibited the breast cancer MCF-7 cell proliferation and induced apoptosis via suppressing PI3/AKT signaling pathway.
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Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL Rowland JH, et al. Cancer treatment and survivorship statistics, 2016. CA 2016;66:271–289.
Deb S, Jene N, Fox SB, Kconfab Investigators. Genotypic and phenotypic analysis of familial male breast cancer shows under representation of the HER2 and basal subtypes in BRCA-associated carcinomas. BMC Cancer 2012;12:510.
Carter LG, D’Orazio JA, Pearson KJ. Resveratrol and cancer: a focus on in vivo evidence. Endocr Related Cancer 2014;21:R209–R225.
Safarzadeh E, Shotorbani SS, Baradaran B. Herbal medicine as inducers of apoptosis in cancer treatment. Adv Pharm Bull 2014;4(Suppl 1):421.
Wink M. Modes of action of herbal medicines and plant secondary metabolites. Medicines 2015;2:251–286.
Jayakumar S, Madankumar A, Asokkumar S, Raghunandhakumar S, Kamaraj S, Divya MG, et al. Potential preventive effect of carvacrol against diethylnitrosamine-induced hepatocellular carcinoma in rats. Mol Cell Biochem 2012;360:51–60.
Slamenova D, Horvathova E, Sramkova M, Marsalkova L. DNA-protective effects of two components of essential plant oils carvacrol and thymol on mammalian cells cultured in vitro. Neoplasma 2007;54:108–112.
Khan I, Bahuguna A, Kumar P, Bajpai VK, Kang SC. In vitro and in vivo antitumor potential of carvacrol nanoemulsion against human lung adenocarcinoma A549 cells via mitochondrial mediated apoptosis. Sci Rep 2018;8:144.
Potočnjak I, Gobin I, Domitrović R. Carvacrol induces cytotoxicity in human cervical cancer cells but causes cisplatin resistance: involvement of MEK-ERK activation. Phytother Res 2018;32:1090–1097.
Bharti R, Dey G, Ojha PK, Rajput S, Jaganathan SK, Sen R, Mandal M. Diacerein-mediated inhibition of IL-6/IL-6R signaling induces apoptotic effects on breast cancer. Oncogene 2016;35:3965–3975.
Al-Fatlawi AA, Rahisuddin I, Ahmad A. Cytotoxicity and proapoptotic activity of carvacrol on humanbreast cancer cell line MCF-7. World J Pharm Sci 2014;2:1218–1223.
Serpeloni JM, Specian AF, Ribeiro DL, Tuttis K, Vilegas W, Martínez-López W, et al. Antimutagenicity and induction of antioxidant defense by flavonoid rich extract of Myrcia bella Cambess, in normal and tumor gastric cells. J Ethnopharm 2015;176:345–355.
Specian AF, Serpeloni JM, Tuttis K, Ribeiro DL, Cilião HL, Varanda EA, et al. LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds. Cytotechnology 2016;68:2729–2744.
Rahman SA, Nur S, Abdul Wahab N, Malek A, Nurestri S. In vitro morphological assessment of apoptosis induced by antiproliferative constituents from the rhizomes of Curcuma zedoaria. Evid Based Complement Alternat Med 2013;2013:257108.
Hezel M, Ebrahimi F, Koch M, Dehghani F. Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain. Micron 2012;43:1031–1038.
Winnicka K, Bielawski K, Bielawska A, Miltyk W. Apoptosis-mediated cytotoxicity of ouabain, digoxin and proscillaridin A in the estrogen independent MDA-MB-231 breast cancer cells. Arch Pharm Res 2007;30:1216–1224.
Mariotti S, Pardini M, Teloni R, Gagliardi MC, Fraziano M, Nisini R. A method permissive to fixation and permeabilization for the multiparametric analysis of apoptotic and necrotic cell phenotype by flow cytometry. Cytometry A 2017;91:1115–1124.
Ribeiro DL, Ciliao HL, Specian AF, Serpeloni JM, Souza MF, Tangerina MM, et al. Chemical and biological characterisation of Machaerium hirtum (Vell.) Stellfeld: absence of cytotoxicity and mutagenicity and possible chemopreventive potential. Mutagenesis 2015;31:147–160.
Kamaraj S, Anandakumar P, Jagan S, Ramakrishnan G, Devaki T. Modulatory effect of hesperidin on benzo(a)pyrene induced experimental lung carcinogenesis with reference to COX-2, MMP-2 and MMP-9. Eur J Pharm 2010;649:320–327.
Şen HS, Şen V, Bozkurt M, Türkçü G, Güzel A, Sezgi C, et al. Carvacrol and pomegranate extract in treating methotrexate-induced lung oxidative injury in rats. Med Sci Monit 2014;20:1983–1990.
Ponraj T, Paulpandi M, Vivek R, Vimala K, Kannan S. Protein regulation and Apoptotic induction in human breast carcinoma cells (MCF-7) through lectin from G. beauts. Int J Biol Macromol 2017;95:1235–1245.
Queiroz EA, Fortes ZB, da Cunha MA, Sarilmiser HK, Dekker AM, Öner ET, et al. Levan promotes antiproliferative and proapoptotic effects in MCF-7 breast cancer cells mediated by oxidative stress. Int J Biol Macromol 2017;102:565–570.
Torii S, Yamamoto T, Tsuchiya Y, Nishida E. ERK MAP kinase in G1 cell cycle progression and cancer. Cancer Sci 2006;97:697–702.
Jia X, Han C, Chen J. Effects of tea on preneoplastic lesions and cell cycle regulators in rat liver. Cancer Epidemiol Prev Biomark 2002;11:1663–1667.
Wu GS, Lu JJ, Guo JJ, Li YB, Tan W, Dang YY, et al. Ganoderic acid DM, a natural triterpenoid, induces DNA damage, G1 cell cycle arrest and apoptosis in human breast cancer cells. Fitoterapia 2012;83:408–414.
Sharpe JC, Arnoult D, Youle RJ. Control of mitochondrial permeability by Bcl-2 family members. Biochim Biophys Acta 2004;1644:107–113.
Kowaltowski AJ, Cosso RG, Campos CB, Fiskum G. Effect of Bcl-2 overexpression on mitochondrial structure and function. J Biol Chem 2002;277:42802–42807.
Balakumaran MD, Ramachandran R, Kalaichelvan PT. Exploitation of endophytic fungus, Guignardia mangiferae for extracellular synthesis of silver nanoparticles and their in vitro biological activities. Microbiol Res 2015;178:9–17.
Fabisiak JP, Borisenko GG, Kagan VE. Quantitative method of measuring phosphatidylserine externalization during apoptosis using electron paramagnetic resonance (EPR) spectroscopy and annexin-conjugated iron. Methods Mol Biology. 2014;1105:613–621.
Kwan YP, Saito T, Ibrahim D, Al-Hassan FM, Ein Oon C, Chen Y, et al. Evaluation of the cytotoxicity, cell-cycle arrest, and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells. Pharm Biol 2016;54:1223–1236.
Chang F, Lee JT, Navolanic PM, Steelman LS, Shelton JG, Blalock WL, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 2003;17:590.
Vara JÁF, Casado E, de Castro J, Cejas P, Belda-Iniesta C, et al. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 2004;30:193–204.
Mitsiades CS, Mitsiades N, Koutsilieris M. The Akt pathway: molecular targets for anti-cancer drug development. Curr Cancer Drug Target 2004;4:235–256.
Chen WL, Barszczyk A, Turlova E, Deurloo M, Liu B, Yang BB, et al. Inhibition of TRPM7 by carvacrol suppresses glioblastoma cell proliferation, migration and invasion. Oncotarget 2015;6:16321.
First author Mr. M. Ashok is grateful to the Indian Council of Medical Research (ICMR) New Delhi, India for the support in the form of Senior Research Fellowship (SRF).
Conflict of Interest
The authors declare that they have no conflict of interest.
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Mari, A., Mani, G., Nagabhishek, S.N. et al. Carvacrol Promotes Cell Cycle Arrest and Apoptosis through PI3K/AKT Signaling Pathway in MCF-7 Breast Cancer Cells. Chin. J. Integr. Med. (2020). https://doi.org/10.1007/s11655-020-3193-5
- breast cancer
- PI3K/AKT signaling pathway