Cytotoxic activity of caffeic acid and gallic acid against MCF-7 human breast cancer cells: An in silico and in vitro study

Document Type : Original Research Article


1 Traditional and Complementary Medicine Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran

2 Cellular and Molecular Research Center, Faculty of Medicine, Sabzevar University of Medical Sciences, Sabzevar, Iran.

3 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.

4 Cellular and Molecular Research Center, Faculty of Medicine, Sabzevar University of Medical Sciences, Sabzevar, Iran

5 Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran

6 Department of Nutrition, Faculty of Medicine, Sabzevar University of Medical Sciences, Sabzevar, Iran.

7 Digestive Oncology Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences, Tehran, Iran

8 Department of Chemistry, School of Sciences, Hakim Sabzevari University, Sabzevar, Iran


Objective: Phenolic compounds have been considered inhibitors of various cancers.
Material and Methods: In this study, caffeic acid and gallic acid were appraised for their possible effects on apoptotic genes expression in a breast cancer cell line in vitro. We also evaluated ligand interaction and ligand binding with estrogen receptor alpha by molecular docking. To determine half maximal inhibitory concentration, MCF-7 cells were treated with different concentrations of caffeic acid and gallic acid by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Furthermore, morphological changes in cells and alterations in P53, Mcl-1 and P21 gene expression were studied by real-time RT-PCR. Also, protein network and different interactions between the desired genes were analyzed using GeneMANIA database.
Results: Evaluation of cell survival by MTT assay revealed that the half-maximal inhibitory concentration values for caffeic acid and gallic acid against MCF-7 cells, were 159 and 18 µg/ml, respectively. These compounds were found to affect P53, Mcl-1 and P21 gene expression; this alteration in gene expression probably occurred along with the activation of intrinsic apoptotic signaling pathway.
Conclusion: Via apoptosis induction, caffeic acid and gallic acid have induce toxic effects and morphological changes in breast cancer cells, suggesting their possible future application as antitumor agents.


Main Subjects

Abbas T, Dutta A. 2009. p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer, 9:400-412.
Beekman AM, Howell LA. 2016. Small-molecule and peptide inhibitors of the pro-survival protein Mcl-1. Chem Med Chem, 11:802–813.
Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, Barretina J, Boehm JS, Dobson J, Urashima M, Mc Henry KT. 2010. The landscape of somatic copy-number alteration across human cancers. Nature, 463:899-905.
Brewster AM, Chavez-MacGregor M, Brown P. 2014. Epidemiology, biology, and treatment of triple-negative breast cancer in women of African ancestry. Lancet Oncol, 15:e625–e634.
Chang BD, Swift ME, Shen M, Fang J, Broude EV, Roninson IB. 2002. Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent. Proc Natl Acad Sci, 99:389–394.
Chen J, Jackson PK, Kirschner MW, Dutta A. 1995. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature, 374:386–388.
Chiang EP, Tsai SY, Kuo YH, Pai MH, Chiu HL, Rodriguez RL, Tang FY. 2014. Caffeic acid derivatives inhibit the growth of colon cancer: involvement of the PI3-K/Akt and AMPK signaling pathways. PLoS One, 9:e99631.
Erster S, Mihara M, Kim RH, Petrenko O, Moll UM. 2004. In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation. Mol Cell Biol, 24:6728–6741.
Forester SC, Choy YY, Waterhouse AL, Oteiza PI. 2014. The anthocyanin metabolites gallic acid, 3-O-methylgallic acid, and 2, 4, 6-trihydroxybenzaldehyde decrease human colon cancer cell viability by regulating pro-oncogenic signals. Mol Carcinog, 53:432–439.
Gartel AL. 2005. The conflicting roles of the cdk inhibitor p21 (CIP1/WAF1) in apoptosis. Leuk Res, 29:1237–1238.
Gartel AL, Tyner AL. 2002. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis 1 supported in part by NIH grant R01 DK56283 (to ALT) for the p21 research and Campus Research Board and Illinois Department of Public Health Penny Severns Breast and Cervical Cancer grant. Mol Cancer Ther, 1:639–649.
Guimaraes TA, Farias LC, Fraga CA, Feltenberger JD, Melo GA, Coletta RD, Souza Santos SH, de Paula A, Guimaraes AL. 2016. Evaluation of the antineoplastic activity of gallic acid in oral squamous cell carcinoma under hypoxic conditions. Anticancer Drugs, 27:407–416.
Guo D, Dou D, Ge L, Huang Z, Wang L, Gu N.  2015. A caffeic acid mediated facile synthesis of silver nanoparticles with powerful anti-cancer activity. Colloids Surfaces B Biointerfaces, 134:229–234.
Halazonetis TD, Gorgoulis VG, Bartek J. 2008. An oncogene-induced DNA damage model for cancer development. Science, 319:1352–1355.
Han Z, Wei W, Dunaway S, Darnowski JW, Calabresi P, Sedivy J, Hendrickson EA, Balan KV, Pantazis P, Wyche JH. 2002. Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin. J Biol Chem, 277:17154–17160.
Hanahan D, Weinberg RA. 2000. The hallmarks of cancer. Cell, 100:57–70.
Huang DCS, Strasser A. 2000. BH3-only proteins—essential initiators of apoptotic cell death. Cell, 103:839–842.
Jia J, Yang M, Chen Y, Yuan H, Li J, Cui X, Liu Z. 2014. Inducing apoptosis effect of caffeic acid 3, 4-dihydroxy-phenethyl ester on the breast cancer cells. Tumor Biol, 35:11781–11789.
Kafi Z, Cheshomi H, Gholami O. 2018. 7-Isopenthenyloxycoumarin, arctigenin, and hesperidin modify myeloid cell leukemia type-1 (Mcl-1) gene expression by hormesis in K562 cell line. Dose-Response, 16:1-6.
Kerr JFR, Wyllie AH, Currie AR. 1972. Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br J Cancer, 26:239-257.
Kim GY, Mercer SE, Ewton DZ, Yan Z, Jin K, Friedman E. 2002. The stress-activated protein kinases p38α and JNK1 stabilize p21Cip1 by phosphorylation. J Biol Chem, 277:29792–29802
Korach KS, Emmen JM, Walker VR, Hewitt SC, Yates M, Hall JM, Swope DL, Harrell JC, Couse JF. 2003. Update on animal models developed for analyses of estrogen receptor biological activity. J Steroid Biochem Mol Biol, 86:387–391.
Kurokawa H, Arteaga CL. 2001. Inhibition of erbB receptor (HER) tyrosine kinases as a strategy to abrogate antiestrogen resistance in human breast cancer. Clin Cancer Res, 7:4436s-4442s.
Lima KG, Krause GC, Schuster AD, Catarina AV, Basso BS, De Mesquita FC, Pedrazza L, Marczak ES, Martha BA, Nunes FB, Chiela EC. 2016. Gallic acid reduces cell growth by induction of apoptosis and reduction of IL-8 in HepG2 cells. Biomed Pharmacother, 84:1282–1290.
Lin CL, Chen RF, Chen JY, Chu YC, Wang HM, Chou HL, Chang WC, Fong Y, Chang WT, Wu CY, Chiu CC. 2012. Protective effect of caffeic acid on paclitaxel induced anti-proliferation and apoptosis of lung cancer cells involves NF-κB pathway. Int J Mol Sci, 13:6236–6245.
Lohr K, Moritz C, Contente A, Dobbelstein M. 2003. p21/CDKN1A Mediates Negative Regulation of Transcription by p53. J Biol Chem, 278:32507–32516.
Martin-Caballero J, Flores JM, García-Palencia P, Serrano M. 2001. Tumor susceptibility of p21Waf1/Cip1-deficient mice. Cancer Res, 61:6234–6238.
Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, Stein KD, Alteri R, Jemal A. 2016. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin, 66:271–289.
Moll UM, Zaika A. 2001. Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett, 493:65–69.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65:55–63.
Nabavi SF, Nabavi SM, Habtemariam S, Moghaddam AH, Sureda A, Jafari M, Latifi AM. 2013. Hepatoprotective effect of gallic acid isolated from Peltiphyllum peltatum against sodium fluoride-induced oxidative stress. Ind Crops Prod, 44:50–55.
Olsen H, Aaby K, Borge GIA. 2009. Characterization and quantification of flavonoids and hydroxycinnamic acids in curly kale (Brassica oleracea L. convar. acephala var. sabellica) by HPLC-DAD-ESI-MS n. J Agric Food Chem, 57:2816–2825.
Pietrzak M, Puzianowska-Kuznicka M. 2008. p53-dependent repression of the human MCL-1 gene encoding an anti-apoptotic member of the BCL-2 family: the role of Sp1 and of basic transcription factor binding sites in the MCL-1 promoter. Biol Chem, 389:383–393.
Poole AJ, Heap D, Carroll RE, Tyner AL. 2004. Tumor suppressor functions for the Cdk inhibitor p21 in the mouse colon. Oncogene, 23:8128-8134.
Prasad NR, Karthikeyan A, Karthikeyan S, Reddy BV. 2011. Inhibitory effect of caffeic acid on cancer cell proliferation by oxidative mechanism in human HT-1080 fibrosarcoma cell line. Mol Cell Biochem, 349:11–19.
Rao K, Indap M, Radhika S, Motiwale L. 2006. Anticancer activity of phenolic antioxidants against breast cancer cells and a spontaneous mammary tumor. Indian J Pharm Sci, 68:470-476.
Rosendahl AH, Perks CM, Zeng L, Markkula A, Simonsson M, Rose C, Ingvar C, Holly JM, Jernström H. 2015. Caffeine and caffeic acid inhibit growth and modify estrogen receptor (ER) and insulin-like growth factor I receptor (IGF-IR) levels in human breast cancer. Clin Cancer Res clincanres, 74:1078-0432.
Sanderson JT, Clabault H, Patton C, Lassalle-Claux G, Jean-François J, Paré AF, Hébert MJ, Surette ME, Touaibia M. 2013. Antiproliferative, antiandrogenic and cytotoxic effects of novel caffeic acid derivatives in LNCaP human androgen-dependent prostate cancer cells. Bioorg Med Chem, 21:7182–7193.
Seresht HR, Albadry BJ, Al-mosawi AK, Gholami O, Cheshomi H. 2019. The cytotoxic effects of thymol as the major component of trachyspermum ammi on breast cancer (MCF-7) cells. Pharm Chem J, 53:1–7.
Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB. 2003. Identification of a cancer stem cell in human brain tumors. Cancer Res, 63:5821–5828.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. JNCI J Natl Cancer Inst, 82:1107–1112.
Tabuchi Y, Matsuoka J, Gunduz M, Imada T, Ono R, Ito M, Motoki T, Yamatsuji T, Shirakawa Y, Takaoka M, Haisa M. 2009. Resistance to paclitaxel therapy is related with Bcl-2 expression through an estrogen receptor mediated pathway in breast cancer. Int J Oncol, 34:313–319.
Thor AD, Liu S, Moore Ii DH, Shi Q, Edgerton SM. 2000. p21 WAF1/CIP1 Expression in breast cancers: associations with p53 and outcome. Breast Cancer Res Treat, 61:33–43.
Vousden KH. 2000. p53: death star. Cell, 103:691–694.
Williams MM, Cook RS. 2015. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget, 6:3519-3530.
Yan J, Liu XL, Han LZ, Xiao G, Li NL, Deng YN, Yin LC, Ling LJ, Yu XY, Tan CL, Huang XP. 2015. Relation between Ki-67, ER, PR, Her2/neu, p21, EGFR, and TOP II-α expression in invasive ductal breast cancer patients and correlations with prognosis. Asian Pac J Cancer Prev, 16:823–829.
Yang W, Velcich A, Lozonschi I, Liang J, Nicholas C, Zhuang M, Bancroft L, Augenlicht LH. 2005. Inactivation of p21WAF1/cip1 enhances intestinal tumor formation in Muc2−/− mice. Am J Pathol, 166:1239–1246.
Ye JC, Hsiao MW, Hsieh CH, Wu WC, Hung YC, Chang WC. 2010. Analysis of caffeic acid extraction from Ocimum gratissimum Linn. by high performance liquid chromatography and its effects on a cervical cancer cell line. Taiwan J Obstet Gynecol, 49:266–271.
Yeh RD, Chen JC, Lai TY, Yang JS, Yu CS, Chiang JH, Lu CC, Yang ST, Yu CC, Chang SJ, Lin HY. 2011. Gallic acid induces G0/G1 phase arrest and apoptosis in human leukemia HL-60 cells through inhibiting cyclin D and E, and activating mitochondria-dependent pathway. Anticancer Res, 31:2821–2832.
Zheng N, Zhang P, Huang H, Liu W, Hayashi T, Zang L, Zhang Y, Liu L, Xia M, Tashiro SI, Onodera S. 2015. ERα down-regulation plays a key role in silibinin-induced autophagy and apoptosis in human breast cancer MCF-7 cells. J Pharmacol Sci, 128:97–107.
Zhu H, Zhang L, Wu S, Teraishi F, Davis JJ, Jacob D, Fang B. 2004. Induction of S-phase arrest and p21 overexpression by a small molecule 2 [3-(2, 3-dichlorophenoxy) propyl] amino] ethanol in correlation with activation of ERK. Oncogene, 23:4984-4992.