Effects of combined 5-Fluorouracil and ZnO NPs on human breast cancer MCF-7 Cells: P53 gene expression, Bcl-2 signaling pathway, and invasion activity

Document Type : Research Paper


1 Clinical Biochemistry Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran

2 Department of Medical Physics and Radiology, Shahrekord University of Medical Sciences, Shahrekord, Iran

3 Institute for Medical Physics, Ambilly, France


Objective(s): The significant contribution of nanoparticles to cancer treatment has attracted therapeutic attention. The present study aimed to evaluate the synergistic effects of 5-fluorouracil (5-FU) and zinc oxide nanoparticles (ZnO NPs) as multimodal drug delivery on human breast cancer MCF-7 cells.
Materials and Methods: In this in-vitro study, the impact of 5-FU and ZnO NPs in the single or combined forms was evaluated on cell viability, colony formation, apoptosis, p53 gene expression, and Bcl-2 signaling protein in MCF-7 breast cancer cell line using several techniques, such as MTT, clonogenic assay, flow cytometry, real-time quantitative polymerase chain reaction, and Western blot.
Results: In this study, 5-FU combined with ZnO NPs showed synergistic effects against MCF-7 within 48 hours. In addition, the combination of 5-FU and ZnO NPs at the respective concentrations of 1 µM and 45 µg/ml exhibited significant apoptosis (79.53%), p53 gene expression (3.6 folds), reduction of cell invasion (9.82%), and plating efficiency (5%), thereby leading to the significant reduction of cell viability (40±0.9%) and decreased Bcl-2 anti-apoptotic protein relative to untreated control cells.
Conclusion: According to the results, the synergistic effects of combined ZnO NPs and 5-FU on MCF-7 human breast cancer cells were exerted via Bcl-2 inhibition and the up-regulation of p53 expression.


1.Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-tieulent J, Jemal A. Global Cancer Statistics. 2012. CA Cancer J Clin. 2015; 65(2): 87–108.
2.Torre LA, Islami F, Siegel RL, Ward EM, Jemal A. Global Cancer in Women: Burden and Trends. Cancer Epidemiol. Biomarkers Prev. 2017; 26(4): 444–457.
3.Smith RA, Andrews KS, Brooks D, Fedew SA, Manassaram-Baptiste D, Saslow D. Brawley O.W, Wender R.C. A review of current American Cancer Society Guidelines and current issues in cancer screening. CA Cancer J Clin. 2018; 68(4): 297–316.
4.Neilson HK, Farris MS, Stone CR, Vaska MM, Brenner DR, Friedenreich CM. Moderate-vigorous recreational physical activity and breast cancer risk. stratified by menopause status. Menopause. 2017; 24(3): 322–344.
5.DeSantis CE, Lin CC, Mariotto AB, Siegel RL, Stein KD, Kramer JL, Alteri R, Robbins A.S, Jemal A. Cancer treatment and survivorship statistics. CA Cancer J Clin. 2014; 64(4): 252–271.
6.Pérez-Herrero E, Fernández-Medarde A. Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy. Eur J Pharm Biopharm. 2015; 93: 52–79.
7.Zhang RX, Lun Wong H, Xue HY, Eoh JY, Wu XY. Nanomedicine of synergistic drug combinations for cancer therapy – Strategies and perspectives. J Control Release. 2016; 240: 489-503.
8.Akpinar B, Bracht E, Reijnders D, Safarikova B, Jelinkova I, Grandien A, Vaculova A, Zhivotovsky B, Olsson M. Oncotarget BS. 5-Fluorouracil-induced RNA stress engages a TRAIL-DISC-dependent apoptosis axis facilitated by p53. Oncotarget. 2015; 6(41): 43679-43681.
9.Bai D-P, Zhang XF, Zhang GL, Huang YF, Gurunathan S. Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells. Int J Nanomedicine. 2017; 12: 6521–6535.
10.Malhotra SPK, Mandal TK. Biomedical applications of Zinc oxide nanomaterials in cancer treatment . A review Scirea J Chem. 2016; 1(2): 67–89.
11.Mishra PK, Mishra H, Ekielski A, Talegaonkar S, Vaidya B. Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications. Drug Discov. 2017; 22(12): 1825–1834.
12.Fu YZ, Yan YY, He M, Xiao QH, Yao WF, Zhao L, Wu HZ, Yu ZJ, Zhou MY, Lv MT. Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway. Oncol Rep. 2016; 35(2): 912–922.
13.Hernández-Vargas H, Ballestar E, Carmona-Saez P, von Kobbe C, Bañón-Rodríguez I, Esteller M, Moreno‐Bueno G, Palacios J. Transcriptional profiling of MCF7 breast cancer cells in response to 5-Fluorouracil: Relationship with cell cycle changes and apoptosis, and identification of novel targets of p53. Int J Cancer. 2006; 119(5): 1164–1175.
14.Senthilraja P, Kathiresan K. In vitro cytotoxicity MTT assay in Vero, HepG2 and MCF-7 cell lines study of Marine Yeast. J Appl Pharm Sci. 2015; 5(03): 80-84.
15.Bendale Y, Bendale V, Paul S. Evaluation of cytotoxic activity of platinum nanoparticles against normal and cancer cells and its anticancer potential through induction of apoptosis. Integr Med Res. 2017; 6: 141–148.
16.Pauzi AZM, Yeap SK, Abu N, Lim KL, Omar AR, Aziz SA, Chow ALT, Subramani T, Tan SG, Alitheen NB. Combination of cisplatin and bromelain exerts synergistic cytotoxic effects against breast cancer cell line MDA-MB-231 in vitro. Chin Med. 2016; 11(1): 46.
17.Roy R, Singh S, Chauhan L, Das M, Tripathi A, Dwivedi PD. Zinc oxide nanoparticles induce apoptosis by enhancement of autophagy via PI3K/Akt/mTOR inhibition. Toxicol Lett. 2014; 227(1): 29-40.
18.Wahab R, Siddiqui MA, Saquib Q, Dwivedi S, Ahmad J, Musarrat J, Al-Khedhairy AA, Shin HS. ZnO nanoparticles induced oxidative stress and apoptosis in HepG2 and MCF-7 cancer cells and their antibacterial activity. Colloids Surf B Biointerfaces. 2014; 117: 267–276.
19.Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72(1–2): 248–254.
20.Heidarian E, Keloushadi M, Ghatreh-Samani K, Jafari-Dehkordi E. Gallic acid inhibits invasion and reduces IL-6 gene expression, pSTAT3, pERK1/2, and pAKT cellular signaling proteins in human prostate cancer DU145 cells. Int J Cancer Manag. 2017; 10(11): 3-6.
21.Chou TC. Drug combination studies and their synergy quantification using the chou-talalay method. Cancer Res. 2010; 70(2): 440–446.
22.Sanad F, Nabih S, Goda M.A. A lot of promise for ZnO-5FU nanoparticles cytotoxicity against breast cancer cell lines. J Nanomed Nanotechnol. 2018; 09(01): 1–8.
23.Panariti A, Miserocchi G, Rivolta I. The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions. Sci App. 2012; 5: 87–100.
24.Deng Y, Zhang H. The synergistic effect and mechanism of doxorubicin-ZnO nanocomplexes as a multimodal agent integrating diverse anticancer therapeutics. Int J Nanomedicine. 2013; 8: 1835–1841.
25.Guo D, Wu C, Jiang H, Li Q, Wang X, Chen B. Synergistic cytotoxic effect of different sized ZnO nanoparticles and daunorubicin against leukemia cancer cells under UV irradiation. J. Photochem. Photobiol B Biol. 2008; 93(3): 119–126.
26.Shoeb M, Singh BR, Khan JA, Khan W, Singh BN, Singh HB, Naqvi AH. ROS-dependent anticandidal activity of zinc oxide nanoparticles synthesized by using egg albumen as a biotemplate. Adv Nat Sci Nanosci Nanotechnol. 2013; 4(3): 1-11.
27.Cheung-Ong K, Giaever G, Nislow C. DNA-damaging agents in cancer chemotherap: serendipity and chemical biology. Chem Biol.2013; 20: 648-659.
28.Arias JL. Novel strategies to improve the anticancer action of 5-fluorouracil by using drug delivery systems. Molecules. 2008; 13(10): 2340–2369.
29.Chandran SP, Natarajan SB, Chandraseharan S, Shahimi MSBM. Nano drug delivery strategy of 5-fluorouracil for the treatment of colorectal cancer. J Cancer Res Pract. 2017; 4(2): 45–48.
30.Leverson JD, Phillips DC, Mitten MJ, Boghaert ER, Diaz D, Tahir SK, Belmont LD, Nimmer P, Xiao Y, Ma X.M. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy. Sci Transl Med. 2015; 7(279): 279ra40.
31.Wang X, Simpson ER, Brown KA. p53: Protection against tumor growth beyond effects on cell cycle and apoptosis. Cancer Res. 2015; 175(23): 5001–5007.
32.Mello SS, Attardi LD. Deciphering p53 signaling in tumor suppression. Curr Opin Cell Biol. 2018; 51: 65–72.
33.Ganeshpurkar A, Saluja AK. The pharmacological potential of rutin. Saudi Pharm J. 2017;25(2):149–164.
34.Mirza MB, Elkady AI, Al-Attar AM, Syed FQ, Mohammed FA, Hakeem KR. Induction of apoptosis and cell cycle arrest by ethyl acetate fraction of Phoenix dactylifera L. (Ajwa dates) in prostate cancer cells. J Ethnopharmacol. 2018; 218: 35–44.
35.Placzek WJ, Wei J, Kitada S, Zhai D, Reed JC, Pellecchia M. A survey of the anti-apoptotic Bcl-2 subfamily expression in cancer types provides a platform to predict the efficacy of Bcl-2 antagonists in cancer therapy. Cell Death Dis. 2010; 1(5):1-9.