Investigation of the cytotoxic/genotoxic potential of the combination of tamoxifen and fluoxetine applied to SKOV3 cells

Öz

Aim: Breast cancer is the most common malignancy in women, and tamoxifen has been used as an effective therapeutic agent for many years in estrogen receptor-positive cases. Fluoxetine is an antidepressant used in the treatment and management of depression and psychiatric disorders. People can be exposed to these drugs through various routes, primarily through medical use. This exposure can affect the genome. Multiple exposures of living organisms to chemicals may result in more severe consequences than single administrations. Therefore, the present study aimed to determine the cytotoxic and genotoxic potential of the combined use of tamoxifen and fluoxetine on ovarian cancer cells. Methods: Tamoxifen doses of 5, 10, 20, 40, and 60 μM and fluoxetine doses of 1, 5, 10, 15, and 20 μM were first administered individually to SKOV3 cells. Subsequently, SKOV3 cells were exposed to the drug combination. Cytotoxicity assessment after exposure was performed using the xCELLigence real-time cell analysis system, and genotoxicity assessment was performed using the alkaline comet assay. Results: Data obtained with the xCELLigence analysis showed that treatment with the drug combination resulted in higher cytotoxicity than single drug applications. Comet analysis showed that drug combinations increased the percentage of damaged cells and the genetic damage index almost twofold compared to single drug exposure. Conclusion: It has been determined that drug combinations can exhibit synergistic effects compared to single applications, leading to more severe toxic effects on human health. These findings necessitate evaluating the potential toxicity of various drug combinations and the synergistic effects of varying concentrations in different ecosystems, particularly wastewater.

Anahtar Kelimeler

• Fluoxetine
• genotoxicity
• cytotoxicity
• tamoxifen
• cancer

SKOV3 hücrelerine uygulanan tamoksifen ve floksetin kombinasyonunun sitotoksik/genotoksik potansiyelinin araştırılması

Öz

Amaç: Meme kanseri, kadınlarda en sık görülen malignite türü olup, bu hastalığın tedavisinde östrojen reseptör pozitif vakalar için tamoksifen uzun yıllardır etkili bir terapötik ajan olarak kullanılmaktadır. Floksetin ise depresyon ve psikiyatrik bozuklukların tedavisi ve kontrolünde kullanılan bir antidepresandır. İnsanlar bu ilaçlara medikal uygulamalar başta olmak üzere farklı yollarla maruz kalabilmektedirler. Bu maruziyet genomu etkileyebilmektedir. Canlıların kimyasallara çoklu maruziyeti tekli uygulamalardan daha riskli sonuçlar doğurabilir. Bu nedenle, sunulan bu çalışmada tamoksifen ve floksetinin beraber kullanımının ovaryum kanser hücreleri üzerindeki sitotoksisite ve genotoksisite potansiyelinin belirlenmesi amaçlanmıştır. Yöntem: Tamoksifenin 5, 10, 20, 40 ve 60 μM’lık dozları ve floksetinin 1, 5, 10, 15 ve 20 μM’lık dozları önce tekli olarak SKOV3 hücrelerine uygulanmıştır. Daha sonra, SKOV3 hücreleri kombinasyon halinde ilaçlara maruz bırakılmıştır. Maruziyetler sonrasında sitotoksisite değerlendirmesi xCELLigence gerçek zamanlı hücre analiz sistemi kullanılarak, genotoksisite değerlendirmesi ise alkalin komet testi kullanılarak gerçekleştirilmiştir. Bulgular: xCELLigence analiziyle elde edilen veriler, kombinasyon şeklindeki uygulamanın tekli ilaç uygulamalarından daha yüksek sitotoksisiteye neden olduğunu göstermiştir. Komet analizi sonucunda ise ilaç kombinasyonlarının hasarlı hücre yüzdesini ve genetik hasar indeksini tekli ilaç maruziyetine oranla neredeyse iki kat arttırdığı gözlemlenmiştir. Sonuç: İlaç kombinasyonlarının tekli uygulamalara kıyasla sinerjistik etki göstererek insan sağlığı üzerinde daha ciddi toksik etkilere yol açabileceği belirlenmiştir. Bu bulgular, farklı ilaç kombinasyonlarının potansiyel toksisitelerinin özellikle atık sular başta olmak üzere farklı ekosistemlerdeki, farklı konsantrasyonların sinerjistik etkilerinin değerlendirilmesini zorunlu kılmaktadır.

Anahtar Kelimeler

• Floksetin
• genotoksisite
• sitotoksisite
• tamoksifen
• kanser

Kaynakça

1. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer Statistics. CA: A Cancer Journal for Clinicians. 2023;73: 17–48. doi:10.3322/caac.21763
2. Yetkin D, Balli E, Ayaz F. Antiproliferative activity of tamoxifene, vitamin D3 and their concomitant treatment. EXCLI Journal. 2021;20: 1394-1406. doi:10.17179/excli2021-3989
3. Küçükgöz-Güleç Ü, Paydaş S, Güzel AB. Tamoksifen Kullanan Hastaların Jinekolojik Yönden Değerlendirilmesi. Arşiv Kaynak Tarama Dergisi. 2021; 30(1): 45-52.
4. Higgins MJ, Baselga J. Targeted therapies for breast cancer. The Journal of Clinical Investigation. 2011;121(10): 3797-3803. doi:10.1172/JCI57152.
5. Ghanavati M, Khorshidi Y, Shadnoush M, ve et al. Tamoxifen use and risk of endometrial cancer in breast cancer patients: A systematic review and dose–response meta-analysis. Cancer Reports. 2023;6:e, 1806. doi:10.1002/cnr2.1806.
6. Bergman L, Beelen MLR, Gallee MPW, Hollema H, Benraadt J, Leeuwen FE. Risk and prognosis of endometrial cancer after tamoxifen for breast cancer. The Lancet. 2000;356(9233): 881-87. doi:10.1016/S0140-6736(00)02677-5
7. Güleç G, Büyükkınacı A. Kanser ve Psikiyatrik Bozukluklar. Psikiyatride Güncel Yaklaşımlar. 2011; 3(2): 343-367.
8. Correia D, Domingues I, Faria M, Oliveira M. Effects of fluoxetine on fish: What do we know and where should we focus our efforts in the future?. Scienceof the Total Environment. 2023;857:159486. doi:10.1016/j.scitotenv.2022.159486

9. Brooks BW, Foran CM, Richards SM, ve et al. Aquatic ecotoxicology of fluoxetine. Toxicology Letters. 2003;142: 169-183. doi:10.1016/S0378-4274(03)00066-3
10. Düsman E, Almeida IV, Mariucci RG, Mantovani MS, Vicentini VEP. Cytotoxicity and mutagenicity of fluoxetine hydrochloride (Prozec), with or without vitamins A and C, in plant and animal model systems. Genetics and Molecular Research. 2014; 13(1): 578-589. doi:10.4238/2014.January.28.3
11. Yaman HM, Erbaş O. Genotoxic effects of commonly used selective serotonin reuptake inhibitors antidepressants. Journal of Experimental and Basic Medical Sciences. 2023;4(3): 170-174. doi:10.5606/jebms.2023.1061
12. Al-Obaidi IA, Al-Shawi NN. Assessment the genotoxic potential of fluoxetine and amitriptyline at maximum therapeutic doses for four-week treatment in experimental male rats. Iraqi Journal of Pharmaceutical Science, 2020;30(1). doi:10.31351/vol30iss1pp81-90
13. Kundi M, Parrella A, Lavorgna M, Criscuolo E, Russo C, Isidori M. Prediction and assessment of ecogenotoxicity of antineoplastic drugs in binary mixtures. Environmental Science and Pollution Research. 2016;23: 14771-14779. doi:10.1007/s11356-015-4884-x
14. Gajski G, Geric M, Domijan AM, Garaj-Vrhovac V. Combined cyto/genotoxic activity of a selected antineoplastic drug mixture in human circulating blood cells.Chemospere. 2016;165: 529-538. doi:10.1016/j.chemosphere.2016.09.058
15. Gajski G, Ladeira C, Geric M, Garaj-Vrhovac V, Viegas S. Genotoxicity assessment of a selected cytostatic drug mixture in human lymphocytes: A study based on concentrations relevant for occupational exposure. Environmental Research. 2018;161: 26-34. doi:10.1016/j.envres.2017.10.044
16. Wieczerzak M, Namiesnik J, Kudlak B. Genotoxicity of selected pharmaceuticals, their binary mixtures, and varying environmental conditions-study with human adenocarcinoma cancer HT29 cell line. Drug and Chemical Toxicology. 2021;44(2): 113-123. doi:10.1080/01480545.2018.1529783
17. Könen Adıgüzel S, Ergene S. In vitro evaluation of the genotoxicity of CeO2 nanoparticles in human peripheral blood lymphocytes using cytokinesis-block micronucleus test, comet assay, and gamma H2AX. Toxicology and Industrial Health. 2018; 34:5. doi:10.1177/0748233717753780
18. Lovecka P, Thimova M, Grznarova P, ve et al. Study of cytotoxic effects of benzonitrile pesticides. BioMed Research International. 2015; Article ID 381264, 9. doi:10.1155/2015/381264
19. Vetten MA, Tlotleng N, Rascher DT, ve et al. Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines. Particle and Fibre Toxicology. 2013;10 (50): 1-15. http://www.particleandfibretoxicology.com/content/10/1/50
20. Lee TH, Chuang LY, Hung WC. Tamoxifen induces p21WAF1 and p27KIP1 expression in estrogen receptor-negative lung cancer cells. Oncogene. 1999;18: 4269-4274. doi:10.1038/sj.onc.1202755
21. Wozniak K, Kolacinska A, Blasinska-Morawiec M, ve et al. The DNA-damaging potential of tamoxifen in breast cancer and normal cells. Archives Toxicol. 2007;81, 519-527. doi:10.1007/s00204-007-0188-3
22. Xie X, Wu MY, Shou LM, ve et al. Tamoxifen enhances the anticancer effect of cantharidin and norcantharidin in pancreatic cancer cell lines through inhibition of the protein kinase C signaling pathway. Oncology Letters. 2015;9: 837-844. doi:10.3892/ol.2014.2711
23. Balça-Silva J, Matias D, Carmo A, ve et al. Tamoxifen in combination with temozolomide induce a synergistic inhibition of PKC-pan in GBM cell lines. Biochim Biophys Acta. 2015;1850 (4): 722-723. doi:10.1016/j.bbagen.2014.12.022
24. Macleod AK, McLaughlin LA, Henderson CJ, Wolf CR. Application of mice humanized for cyp2d6 to the study of tamoxifen metabolism and drug–drug ınteraction with antidepressants. Drug metabolısm and dısposıtıon. 2017; 45:17–22. doi:10.1124/dmd.116.073437
25. Sanaei M, Kavoosi F, Atashpour S, Haghighat S. Effects of Genistein and Synergistic Action in Combination with Tamoxifen on the HepG2 Human Hepatocellular Carcinoma Cell Line. Asian pacific Journal at Center Prevention. 2017;18(9): 2381-2385. doi:10.22034/apjcp.2017.18.9.2381
26. Yetkin D, Ballı E, Bayrak G, Kibar D, Türkegün M. The ınvestigation of the effects of tamoxifen and vitamin d combination on the expression of p53, bcl-2 and bax and cell cycle in mcf-7 cell line. Proceedings. 2018;2(25): 1527. doi:10.3390/proceedings2251527.
27. Üstüner D. Fluoksetin’in sitogenetik olarak analizi. Tübav Bilim Dergisi. 2010;3(3):276-281.
28. Lee CS, Kim YJ, Jang ER, Kim W, Myung SC. Fluoxetine Induces Apoptosis in Ovarian Carcinoma Cell Line OVCAR-3 Through Reactive Oxygen Species-Dependent Activation of Nuclear Factor-kB. Journal Compilation 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology. 2009; 106: 446–453. doi:10.1111/j.1742-7843.2009.00509.x
29. Draz EI, Emara AM, Saad KM, Badaway A, El-Nabi SEH, Abd-Elgelil H. Genotoxicity of Some Commonly Used Antidepressants (Fluoxetine, Sertraline and Clomipramine). Mansoura Journal of Forensic Medicine and Clinical Toxicology. 2009;17(2):63-78. doi:10.21608/mjfmct.2009.61873
30. Alzahrani HAS. Sister chromatid exchanges and sperm abnormalities produced by antidepressant drug fluoxetine in mouse treated in vivo. European Review for Medical Pharmacological Sciences. 2012; 16 (15); 2154-2161.
31. Marcinkute M, Afshinjavid S, Fatokun AA, Javid FA. Fluoxetine selectively induces p53-independent apoptosis in human colorectal cancer cells. European Journal of Pharmacology. 2019;857: 172441. doi:10.1016/j.ejphar.2019.17244