The effect of thymoquinone on cell proliferation, 8-hydroxy-2 -deoxyguanosine level and expression changes of DNA repair and oxidative stress-related genes in MCF-7 breast cancer cells

Öz

Purpose: The aim of this study was to determine the effects of thymoquinone on cell proliferation, 8-hydroxy-2' -deoxyguanosine level and expression changes of oxidative stress and DNA repair-related genes in MCF-7 breast cancer cells. Material methods: Cell proliferation in MCF-7 cells after thymoquinone exposure was determined by MTT assay. 8-hydroxy-2' -deoxyguanosine protein concentration was measured by ELISA assay. Total RNA isolation from control and thymoquinone treated cell was performed by Trizol and cDNA was synthesized. mRNA expression changes of OGG1, NEIL-1, CRCC1 SOD2, CAT and NRF-2 were also determined in RT-PCR using SYBER Green method. Results: In this study, the IC50 dose of thymoquinone in MCF-7 cells was determined as 7.867 μM at 24th hour. It was investigated that thymoquinone inhibited 8-hydroxy-2' -deoxyguanosine level in breast cancer cells according to RT-PCR results, thymoquinone increased XRRC1 expression 4.71-fold and catalase expression 6.68-fold in breast cancer cells. Conclusion: In conclusion, TQ inhibits MCF-7 cell proliferation under in vitro conditions. It also alters the expression of genes associated with DNA repair and oxidative stress and acts through this oxidative stress mechanism. This study contributes to the existing literature and provides preliminary data for more comprehensive studies.

Anahtar Kelimeler

• Thymoquinone
• MCF-7 cells
• breast cancer
• DNA repair genes
• oxidative stress

Timokinonun MCF-7 meme kanseri hücrelerinde hücre proliferasyonu, 8-hidroksi-2' –deoksiguanozin seviyesi ile DNA tamiri ve oksidatif stres ilişkili genlerin ekspresyon değişimlerine etkisi

Öz

Amaç: Bu çalışmanın amacı, timokinonun MCF-7 meme kanseri hücrelerinde hücre proliferasyonu, 8-hidroksi-2' -deoksiguanozin düzeyi ve oksidatif stres ve DNA onarımı ile ilişkili genlerin ekspresyon değişiklikleri üzerindeki etkilerini belirlemektir. Gereç ve yöntem: Timokinon maruziyetinden sonra MCF-7 hücrelerinde hücre proliferasyonu MTT testi ile belirlendi. 8-hidroksi-2' -deoksiguanozin protein konsantrasyonu ELISA testi ile ölçüldü. Kontrol ve timokinon uygulanan hücrelerden Trizol ile total RNA izolasyonu yapıldı ve cDNA sentezlendi. OGG1, NEIL-1, CRCC1 SOD2, CAT ve NRF-2'nin mRNA ekspresyon değişiklikleri de SYBER Green yöntemi ile RT-PCR'da belirlendi. Bulgular: Bu çalışmada, MCF-7 hücrelerinde timokinonun IC50 dozu 24. saatte 7,867 μM olarak belirlenmiştir. Timokinonun meme kanseri hücrelerinde 8-hidroksi-2' -deoksiguanozin seviyesini inhibe ettiği araştırılmıştır. Ayrıca RT-PCR sonuçlarına göre, timokinon meme kanseri hücrelerinde XRRC1 ekspresyonunu 4,71 kat ve katalaz ekspresyonunu 6,68 kat artırmaktadır. Sonuç: Sonuç olarak, TQ in vitro koşullar altında MCF-7 hücre proliferasyonunu inhibe eder. Ayrıca DNA onarımı ve oksidatif stres ile ilişkili genlerin ekspresyonunu değiştirir ve bu oksidatif stres mekanizması yoluyla etki eder. Bu çalışma mevcut literatüre katkıda bulunmakta ve daha kapsamlı çalışmalar için ön veri sağlamaktadır.

Anahtar Kelimeler

• Timokinon
• MCF-7 hücreleri
• meme kanseri
• DNA onarım genleri
• oksidatif stres

Kaynakça

1. 1. Brown JS, Amend SR, Austin RH, Gatenby RA, Hammarlund EU, Pienta KJ. Updating the definition of cancer. Mol Cancer Res. 2023;21(11):1142-1147. doi:10.1158/1541-7786.MCR-23-0411
2. 2. Khan SU, Fatima K, Malik F, Kalkavan H, Wani A. Cancer metastasis: molecular mechanisms and clinical perspectives. Pharmacol Ther. 2023;250:108522. doi:10.1016/j.pharmthera.2023.108522 3. Park JH, Pyun WY, Park HW. Cancer metabolism: phenotype, signaling and therapeutic targets. Cells. 2020;9(10):2308. doi:10.3390/cells9102308
3. 4. Zhang S, Xiao X, Yi Y, et al. Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets. Signal Transduct Target Ther. 2024;9(1):149. doi:10.1038/s41392-024-01848-7
4. 5. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12-49. doi:10.3322/caac.21820
5. 6. Maroufi NF, Ashouri N, Mortezania Z, et al. The potential therapeutic effects of melatonin on breast cancer: an invasion and metastasis inhibitor. Pathol Res Pract. 2020;216(10):153226. doi:10.1016/j.prp.2020.153226
6. 7. Moo TA, Sanford R, Dang C, Morrow M. Overview of breast cancer therapy. PET Clin. 2018;13(3):339-354. doi:10.1016/j.cpet.2018.02.006
7. 8. Senapati S, Mahanta AK, Kumar S, Maiti P. Controlled drug delivery vehicles for cancer treatment and their performance. Signal Transduct Target Ther. 2018;3:7. doi:10.1038/s41392-017-0004-3
8. 9. Shabani H, Karami MH, Kolour J, et al. Anticancer activity of thymoquinone against breast cancer cells: mechanisms of action and delivery approaches. Biomed Pharmacother. 2023;165:114972. doi:10.1016/j.biopha.2023.114972

9. 10. Liu B, Zhou H, Tan L, Siu KTH, Guan XY. Exploring treatment options in cancer: tumor treatment strategies. Signal Transduct Target Ther. 2024;9(1):175. doi:10.1038/s41392-024-01856-7
10. 11. Nahata A. Anticancer agents: a review of relevant information on important herbal drugs. Int J Clin Pharmacol Toxicol. 2017;6(2):250-255. doi:10.19070/2167-910x-1700042
11. 12. Blowman K, Magalhães M, Lemos M, Cabral C, Pires IM. Anticancer properties of essential oils and other natural products. Evid Based Complement Alternat Med. 2018;2018:3149362. doi:10.1155/2018/3149362
12. 13. Secme M, Dodurga Y. Usnic acid inhibits cell proliferation and downregulates lncRNA UCA1 expression in Ishikawa endometrial cancer cells. Nat Prod Biotechnol. 2021;1(1):28-37.
13. 14. Yuan M, Zhang G, Bai W, Han X, Li C, Bian S. The role of bioactive compounds in natural products extracted from plants in cancer treatment and their mechanisms related to anticancer effects. Oxid Med Cell Longev. 2022;2022:1429869. doi:10.1155/2022/1429869
14. 15. Dajani EZ, Shahwan TG, Dajani NE. Overview of the preclinical pharmacological properties of Nigella sativa (black seeds): a complementary drug with historical and clinical significance. J Physiol Pharmacol. 2016;67(6):801-817.
15. 16. Tavakkoli A, Mahdian V, Razavi BM, Hosseinzadeh H. Review on clinical trials of black seed (Nigella sativa) and its active constituent, thymoquinone. J Pharmacol Phytother. 2017;20(3):179-193. doi:10.3831/KPI.2017.20.021
16. 17. Alshaibi HF, Aldarmahi NA, Alkhattabi NA, Alsufiani HM, Tarbiah NI. Studying the anticancer effects of thymoquinone on breast cancer cells through natural killer cell activity. Biomed Res Int. 2022;2022:9218640. doi:10.1155/2022/9218640
17. 18. Gholamnezhad Z, Havakhah S, Boskabady MH. Preclinical and clinical effects of Nigella sativa and its constituent, thymoquinone: a review. J Ethnopharmacol. 2016;190:372-386. doi:10.1016/j.jep.2016.06.061
18. 19. Rooney S, Ryan MF. Effects of alpha-hederin and thymoquinone, constituents of Nigella sativa, on human cancer cell lines. Anticancer Res. 2005;25(3B):2199-2204.
19. 20. Imran M, Rauf A, Khan IA, et al. Thymoquinone: a novel strategy to combat cancer: a review. Biomed Pharmacother. 2018;106:390-402. doi:10.1016/j.biopha.2018.06.159
20. 21. Zhao ZX, Li S, Liu LX. Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways. World J Gastroenterol. 2024;30(21):2793-2816. doi:10.3748/wjg.v30.i21.2793
21. 22. Gulbay G, Secme M, Ilhan H. Exploring the potential of thymoquinone-stabilized selenium nanoparticles: in HEC1B endometrial cancer cells revealing enhanced anticancer efficacy. ACS Omega. 2023;8(42):39822-39829. doi:10.1021/acsomega.3c06028
22. 23. Sohrabi B, Qadbeigi M, Sabouni F, Hamta A. Thymoquinone nanoparticle induces apoptosis and cell migration retardation through modulating of SUMOylation process genes in breast cancer cell line. Iran J Biotechnol. 2024;22(1):e3676. doi:10.30498/ijb.2024.390400.3676
23. 24. Almajali B, Al Jamal HAN, Taib WRW, et al. Thymoquinone, as a novel therapeutic candidate of cancers. Pharmaceutics. 2021;14(4):369. doi:10.3390/ph14040369
24. 25. Bashmail HA, Alamoudi AA, Noorwali A, et al. Thymoquinone synergizes gemcitabine anti-breast cancer activity via modulating its apoptotic and autophagic activities. Sci Rep. 2018;8(1):11674. doi:10.1038/s41598-018-30046-z
25. 26. Motaghed M, Al Hassan FM, Hamid SS. Cellular responses with thymoquinone treatment in human breast cancer cell line MCF-7. Phcog Res. 2013;5(3):200-206. doi:10.4103/0974-8490.112428
26. 27. Talib WH. Regressions of breast carcinoma syngraft following treatment with piperine in combination with thymoquinone. Sci Pharm. 2017;85(3):27. doi:10.3390/scipharm85030027
27. 28. Barkat MA, Harshita, Ahmad J, Khan MA, Beg S, Ahmad FJ. Insights into the targeting potential of thymoquinone for therapeutic intervention against triple-negative breast cancer. Curr Drug Targets. 2018;19(1):70-80. doi:10.2174/1389450118666170612095959
28. 29. Woo CC, Hsu A, Kumar AP, Sethi G, Tan KH. Thymoquinone inhibits tumor growth and induces apoptosis in a breast cancer xenograft mouse model: the role of p38 MAPK and ROS. PLoS One. 2013;8(10):e75356. doi:10.1371/journal.pone.0075356
29. 30. Boiteux S, Radicella JP. The human OGG1 gene: structure, functions, and its implication in the process of carcinogenesis. Arch Biochem Biophys. 2000;377(1):1-8. doi:10.1006/abbi.2000.1773
30. 31. Singh B, Chatterjee A, Ronghe AM, Bhat NK, Bhat HK. Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated with inhibition of oxidative DNA damage in estrogen-induced breast cancer. BMC Cancer. 2013;13:253. doi:10.1186/1471-2407-13-253
31. 32. Cooke MS, Olinski R, Evans MD. Does measurement of oxidative damage to DNA have clinical significance? Clin Chim Acta. 2006;365(1-2):30-49. doi:10.1016/j.cca.2005.09.009
32. 33. Evans MD, Dizdaroglu M, Cooke MS. Oxidative DNA damage and disease: induction, repair and significance. Mutat Res. 2004;567(1):1-61. doi:10.1016/j.mrrev.2003.11.001
33. 34. Vomund S, Schäfer A, Parnham MJ, Brüne B, von Knethen A. Nrf2, the master regulator of anti-oxidative responses. Int J Mol Sci. 2017;18(12):2772. doi:10.3390/ijms18122772
34. 35. Kumar H, Kumar RM, Bhattacharjee D, Somanna P, Jain V. Role of Nrf2 signaling cascade in breast cancer: strategies and treatment. Front Pharmacol. 2022;13:720076. doi:10.3389/fphar.2022.720076
35. 36. Xue W, Liu Y, Xin N, et al. Nei endonuclease VIII-like1 (NEIL1) inhibits apoptosis of human colorectal cancer cells. Biomed Res Int. 2020;2020:5053975. doi:10.1155/2020/5053975
36. 37. Hussain AR, Ahmed M, Ahmed S, et al. Thymoquinone suppresses growth and induces apoptosis via generation of reactive oxygen species in primary effusion lymphoma. Free Radic Biol Med. 2011;50(8):978-987. doi:10.1016/j.freeradbiomed.2010.12.034
37. 38. Abdel Daim MM, Sayed AA, Abdeen A, et al. Piperine enhances the antioxidant and anti-inflammatory activities of thymoquinone against microcystin-LR-induced hepatotoxicity and neurotoxicity in mice. Oxid Med Cell Longev. 2019;2019:1309175. doi:10.1155/2019/1309175