Investigation of the Effect of Carbonic Anhydrase-IX Enzyme Inhibition with Acetazolamide on Glutathione Reductase and Glutathione Peroxidase Activities

Abstract

Oxidative stress is one of the important parameters for renal cancer. The antioxidant system plays a prominent role and resists oxidative stress in the development of renal cancer. The endogenous antioxidants GPx and GR, are defined as crucial enzymes in the antioxidant system of the kidneys. The main purpose of this study is to determine the effect of AZA, a carbonic anhydrase classical enzyme inhibitor in the renal cancer cell line CAKI-2 to evaluate the GPx and GR enzyme activities. The optimal dose of AZA was found to be 8.65 μM at the 48th hour by WST-1 cytotoxicity test. ELISA test was performed to determine the effect of AZA on CA-IX enzyme in CAKI-2 cells. All measurements were performed at 340 nm by Epoch™ Microplate Spectrophotometer using “Glutathione Reductase Assay Kit” and “Glutathione Peroxidase Activity Assay Kit”. After AZA application, an increase was observed in GR and GPx enzyme activities (p≤0.05). As a consequence, AZA may be an important therapeutic agent for the glutathione mechanism related to renal cancer. Our study makes a valuable contribution to the literature due to the importance of the CA-IX enzyme related to renal cancer. The investigation of other endogenous antioxidant enzymes in renal cancer may be designed for further prospective studies.

Keywords

• Acetazolamide
• Carbonic anhydrase
• Glutathione peroxidase
• Glutathione reductase
• Renal Cancer

Karbonik Anhidraz-IX Enziminin Asetazolamid ile İnhibisyonunun Glutatyon Redüktaz ve Glutatyon Peroksidaz Aktiviteleri Üzerine Olan Etkisinin İncelenmesi

Abstract

Oksidatif stres, renal kanser için önemli parametrelerden birisidir. Antioksidan sistem renal kanser oluşumunda devreye girerek oksidatif strese karşı koyar. Endojen antioksidanlar olarak tanımlanan GR ve GPx, böbreklerin antioksidan sistemindeki önemli enzimlerdir. Renal kanserdeki önemli parametrelerden biri olan CA-IX, bir pH pompası olarak görev yaparak tümör mikroçevresinin asidifikasyonuna sebep olur ve karsinogenezde rol oynar. Çalışmamızın temel amacı, bir karbonik anhidraz enzim inhibitörü olan AZA’nın glutatyon mekanizması üzerine olan etkisinin renal kanserde incelenmesidir. Deneysel çalışmalarda öncelikle renal kanser hücre hattı olan CAKI-2 çoğaltılarak WST-1 sitotoksisite testi ile AZA’nın uygun dozu 48. saatte 8.65 μM olarak bulunmuştur. AZA’nın CAKI-2 hücrelerinde CA-IX enzimi üzerine olan etkisi belirlenmek için ELISA testi yapılmıştır. CAKI-2 hücrelerine AZA uygulandıktan sonra GR ve GPx üzerine olan etkisini belirlemek için “Glutathione Reductase Assay Kit” ve “Glutathione Peroxidase Assay Kit” kullanılarak Epoch™ Microplate Spectrophotometer cihazında 340 nm’de ölçüm yapılmıştır. AZA uygulaması sonrası GR ve GPx enzim aktivitelerinde artış görülmüştür (p≤0.05). Çalışmanın sonucu olarak AZA inhibisyonunun, renal kanserde glutatyon mekanizmasının devreye girmesi için önemli bir ajan olabileceği söylenebilir. Renal kanserde hem CA-IX enziminin önemli bir terapötik biyobelirteç olması hem de glutatyon mekanizmasının bu kanser türündeki öneminden dolayı çalışmamız literatüre önemli bir katkı sağlamaktadır. Bu çalışmanın devamı niteliğinde olması planlanan diğer endojen antioksidan enzim aktivitelerinin renal kanserde araştırılması yeni terapötik yaklaşımların geliştirilmesini sağlayacaktır.

Keywords

• Asetazolamid
• Glutatyon peroksidaz
• Glutatyon redüktaz
• Karbonik anhidraz
• Renal Kanser

References

1. Kumari S, Badana AK, Malla R. Reactive oxygen species: a key constituent in cancer survival. Biomarker Insights. 2018; 13:1177271918755391.
2. Saikolappan S, Kumar B, Shishodia G, Koul S, Koul HK. Reactive oxygen species and cancer: A complex interaction. Cancer letters.2019; 452:132-143.
3. Veith A, Moorthy B. Role of cytochrome P450s in the generation and metabolism of reactive oxygen species. Current opinion in toxicology. 2018; 7, 44-51.
4. Gulcin İ. Antioxidants and antioxidant methods: An updated overview. Archives of toxicology. 2020; 94(3):651-715.
5. Harris IS, DeNicola GM. The complex interplay between antioxidants and ROS in cancer. Trends in cell biology. 2020; 30(6):440-451.
6. Couto N, Wood J, Barber J. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free radical biology and medicine.2016; 95:27-42.
7. Adwas AA, Elsayed A, Azab AE, Quwaydir FA. Oxidative stress and antioxidant mechanisms in human body. J. Appl. Biotechnol. Bioeng.2019; 6(1):43-47.
8. Ighodaro OM, Akinloye OA. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alexandria journal of medicine.2018; 54(4):287-293.

9. Ali SS, Ahsan H, Zia MK, Siddiqui T,Khan FH. Understanding oxidants and antioxidants: Classical team with new players. Journal of food biochemistry. 2020; 44(3):e13145.
10. Supuran CT. Carbonic anhydrase inhibitors and their potential in a range of therapeutic areas. Expert opinion on therapeutic patents. 2018; 28(10):709-712.
11. Kumar S, Rulhania S, Jaswal S, Monga V. Recent advances in the medicinal chemistry of carbonic anhydrase inhibitors. European Journal of Medicinal Chemistry.2021; 209: 112923.
12. Cazzamalli S, Dal Corso A,Neri D. Acetazolamide serves as selective delivery vehicle for dipeptide-linked drugs to renal cell carcinoma. Molecular cancer therapeutics.2016; 15(12):2926-2935.
13. Pavlović I, Pejić S, Radojević-Škodrić S, Todorović A, Stojiljković V, Gavrilović L, Popović N, Basta-Jovanović G, Džamić Z, Pajović SB. The effect of antioxidant status on overall survival in renal cell carcinoma. Archives of Medical Science.2020;16(1): 94.
14. https://www.sigmaaldrich.com/technicaldocuments/protocols/biology/roche/cell- proliferation-reagent-wst-1.html. 9 Nisan 2021.
15. Nabi S, Kessler ER, Bernard B, Flaig TW, Lam ET. Renal cell carcinoma: a review of biology and pathophysiology. F1000Research. 2018;7:307.
16. Ivanov, S., Liao, S. Y., Ivanova, A., Danilkovitch-Miagkova, A., Tarasova, N., Weirich, G., ... & Stanbridge, E. J. (2001). Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. The American journal of pathology, 158(3), 905-919.
17. Pastorekova, S., Ratcliffe, P. J., & Pastorek, J. (2008). Molecular mechanisms of carbonic anhydrase IX‐mediated pH regulation under hypoxia. BJU international, 101, 8-15.
18. Ho WJ, Simon MS, Yildiz VO, Shikany JM, Kato I, Beebe‐Dimmer JL, CetnarJP, Bock CH. Antioxidant micronutrients and the risk of renal cell carcinoma in the Women's Health Initiative cohort. Cancer, 2015;121(4):580-588.
19. Liao SY, Aurelio ON, Jan K, Zavada J SE. Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res. 1997; 57(14):2827-31.
20. Frederiks WM, Bosch KS, Hoeben KA, van Marle J, Langbein S. Renal cell carcinoma and oxidative stress: the lack of peroxisomes. Acta histochemica. 2010; 112(4), 364-371.
21. Ganesamoni R, Bhattacharyya S, Kumar S, Chauhan A, Mete UK, Agarwal MM, Mavuduru R, Kaushik G, Mandal AK, Singh SK. Status of oxidative stress in patients with renal cell carcinoma. J Urol. 2012;187:1172-6.
22. Pirinççi N, Kaba M, Geçit İ, Güneş M, Yüksel, MB, TanıkS, Arslan A, Demir, H. Serum prolidase activity, oxidative stress, and antioxidant enzyme levels in patients with renal cell carcinoma. Toxicology and Industrial Health. 2016; 32(2):193-199.
23. Lusini L, Tripodi SA, Rossi R, Gıannerini F, Giustarini G, Del Vecchio MT, Barbanti G, Cintorino M, Tosi P, Di Simplicio P. Altered (glutathione anti-oxidant metabolism during tumor progression in human renal-cell, carcinoma. Int. J. Cancer. 2001;91:55-59.
24. Pljesa-Ercegovac M, Mimic-Oka J, DragicevicD, Savic-Radojevic A, Opacic M, Pljesa S, Radosavljevic R, Simic T. Altered antioxidant capacity in human renal cell carcinoma: role of glutathione associated enzymes. In Urologic Oncology: Seminars and Original Investigations. 2008;26(2):175-181.
25. Cheng, Y, Xu T, Li S,Ruan H. GPX1, a biomarker for the diagnosis and prognosis of kidney cancer, promotes the progression of kidney cancer. Aging (Albany NY). 2019; 11(24):12165.