Targeting Ras Signaling via Carbonic Anhydrase IX Inhibition: The Differential Impact of Acetazolamide on Clear Cell Renal Carcinoma and Melanoma Cells

Year 2025, Volume: 15 Issue: 3, 244 - 251, 31.12.2025

Emine Terzi , Beyza Ecem Oz Bedir , Ahmet Çarhan , Özen Özensoy Güler

https://doi.org/10.26650/experimed.1703562

Abstract

Objective: To investigate the effect of carbonic anhydrase IX (CA-IX) inhibition by acetazolamide (AAZ) on Ras protein expression in clear cell renal carcinoma and melanoma cell lines.

Materials and Methods: CAKI-2 (clear cell renal carcinoma) and A375 (melanoma) cell lines were cultured and treated with AAZ, rapamycin (RAPA), or vemurafenib (VMF). Cytotoxicity was assessed using the WST-1 assay. CA-IX inhibition was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunofluo rescence staining. Ras protein expression levels were determined by Western blot analysis.

Results: In CAKI-2 cells, there was no significant change in Ras protein expression following AAZ or RAPA treatment (p>0.05). In A375 melanoma cells, AAZ treatment significantly reduced Ras protein levels compared with both the untreated control and VMF-treated groups (p<0.001).

Conclusion: AAZ significantly reduced Ras protein expression in A375 melanoma cells. These findings provide evidence supporting the role of AAZ in regulating Ras-MAPK signaling in melanoma.

Keywords

CA-IX , Clear cell renal carcinoma , MAPK , Melanoma , mtor

Ethical Statement

Ethical approval: This study was conducted entirely using established human cancer cell lines and did not involve human participants or animal subjects. Therefore, ethical committee approval was not required.

Project Number

TUBITAK-Project number: 569337

References

• 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018; 68(1): 7-30.
• 2. Morris MR, Latif F. The epigenetic landscape of renal cancer. Nat Rev Nephrol 2017; 13(1): 47-60.
• 3. Capitanio U, Montorsi F. Renal cancer. Lancet 2016; 387(10021): 894-906.
• 4. Wu R, Wang K, Gai Y, Li M, Wang J, Wang C, et al. Nanomedicine for renal cell carcinoma: imaging, treatment and beyond. J Nanobiotechnology 2023; 21(1): 3.
• 5. Saginala K, Barsouk A, Aluru JS, Rawla P, Barsouk A. Epidemiology of melanoma. Med Sci (Basel) 2021; 9 (4): 63.
• 6. Wagstaff W, Mwamba RN, Grullon K, Armstrong M, Zhao P, Hendren-Santiago B, et al. Melanoma: Molecular genetics, metastasis, targeted therapies, immunotherapies, and therapeutic resistance. Genes Dis 2022; 9(6): 1608-23.
• 7. Lopes J, Rodrigues CM, Gaspar MM, Reis CP. Melanoma management: From epidemiology to treatment and latest advances. Cancers (Basel) 2022; 14(19): 4652.
• 8. Alterio V, Di Fiore A, D’Ambrosio K, Supuran CT, De Simone G. Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? Chem Rev 2012; 112(8): 4421-68.
• 9. Supuran CT. Structure and function of carbonic anhydrases. Biochem J 2016; 473(14): 2023-32.
• 10. Chegwidden WR, Carter ND. Introduction to the carbonic anhydrases. EXS 2000; (90): 14-28.
• 11. Hirakawa Y, Senda M, Fukuda K, Yang Yu H, Ishida M, Taira M, et al. Characterization of a novel type of carbonic anhydrase that acts without metal cofactors. BMC Biol 2021; 19: 105.
• 12. Kumar S, Rulhania S, Jaswal S, Monga V. Recent advances in the medicinal chemistry of carbonic anhydrase inhibitors. Eur J Med Chem 2021; 209: 112923.
• 13. Zamanova S, Shabana AM, Mondal UK, Ilies MA. Carbonic anhydrases as disease markers. Expert Opin Ther Pat 2019; 29(7): 509-33.
• 14. Ward C, Meehan J, Gray M, Kunkler IH, Langdon SP, Argyle DJ. Carbonic anhydrase IX (CAIX), cancer, and radiation responsiveness. Metabolites 2018; 8(1): 13.
• 15. Zaman A, Wu W, Bivona TG. Targeting oncogenic BRAF: past, present, and future. Cancers (Basel) 2019; 11(8): 1197.
• 16. Akocak S, Lolak N, Vullo D, Durgun M, Supuran CT. Synthesis and biological evaluation of histamine Schiff bases as carbonic anhydrase I, II, IV, VII, and IX activators. J Enzyme Inhib Med Chem 2017; 32(1): 1305-12.
• 17. Waheed A, Sly WS. Carbonic anhydrase XII functions in health and disease. Gene 2017; 623: 33-40.
• 18. Swietach P, Vaughan-Jones RD, Harris AL, Hulikova A. The chemistry, physiology and pathology of pH in cancer. Cancer Metastasis Rev 2007; 26(2): 299-310.
• 19. McDonald PC, Winum JY, Supuran CT, Dedhar S. Recent developments in targeting carbonic anhydrase IX for cancer therapeutics. Oncotarget 2012; 3(1): 84-97.
• 20. Supuran CT. Carbonic anhydrase inhibitors as emerging drugs for the treatment of obesity. Expert Opin Emerg Drugs 2008; 13(3): 383-92.
• 21. Han CW, Jeong MS, Jang SB. Structure, signaling and the drug discovery of the Ras oncogene protein. BMB Rep 2017; 50(7): 355.
• 22. Saxena N, Lahiri SS, Hambarde S, Tripathi RP. RAS: target for cancer therapy. Cancer Invest 2008; 26(9): 948-55.
• 23. Khan I, Rhett JM, O'Bryan JP. Therapeutic targeting of RAS: new hope for drugging the “undruggable”. Biochim Biophys Acta Mol Cell Res 2020; 1867(2): 118570.
• 24. Pucciarelli D, Lengger N, Takáčová M, Csaderova L, Bartosova M, Breiteneder H, et al. Hypoxia increases the heterogeneity of melanoma cell populations and affects the response to vemurafenib. Mol Med Rep 2016; 13(4): 3281-8.
• 25. Supuran CT. Carbonic anhydrase inhibitors as emerging agents for the treatment and imaging of hypoxic tumors. Expert Opin Investig Drugs 2018; 27(12): 963-70.
• 26. Parkkila S, Rajaniemi H, Parkkila AK, Kivela J, Waheed A, Pastorekova S, et al. Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro. Proc Natl Acad Sci 2000; 97(5): 2220-4.
• 27. Koike H, Nitta T, Sekine Y, Arai S, Furuya Y, Nomura M, et al. YM155 reverses rapamycin resistance in renal cancer by decreasing survivin. J Cancer Res Clin Oncol 2014; 140(10): 1705-13.
• 28. Chen K, Zhang Y, Qian L, Wang P. Emerging strategies to target RAS signaling in human cancer therapy. J Hematol Oncol 2021; 14(1): 116.
• 29. Akbani R, Akdemir KC, Aksoy BA, Albert M, Ally A, Amin SB, et al. Genomic classification of cutaneous melanoma. Cell 2015; 161: 1681-96.
• 30. Millet A, Martin AR, Ronco C, Rocchi S, Benhida R. Metastatic melanoma: insights into the evolution of the treatments and future challenges. Med Res Rev 2017; 37(1): 98-148.
• 31. Mann GJ, Pupo GM, Campain AE, Carter CD, Schramm SJ, Pianova S, et al. BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in patients with stage III melanoma. J Invest Dermatol 2013; 133: 509-17.
• 32. Devitt B, Liu W, Salemi R, Wolfe R, Kelly J, Tzen CY, et al. Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma. Pigment Cell Melanoma Res 2011; 24(4): 666-72.
• 33. Li F, Aljahdali IA, Zhang R, Nastiuk KL, Krolewski JJ, Ling X. Kidney cancer biomarkers and targets for therapeutics: survivin (BIRC5), XIAP, MCL-1, HIF1α, HIF2α, NRF2, MDM2, MDM4, p53, KRAS and AKT in renal cell carcinoma. J Exp Clin Cancer Res 2021; 40(1): 1-3
• 34. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011; 475(7354): 106-9.
• 35. Banumathy G, Cairns P. Signaling pathways in renal cell carcinoma. Cancer Biol Ther 2010; 10(7): 658-64.
• 36. Takai T, Tsujino T, Yoshikawa Y, Inamoto T, Sugito N, Kuranaga Y, et al. Synthetic miR-143 exhibited an anti-cancer effect via the downregulation of K-RAS networks of renal cell cancer cells in vitro and in vivo. Mol Ther 2019; 27(5): 1017-27.
• 37. Linehan WM, Ricketts CJ. The metabolic basis of kidney cancer. Semin Cancer Biol 2013; 23(1): 46-55.