Anticancer effect of new carbonic anhydrase 9 inhibitors in glioblastoma cells

Abstract

Introduction: Carbonic anhydrase 9 (CA9) is a membrane enzyme, a regulator of intracellular and extracellular pH, overexpressed in cells in a hypoxic environment [1]. Solid tumors, adapted to hypoxia, have large quantities of the CA9 and the increased expression correlates with tumor patients’ poor prognosis, tumor malignancy, and resistance to drugs [2]. In glioblastoma, hypoxia promotes the spreading of cancer cells into the brain tissue, to evade the environment with low oxygen levels [3]. Inhibitors of CA9 have previously been investigated for anticancer drugs [2]. In the present study, we evaluated the anticancer properties of three CA9 inhibitors (AFA-30, AFA-40 and AFA-49), phosphonium salts derived from coumarin, in sensitive (U87) and chemoresistant (U87-TxR) human glioblastoma cell lines. Materials & Methods: The effect of CA9 inhibitors on cell growth, either alone or in combination with tariquidar was determined by sulforhodamine B assay. Flow cytometry was used for the assessment of change in intracellular pH by BCECF staining, and the rhodamine 123 assay of P-gp activity. Changes in the expression of CA9, CA12, and ABCB1 genes were analyzed by qPCR. Results: The three compounds inhibited cell growth of both sensitive (U87) and resistant (U87-TxR) cells in 48 h treatments, in both hypoxic (1% O2) and normoxic (20% O2) conditions. However, compared to U87 (IC50 range 1 - 5 μM), the U87-TxR were less sensitive to the compounds’ growth inhibition effect (IC50 range 8 – 30 μM). U87-TxR cells are characterized by the increased expression of the P-gp extruding pump. When tariquidar, a P-gp inhibitor, was applied in combination with CA9 inhibitors, U87-TxR cells were sensitized to these compounds. In the P-gp activity assay, we demonstrated that compounds (5 – 50 μM) increase a P-gp substrate accumulation – rhodamine 123. Further, gene expression of ABCB1 was increased 2 – 8 times in U87, following treatment. In 24 h treatments, these CA9 inhibitors decreased intracellular pH. Moreover, the 24 h treatments resulted in decreased expression of CA9 and CA12. Conclusion: The three CA9 inhibitors here described have significant anticancer effects in glioblastoma cells and show potential for further pre-clinical investigation, especially in tumors with emphasized hypoxic zones contributing to increased malignancy, such as glioblastomas. 1. Mussi, S., et al., Antiproliferative effects of sulphonamide carbonic anhydrase inhibitors C18, SLC-0111 and acetazolamide on bladder, glioblastoma and pancreatic cancer cell lines. J Enzyme Inhib Med Chem, 2022. 37(1): p. 280-286. 2. Kalinin, S., et al., Carbonic Anhydrase IX Inhibitors as Candidates for Combination Therapy of Solid Tumors. Int J Mol Sci, 2021. 22(24). 3. Monteiro, A.R., et al., The Role of Hypoxia in Glioblastoma Invasion. Cells, 2017. 6(4). Funding: This research was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (ref. number 451-03-68/2020-14/200007).