Abstract:
To investigate the intrinsic relation between carbonic anhydrase inhibition and anticancer activity, we have prepared four sets of diaryl urea molecules and tested for the inhibition of hCA-IX and XII on two breast cancer cell lines. Among 21 compounds, compound J2 (with -SO2NH2 group) and J16 (without -SO2NH2 group) showed the best activity under normoxic and hypoxic conditions. The IC50 values of J16 for MDA-MB-231 and MCF-7 cells, under normoxic condition were 6.3 and 3.7 µM respectively, which are 1.9/3.3 and 15.8 times better than U-4-Nitro and SLC-0111 respectively. Whereas, under the hypoxic condition the corresponding values were 12.4 and 1.1 µM (MDA-MB-231 and MCF-7 cells respectively), which are equal/8 times better than U-4-Nitro. Whereas, J2 showed better IC50 value than U-4-Nitro (6.3 µM) under normoxic condition for both MDA-MB-231 and MCF-7 cells (1.9/2.7 times). Compound J2 inhibits the activity of hCA-IX and XII in nanomolar concentration [Ki values 4.09 and 9.10 nM respectively with selectivity ratio of 1.8 and 0.8 with hCA-II]. The crystal structure and modelling studies demonstrates that the inhibition of CAs arises due to the blocking of the CO2 coordination site of zinc in its catalytic domain. However, J16 was found to be unable to inhibit the activity of hCAs (Ki > 89000 nM). qPCR and western blot analysis showed a significant reduction (1.5 to 20 fold) of the transcription and expression of HIF1A, CA9 and CA12 genes in presence of J2 and J16. Both J2 and J16 found to reduce accumulation of HIF-1α protein by inhibiting the chaperone activity of hHSP70 with IC50 values of 19.4 and 15.3 µM respectively. Perturbation of the hCA-IX and XII activity by binding at active site or by reduced expression or by both leads to the decrease of intracellular pH, which resulted in concomitant increase of reactive oxygen species by 2.6/2.0 (MCF-7) and 2.9/1.8 (MDA-MB-231) fold for J2/J16. Increased cyclin D1 expression in presence of J2 and J16 was presumed to be indirectly responsible for the apoptosis of the cancer cells. Expression of the other apoptosis markers Bcl-2, Bim, caspase 9 and caspase 3 substantiated the apoptosis mechanism. However, decreased transcription/expression of HIF1A/HIF-1α and hCA-IX/XII also implies the inhibition of the extracellular signal-regulated kinase pathway by J2 and J16.