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.
摘要：
探讨碳酸酐酶抑制与抗癌活性的内在关系,我们制备了四组二芳基脲分子，并测试了hCA-IX和XII对两种乳腺癌细胞系的抑制作用。在21个化合物中，化合物J2(具有-SO2NH2基团)和J16(不具有-SO2NH2基团)在常氧和低氧条件下显示出最佳活性。J16对MDA-MB-231和MCF-7细胞的IC50值，在常氧条件下分别为6.3和3.7µM，分别比U-4-硝基和SLC-0111好1.9/3.3和15.8倍。然而,在低氧条件下，相应的值分别为12.4和1.1μM（MDA-MB-231和MCF-7细胞），比U-4-硝基好8倍。然而,在常氧条件下，对于MDA-MB-231和MCF-7细胞，J2显示出比U-4-硝基（6.3µM）更好的IC50值（1.9/2.7倍）。化合物J2以纳摩尔浓度抑制hCA-IX和XII的活性[Ki值分别为4.09和9.10nM,hCA-II的选择性比为1.8和0.8]。晶体结构和建模研究表明，CA的抑制作用是由于锌在其催化域中的CO2配位位点的阻断而引起的。然而,发现J16不能抑制hCA的活性(Ki>89000nM)。qPCR和蛋白质印迹分析显示HIF1A的转录和表达显着减少（1.5至20倍），CA9和CA12基因中存在J2和J16。发现J2和J16均通过抑制hHSP70的伴侣活性来减少HIF-1α蛋白的积累，IC50值分别为19.4和15.3µM。hCA-IX和XII活性通过结合在活性位点或通过降低表达或通过这两者引起细胞内pH降低的扰动,这导致J2/J16的活性氧种类同时增加2.6/2.0倍(MCF-7)和2.9/1.8倍(MDA-MB-231)。推测在J2和J16存在下细胞周期蛋白D1表达的增加是癌细胞凋亡的间接原因。其他凋亡标志物Bcl-2，Bim的表达，caspase9和caspase3证实了细胞凋亡机制。然而,HIF1A/HIF-1α和hCA-IX/XII的转录/表达降低也意味着J2和J16对细胞外信号调节激酶途径的抑制。
