Checkpoint Kinase

Antitumor efficacy of a monoclonal antibody that inhibits the activity of cancer-associated carbonic anhydrase XII

Antitumor efficacy of a monoclonal antibody that inhibits the activity of cancer-associated carbonic anhydrase XII. than the other cancer-associated CA, CAIX. The aim of this study is to evaluate CAXII inhibitors as selective chemosensitizers in MDR tumor models. Eight test inhibitors with variable CA inhibition profiles and variable physicochemical properties were selected to establish the potential of CAXII inhibitors to indirectly inhibit Pgp activity to resensitize MDR cells to doxorubicin. We show that CAXII inhibitors have very good chemosensitizing efficacy, and increase the effectiveness of the chemotherapeutic drug doxorubicin up to 4.4-fold. This correlated with high expression of both CAXII and Pgp and values PQ 401 of compounds 1C8 and the established CA inhibitor acetazolamide (AZA) (nM)bassays. In these experimental conditions, compounds 1, 2 and 4 increased the intracellular accumulation of doxorubicin, a Pgp substrate, in cells with high expression of both CAXII and Pgp (Supplementary Figure S1), such as HT29/DX, A549/DX, MDA-MB-231, TUBO, JC, U2OS/DX and SaOS/DX cells (Figure 2AC2J). The compounds had no effect on cells with detectable levels of just one of these two proteins expressed (Supplementary Figure S1), such as HT29, A549, MCF7, SKBR3, T74D, U2OS and SaOS cells (Figure 2AC2J). The expression of CAIX did not influence the effects of the compounds on the intracellular doxorubicin accumulation in all cell lines tested. Compound 3 (CAXII = 4). Versus doxorubicin alone (C): *p < 0.05; for cells treated with compounds 1C8 or tariquidar, doxorubicin-resistant cells versus the corresponding doxorubicin-sensitive cells: p < 0.05. In accordance with the correlation of CAXII expression and cancer cell proliferation [14], compounds 1, 2 and 4 reduced the viability of CAXII-positive cell lines. The reduction in viability for individual compounds was: 31 6% in HT29/DX cells, 28 10% in A549 cells, 38 7% in A549/DX cells, 33 12% in T74D cells, 36 11% in MDA-MB-231 cells, 28 7% in TUBO cells, 30 10% in JC cells, 27 8% in U2OS/DX cells, 32 7% in SaOS/DX cells (< 0.05 for all cell lines; = 4). In contrast, PQ 401 the compounds were devoid of any effects on viability in cells with low or undetectable levels of CAXII, including HT29, MCF7, SKBR3, U2OS, SaOS cell lines (not shown). As expected, doxorubicin reduced viability in cells with undetectable or low levels of Pgp, i.e. HT29, A549, MCF7, SKBR3, T74D, U2OS and PQ 401 SaOS cells; in these doxorubicin-sensitive cell lines the compounds did Mouse monoclonal to CD4 not exert additive effects on viability compared to doxorubicin treatment alone (not shown). In contrast, HT29/DX, A549/DX, MDA-MB-231, TUBO, JC, U2OS/DX, SaOS/DX cells, which are positive for both Pgp and CAXII (Supplementary Figure S1), were unresponsive to doxorubicin alone not shown. Compounds 1, 2 and 4 restored doxorubicin efficacy and further reduced cell viability. The differences in cell viability between cells treated with compounds alone and cells co-treated with compounds plus doxorubicin were: 38 6% in HT29/DX cells, 22 8% in A549 cells, PQ 401 38 7% in A549/DX cells, 18 7% in T74D cells, 34 10% in MDA-MB-231 cells, 22 8% in TUBO cells, 29 8% in JC cells, 27 9% in U2OS/DX cells, 27 7% in SaOS/DX cells, (< 0.05; = 4). These differences suggest that the decreased viability of cells co-treated with CAXII inhibitors and doxorubicin was due to the increased doxorubicin accumulation with added compound 1, 2 or 4 and/or to a synergistic effect of compound 1, 2 or 4 and doxorubicin, rather than to cytotoxicity exerted by the PQ 401 CAXII inhibitors themselves. Accordingly, the doxorubicin IC50 was significantly reduced by the co-treatment with the CAXII inhibitors in these cell lines. Co-treatment with compounds 1, 2 and 4 had the same efficacy as treatment with tariquidar (Figure 3AC3J) in resensitizing cells to doxorubicin (Table ?(Table2).2). Notably, in CAXII-negative MCF7 and SKBR3 cells that overexpress Pgp (Supplementary Figure S3A), the compounds did not increase the intracellular retention of doxorubicin (Supplementary Figure S3B). Lastly, compounds 1, 2 and 4 did not exert any cytotoxic effect (Supplementary Figure S4B) in not-transformed human epithelial colon CCD-Co-18 cells, epithelial lung BEAS-2B cells, epithelial breast MCF10A cells or fibroblasts that do not have detectable levels of CAXII (Supplementary Figure S4A). Collectively these results demonstrate that compounds 1, 2 and 4 are cytotoxic agents against CAXII-positive cancer.