Experiments in this figure were repeated four times, and similar results were obtained. Next, we analyzed the potential activity of SF2523 in other RCC cells. cells. As described in our previous studies [10, 11, 14], two lines of primary human RCC cells, namely RCC-1/2, as well as the other established RCC cell line, A498, were treated with SF2523 (at 1 M). CCK-8 assay results in Figure ?Figure1E1E confirmed that SF2523 was anti-survival/cytotoxic to both A498 cells and two lines of the primary RCC cells. The CCK-8 OD was reduced significantly following SF2523 (1 M, 72 hours) treatment in the RCC cells (Figure ?(Figure1E).1E). Furthermore, proliferation of the RCC cells, again tested by the BrdU ELISA assay, was also inhibited by SF2523 (1 M, 48 hours) (Figure ?(Figure1F).1F). These results suggest that SF2523 was cytotoxic and anti-proliferative to both established and primary human RCC cells. The potential effect of SF2523 to non-cancerous renal cells was also tested. HK-2 tubule epithelial cells and the primary human renal epithelial cells were cultured (see CFTRinh-172 the previous study ) and treated with SF2523 (1 M). Intriguingly, CCK-8 assay results in Figure ?Figure1G1G showed that SF2523 treatment (1 M, 72 hours) was non-cytotoxic to the renal epithelial cells. The CCK-8 OD was almost unchanged before and after SF2523 treatment (Figure ?(Figure1G).1G). Meanwhile, the BrdU incorporation was also not significantly changed by SF2523 treatment (1 M, 48 hours) in epithelial cells (Figure ?(Figure1H).1H). These results indicate that SF2523 was uniquely non-cytotoxic to normal renal epithelial cells. SF2523 induces profound apoptosis activation in RCC cells Apoptosis induction is a major reason of cancer cell growth inhibition/cell death [28C32]. A number of anti-cancer agents provoke cell apoptosis to kill cancer cells [29C31, 33, 34]. Over-production of single strand DNA (ssDNA) is often detected as the indicator of cell apoptosis. Here we show that SF2523 treatment dose-dependently increased ssDNA content in 786-O RCC cells (Figure ?(Figure2A).2A). Further, SF2523 (1 M) increased activities of caspase-3 and caspase-9 in 786-O cells (Figure ?(Figure2B).2B). Meanwhile, cleavages of caspase-3 and PARP (poly (ADP-ribose) polymerase) were observed in SF2523 (1 M)-treated cells (Figure ?(Figure2C).2C). Additionally, SF2523 (1 M) significantly increased the number of Annexin V-labeled (Figure ?(Figure2D)2D) and TUNEL-stained (Figure ?(Figure2E)2E) 786-O cells. Open in CFTRinh-172 a separate window Figure 2 SF2523 provokes RCC cell apoptosisEstablished human RCC cell lines (786-O and A498), the CFTRinh-172 primary human RCC cells (RCC-1/2 lines), HK-2 tubular epithelial cells as well as the primary human renal epithelial cells (Renal Epi) were treated with indicated concentration of SF2523 for the applied time; Cell apoptosis was tested by the assays mentioned in the text (A, B, D-G); Expressions of cleaved-caspase-3 (Cle-Cas-3) and cleaved-PARP (Cle-PARP) were also tested, with ERK1 as the loading control (C, for 786-O cells). Data were expressed as mean standard deviation (SD, n=5). The data in this figure were summarizing one set of experiment. *< 0.05 vs. untreated control group (C). Vehicle control (0.1% of DMSO) failed to change apoptosis of IL-10C the tested cells. Experiments in this figure were repeated three times, and similar results were obtained. TUNEL assay was also employed to test the potential activity of SF2523 on other RCC cells. Results in Figure ?Figure1F1F clearly showed that SF2523 (1 M, 48 hours) treatment significantly increased the number of TUNEL staining in A498 cells and in two lines of the primary human RCC cells. Thus, SF2523 is also pro-apoptotic in these RCC cells. On the other hand, the very same SF2523 treatment (1 M, 48 hours) in HK-2 cells and the primary human renal epithelial cells was unable to induce significant apoptosis (TUNEL assay, Figure ?Figure1G),1G), again showing a selective response of this compound only to the cancer cells..