(A) and (B) T24 and EJ cells were exposed to the indicated concentration of PPM-18 for 24?h, and the expression of phospho-AMPK, AMPK, phospho-mTORC1, phospho-P70S6K, phospho-PI3K, PI3K, phospho-AKT, and AKT were analyzed by western blot
(A) and (B) T24 and EJ cells were exposed to the indicated concentration of PPM-18 for 24?h, and the expression of phospho-AMPK, AMPK, phospho-mTORC1, phospho-P70S6K, phospho-PI3K, PI3K, phospho-AKT, and AKT were analyzed by western blot. able to induce autophagy and apoptosis in Choline Chloride bladder cancer cells AMPK activation. Moreover, reactive oxygen species (ROS) were notably accumulated in PPM-18Ctreated bladder cancer cells, and treatment with ROS scavengers not only eliminated ROS production but also abrogated AMPK activation, which eventually rescued bladder cancer cells from PPM-18Ctriggered autophagy and apoptotic cell death. In bladder cancer xenografts, the anticancer activities of PPM-18, including suppressing the growth of tumors and inducing autophagy and apoptosis in tumor cells, were also established. Collectively, this study was the first to demonstrate the anticancer effect of PPM-18 on bladder cancer cells and through eliciting autophagy and apoptosis ROS and AMPK pathways, which might provide new insights into the potential utilization of PPM-18 for future bladder cancer Choline Chloride treatment. and = 7) and PPM-18Ctreated group (= 7). PPM-18 (10?mg/kg) was dissolved in saline and administered each day by intratumoral injection for 30?days, while controls were injected with the equivalent volume of saline. The tumor sizes and weights of the mice were measured every three days. Tumor volume was calculated by the formula: length width2/2. After 30?days of treatment, the mice were sacrificed, and the tumors were excised and fixed for immunohistochemistry (IHC), TUNEL staining and ROS detecting. In addition, several important organs, including the heart, liver, spleen, lung, and kidney, were also fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. The sections were then subjected to hematoxylin and eosin staining (H&E). Statistical Analysis All data were presented as the mean SD of at least three times independent experiments and analyzed using the GraphPad Prism 6 software. Differences between groups were measured using Students test. The statistical significance in the figures was regarded as * 0.05, ** 0.01, and *** 0.001. Results PPM-18 Suppresses the Proliferation Choline Chloride of Bladder Cancer Cells To investigate whether PPM-18 inhibits the proliferation of bladder cancer cells, we first assessed the effect of PPM-18 on the viability of bladder cancer cells. As shown in Figures 1A,B, PPM-18 remarkably reduced the viability of bladder cancer T24 and EJ cells in a dose- or time-dependent manner. Moreover, significant morphological alterations were exhibited in PPM-18Ctreated Choline Chloride T24 and EJ cells (Figure 1C). To further determine the inhibitory growth effect of PPM-18 on bladder cancer cells, BrdU and cellular colony assays were performed. As shown in Figure 1D, PPM-18 dose-dependently repressed the BrdU incorporation in T24 and EJ cells. Moreover, cellular colony formation was markedly inhibited following exposure to the increasing concentration of PPM-18 (Figure 1E). These results indicate that PPM-18 is capable of suppressing the proliferation of bladder cancer cells. In addition, the anticancer effect of PPM-18 on other human solid tumor cells was also confirmed by its capacity of reducing cell viability and inhibiting cellular colony formation (Supplementary Figures S1A,B), suggesting that PPM-18 exerts anticancer activity TSPAN17 in a broad spectrum of human cancer cells. We next evaluated the anticancer efficiency of PPM-18 through IC50 values. As shown in Supplementary Table S1, we found that bladder cancer cells were more sensitive to PPM-18 treatment compared with other human solid tumor cells. To investigate whether PPM-18 has cytotoxic effect on human normal cells, SV-HUC-1 (human bladder immortalized epithelium cells), L02 (human normal liver cells), and HEK293T (human embryonic kidney cells) were employed in this study. As shown in Supplementary Figure S2A, the cell viability of SV-HUC-1, L02, and HEK293T nearly remained intact after exposure to the indicated concentration of PPM-18, implying that PPM-18, at a range of concentration, exerts lower cytotoxicity Choline Chloride in human normal cells compared to most cancer cells. Open in a separate window FIGURE 1 PPM-18 inhibits the growth of bladder cancer cells. (A) Bladder cancer T24 and EJ cells were dose-dependently treated with PPM-18 for 24?h, and the cell viability was.