Dasatinib induces apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in bladder cancer cells

Abstract

Purpose: Bladder cancer is a common genitourinary malignant disease worldwide. Dasatinib is a small molecule inhibitor of Src family kinases. We investigated the anticancer effect and putative molecular mechanisms of dasatinib on T24 and cisplatin-resistant T24R2 human bladder cancer cells.

Materials and methods: Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and colony formation in dasatinib treated bladder cancer cells. Flow cytometry was used to determined cell cycle arrest and apoptosis. The expression of apoptosis and autophagy related proteins were detected by western blot analysis.

Results: In bladder cancer cells, dasatinib significantly reduced cell proliferation, colony formation, and induced G1-phase arrest. Dasatinib triggered apoptosis along with an increased expression of apoptosis-related genes (caspases, PARP, and cytochrome c). Down-regulation of Bcl-2 and up-regulation of Bad, which are hallmarks of apoptosis, were found to play a dominant role in mediating the effects of dasatinib treatment. We further showed that dasatinib inhibits p-Src, p-PI3K, p-Akt, and p-mTOR in bladder cancer cells. Dasatinib also increased the expression of markers of autophagy flux such as LC3-II and p62.

Conclusions: These results confirmed that dasatinib is a potent chemotherapeutic drug which induces apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in bladder cancer cells.

Keywords: Anticancer; Apoptosis; Autophagy; Bladder cancer; Dasatinib.

Fig. 1. The structure of dasatinib.

Fig. 2. Effects of dasatinib on viability of T24 and T24R2 cells. The cells were incubated with dasatinib for 24 hours (A) and 48 hours (B). The cell viability was measured by the Cell Counting Kit-8 assay. The data are represented as mean±standard deviation of three independent experiments.

^*Statistically significant difference with the untreated control (p<0.05).

Fig. 3. Effects of dasatinib in T24 and T24R2 cells according to a colony formation assay. The cells were incubated with dasatinib for 24 hours and were then cultured in the fresh medium for 10–14 days to allow colonies to form. (A) A photograph and the numbers of colonies in T24 cells. (B) A photograph and the numbers of colonies in T24R2 cells. The data are represented as mean±standard deviation of three independent experiments. ^*Statistically significant difference with the untreated control (p<0.05).

Fig. 4. Effects of dasatinib on cell cycle progression in T24 and T24R2 cells. The cells were incubated with dasatinib for 24 hours. (A) Flow cytometric DNA content histogram of T24. (B) Quantitative measures of cycle alterations in T24. (C) Flow cytometric DNA content histogram of T24R2. (D) Quantitative measures of cycle alterations in T24R2. Cellular DNA was stained with propidium iodide and flow cytometry analysis was performed to evaluate the cell cycle distribution. The data are represented as mean±standard deviation of three independent experiments. ^*Statistically significant difference with the untreated control (p<0.05).

Fig. 5. Effects of dasatinib on expression of apoptosis-related proteins in T24 (A) and T24R2 (B) cells. The cells were incubated with dasatinib for 24 hours. Protein expression was analyzed by western blotting. Equal amounts of total proteins were loaded onto the gels. The data are represented as mean±standard deviation of three independent experiments. ^*Statistically significant difference with the untreated control (p<0.05).

Fig. 6. Effects of dasatinib on expression of cell cycle and autophagy-related proteins in T24 (A) and T24R2 (B) cells. The cells were incubated with dasatinib for 24 hours. Protein expression was analyzed by western blotting. Equal amounts of total proteins were loaded onto the gel. The data are represented as mean±standard deviation of three independent experiments. ^*Statistically significant difference with the untreated control (p<0.05).

Fig. 7. Dasatinib mediated apoptosis and autophagy of T24 (A) and T24R2 (B) cells by suppression of the PI3K/Akt/mTOR pathway. The cells were incubated with dasatinib for 24 hours. Protein expression was analyzed by western blotting. Equal amounts of total proteins were loaded onto the gels. The data are represented as mean±standard deviation of three independent experiments. ^*Statistically significant difference with the untreated control (p<0.05).

Fig. 8. Schematic illustration showing the mechanisms underlying the anticancer effects of dasatinib in bladder cancer cells.