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
Graphical Abstract
INTRODUCTION
Protein tyrosine kinases (PTKs) are enzymes that transfer a phosphate group to a protein, while phosphatases remove a phosphate group from a protein. Since most PTKs promote cell proliferation, survival, and migration when constitutively overexpressed or activated, they are also associated with oncogenesis. The following two classes of PTKs are present in cells: the transmembrane receptor PTKs and non-receptor PTKs (NRTKs) [1,2].
The Src family of tyrosine kinases is the largest group of non-receptor tyrosine kinases [2]. Src kinase is known as a proto-oncogene which is overexpressed in various human cancers including those of colon, breast, pancreas, and brain [3,4]. Src activity is much higher in metastatic tissues compared to normal tissues [5]. Src enhances the activity of multiple oncogenic signaling pathways including phosphatidylinositol 3-kinase/AKT, c-Myc, and the Ras/Raf pathway [6]. Therefore, Src is engaged in multiple cellular processes and plays a central role in influencing cell signaling activities involved in oncogenesis [7]. It regulates a broad spectrum of cellular events including cell differentiation, apoptosis, proliferation, survival, motility, invasion, and epithelial-to-mesenchymal transition [2,4,7]. Consequently, studies to develop drugs targeting Src family kinases for cancer therapy are important.
Dasatinib (formerly known as BMS-354825) is an orally bioavailable small-molecule multi-targeted kinase inhibitor and has been approved by the U.S. Food and Drug Administration to treat chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia [6,8]. It is a dual Src/Abl kinase inhibitor which has demonstrated anti-proliferative activity against multiple tumor types [9]. Dasatinib inhibited cell proliferation and migration in the urothelial bladder cancer cell line and its gemcitabine-resistant sub-line and reduced tumor growth and muscle invasion in orthotopic xenografts [3]. Hahn et al. [10] reported that neoadjuvant dasatinib was feasible and safe in patients with muscle-invasive urothelial carcinoma of the bladder cancer.
However, the anticancer effect of dasatinib and the role of autophagy, a type II programmed cell death, in dasatinib-treated bladder cancer cells has not been reported. Here, we investigated the mechanisms of dasatinib in bladder cancer cell line and its cisplatin-resistant cell line.
MATERIALS AND METHODS
1. Cell lines and reagents
T24 bladder cancer cells were obtained from American Type Culture Collection. Cisplatin-resistant T24R2 cells (resistant to 2 µg/mL cisplatin) were generated by serial desensitization [11]. The cells were cultured in RPMI-1640 medium (Gibco/Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco/Invitrogen) in a humidified atmosphere of 95% air and 5% CO2 at 37℃. Dasatinib was purchased from Selleckchem (Fig. 1). It was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and was then diluted to obtain the working concentration. The final concentration of DMSO in the culture media was 0.1% (v/v). Medium containing 0.1% DMSO was used as a control.
Fig. 1. The structure of dasatinib.
2. Cell Counting Kit-8 (CCK-8) assay
For cell proliferation assay, bladder cancer cells were seeded in 96-well plates at 2×103 cells per well. After incubation for 24 hours, the cells were treated with dasatinib for 24 hours and 48 hours. At the end of the drug treatment, 10 µL of the CCK-8 solution (Dojindo Molecular Technologies) was added to the plates, incubated for 4 hours. Absorbance was measured at 450 nm on an ELISA plate reader (Molecular Devices). Cell viability was calculated as a percentage of viable cells in the total population.
3. Colony formation assay
Cells were seeded at 4×102 cells/well in 6-well plates and incubated with dasatinib for 24 hours. The cells were cultured for another 10–14 days in dasatinib-free medium. The colonies were fixed with methanol and stained with a 0.1% crystal violet dye at room temperature for 1 hour. After staining, the plates were washed with distilled water and then dried. The plates were photographed, and colonies greater than 0.2 mm in diameter were counted.
4. Cell cycle analysis
A total of 3×105 cells were seeded in a 60 mm dish and incubated with dasatinib for 24 hours. Cells were then collected, washed with phosphate-buffered saline, and fixed with 70% ethanol for 2 hours at 4℃. Next, the cells were stained with a propidium iodide (PI; Sigma Aldrich) solution for 30 minutes at 37℃. Cell cycle distribution was determined on a FACSCalibur instrument (BD Biosciences). The resultant data were analyzed using the BD CellQuest Pro software (BD Biosciences).
5. Western blotting analysis
Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer, consisting of 50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentration from cell extracts was measured by Bradford protein assay kit (Bio-Rad), and equal amounts of total protein (30 µg) was separated by SDS-PAGE (8%–12%) and the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked in 5% (w/v) non-fat dry milk for 1 hour at room temperature, and then incubated with primary antibodies (PARP, cleaved caspases-3, -8, -9, cytochrome c, Bcl-2, Bad, cyclin A, cyclin B, cyclin D, cyclin E1, Src, phosphorylated-Src [Typ416], mTOR, phosphorylated-mTOR [Ser2448], phosphoinositide 3-kinase [PI3K], phosphorylated-PI3 Kinase p85 [Tyr485]/p55 [Tyr199], protein kinase B [Akt], phosphorylated-Akt [Ser473], p62, and Atg5 [Cell Signaling Technology]; LC3 [Novus Biological]) at 4℃ overnight. After incubation with secondary antibodies for 1 hour, membranes were visualized using the enhanced chemiluminescence western blot substrate kit (Pierce).
6. Statistical analysis
All statistical analyses were performed using IBM SPSS Statistics software version 20 (IBM Corp.). The measured values were presented as the mean±standard deviation of at least three independent experiments performed in triplicates. Differences were analyzed using the Tukey’s multiple-range test, and significance was set at p<0.05.
RESULTS
1. Treatment with dasatinib suppresses proliferation of bladder cancer cells
Bladder cancer cells were treated with dasatinib (0, 0.01, 0.1, 0.5, 1, 2.5, 5, 10, 25, or 50 µM) and cell viability was measured by CCK-8 assay. Dasatinib significantly inhibited cell proliferation in a dose-dependent manner compared with untreatment control cells (Fig. 2). T24R2 cells showed more sensitivity to dasatinib compared to parental T24 cells at 24 hours. However, T24 and T24R2 cells showed similar growth curve at 48 hours. We confirmed the ability of dasatinib to suppress cell proliferation by assessing its impact on colony formation from a single cell. Upon 10 µM dasatinib treatment, the colony numbers of T24 and T24R2 cells were significantly reduced by 75.41% and 90.20% respectively, compared to the untreated control (Fig. 3). These results suggest that, with regard to proliferation, T24R2 cells more sensitive to dasatinib treatment than parental T24 cells.
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).
2. Treatment with dasatinib induces cell cycle alteration in bladder cancer cells
Flow cytometry was performed to assess changes in the cell cycle and apoptosis, in bladder cancer cells. As shown in Fig. 4, dasatinib treatment significantly increased the percentage of cells in the sub-G1 cell phase in T24 and T24R2 cells. Treatment with 5 µM dasatinib significantly increased the percentage of apoptotic cells as compared to control cells. These results showed that dasatinib treatment significantly increased the sub-G1 population, corresponding to apoptotic cells, in bladder cancer cells. Furthermore, treatment of both T24 and T24R2 cells with ≥0.1 µM dasatinib significantly increased the percentage of cells in the G1 phase compared to the untreated control. We found that treatment with dasatinib strongly altered cell cycle progression of bladder cancer cell lines.
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).
3. Treatment with dasatinib changes the expression of proteins regulating apoptosis in bladder cancer cells
We evaluated the effect of dasatinib treatment on the expression of crucial proteins involved in apoptosis by western blot analysis. As shown in Fig. 5, the expression levels of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, fragmented PARP, cytochrome c, and pro-apoptotic protein Bad were markedly increased after treatment with dasatinib, whereas the expression of the anti-apoptotic protein Bcl-2 was remarkably reduced in bladder cancer cells indicating that dasatinib induced apoptosis in bladder cancer cells.
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).
4. Treatment with dasatinib changes expression of cell cycle and autophagy regulating proteins in bladder cancer cells
Furthermore, we measured the expression levels of cyclins as an index of the G1 phase arrest. Dasatinib treatment markedly decreased the expression of cyclin A, B, and D (Fig. 6). To elucidate the role of dasatinib in autophagy, the levels of LC3, Atg5, and p62, which are known markers of autophagy, were measured by western blot analysis. As illustrated in Fig. 7, expression of Atg5 and the ratio of LC3-II/LC3-I increased gradually in a dose-dependent manner following dasatinib treatment in both T24 and T24R2 cells. p62, an indicator of lysosome degradation, has been used for monitoring autophagic flux [12]. Our results showed a marked decline in p62 levels in T24 cells upon dasatinib treatment in a dose-dependent pattern, compared with the control cells. Consequently, our findings indicate that dasatinib triggers autophagy in bladder cancer cells.
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).
5. Treatment with dasatinib affected PI3K/AKT/mTOR pathway in bladder cancer cells
To further understand the molecular mechanism of the anti-tumor effect of dasatinib, we examined the effect of dasatinib on the PI3K/Akt/mTOR pathway. As shown in Fig. 7, dasatinib significantly down-regulated the levels of phosphorylated PI3K, Akt, and mTOR in a concentration-dependent manner but did not changed the expression of total PI3K, Akt, and mTOR. In addition, dasatinib significantly suppressed the expression of phosphorylated Src in bladder cancer cells. The expression of total Src did not change significantly. Therefore, dasatinib could be a serve as a potential agent for regulating PI3K/AKT/mTOR pathway in bladder cancer.
DISCUSSION
Bladder cancer is the ninth most frequent malignant disease and thirteenth most common cause of cancer death worldwide [13,14]. Incidence rates of bladder cancer is three to four times higher in men compared to women. The main risk factors include increasing age, tobacco smoking and occupational hazards involving processing of paint, dye, metal, and petroleum products [14,15]. Most non-muscle invasive bladder cancer can be effectively treated by surgery. However, advanced bladder cancer develops a muscle-invasive pattern resulting in lethal disease without effective treatment [13]. Although platinum-based chemotherapy is the standard treatment of choice for advancer bladder cancer, chemoresistance limits its long-term curative effect [16]. Therefore, novel and potent therapeutic agents are required for the treatment of bladder cancer.
The Src protein is a non-receptor tyrosine kinases and was the first identified proto-oncogene. Increased Src expression and/or activity have been described in many different human tumors including those of colon, breast, pancreas, and brain [3,5]. Src regulates key cellular processes such as proliferation, survival, adhesion and motility [5,7]. Kong et al. [17] revealed that Src siRNA significantly reduced TGF-β-induced cell migration and invasion of T24 cells. Tyrosine at site 419 (Y419) in human is Src has been known as the classical activation site, which is most commonly targeted in cell line studies to investigate the functional relevance of Src kinase activation [5,7].
Dasatinib is a multi-targeted tyrosine kinase inhibitor which targets important oncogenic pathways including SRC family kinases [18]. Chang and Wang [19] reported that dasatinib inhibited proliferation, adhesion, migration, and invasion of hepatocellular carcinoma cells by suppressing Src tyrosine kinase, affecting SFK/FAK, and PI3K/PTEN/Akt pathways.
In A549 cells, dasatinib significantly increased the proportion of cells in G1 phase, suggesting the direct arrest of cell cycle progression by inhibiting Src in cancer cells [20]. Our data demonstrated that dasatinib results in antiproliferation, cell cycle arrest at G1 phase, and induction of apoptosis and autophagy in bladder cancer cells. This effect of dasatinib appears to be mediated by inhibition of Src phosphorylation (Y419).
Apoptosis is considered as protective mechanism against cancer development [21]. Targeting and inducing apoptosis is an interesting strategy for cancer therapy, since the induction of apoptosis shifts the treatment effect from cytostatic to cytotoxic [22]. To better understand the effects of dasatinib treatment on apoptosis, we performed western blot analysis. Dasatinib was effective in activation of cleaved caspases-3, cleaved caspase-8, cleaved caspase-9, fragmented PARP, and cytochrome c, indicating that dasatinib induced apoptosis in both T24 and T24R2 cells. These results suggest that dasatinib-induced apoptosis plays a vital role in the inhibition of T24 and T24R2 cells, as apoptosis was considered as a protective mechanism against cancer development.
Similar to apoptosis, autophagy is also an important form of programmed cell death, which prevents survival and proliferation of malignant cells [23]. Autophagy is an intracellular degradative mechanism for eliminating damaged organelles and long-lived proteins. The conversion of LC3-I to LC3-II is a key step in mammalian autophagy, and the number of LC3-II puncta correlates with the number of autophagosomes. The level of p62, a multifunctional protein that targets proteins to degradation by proteasomes and autophagy, is also correlated with the extent of autophagy flux progression [24]. Recent studies have demonstrated that autophagy might be a potential antitumor mechanism [25]. Therefore, the expression levels of LC3-I, LC3-II, p62, and Atg5, proteins related to autophagy, in response to dasatinib treatment was examined. Our experiments showed that dasatinib induced autophagy by up-regulating the expression levels of LC3-II and Atg5 and down-regulating the expression level of p62, in bladder cancer cells.
Finally, we examined the signaling pathway upstream of apoptosis and autophagy that might be responsible for the anticancer efficacy of dasatinib. The PI3K/Akt/mTOR pathway is an essential signaling pathway involved in the regulation of the cell cycle, and its increased activation prevents apoptosis and autophagy and promotes tumor cell survival, proliferation, and motility in many cancers [26,27]. Accordingly, in this study, we also examined the PI3K/Akt/mTOR pathway that might be affected by Src. We found that phosphorylated PI3K, Akt, and mTOR were significantly down-regulated in both T24 and T24R2 cells, and apoptotic and autophagic proteins were activated after dasatinib treatment. However, unlike the other pathways, p-Akt/Akt ratio in T24R2 cells was significantly reduced only at 5 µM dasatinib. According to the study of Chang and Wang [19], the expression of p-Akt in Sk-Hep1 and HepG2 was significantly reduced only at 10 µM of dasatinib, showing the same pattern as in our study. However, in HLE cells, p-Src and p-Akt expression were reduced in a concentration-dependent manner by dasatinib, suggesting that the p-Akt expression pattern was probably related to cell types. Consistent with our findings, Chang et al. [28] reported that dasatinib exhibits anti-tumor effects by inhibition of PI3K/Akt/mTOR pathway in a rare muscle-invasive bladder cancer with combined EGFR gene amplification and PTEN deletion. Additionally, inhibition of Src by dasatinib could suppress the activation of PI3K/Akt signaling pathway in lung cancer cells A549 [20]. Pang and Zhang [29] also demonstrated that dasatinib enhances autophagy through Akt/mTOR pathway in schwannoma cells HEI-193. Thus, targeting PI3K/Akt/mTOR pathway not only triggers apoptosis, but also induces autophagy in bladder cancer cells (Fig. 8). However, since this study has limitations conducted in the in vitro model, additional experiments on safety and anticancer effects using an in vivo model are required.
Fig. 8. Schematic illustration showing the mechanisms underlying the anticancer effects of dasatinib in bladder cancer cells.
CONCLUSIONS
In summary, this study demonstrates that dasatinib markedly enhanced its anticancer effect by suppressing cell proliferation, promoting cell cycle arrest at G1 phase, and inducing apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in bladder cancer cells. Our findings indicate that dasatinib could serve as a potential agent for the treatment of bladder cancer.
Footnotes
CONFLICTS OF INTEREST: The authors have nothing to disclose.
FUNDING: This study was supported by grant number 800-20170053 from the Alvogen Korea, and by grant number 02-2023-0020 from the Seoul National University Bundang Hospital Research Fund.
- Research conception and design: Sangchul Lee.
- Data acquisition: Jin-Nyoung Ho and Danhyo Kim.
- Statistical analysis: Hoyoung Ryu.
- Data analysis and interpretation: Jin-Nyoung Ho and Danhyo Kim.
- Drafting of the manuscript: Jin-Nyoung Ho and Hoyoung Ryu.
- Critical revision of the manuscript: Seok-Soo Byun.
- Obtaining funding: Seok-Soo Byun.
- Administrative, technical, or material support: Seok-Soo Byun and Sangchul Lee.
- Supervision: Seok-Soo Byun and Sangchul Lee.
- Approval of the final manuscript: all authors.
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