Proteomics Analysis of Tangeretin-Induced Apoptosis through Mitochondrial Dysfunction in Bladder Cancer Cells

Abstract

Tangeretin is one of the most abundant compounds in citrus peel, and studies have shown that it possesses anti-oxidant and anti-cancer properties. However, no study has been conducted on bladder cancer cells. Bladder cancer has the second highest mortality rate among urological cancers and is the fifth most common malignancy in the world. Currently, combination chemotherapy is the most common approach by which to treat patients with bladder cancer, and thus identifying more effective chemotherapeutic agents that can be safely administered to patients is a very important research issue. Therefore, this study investigated whether tangeretin can induce apoptosis and identified the signaling pathways of tangeretin-induced apoptosis in human bladder cancer cells using two-dimensional gel electrophoresis (2DGE). The results of the study demonstrated that 60 μM tangeretin reduced the cell survival of a BFTC-905 bladder carcinoma cell line by 42%, and induced early and late apoptosis in the cells. In this study 2DGE proteomics technology identified 41 proteins that were differentially-expressed in tangeretin-treated cells, and subsequently LC⁻MS/MS analysis was performed to identify the proteins. Based on the functions of the differentially-expressed proteins, the results suggested that tangeretin caused mitochondrial dysfunction and further induced apoptosis in bladder cancer cells. Moreover, western blotting analysis demonstrated that tangeretin treatment disturbed calcium homeostasis in the mitochondria, triggered cytochrome C release, and activated caspase-3 and caspase-9, which led to apoptosis. In conclusion, our results showed that tangeretin-induced apoptosis in human bladder cancer cells is mediated by mitochondrial inactivation, suggesting that tangeretin has the potential to be developed as a new drug for the treatment of bladder cancer.

Keywords: apoptosis; mitochondrial dysfunction; proteomics; tangeretin.

Figure 1

Cell survival of J82, BFTC-905, T24, and RT4 bladder cancer cell lines after tangeretin treatment. (A) Chemical structure of tangeretin. (B) Cytotoxicity was measured using an MTT assay. # p < 0.05, * p < 0.001.

Figure 2

Effect of tangeretin on the cellular behavior of BFTC-905 cells. (100× magnification) (A) Effect of tangeretin on cell viability. # p < 0.05, * p < 0.001. (B) Change in cell morphology after tangeretin treatment. (C) Effect of tangeretin on wound-healing. (D) Effect of tangeretin in a transwell migration assay.

Figure 3

Tangeretin-induced apoptosis in BFTC-905 cells. (A) TUNEL/DAPI staining of cells after tangeretin (0, 20, 40, and 60 μM) treatment. Scale bars = 50 μm. (B) Annexin V/PI labeling with flow cytometry analysis indicated the percentages of cells in early and late apoptosis after tangeretin treatment.

Figure 4

Two-dimensional gel electrophoresis analysis of BFTC-905 cells without and with 60 μM tangeretin treatment. (A,C) Control, 60 μM tangeretin treatment (B,D). (E) Validation of six differentially-expressed proteins by western blotting from BFTC-905 cells following tangeretin treatment (0, 20, 40, 60 μM).

Figure 5

Changes in mitochondrial proteins, Bcl-2 family proteins, and caspases in BFTC-905 cells treated with different concentrations of tangeretin. AIF: apoptosis inducing factor.

Figure 6

Effect of mitochondrial inhibitors on the cell viability and Bcl-2 family protein expression in tangeretin-treated BFTC-905 cells. (A) Cell viability and (B) western blotting analysis. Cells were pre-treated with mitochondrial inhibitors trifluoperazine (TFZ), aristolochic acid (ArA), and cyclosporine A (CyA), at the indicated concentrations, followed by 24 h of tangeretin treatment. # p < 0.05.

Figure 7

Cell survival of BFTC-905 bladder cancer cells after tangeretin and cisplatin treatment. (A) Cytotoxicity was measured using an MTT assay. # p < 0.05, * p < 0.001. (B) Validation of Bcl-2 family proteins by western blotting from BFTC-905 cells following tangeretin and cisplatin treatment.

Figure 8

Tangeretin-induced apoptotic pathway in BFTC-905 bladder cancer cells. Based on the results of our study, the apoptosis process caused by tangeretin is mediated by mitochondrial dysfunction and caspase activation.