Blockade of the STAT3/BCL-xL Axis Leads to the Cytotoxic and Cisplatin-Sensitizing Effects of Fucoxanthin, a Marine-Derived Carotenoid, on Human Bladder Urothelial Carcinoma Cells

Abstract

Bladder cancer is a globally prevalent urological malignancy, with transitional carcinoma (TCC) representing the majority of cases. Cisplatin is the primary drug for metastatic bladder cancer chemotherapy; however, its application is limited by nephrotoxicity and resistance. Signal Transducer and Activator of Transcription 3 (STAT3) is an oncogenic transcription factor often overactivated in various cancers, making it an appealing drug target. Fucoxanthin, a marine carotenoid, has significant anticancer properties. This study explored Fucoxanthin's cytotoxic effects and its potential to potentiate the efficacy of Cisplatin, along with the mechanisms underlying these effects, on human bladder TCC cells. We demonstrated that Fucoxanthin is cytotoxic to bladder TCC cells by inducing apoptosis, evidenced by z-VAD-fmk-mediated annulment of Fucoxanthin's cytotoxicity. Furthermore, Fucoxanthin reduced the levels of inherent or interleukin-6-induced tyrosine 705-phosphorylated STAT3 accompanied by downregulating BCL-xL, a well-established STAT3 target. Notably, ectopic expression of STAT3-C, a dominant-active STAT3 mutant, or BCL-xL thwarted Fucoxanthin's proapoptotic and cytotoxic actions. Moreover, Fucoxanthin at subtoxic dosages enhanced the susceptibility to Cisplatin-induced apoptosis of bladder TCC cells initially resistant to Cisplatin. Remarkably, this Cisplatin-sensitizing effect of Fucoxanthin was abrogated when cells ectopically expressed STAT3-C or BCL-xL. Overall, for the first time, we proved that the proapoptotic, cytotoxic, and Cisplatin-sensitizing effects of Fucoxanthin on human bladder TCC cells are attributed to the blockade of the STAT3/BCL-xL axis. Our findings highlight that targeting the STAT3/BCL-xL axis is a promising strategy to eliminate bladder TCC cells and facilitate Cisplatin sensitization, and further support the potential of incorporating Fucoxanthin into Cisplatin-based chemotherapy for treating bladder cancer.

Keywords: BCL-xL; STAT3; apoptosis; bladder cancer; cisplatin; fucoxanthin.

Figure 1

Fucoxanthin’s cytotoxic effect on human bladder TCC cells. (A) Cell viability assay. Human bladder TCC cell lines TCCSUP and T24 were treated for 48 h with 0 μM to 200 μM of Fucoxanthin or Cisplatin, a common chemotherapy drug for bladder cancer treatment, followed by cell viability determination using MTT assay as detailed in the Section 4. (B) Clonogenicity assay. TCCSUP and T24 cells were treated with Fucoxanthin (0, 60, 120 μM) for 24 h, followed by drug-free incubation for 14 days to form colonies. **: p < 0.01; ***: p < 0.001.

Figure 2

Apoptosis is the primary mechanism of Fucoxanthin’s cytotoxic action on human bladder TCC cells. (A) Fucoxanthin dose-dependently triggers PARP cleavage. TCCSUP and T24 cells were treated with Fucoxanthin (0, 60, 120 μM) for 24 h, followed by immunoblotting to detect the levels of cleaved PARP (c-PARP), a canonical biomarker of apoptosis. (B) Fucoxanthin increases the levels of annexin V-positively stained cell population. TCCSUP and T24 cells were treated with Fucoxanthin (0, 60, 120 μM) for 24 h, followed by flow cytometry analysis to assess the levels of annexin V-stained (i.e., apoptotic) cell population, represented by scatter plots (upper image) and histograms (lower image). (C) Validation of apoptosis blockage by the absence of PARP cleavage. TCCSUP and T24 cells were pre-treated with the pan-caspase inhibitor z-VAD-fmk (50 μM) for 2 h, followed by 24 h treatment with Fucoxanthin. The levels of cleaved PARP were detected using immunoblotting. (D) Blockade of apoptosis nullifies Fucoxanthin-induced cytotoxicity. Fucoxanthin-treated TCCSUP and T24 cells with z-VAD-fmk pre-treatment were evaluated for their colony-forming abilities. All immunoblotting used the levels of β-actin as a control for equal loading. *: p < 0.05; ***: p < 0.001.

Figure 2

Apoptosis is the primary mechanism of Fucoxanthin’s cytotoxic action on human bladder TCC cells. (A) Fucoxanthin dose-dependently triggers PARP cleavage. TCCSUP and T24 cells were treated with Fucoxanthin (0, 60, 120 μM) for 24 h, followed by immunoblotting to detect the levels of cleaved PARP (c-PARP), a canonical biomarker of apoptosis. (B) Fucoxanthin increases the levels of annexin V-positively stained cell population. TCCSUP and T24 cells were treated with Fucoxanthin (0, 60, 120 μM) for 24 h, followed by flow cytometry analysis to assess the levels of annexin V-stained (i.e., apoptotic) cell population, represented by scatter plots (upper image) and histograms (lower image). (C) Validation of apoptosis blockage by the absence of PARP cleavage. TCCSUP and T24 cells were pre-treated with the pan-caspase inhibitor z-VAD-fmk (50 μM) for 2 h, followed by 24 h treatment with Fucoxanthin. The levels of cleaved PARP were detected using immunoblotting. (D) Blockade of apoptosis nullifies Fucoxanthin-induced cytotoxicity. Fucoxanthin-treated TCCSUP and T24 cells with z-VAD-fmk pre-treatment were evaluated for their colony-forming abilities. All immunoblotting used the levels of β-actin as a control for equal loading. *: p < 0.05; ***: p < 0.001.

Figure 3

Blockade of STAT3 activation accounts for Fucoxanthin’s proapoptotic and cytotoxic effects on human bladder TCC cells. (A) Fucoxanthin inhibits constitutively active STAT3. TCCSUP and T24 cells after 24 h treatment with Fucoxanthin (0, 60, 120 μM) were subjected to immunoblotting for the levels of active STAT3, revealed by Tyr 705 phosphorylation (p-STAT3 (Tyr 705)), and the levels of BCL-xL, a well-known STAT3 transcriptional target. (B) Fucoxanthin inhibits IL-6-induced activation of STAT3. TCCSUP and T24 cells were stimulated with IL-6 (100 ng/mL) for 30 min, followed by 24 h treatment with Fucoxanthin and then immunoblotting for p-STAT3 (Tyr 705) levels. (C) Ectopic expression of STAT3-C, a dominant-active mutant of STAT3, abolished Fucoxanthin-induced PARP cleavage and BCL-xL downregulation. The vector control and STAT3-C stable clones of T24 cells were treated with Fucoxanthin for 24 h, followed by gauging the levels of c-PARP and BCL-xL using immunoblotting. (D) Fucoxanthin failed to enhance annexin V-positive cell population levels of T24 STAT3-C stable clones. The annexin V-stained cells were determined using flow cytometry analysis. (E) Fucoxanthin was ineffective in blocking T24 STAT3-C stable clones to form colonies revealed by clonogenicity assay. All immunoblotting used the levels of β-actin as a control for equal loading. **: p < 0.01; ***: p < 0.001.

Figure 3

Blockade of STAT3 activation accounts for Fucoxanthin’s proapoptotic and cytotoxic effects on human bladder TCC cells. (A) Fucoxanthin inhibits constitutively active STAT3. TCCSUP and T24 cells after 24 h treatment with Fucoxanthin (0, 60, 120 μM) were subjected to immunoblotting for the levels of active STAT3, revealed by Tyr 705 phosphorylation (p-STAT3 (Tyr 705)), and the levels of BCL-xL, a well-known STAT3 transcriptional target. (B) Fucoxanthin inhibits IL-6-induced activation of STAT3. TCCSUP and T24 cells were stimulated with IL-6 (100 ng/mL) for 30 min, followed by 24 h treatment with Fucoxanthin and then immunoblotting for p-STAT3 (Tyr 705) levels. (C) Ectopic expression of STAT3-C, a dominant-active mutant of STAT3, abolished Fucoxanthin-induced PARP cleavage and BCL-xL downregulation. The vector control and STAT3-C stable clones of T24 cells were treated with Fucoxanthin for 24 h, followed by gauging the levels of c-PARP and BCL-xL using immunoblotting. (D) Fucoxanthin failed to enhance annexin V-positive cell population levels of T24 STAT3-C stable clones. The annexin V-stained cells were determined using flow cytometry analysis. (E) Fucoxanthin was ineffective in blocking T24 STAT3-C stable clones to form colonies revealed by clonogenicity assay. All immunoblotting used the levels of β-actin as a control for equal loading. **: p < 0.01; ***: p < 0.001.

Figure 4

BCL-xL downregulation accounts for Fucoxanthin’s proapoptotic and cytotoxic effects on human bladder TCC cells. (A) Ectopic BCL-xL expression sabotages Fucoxanthin-induced PARP cleavage. The vector control and BCL-xL stable clones of T24 cells were treated with Fucoxanthin for 24 h, then were gauged for the levels of c-PARP and BCL-xL using immunoblotting. (B) T24 BCL-xL stable clones resist Fucoxanthin-induced upregulation of annexin V-positive cell population, assessed by flow cytometry analysis. (C) T24 BCL-xL stable clones exhibit resilience against Fucoxanthin’s inhibitory effect on colony-forming capacity. All immunoblotting used the levels of β-actin as a control for equal loading. **: p < 0.01; ***: p < 0.001.

Figure 5

Fucoxanthin potentiates Cisplatin’s efficacy in human bladder TCC cells initially resistant to Cisplatin by blocking the STAT3/BCL-xL axis. (A) TCCSUP and T24 cells were treated for 24 h with Fucoxanthin (0, 30, 60, 120 μM), Cisplatin (0, 30, 60, 120 μM), or combinations of Fucoxanthin with Cisplatin, followed by gauging the percentage of viability inhibition compared to drug-free controls using MTT assay. (B) Fucoxanthin at a subtoxic dosage significantly enhances the levels of Cisplatin-induced apoptosis. TCCSUP cells were treated for 24 h with Fucoxanthin (60 μM), Cisplatin (120 μM), or a combination of Fucoxanthin with Cisplatin, then the levels of annexin V-positive (apoptotic) cell populations were scored by flow cytometry analysis. Likewise, combined treatment with Fucoxanthin (60 μM) markedly uplifts the apoptotic cell population of T24 cells triggered by Cisplatin (60 μM) treatment alone. (C) Ectopic expression of STAT3-C or BCL-xL negates the Cisplatin-sensitizing effect of Fucoxanthin. The vector control, STAT3-C, or BCL-xL stable clones of T24 cells were subjected to 24 h treatment with Fucoxanthin (60 μM), Cisplatin (60 μM), or the combination of both. MTT assay was then employed to assess the percentage of cell survival relative to the viability of the vector control under these treatments. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 5

Fucoxanthin potentiates Cisplatin’s efficacy in human bladder TCC cells initially resistant to Cisplatin by blocking the STAT3/BCL-xL axis. (A) TCCSUP and T24 cells were treated for 24 h with Fucoxanthin (0, 30, 60, 120 μM), Cisplatin (0, 30, 60, 120 μM), or combinations of Fucoxanthin with Cisplatin, followed by gauging the percentage of viability inhibition compared to drug-free controls using MTT assay. (B) Fucoxanthin at a subtoxic dosage significantly enhances the levels of Cisplatin-induced apoptosis. TCCSUP cells were treated for 24 h with Fucoxanthin (60 μM), Cisplatin (120 μM), or a combination of Fucoxanthin with Cisplatin, then the levels of annexin V-positive (apoptotic) cell populations were scored by flow cytometry analysis. Likewise, combined treatment with Fucoxanthin (60 μM) markedly uplifts the apoptotic cell population of T24 cells triggered by Cisplatin (60 μM) treatment alone. (C) Ectopic expression of STAT3-C or BCL-xL negates the Cisplatin-sensitizing effect of Fucoxanthin. The vector control, STAT3-C, or BCL-xL stable clones of T24 cells were subjected to 24 h treatment with Fucoxanthin (60 μM), Cisplatin (60 μM), or the combination of both. MTT assay was then employed to assess the percentage of cell survival relative to the viability of the vector control under these treatments. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 6

Schematic model of the cytotoxic and Cisplatin-sensitizing effects of Fucoxanthin on human bladder TCC cells elucidated in this study. To exert its cytotoxic effect on human bladder TCC cells, Fucoxanthin suppresses the antiapoptotic STAT3/BCL-xL signaling axis to trigger apoptosis, leading to the elimination of cancer cells and sensitization of Cisplatin efficacy. The image of Fucoxanthin was acquired from Wikipedia. (Image source: Fucoxanthin (2 September 2024) in Wikipedia, https://en.wikipedia.org/wiki/Fucoxanthin)