Stellettin B Induces Cell Death in Bladder Cancer Via Activating the Autophagy/DAPK2/Apoptosis Signaling Cascade

Abstract

Bladder cancer (BC) is one of the most prevalent cancers worldwide. However, the recurrence rate and five-year survival rate have not been significantly improved in advanced BC, and new therapeutic strategies are urgently needed. The anticancer activity of stellettin B (SP-2), a triterpene isolated from the marine sponge Rhabdastrella sp., was evaluated with the MTT assay as well as PI and Annexin V/7-AAD staining. Detailed mechanisms were elucidated through an NGS analysis, protein arrays, and Western blotting. SP-2 suppressed the viability of BC cells without severe toxicity towards normal uroepithelial cells, and it increased apoptosis with the activation of caspase 3/8/9, PARP, and γH2AX. The phosphorylation of FGFR3 and its downstream targets were downregulated by SP-2. Meanwhile, it induced autophagy in BC cells as evidenced by LC3-II formation and p62 downregulation. The inhibition of autophagy using pharmacological inhibitors or through an ATG5-knockout protected RT-112 cells from SP-2-induced cell viability suppression and apoptosis. In addition, the upregulation of DAPK2 mRNA and protein expression also contributed to SP-2-induced cytotoxicity and apoptosis. In RT-112 cells, an FGFR3-TACC3-knockout caused the downregulation of DAPK2, autophagy, and apoptosis. In conclusion, this is the first study demonstrating that SP-2 exhibits potent anti-BC activity by suppressing the FGFR3-TACC3/Akt/mTOR pathway, which further activates a novel autophagy/DAPK2/apoptosis signaling cascade.

Keywords: DAPK2; apoptosis; autophagy; bladder cancer; stellettin B.

Figure 1

SP-2 selectively suppressed cell viability in bladder cancer (BC) cells. BC cells (RT-112, J82, UMUC3, and RT4) and normal uroepithelial cells (SV-HUC-1) were treated with different concentrations of SP-2 for 48 h (A) and 72 h (B), and cell viability was determined through MTT assay. * p < 0.05, *** p < 0.001, and **** p < 0.0001 compared with control group.

Figure 2

SP-2 induced accumulation of cells in the sub-G1 phase and apoptosis in RT-112 cells. (A–C) SP-2 induced sub-G1 cell population in RT-112 cells. Cells were treated with SP-2 at different concentrations for 48 h then were stained with PI, and cell cycle distribution was detected through flow cytometry (A). Quantitative data (B,C) are based on flow cytometry histograms and are presented as mean ± S.D. (D,E) SP-2 induced annexin-V-positive apoptotic cells in RT-112 cells. Quantitative data of apoptotic cells are presented as mean ± S.D. (E). Cells were treated with SP-2 at different concentrations for 48 h and stained with Annexin V/7-AAD. Apoptotic cells were detected through flow cytometry. (F) SP-2 increased levels of the cleaved forms of PARP; caspase 3, 8, and 9; and γH2AX in a concentration-dependent manner. Cells were treated with the indicated concentrations of SP-2 for 48 h, and cell lysates were immunoblotted using the indicated antibodies. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with control group.

Figure 3

The effects of SP-2 on the phosphorylation profiles in RT-112 cells. (A,B) SP-2 affected the phosphorylation of various receptor tyrosine kinases (RTKs) (A) and protein kinases (B) in RT-112 cells. Cells were treated with SP-2 (0.5 μM) for 36 h, and cell lysates were applied to phosphoprotein array analysis. Protein dots in the blue box indicate increased phosphorylation, and protein dots in the red box indicate decreased phosphorylation after SP-2 treatment. (C) SP-2 significantly downregulated p-FGFR3 and its downstream signaling pathways. Cells were treated with different concentrations of SP-2 for the indicated times, and cell lysates were immunoblotted using the indicated antibodies.

Figure 4

SP-2 induced autophagy in RT-112 cells. (A) SP-2 upregulated autophagy and downregulated steroid biosynthesis according to NGS-based pathway analysis. Cells were treated with 0.5 μM SP-2 for 24 h. The data were analyzed through NGS as described in Materials and Methods. (B,C) Expression levels of mRNAs (B) and proteins (C) of ATG9B and DAPK2 with SP-2 treatment. Cells were treated with various concentrations of SP-2 for 24 h, and mRNA levels and proteins were analyzed through RT-qPCR and Western blotting. (D) Concentration-dependent effect of SP-2 on the conversion of endogenous LC3-I to LC3-II. Cells were treated with different concentrations of SP-2 for 24 h and 48 h, and cell lysates were immunoblotted using the indicated antibodies. * p < 0.05 and **** p < 0.0001 compared with the control (CTL) group.

Figure 5

Knockout of ATG5 reduced SP-2-induced sub-G1 accumulation and apoptosis in RT-112 cells. (A,B) Inhibition of autophagy with either CQ (A) or 3-MA (B) reduced SP-2-induced apoptosis in RT-112 cells. The cells were treated with SP-2 for 48 h in the presence or absence of an autophagy inhibitor (CQ or 3-MA), and cell lysates were analyzed through Western blotting. (C–E) ATG5-knockout reduced SP-2-induced cytotoxicity (C), apoptosis (D), and accumulation in the sub-G1 phase (E) in RT-112 cells. Cells were treated with indicated concentrations of SP-2 for 48 h, and cell viability (C) and cell cycle distribution (E) were determined with MTT assay and through flow cytometry, respectively. * p < 0.05, ** p < 0.01, and *** p < 0.001. Cell lysates from 48 h SP-2 treatment were analyzed through Western blotting (D).

Figure 6

DAPK2 potentiated SP-2-induced apoptosis in RT-112 cells. (A) SP-2 induced autophagy and the DAPK2 level in a time-dependent manner. (B) ATG5-knockout reduced the upregulation of DAPK2 in RT-112 cells. Cells were treated with different concentrations of SP-2 for 48 h, and cell lysates were immunoblotted using the indicated antibodies. (C–E) DAPK2-knockdown protected SP-2-induced cytotoxicity (C) and apoptosis (D,E) in RT-112 cells. Cells were treated with different concentrations of SP-2 for 48 h, and cell viability and apoptosis were determined with MTT assay and through Western blotting, respectively. * p < 0.05 and ** p < 0.01.

Figure 7

FGFR3-TACC3 fusion was essential to SP-2-induced autophagy and apoptosis in RT-112 cells. (A) FGFR3-TACC3 knockout significantly reversed SP-2-reduced cell viability. Cells were treated with different concentrations of SP-2 for 48 h, and cell viability was determined with MTT assay. * p < 0.05 and *** p < 0.001. (B) FGFR3-TACC3 knockout decreased SP-2-indcued autophagy. Cells were treated with different concentrations of SP-2 for 48 h, and cell lysates were immunoblotted using the indicated antibodies. (C) FGFR3-TACC3 knockout protected RT-112 cells from SP-2-induced apoptosis. Cells were treated with different concentrations of SP-2 for 48 h, and cell lysates were immunoblotted using the indicated antibodies.