Sulfated polysaccharide of Sepiella maindroni ink targets Akt and overcomes resistance to the FGFR inhibitor AZD4547 in bladder cancer

Abstract

Rapid appearance of resistance to fibroblast growth factor receptor (FGFR) inhibitors hampers targeted regimens in bladder cancer. In the present study, we evaluated whether SIP-SII, a sulphated derivative of the polysaccharide in Sepiella maindroni (spineless cuttlefish) ink used in traditional Chinese medicine, could attenuate resistance to FGFR inhibition in bladder cancer cells. In vitro assays indicated that SIP-SII reduced cell viability and migration, restricted cell cycle progression, and increased apoptosis in parallel with decreased AKT phosphorylation and downregulation of CDK4, MMP2, and Bcl-2 in RT112 and JMSU1 cells. Synergistic effects on cell viability were observed when SIP-SII was combined with the small-molecule FGFR inhibitor AZD4547. Specific Akt targeting by SIP-SII was suggested by the fact that neither Akt knockdown nor the selective PI3K inhibitor BKM120 enhanced the inhibitory effects of SIP-II, while expression of a constitutively active Akt mutant rescued SIP-SII effects. Furthermore, subcutaneous transplantation of RT112 xenografts confirmed the superiority and tolerability of combined SIP-SII and AZD4547 administration over monotherapy regimens. The present study thus provides pre-clinical evidence of the ability of SIP-SII to improve FGFR-targeted therapies for bladder cancer by inhibiting Akt.

Keywords: AZD4547; FGFR therapy resistance; Sulfated polysaccharide of ink Sepiella maindroni; bladder cancer; xenograft.

Figure 1

SIP-SII impairs proliferation and migration and attenuates Akt signaling in bladder cancer cells. (A) Half-maximum inhibitory concentration (IC₅₀) of test drugs evaluated through the MTT viability assay. (B) Cell viability (MTT) assay results for RT112 and JMSU1 cells treated respectively with 2.5 μM or 5μM SIP-SII for the indicated time-points. Cell migration assay results for RT112 cells (C) and JMSU1 cells (D) treated respectively with 2.5 μM or 5.0 μM SIP-SII for 24 h. Representative images at 200x magnification. Data are mean ± SD (error bars) of three experiments performed in triplicate. **P < 0.01 vs. DMSO (control); n = 3. (E) Western blot analysis of total Akt, phospho-Akt, CDK4, Bcl-2, and MMP2 24 h post-exposure to SIP-SII (RT112: 2.5 μM; JMSU1: 5.0 μM).

Figure 2

SIP-SII hampers proliferation and migration of bladder cancer cells in an Akt-dependent manner. (A) Cell viability (MTT) assay results for RT112 and JMSU1 cells treated with negative control siRNA (NC), 5.0 μM SIP-SII, or dual treatment with SIP-SII and Akt siRNA (si-Akt) for 24 h. Cell migration assay results for RT112 cells (B) and JMSU1 cells (C) treated with SIP-SII alone or in combination with si-Akt for 24 h. Representative images at 200x magnification. Data are mean ± SD (error bars) of three experiments performed in triplicate. **P < 0.01 vs. DMSO; n = 3. Western blot analyses of RT112 (D) and JMSU1 (E) cells treated with DMSO, SIP-SII, or the combination of SIP-SII and si-Akt for 24 h.

Figure 3

Overexpression of Akt DD abrogates SIP-SII inhibitory effects. (A) Cell viability assay results for RT112 and JMSU1 cells treated with the FLAG-HA empty vector and DMSO (control), 5.0 μM SIP-SII, or SIP-SII combined with Akt DD (PKB T308D S473D) for 24 h. Cell migration assay results for RT112 (B) and JMSU1 cells (C) treated with SIP-SII alone or in combination with Akt DD for 24 h. Representative images at 200x magnification. Data are mean ± SD (error bars) of three experiments performed in triplicate. **P < 0.01 vs. DMSO; n = 3. Western blot analyses in RT112 (D) and JMSU1 (E) cells treated with SIP-SII alone or in combination with Akt DD for 24 h.

Figure 4

Inhibition of cell proliferation and migration by dual treatment with SIP-SII and AZD4547. (A) Results of cell viability assays in RT112 and JMSU1 cells cultured with DMSO, 5.0 μM SIP-SII, 100 nM AZD4547, or SIP-SII and AZD4547 combined. Transwell migration assay results for RT112 (B) and JMSU1 (C) cells treated with DMSO, 5.0 μM SIP-SII, 100 nM AZD4547, or the combination of SIP-SII and AZD4547 for 24 h. Representative images at 200x magnification. Cell cycle analyses of RT112 (D) and JMSU1 (E) cells treated with DMSO, 5.0 μM SIP-SII, 100 nM AZD4547, or dual treatment with SIP-SII and AZD4547. Flow cytometry was performed 24 h post-treatment in PI-stained cells. JC-1 apoptosis assay results in RT112 (F) and JMSU1 (G) cells treated (24 h) with DMSO, 5.0 μM SIP-SII, 100 nM AZD4547, or the combination of SIP-SII and AZD4547. Data are mean ± SD (error bars) of three individual experiments. **P < 0.01 vs. DMSO; ##P < 0.01 vs. SIP-SII; ^^P < 0.01 vs. AZD4547; n = 3.

Figure 5

Dual treatment with SIP-SII and AZD4547 potentiates the inhibition of Akt and downstream effectors. (A) Western blot analysis of RT112 and JMSU1 cells exposed (24 h) to DMSO, 5.0 μM SIP-SII, 100 nM AZD4547, or SIP-SII combined with AZD4547. (B) Western blot analysis of RT112 and JMSU1 cells treated for 24 h with DMSO, 5.0 μM SIP-SII, 100 nM AZD4547, or SIP-SII combined with AZD4547 in the presence of 0.5 μM BKM120. Akt in, Akt inhibitor BKM120. ** vs. Con, ## vs. AKT IN, ^^ vs. AKT IN+SIP, $$ vs. AKT IN+AZD. **, ##, ^^ and $$ indicated P < 0.01, n=3. (C) Cell viability assay results. (D) Transwell migration assay results. Representative images at 200x magnification. Data are mean ± SD (error bars) of three individual experiments. **P < 0.01 vs. control; n = 3.

Figure 6

Combination of SIP-SII and AZD4547 enhances growth suppression of RT112 xenografts. Nude mice were randomly divided into four experimental groups after subcutaneous injection of RT112 cells. (A) Tumor volume measurements. (B) Tumor weight measurements after euthanasia on day 28 post-treatment initiation. (C) Body weight measurements. ***P < 0.001 vs. DMSO control; n = 3 per group. (D) Immunohistochemical staining of phospho-Akt in excised tumors (vehicle, SIP-SII, AZD4547, and combination therapy). Representative images at 100x magnification. (E) Western blot analysis of tumor samples.