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

SIP-SII impairs proliferation and migration and attenuates Akt signaling in bladder cancer cells

To assess the effects of SIP-SII on bladder cancer cell viability, dose-response experiments were performed on RT112 and JMSU1 cells using the MTT assay. Viability was reduced by approximately 50% when RT112 and JMSU1 cells were treated with 6.73 μM and 7.39 μM SIP-SII, respectively (Figure 1A). Hence, IC₅₀ concentrations of 2.5 μM and 5 μM were respectively selected to verify SIP-SII’s inhibitory effects on cell growth. For the FGFR inhibitor AZD4547, IC₅₀ values of 1.25 μM and 1.28 μM were estimated for RT112 and JMSU1 cells, respectively (Figure 1A). Time-course viability experiments were further conducted on RT112 and JMSU1 cells exposed to different concentrations of SIP-SII for 12, 24, 36, or 48 h. As shown in Figure 1B, SIP-SII suppressed cell growth in a dose- and time-dependent manner.

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).

The transwell migration assay was next performed to determine the effects of SIP-SII on cell migration. After incubation with SIP-SII for 24 h, the number of migrating RT112 cells decreased significantly compared to the control group (Figure 1C). Similar results were observed in JMSU1 cells (Figure 1D). Western blotting analysis was further used to detect the expression of total Akt, phospho-Akt, CDK4, Bcl-2, and MMP2. As shown in Figure 1E, after exposure to SIP-SII, the levels of phospho-Akt, CDK4, Bcl-2, and MMP2 declined in a dose-dependent manner, while total Akt showed little variation. Densitometric gel analyses confirmed these results (Supplementary Figure 1A). These data showed that SIP-SII inhibits bladder cancer cell proliferation and migration, while decreasing Akt activation and downstream signaling.

SIP-SII hampers proliferation and migration of bladder cancer cells in an Akt-dependent manner

Previous studies reported that SIP-SII exhibited multiple anti-tumor effects such as suppression of EGFR, FGF, and intercellular adhesion molecule (ICAM)-mediated pathways in ovarian cancer cells and epidermoid carcinoma cells [13] [15]. To verify that Akt inhibition mediates the inhibitory effects of SIP-SII on bladder cancer cells, Akt-targeted siRNAs (si-Akt) were introduced into RT112 and JMSU1 cells. The silencing efficiency of si-Akt was verified by western blotting (data not shown). Cells were treated with control siRNA, 5μM SIP-SII, or the combination of SIP-SII and si-Akt, and MTT and transwell assays were performed 24 h later. SIP-SII alone and in combination with si-Akt repressed cell viability relative to the control group. However, dual treatment did not amplify the inhibition induced by SIP-SII alone, both on cell growth (Figure 2A) or cell migration (Figure 2B and 2C). Western blotting was further performed to determine the activation of Akt and the expression of effector molecules. As shown in Figure 2D and 2E, the decrease in phospho-Akt expression was identical in SIP-SII-treated cells, either when applied alone or in combination with si-Akt, whereas total Akt expression fell significantly only in the latter. Three independent experiments (Supplementary Figure 1B) showed that CDK4 and MMP2 decreased by 16%, Bcl-2 decreased by 25%, phospho-AKT was reduced by 70%, and total AKT expression declined 75% in the combination group. These results indicated that SIP-SII suppressed cell growth and migration by inhibiting Akt activation.

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.

Akt overexpression reverses SIP-SII effects on bladder cancer cells

To further verify that the inhibitory effects of SIP-SII on bladder cancer cell growth and migration relied on inactivation of Akt, a constitutively active Akt mutant, Akt T308D S473D (Akt DD), was introduced into RT112 and JMSU1 cells. Transfected cells were then treated with 5 μM SIP-SII for 24 h, and the MTT assay was performed to assess cell viability. As shown in Figure 3A, Akt DD completely reversed SIP-SII-induced inhibition of cell viability. Moreover, Akt DD expression also attenuated the inhibition of cell migration elicited by SIP-SII (Figure 3B and 3C). Akt DD transfection efficiency was confirmed by western blotting (Figure 3D and 3E). After transfection, the expression of CDK4, Bcl-2, and MMP2 was restored in parallel with hyperactivation of Akt (Supplementary Figure 1C). These data strongly suggest that the inhibitory actions of SIP-SII on bladder cancer cells are dependent on Akt inhibition.

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.

Dual treatment with SIP-SII and AZD4547 enhances the anticancer effects elicited by single inhibitors

Resistance to AZD4547 in RT112 and JMSU1 cells, carrying respectively a FGFR3-TACC3 translocation and FGFR1 amplification, is mediated by constitutively active PI3K/Akt signaling [7, 16]. To investigate the potential of SIP-SII in overcoming AZD4547 resistance, RT112 and JMSU1 cells were exposed to these inhibitors, either singly or in combination (Table 1), and the MTT assay was performed to determine compounded drug effects through estimation of the combination index (Table 2). The IC₅₀ of combined drug exposure was lower than that measured for AZD4547 alone both in RT112 cells (0.43 μM vs 1.25 μM) and in JMSU1 cells (0.47 μM vs 1.28 μM). In turn, CI values were 0.7037 for RT112 and 0.7407 for JMSU1 (Table 2, indicating that the effects of the drug combination were synergistic. These results are exemplified in Figure 4A, showing enhanced inhibition of cell viability by combined treatment, compared with single exposure to SIP-SII (5 μM) or AZD4547 (100 nM).

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.

Transwell assays were also performed to assess the effects of the combined inhibitors on cell migration. As shown in Figure 4B and 4C, migration declined by 70% after combination treatment, compared to the 50% reduction elicited by SIP-SII and AZD4547 separately. Furthermore, as sown in Figure 4D, cell cycle analysis indicated that the proportion of RT112 cells in G1 was significantly lower after combined drug treatment than after AZD4547 treatment alone (0.75% vs. 0.84% respectively; P < 0.01). Similar results were observed in JMSU1 cells (0.65% vs. 0.82% respectively; P < 0.01, Figure 4E). Additionally, the JC-1 assay was performed to evaluate apoptotic rates post-exposure to single or dual inhibitors. As shown in Figure 4F, the percentage of apoptotic RT112 cells increased from 0.69% in the AZD4547 group to 1.68% in the combination group (p < 0.01). Similar results were obtained in JMSU1 cells (0.93% vs. 1.84%, respectively; P < 0.01, Figure 4G). These results indicate that combination therapy decreased bladder cancer cell migration and promoted cell cycle arrest and apoptosis to a larger extent than AZD4547 monotherapy.

SIP-SII specifically targets Akt

The effects of combined SIP-SII and AZD4547 treatment on Akt activation and expression of downstream signaling molecules were explored through western blot. As shown in Figure 5A, the expression of phospho-Akt, CDK4, Bcl-2, and MMP2 in RT112 cells was downregulated to a greater by SIP-SII and AZD4547 combined, compared to AZD4547 alone. Similar results were obtained in JMSU1 cells. These results were validated by gel analysis (Supplementary Figure 1D), indicating that combination therapy might overcome AZD4547 resistance by blocking Akt-mediated pathways. To further explore SIP-SII specificity, the selective PI3K inhibitor BKM120 was used to inhibit AKT activation. RT112 cells were treated for 24 h with DMSO (vehicle), 5 μM SIP-SII, 100 nM AZD4547, or SIP-SII combined with both AZD4547 and 0.5 μM BKM120. Western blots results showed that phospho-AKT, CDK4, Bcl-2, and MMP2 decreased significantly, while total AKT showed little change in cells treated with the three inhibitors (Figure 5B). Addition of BKM120 did not further enhance the suppression of AKT phosphorylation elicited by SIP-SII, indicating specific inhibition of Akt signaling by SIP-SII. These findings were corroborated by gel analysis (Supplementary Figure 1E). In addition, the effects of the combined inhibitors on cell growth were evaluated through MTT assays. Figure 5C shows that the combination of BKM120 and AZD4547 enhanced growth inhibition compared to AZD4547 alone. In contrast, dual treatment with BKM120 and SIP-SII neither enhanced nor reduced the suppression induced by SIP-SII. On the other hand, migration assay results indicated that dual treatment with BKM120 and AZD4547 promoted a stronger inhibition than that elicited by AZD4547 alone, whereas the inhibition induced by combined BKM120 and SIP-SII exposure was similar to that produced by SIP-SII alone. These results further imply that SIP-SII inhibits bladder cancer cell proliferation and migration by targeting Akt.

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.

Combination of SIP-SII and AZD4547 enhances growth inhibition of RT112 xenografts

To assess whether the SIP-SII and AZD4547 combinatorial regimen suppresses tumor growth in vivo, subcutaneous RT112 xenografts were generated in C57BL/6 mice. When tumors reached a mean volume of ~100 mm³, mice were divided into four groups and treated respectively with saline, SIP-SII, AZD4547, or the combination of SIP-SII and AZD4547. As shown in Figure 6A, tumor growth was initially reduced by each inhibitor alone, but the effects diminished gradually over time. In contrast, sustained tumor regression was observed during combined SIP-SII and AZD4547 administration. In parallel with decreased tumor volumes, tumor weights in the drug combination group were significantly reduced compared to the control group (0.47 g vs. 1.7 g; P < 0.01) while those in the monotherapy groups showed a more modest reduction (0.84 g and 0.86 g for SIP-SII and AZD4547, respectively; P < 0.01; Figure 6B). Body weights were not significantly altered by any treatment, suggesting that the interventions were well tolerated (Figure 6C).

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.

After tumor excision, the expression of phospho-Akt was verified by immunohistochemistry. As shown in Figure 6D, phosphorylation of Akt decreased remarkably in the SIP-SII group and in the combined treatment group. Furthermore, western blots demonstrated a stronger decrease in the expression of CDK4, Bcl-2, and MMP2 in the combination group, compared to each single treatment (Figure 6E and Supplementary Figure 1F). Thus, results of in vivo experiments suggested that combination of SIP-SII and AZD4547 induced gradual and sustained tumor regression by concomitant inhibition of FGFR and Akt.

Cell lines, transfection reagents, drugs, and antibodies

Human bladder cancer RT112 (ACC-418) and JMSU1 (ACC-505) cells were obtained from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH (Inhoffenstraße, Braunschweig, Germany). RT112 cells carry a FGFR3-TACC3 translocation, while JMSU1 cells harbor FGFR1 amplification [7]. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humid incubator containing 5% CO₂ and 95% air.

Control siRNA (sc-37007) and Akt1/2 siRNA (sc-43609) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). The selective FGFR inhibitor AZD4547 (S2801) was purchased from Selleckchem (Houston, TX, USA). SIP-SII was prepared following the protocol described previously [11]. HA PKB T308D S473D pcDNA3 (Akt DD) was a gift from Jim Woodgett (Addgene plasmid #14751; http://n2t.net/addgene:14751; RRID: Addgene_14751) [30]. Empty vector FLAG-HA-pcDNA3.1- was a gift from Adam Antebi (Addgene plasmid #52535; http://n2t.net/addgene:52535; RRID: Addgene_52535) [31]. The selective PI3K inhibitor BKM120 (S2247) was purchased from Selleck Chemicals Inc. (Shanghai, China). Antibodies against p-Akt1/2/3 (sc-514032), Akt1/2/3 (sc-81434), Cdk4 (sc-70832), Bcl-2 (sc-509), MMP-2 (sc-13594), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Cell viability analysis

RT112 or JMSU1 cells were seeded in 96-well plates (5x10³ cells/well) and allowed to attach overnight. Next, cells were treated with SIP-SII, AZD4547, or the combination of SIP-SII and AZD4547 for 12, 24, 36, 48, or 72 h, after which 20 μL of a 5 mg/mL stock of methylthiazolyldiphenyl-tetrazolium bromide (MTT, 298-93-1, Sigma-Aldrich Inc., St. Louis, MO, USA) was added to the medium. Cells were further incubated at 37 °C for 4 h, centrifuged, and the pellet was dissolved in 150 μL of DMSO. Absorbance was measured at 570 nm on a microplate reader (Bio-Rad 680 XR, Hercules, CA, USA). The combination index (CI) of SIP-SII and AZD4547 was calculated using the Chou-Talalay method, where CI < 1, CI = 1, and CI > 1 indicate synergism, additive effect, and antagonism in drug combinations, respectively [32].

Transwell migration assay

The transwell migration assay was performed as reported before, using membranes with 8 μm pore size [33]. In brief, 3x10⁴ bladder cancer cells suspended in 50 μL of serum-free DMEM were seeded in the upper chambers and treated with test compounds. The lower wells were filled with 600 μL of DMEM containing 10% FBS. After a 24 h incubation, cells on the upper surface of the membrane were removed with cotton swabs and cells that migrated to the lower surface were fixed, stained, and counted under an inverted microscope. Five random fields in each group were recorded.

Western blotting

Attached cells were washed twice with ice-cold PBS and then lysed with lysis buffer for 30 min on ice. Total protein content (for cell and tissue samples) was determined using a BCA protein assay kit (ab102536, Abcam, Cambridge, UK). Equal amounts of proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in TBS-T for 1 h and incubated with specific primary antibodies (1:1000) at 4 °C with gentle shaking overnight. Membranes were washed three times with TBS-T, reacted with secondary antibodies conjugated to HRP (1:2000; ab205719 and ab205718, Abcam, Cambridge, UK), and antibody:protein complexes detected by enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.). Data obtained from three independent experiments were analyzed with ImageJ software (v 1.52p).

Cell cycle analysis

Cell cycle distribution analysis was performed using the Propidium Iodide (PI) Flow Cytometry Kit (ab139418, Abcam, Cambridge, UK) following the manufacturer’s protocol. Untreated cells were used as control. Cells were prepared at a density of 1x10⁴ per well in 6 well plates and exposed to test reagents for 24 h at 37ºC. After harvesting and preparation of single-cell suspensions, cells were fixed, stained with PI, and analyzed on a FACSCalibur cytometer (BD Biosciences, San Jose, CA, US). Cell cycle distribution analysis was performed on three separate experiments using BD CellQuest™ Pro Analysis software (BD Biosciences, San Jose, CA, US).

Apoptosis analysis

For apoptosis analysis, cells (2x10⁵) were seeded in 6-well plates and allowed to attach overnight. Experimental treatments were applied, and cells were then harvested and stained with the mitochondrial membrane potential reporter JC-1 (ab141387, Abcam, Cambridge, UK). JC-1 fluorescence was assessed by flow cytometry in three individual experiments.

Xenograft model

Animal experiments were approved by the Institutional Animal Care and Use Committee of Shengjing Hospital of China Medical University (Shenyang, China). Nude mice (male, 18–22 g) were obtained from the Experimental Animal Centre of Shengjing Hospital of China Medical University. RT112 cells (5×10⁶ in 0.2 mL of saline) were subcutaneously injected into the right axilla of mice. When tumor volumes reached ~100 mm³ mice were randomly divided into four groups (n = 3 per group): control (saline, vehicle), SIP-SII (30 mg/kg/d), AZD4547 (30 mg/kg/d), and combination of SIP-SII and AZD4547 (SIP + AZD). All drugs were administered by intraperitoneal injection (0.2 mL) every day for 28 days. Tumor volumes and body weights were documented every other day. Mice were euthanized by cervical dislocation under isoflurane anaesthesia 24 h after the last drug injection.

Immunohistochemistry

Tumors were harvested, fixed with 10% neutral buffered formalin, and embedded in paraffin. Representative tumor sections were incubated with a p-Akt-Ser473 antibody (sc-514032, Santa Cruz Biotechnology, Inc., Dallas, Texas, US) and immunoreactivity detected using the Ultra-Sensitive^TM SAP IHC staining kit (KIT-7710, Maixin Biotech., Fuzhou, China).

Statistical analysis

Data were analyzed using GraphPad Prism 7.00 (GraphPad Software, San Diego, CA, USA) and are expressed as the mean ± standard deviation (SD). Multiple comparisons were performed using two-way analysis of variance (ANOVA) and Tukey's multiple comparisons test. Multiple comparisons of tumor weights were performed by one-way ANOVA and Tukey's multiple comparisons test. P < 0.05 was considered statistically significant.