9-ING-41, a small molecule inhibitor of GSK-3beta, potentiates the effects of anticancer therapeutics in bladder cancer

Abstract

Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine kinase, has been identified as a potential therapeutic target in human bladder cancer. In the present study, we investigated the antitumor effect of a small molecule GSK-3β inhibitor, 9-ING-41, currently in clinical studies in patients with advanced cancer, in bladder cancer cell lines. We found that treatment with 9-ING-41 leads to cell cycle arrest, autophagy and apoptosis in bladder cancer cells. The autophagy inhibitor chloroquine potentiated the antitumor effects of 9-ING-41 when tested in combination studies. Our findings also demonstrate that 9-ING-41 enhanced the growth inhibitory effects of gemcitabine or cisplatin when used in combination in bladder cancer cells. Finally, we found that 9-ING-41 sensitized bladder cancer cells to the cytotoxic effects of human immune effector cells. Our results provide a rationale for the inclusion of patients with advanced bladder cancer in clinical studies of 9-ING-41.

Conflict of interest statement

9-ING-41 has been licensed to Actuate Therapeutics, Inc. Andrew Mazar and Andrey Ugolkov hold an equity interest in Actuate Therapeutics, Inc. Daniel Schmitt and Francis Giles are employees of, and hold an equity interest in, Actuate Therapeutics, Inc. Hiroo Kuroki, Tsutomu Anraku, Akira Kazama, Vladimir Bilim, Masayuki Tasaki and Yoshihiko Tomita declare no competing interests.

Figures

Figure 1

Treatment with 9-ING-41 decreases proliferation and survival of bladder cancer cells. (A) Relative cell viability of bladder cancer cells treated with the indicated doses of 9-ING-41 for 96 hours was measured by MTS assay. *P < 0.05 by one-way ANOVA with Tukey post-hoc test. (B) BrdU colometric assay was done using bladder cancer cells treated with 9-ING-41 at indicated concentrations for 48 hours. Columns, mean; bars, SD. *P < 0.05 by one-way ANOVA with Tukey post-hoc test. (C) Flow cytometry was performed using T24, HT1376 and RT4 bladder cancer cells treated with 10 μM of 9-ING-41 for 72 hours. Cell cycle distribution and fraction of sub-G1, G1, S and G2 population are shown.

Figure 2

Antitumor effects of 9-ING-41 therapy in bladder cancer cells. (A) Bladder cancer cells were treated with indicated concentration of 9-ING-41 for 96 hrs. Post-treatment, whole cell lysate was prepared from the cells, separated by SDS-PAGE (20 μg/well), transferred to PVDF membrane, and immunoblotted as indicated. (B) T24 and HT1376 bladder cancer cells were treated with 9-ING-41 for 72 hours and mRNA expression of Bcl-2 and XIAP was analyzed by RT-PCR. Columns, mean; bars, SD. *P < 0.05 by one-way ANOVA with Tukey post-hoc test. (C) Activity of caspase-3 was detected in 9-ING-41-treated bladder cancer cells using colorimetric CaspACE Assay System. Columns, mean; bars, SD. *P < 0.05 by one-way ANOVA with Tukey post-hoc test.

Figure 3

Inhibition of autophagy potentiates the antitumor effects of 9-ING-41 in bladder cancer cells. (A) Brightfield images of T24 cancer cells treated with Vehicle (Control) and 9-ING-41 5 μM for 24 hours. (B) T24 bladder cancer cells were treated with 9-ING-41 10 μM for 48 hrs. Post-treatment, whole cell lysate was prepared from the cells, separated by SDS-PAGE (20 μg/well), transferred to PVDF membrane, and immunoblotted as indicated. (C) Relative cell growth was measured by MTS assay in bladder cancer cell lines T24 and HT1376 treated with 9-ING-41 in combination with autophagy inhibitor chloroquine for 72 hours as indicated. Columns, mean; bars, SD. *P < 0.05 by one-way ANOVA with Tukey post-hoc test.

Figure 4

Pharmacological inhibition of GSK-3β with 9-ING-41 potentiates the effect of anticancer therapeutics in bladder cancer cells. (A,B) Relative cell growth was measured by MTS assay in bladder cancer cell lines T24 and HT1376 treated with 9-ING-41 in combination with gemcitabine (A) and cisplatin (B) for 3 hours as indicated. After the treatment, drugs were replaced with fresh culture medium and relative cell growth was measured by MTS assay after 72 hours. Columns, mean; bars, SD. *P < 0.05 by one-way ANOVA with Tukey post-hoc test.

Figure 5

Treatment with 9-ING-41 sensitizes bladder cancer cells to cytotoxic effects of human immune cells. T24 (A) and HT1376 (B) bladder cancer cells were treated with 9-ING-41 for 48 hrs as indicated, harvested and mixed with human immune cells. LDH activity of supernatant was measured for evaluation of cytotoxicity. Columns, mean; bars, SD. Statistical analysis was performed using unpaired t-test. *P < 0.05.