Clinically relevant GSK‑3β inhibitor 9‑ING‑41 is active as a single agent and in combination with other antitumor therapies in human renal cancer

Tsutomu Anraku, Hiroo Kuroki, Akira Kazama, Vladimir Bilim, Masaaki Tasaki, Daniel Schmitt, Andrew Mazar, Francis J Giles, Andrey Ugolkov, Yoshihiko Tomita, Tsutomu Anraku, Hiroo Kuroki, Akira Kazama, Vladimir Bilim, Masaaki Tasaki, Daniel Schmitt, Andrew Mazar, Francis J Giles, Andrey Ugolkov, Yoshihiko Tomita

Abstract

Glycogen synthase kinase‑3 (GSK‑3), a serine/threonine kinase, is involved in a broad range of pathological processes including cancer. GSK‑3 has two isoforms, GSK‑3α and GSK‑3β, and GSK‑3β has been recognized as a therapeutic target for the development of new anticancer drugs. The present study aimed to investigate the antitumor effects of 9‑ING‑41, which is a maleimide‑based ATP‑competitive small molecule GSK‑3β inhibitor active in patients with advanced cancer. In renal cancer cell lines, treatment with 9‑ING‑41 alone induced cell cycle arrest and apoptosis, and autophagy inhibitors increased the antitumor effects of 9‑ING‑41 when used in combination. Treatment with 9‑ING‑41 potentiated the antitumor effects of targeted therapeutics and increased the cytotoxic effects of cytokine‑activated immune cells on renal cancer cell lines. These results provided a compelling rationale for the inclusion of patients with renal cancer in studies of 9‑ING‑41, both as a single agent and in combination with current standard therapies.

Figures

Figure 1
Figure 1
Treatment with 9-ING-41 inhibits the proliferation and survival of RCC cells. (A) RCC cells were treated with the indicated concentrations of 9-ING-41 for 96 h, and protein expression was analyzed by western blotting. (B) Relative cell proliferation was measured by MTS assay in RCC cells treated with the indicated doses of 9-ING-41 for 24, 48, 72 and 96 h. Differences were analyzed by one-way ANOVA. (C) BrdU colorimetric assay was performed in RCC cells treated with diluent (DMSO) or 9-ING-41 at indicated concentrations for 48 h. *P<0.05, **P<0.01 and ***P<0.001. RCC, renal cell carcinoma; GS, glycogen synthase; p, phosphorylated; GSK-3β, glycogen synthase kinase-3β; GI50, concentration that inhibits cell proliferation by 50%; BrdU, 5-bromo-2-deoxyuridine; OD, optical density.
Figure 1
Figure 1
Treatment with 9-ING-41 inhibits the proliferation and survival of RCC cells. (A) RCC cells were treated with the indicated concentrations of 9-ING-41 for 96 h, and protein expression was analyzed by western blotting. (B) Relative cell proliferation was measured by MTS assay in RCC cells treated with the indicated doses of 9-ING-41 for 24, 48, 72 and 96 h. Differences were analyzed by one-way ANOVA. (C) BrdU colorimetric assay was performed in RCC cells treated with diluent (DMSO) or 9-ING-41 at indicated concentrations for 48 h. *P<0.05, **P<0.01 and ***P<0.001. RCC, renal cell carcinoma; GS, glycogen synthase; p, phosphorylated; GSK-3β, glycogen synthase kinase-3β; GI50, concentration that inhibits cell proliferation by 50%; BrdU, 5-bromo-2-deoxyuridine; OD, optical density.
Figure 1
Figure 1
Treatment with 9-ING-41 inhibits the proliferation and survival of RCC cells. (A) RCC cells were treated with the indicated concentrations of 9-ING-41 for 96 h, and protein expression was analyzed by western blotting. (B) Relative cell proliferation was measured by MTS assay in RCC cells treated with the indicated doses of 9-ING-41 for 24, 48, 72 and 96 h. Differences were analyzed by one-way ANOVA. (C) BrdU colorimetric assay was performed in RCC cells treated with diluent (DMSO) or 9-ING-41 at indicated concentrations for 48 h. *P<0.05, **P<0.01 and ***P<0.001. RCC, renal cell carcinoma; GS, glycogen synthase; p, phosphorylated; GSK-3β, glycogen synthase kinase-3β; GI50, concentration that inhibits cell proliferation by 50%; BrdU, 5-bromo-2-deoxyuridine; OD, optical density.
Figure 2
Figure 2
9-ING-41 induces cell cycle arrest and apoptosis in RCC cells. (A) Flow cytometry was performed in ACHN and KRCY renal cancer cells treated with 5 µM 9-ING-41 for 48 and 96 h. (B) Cell cycle distribution and rate of sub-G1, G1, S and G2 populations are presented following treatment of RCC cells with 9-ING-41 as indicated. RCC, renal cell carcinoma; FL2-A, total cell fluorescence area.
Figure 2
Figure 2
9-ING-41 induces cell cycle arrest and apoptosis in RCC cells. (A) Flow cytometry was performed in ACHN and KRCY renal cancer cells treated with 5 µM 9-ING-41 for 48 and 96 h. (B) Cell cycle distribution and rate of sub-G1, G1, S and G2 populations are presented following treatment of RCC cells with 9-ING-41 as indicated. RCC, renal cell carcinoma; FL2-A, total cell fluorescence area.
Figure 3
Figure 3
Treatment with 9-ING-41 suppresses the expression of cyclin-dependent kinases and anti-apoptotic proteins in RCC cells. (A and B) RCC cells were treated with the indicated concentrations of 9-ING-41 for 96 h, and protein expression was analyzed by western blotting. (C) Reverse transcription-quantitative PCR was performed in RCC cells following treatment with the indicated doses of 9-ING-41 for 72 h. Gene expression of Bcl-2 and E2F-1 was significantly decreased in cells treated with 9-ING-41 compared with the control cells. *P<0.05. RCC, renal cell carcinoma; E2F-1, E2F transcription factor 1; CDK1, cell cycle controller 2; XIAP, X-linked inhibitor of apoptosis; PARP, poly (ADP-ribose) polymerase.
Figure 3
Figure 3
Treatment with 9-ING-41 suppresses the expression of cyclin-dependent kinases and anti-apoptotic proteins in RCC cells. (A and B) RCC cells were treated with the indicated concentrations of 9-ING-41 for 96 h, and protein expression was analyzed by western blotting. (C) Reverse transcription-quantitative PCR was performed in RCC cells following treatment with the indicated doses of 9-ING-41 for 72 h. Gene expression of Bcl-2 and E2F-1 was significantly decreased in cells treated with 9-ING-41 compared with the control cells. *P<0.05. RCC, renal cell carcinoma; E2F-1, E2F transcription factor 1; CDK1, cell cycle controller 2; XIAP, X-linked inhibitor of apoptosis; PARP, poly (ADP-ribose) polymerase.
Figure 3
Figure 3
Treatment with 9-ING-41 suppresses the expression of cyclin-dependent kinases and anti-apoptotic proteins in RCC cells. (A and B) RCC cells were treated with the indicated concentrations of 9-ING-41 for 96 h, and protein expression was analyzed by western blotting. (C) Reverse transcription-quantitative PCR was performed in RCC cells following treatment with the indicated doses of 9-ING-41 for 72 h. Gene expression of Bcl-2 and E2F-1 was significantly decreased in cells treated with 9-ING-41 compared with the control cells. *P<0.05. RCC, renal cell carcinoma; E2F-1, E2F transcription factor 1; CDK1, cell cycle controller 2; XIAP, X-linked inhibitor of apoptosis; PARP, poly (ADP-ribose) polymerase.
Figure 4
Figure 4
Pharmacological inhibition of GSK-3 with 9-ING-41 potentiates the effect of anticancer therapeutics in RCC cells. Relative cell proliferation was measured by MTS assay in RCC cell lines (A and B) ACHN and (C and D) Caki-1 treated with 9-ING-41 in combination with (A and C) targeted therapeutics (sorafenib, sunitinib, pazopanib and cabozantinib) or (B and D) autophagy inhibitors (chloroquine and bafilomycin) for 72 h as indicated. RCC, renal cell carcinoma.
Figure 4
Figure 4
Pharmacological inhibition of GSK-3 with 9-ING-41 potentiates the effect of anticancer therapeutics in RCC cells. Relative cell proliferation was measured by MTS assay in RCC cell lines (A and B) ACHN and (C and D) Caki-1 treated with 9-ING-41 in combination with (A and C) targeted therapeutics (sorafenib, sunitinib, pazopanib and cabozantinib) or (B and D) autophagy inhibitors (chloroquine and bafilomycin) for 72 h as indicated. RCC, renal cell carcinoma.
Figure 5
Figure 5
Treatment with 9-ING-41 sensitizes renal cancer cells to the cytotoxic effects of human immune cells. ACHN and Caki-1 renal cancer cells were treated with 9-ING-41 as indicated, harvested and mixed with activated human immune cells. Lactate dehydrogenase activity of the supernatants was measured and cytotoxicity was calculated. The effector:target ratio indicates the proportion of immune to cancer cells. *P<0.05.

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