9-ING-41, a small-molecule glycogen synthase kinase-3 inhibitor, is active in neuroblastoma

Abstract

Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3β (GSK-3β) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3β expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3β inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.

Figures

Figure 1. Expression of GSK-3β in human neuroblastoma

(a–d) Representative pictures of GSK-3β expression in human neuroblastoma. GSK-3β is overexpressed in cancer cells in human neuroblastoma tumors obtained from two cancer patients (a, b) and in xenograft tumors established from SK-N-BE(2) and SK-N-DZ neuroblastoma cell lines (c, d).

Figure 2. Treatment with GSK-3 inhibitor 9-ING-41 suppresses growth and viability of neuroblastoma cells

(a) SK-N-DZ neuroblastoma cells were treated with 5 µM AR-A014418, 5 µM TDZD-8 and 0.1 µM 9-ING-41 for 48 hours. Cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to the indicated proteins. (b) SK-N-DZ and SK-N-BE(2) neuroblastoma cells were treated with 0.1 µM and 1 µM 9-ING-41, respectively. 48 hrs post-treatment, cell lysate was prepared and processed as described in (a). (c) SK-N-BE(2) neuroblastoma cells were treated with 9-ING-41, CPT-11 or combination of 9-ING-41 with CPT-11 for 5 hours as indicated. After the treatment, drugs were replaced with fresh media and relative cell growth was measured by MTS assay after 72 hours. Columns, mean cell viability; bars, SE.

Figure 3. Antitumor effects of 9-ING-41 and CPT-11 treatment in SK-N-BE(2) xenograft model

(a) SK-N-BE(2) neuroblastoma cells were inoculated subcutaneously to 8 nude mice (1 tumor per mouse). 4 weeks post-transplantation; tumors were size matched and mice were randomized into 4 treatment groups: control (DMSO; n=2 mice), GSK-3 inhibitor 9-ING-41 (70 mg/kg; n=2 mice), CPT-11 (5 mg/kg; n=2 mice), and CPT-11+9-ING-41 (n=2 mice). Vehicle (DMSO) or drugs were injected as shown by arrows. Points, mean tumor volume; bars, SE. (b) The weight of resected SK-N-BE(2) tumors was measured. Columns, mean tumor weight; bars, SE. (c) Representative pictures of SK-N-BE(2) tumors from each group of animals.

Figure 4. Treatment with combination of CPT-11 and 9-ING-41 leads to an increased apoptosis and a regression of SK-N-DZ xenograft tumors

SK-N-DZ neuroblastoma cells were inoculated subcutaneously to 20 nude mice (1 tumor per mouse). Tumors were size matched and mice were randomized into 4 treatment groups: control (DMSO; n=5 mice), CPT-11 (5 mg/kg, n=5 mice), 9-ING-41 (70 mg/kg, n=5 mice) and CPT-11+9-ING-41 (n=5 mice). (a) Vehicle (DMSO) or drugs were injected as shown by arrows. Points, mean tumor volume; bars, SE. (b) The weight of resected tumors was measured. Columns, mean tumor weight; bars, SE. (c) Representative pictures of tumors from each group of animals. (d) The percentage of apoptotic cells was determined by TUNEL staining. Columns, mean apoptotic cells; bars, SE. (e) Representative pictures of TUNEL staining of SK-N-DZ neuroblastoma xenograft tumors treated as indicated.