Inhibition of GSK-3 induces differentiation and impaired glucose metabolism in renal cancer

Abstract

Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell-cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases, including type II diabetes, Alzheimer disease, bipolar disorder, inflammation, and cancer. Consequently, it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacologic and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent antiproliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The antiproliferative activity is most likely caused by G(0)-G(1) and G(2)-M phase arrest as evident from cell-cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. In addition, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft renal cell carcinoma tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells.

Figures

Figure 1. Inhibition of GSK-3 decreases renal cancer cell proliferation in vitro

A. Chemical structure of 9-ING-41, a maleimide-based GSK-3 inhibitor and its parent compound Staurosporine. B. Renal cancer cells 786-O and A498 were checked for GSK-3 and Glycogen synthase (GS) levels. β-Actin levels served as loading control. C & D. siRNA mediated downregulation of GSK-3β showed a slight yet significant inhibition of proliferation in both 786-O and A498 cells (** denotes p<0.01). Proliferation was measured using 3H-thymidine incorporation assay. E. Western blot analysis of siRNA mediated downregulation of GSK-3β in 786-O and A498 cells. F & G. 786-O and A498 cells were treated with increasing concentrations of 9-ING-41, a novel GSK-3 inhibitor, for 72 hours. The cell proliferation was measured using 3H-thymidine incorporation assay. The figures are representative of three separate experiments (in triplicates) with similar results.

Figure 2. 9-ING-41 induces cell cycle arrest in renal cancer cells

A. Analysis of apoptosis induction property of 9-ING-41 in renal cancer cells by flow cytometry. Cells were treated with increasing concentrations of 9-ING-41 for 48 hours. Apoptosis was measured by Annexin-FITC/PI method. Dead cells (PI-positive) were differentiated from late apoptotic cells (Annexin-positive and PI-positive), early apoptotic cells (Annexin-positive and PI-negative) and live cells (Annexin-negative and PI-negative). The figures are representative of three separate experiments with similar results. B. Cell cycle analysis of renal cancer cells treated with increasing doses of 9-ING-41 for 24 hours. DNA content was analyzed by flow cytometry. The figures are representative of three separate experiments with similar results. C & D. 786-O and A498 cells were treated with increasing concentrations of 9-ING-41 for 24 hours. 9-ING-41 inhibits the expression of cyclin D in a dose dependent manner. Additionally, 9-ING-41 inhibits the phosphorylation and consequent degradation of p21, a cdk inhibitor that directly inhibits the activity of cyclin D/Cdk4/6 complex. β-Actin levels served as loading control. E. Similar results were obtained upon siRNA mediated downregulation of GSK-3β in both the cancer cells. However, cyclin D levels slightly increased here.

Figure 3. GSK-3 inhibition induces differentiation in renal cancer cells

A & B. 786-O and A498 cells were treated with increasing concentrations of 9-ING-41 for 48 hours. 9-ING-41 induced Ksp-Cadherin expression and inhibited Id-1 expression in a dose dependent manner in both the cancer cells. β-Actin levels served as loading control. C. Similar results were obtained upon siRNA mediated downregulation of GSK-3β in both the cancer cells. D & E. 786-O and A498 cells were simultaneously treated with GSK-3β siRNA and 9-ING-41 to evaluate any additive or synergistic effects on Ksp-Cadherin or Id-1 expression.

Figure 4. Inhibition of GSK-3 increases intracellular glucose storage

A & B. 786-O and A498 cells were treated with increasing doses of 9-ING-41 for 24 hours. Intracellular glucose storage was measured using Amplex Red glucose/glucose oxidase assay kit as per manufacturer's protocol. The values were expressed as RFU per mg protein. There was a dose-dependent increase in intracellular glucose in both the renal cancer cells. C & D. siRNA mediated downreluation of GSK-3β also increased intracellular glucose storage in both the cells. The figures are representative of three separate experiments (in triplicates) with similar results. E. Western blot analysis of siRNA mediated downregulation of GSK-3β in 786-O and A498 cells.

Figure 5. GSK-3 inhibition affects energy homeostasis and induces autophagy

A & B. 786-O and A498 cells were treated with increasing concentrations of 9-ING-41 for 24–48 hours. 9-ING-41 induced phosphorylation of both AMPK-α and AMPK-β and inhibited mTOR phosphorylation in both the renal cancer cells. Induction of autophagy was indicated by an increase in LC3 levels. β-Actin levels served as loading control. C. Similar results were obtained upon siRNA mediated downregulation of GSK-3β in both the cancer cells, albeit the effects were slightly less in comparison with 9-ING-41. D. Confocal images showing upregulation of LC3 level in 9-ING-41 treated cells. Bar length= 50 micron.

Figure 6. GSK-3 inhibition inhibits tumor growth in Nude mice

A & B. In two different experiments, 6–8 weeks old male nude Mice received subcutaneous injections of 5 × 106 786-O and A498 cells respectively. Tumors were allowed to grow for 21 days before the initiation of single-agent treatment with 9-ING-41 (20 mg/kg) in PBS containing 50% PEG-400. The control group received only PBS containing 50% PEG-400. After 4 weeks of treatment, significant reduction in tumor weight was observed in both experiments (* denotes p<0.05). C. H & E, Ksp-Cadherin and Ki67 immunohistochemical staining in formalin fixed tissue sections obtained from tumors of control and treatment group. Bar length= 200 micron. D & E. Quantification of Ki67 stained nuclei in 786-O and A498 tissue sections respectively. (** denotes p<0.01). F& G. Western blot analysis with lysates obtained from representative tumor samples of vehicle treated and 9-ING-41 treated groups to correlate with key in vitro results.