Suppression of Glycogen Synthase Kinase 3 Activity Reduces Tumor Growth of Prostate Cancer In vivo

BACKGROUND. Glycogen synthase kinase 3 (GSK-3) has been regarded as a potential therapeutic target for multiple human cancers. We previously reported that suppression of GSK-3 activity with lithium chloride (LiCl) or small chemical inhibitors impaired cellular DNA synthesis and reduced cell proliferation in prostate cancer cells. Therefore, in this study, we extended this in vitro findings to in vivo settings in order to establish a proof of concept that inhibition of GSK-3 activity is feasible in suppressing tumor growth of prostate cancer in vivo. METHODS. In this study, we used three GSK-3 inhibitors, LiCl, TDZD-8, and L803-mts, which are structurally unrelated and non-ATP competitive. Human prostate cancer cell lines PC-3 and C4-2 were used for nude mouse xenograft models. The autochthonous transgenic prostate cancer TRAMP mice were used for testing GSK-3 inhibitor's effect on tumor development. Anti-Ki-67 and BrdU immunohistochemistry was used to determine cell proliferation. The pE2F-TA-LUC (E2F-LUC) luciferase reporter assay and gene specific small interferencing RNA technique were used to examine C/EBP involvement in GSK-3 inhibitor-induced E2F-1 suppression. RESULTS. Using mouse xenograft models, we demonstrated that LiCl and TDZD-8 significantly suppressed tumor development and growth of subcutaneous xenografts derived from human prostate cancer cells. Similarly, in the TRAMP mice, TDZD-8 and L803-mts reduced the incidence and tumor burden in the prostate lobes. Consistent with our previous in vitro findings, GSK-3 inhibitors significantly reduced BrdU incorporation and Ki67-positive cells in xenograft tumors and mouse cancerous prostates compared to the control. Further analysis revealed that following GSK-3 inhibition, C/EBP alpha, a negative cell cycle regulator, was remarkably accumulated in xenograft tumors or in cultured prostate cancer cells. Meanwhile, knocking down C/EBP alpha expression abolished GSK-3 inhibition-induced suppression of E2F1 transactivation, suggesting that C/EBP alpha accumulation is involved in GSK-3 inhibition-induced anti-tumor effect. CONCLUSION. Taken together, these results suggest that GSK-3 inhibition has the potential as a therapeutic strategy for prostate cancer intervention, although further pre-clinical and clinical testing are desirable. Prostate 71: 835-845, 2011. (C) 2010 Wiley-Liss, Inc.