A novel Jak2/STAT3 pathway inhibitor promotes apoptosis and blocks growth of bladder cancer cells. Free

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Background:The second most common type of cancer in genitourinary system is bladder cancer. IL-6 is strongly associated with malignant phenotype of bladder tissues. IL-6 potently activates signal transducers and activators of transcription 3 (STAT3), a member of the family of latent cytoplasmic transcription factors that transmit signals from the cell membrane to the nucleus. STAT3, is also constitutively activated by aberrant activation of upstream tyrosine kinases in multiple cancer cell lines as well as in human tumors. STAT3 activation is known to promote tumor cell proliferation, survival, angiogenesis, and metastasis in vivo. In bladder cancer, IL-6 strongly promotes mitogenic and angiogenic signaling through phosphorylation of Jak2 and then STAT3, which are potential novel targets for drug development. Currently available inhibitors of Jak2/STAT3 pathway including AG490 block Jak2/STAT3 phosphorylation only at high concentrations (50-100 µM). Interestingly, AG490 shows striking structural similarity to natural products of the caffeic acid family. We demonstrated that caffeic acid benzyl ester and AG490 have similar in vitro activity. This led to the design and synthesis of a novel inhibitor, WP1066, which has far greater potency. Our preliminary studies show that WP1066 blocks Jak2/STAT3 phosphorylation and related downstream transcription of pro-survival proteins.

Aim: Determine the effects of novel Jak2/STAT3 inhibitor WP1066 on bladder cancer cells both in vitro and in vivo.

Methods and Results: The apoptosis and cytotoxic effect of WP1066 was determined with fluorescein conjugate of Annexin V by flow cytometry and MTS assays, respectively in four moderate to poorly differentiated bladder cancer cell lines. WP1066 strongly induced cytotoxicity and apoptosis of four bladder cancer cells with IC50 as low as 2 μM. WP1066 (5 μM) potently inhibited constitutively activated Jak2/STAT3 phosphorylation and IL-6-stimulated STAT3 phosphorylation in bladder cancer cells as determined by western blots. WP1066 (5 μM) also blocked expression of STAT-3 dependent antiapoptotic proteins including survivin and Bcl-xL. Next, we determined the in vivo effect of WP1066 in a murine subcutaneous xenograft model of bladder cancer. WP1066 was well tolerated in athymic nu/nu mice with no effects of organ damage or changes in white blood cell differential counts. WP1066 reduced tumor volume by 4 fold as compared with the solvent.

Conclusion: We showed that WP1066 is a novel therapeutic agent in bladder cancer by effectively blocking JAK2/STAT3-mediated signaling pathways.