Purpose: Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector^based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model. Experimental Design: To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector^based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo. Results: Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil domain, respectively, strongly suppressed the expression of Stat3 in PC3 and LNCaP cells. The Stat3-1 siRNA, which targeted the DNA-binding domain, exerted no effect on Stat3 expression, indicating that the gene silencing efficiency of siRNA may be dependent on the local structure of Stat3 mRNA. The Stat3 siRNAs down-regulated the expression of Bcl-2 (an antiapoptotic protein), and cyclin D1 and c-Myc (cell growth activators) in prostate cancer cells. Inhibition of Stat3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro and in tumors implanted in nude mice. Conclusions:These data indicate that Stat3 signaling is a promising molecular target for prostate cancer therapy and that vector-based Stat3 siRNA may be useful as a therapeutic agent for treatment of prostate cancer.Prostate cancer is the most common cancer and the second leading cause of cancer-related deaths among men in Western countries (1, 2). More men are currently diagnosed at the early stages of prostate cancer and can be effectively treated by surgery or radiation. However, in one third of the

Purpose: Persistent activation of signal transducers and activators of transcription 3 (Stat3) and its overexpression contribute to the progression and metastasis of several different tumor types. For this reason, Stat3 is a reasonable target for RNA interference^mediated growth inhibition. Blockade of Stat3 using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth in mice. However, RNA interference does not fully ablate target gene expression in vivo, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Stat3-specific shRNA, we applied a combination treatment involving gene associated with retinoid-IFN^induced mortality 19 (GRIM-19), another inhibitor of STAT3, along with shRNA. Experimental Design: The coding sequences for GRIM-19, a cellular STAT3-specific inhibitor, and Stat3-specific shRNAs were used to create a dual expression plasmid vector and used for prostate cancer therapy in vitro and in mouse xenograft models in vivo. Results: The coexpressed Stat3-specific shRNA and GRIM-19 synergistically and more effectively suppressed prostate tumor growth and metastases when compared with treatment with either single agent alone. Conclusion: The simultaneous use of two specific, but mechanistically different, inhibitors of STAT3 activity exerts enhanced antitumor effects.

Aim: To determine the inhibitory effect of the synthetic STAT3 siRNA on the expression of STAT3 gene in human laryngeal cancer cell lines Hep2 and to investigate the effect of STAT3 siRNA on growth and apoptosis in Hep2 cells. Methods: A pair of DNA templates coding siRNA against STAT3-mRNA was synthesized to reconstruct plasmid of pSilencer1.0-U6 siRNA-STAT3. Hep2 cells were transfected with RPMI-1640 media (untreated), plasmid (empty), and STAT3 siRNA, respectively. Northern blot and Western blot analysis of STAT3 and pTyr-STAT3 expression in Hep2 cells and Western blot analysis of Bcl-2 expression in the Hep2 cell was performed 72 h after transfection. MTT, flow cytometry, and AO/EB assay were used for determination of cells proliferation and apoptosis in Hep2 cells. Results: pTyr-STAT3 was markedly expressed in untreated Hep2 cells and the vector-treated Hep2 cells, whereas pTyr-STAT3 expression was significantly reduced in STAT3 siRNA-transfected Hep2 cells, indicating that STAT3 siRNA inhibited the activity of STAT3. Transfection of Hep2 cells with STAT3 siRNA significantly inhibited STAT3 expression at both mRNA and protein level in Hep2 cells and the inhibition was characterized by time-dependent transfection. Treatment of Hep2 cells with STAT3 siRNA resulted in dose-dependent growth inhibition of Hep2, this significantly increased apoptotic cell rate, and decreased Bcl-2 expression level in Hep2 cells. STAT3 siRNA had an effect on induction of either early or late stage apoptosis. Conclusion: This study demonstrates that STAT3 siRNA effectively inhibits STAT3 gene expression in Hep2 cells leading to growth suppression and induction of apoptosis in Hep2 cells. The use of siRNA technique may provide a novel therapeutic approach to treat laryngeal cancer and other malignant tumors expressing constitutively activated STAT3.

Protocadherins are cell-adhesion molecules with 6 or 7 cadherin motifs in their extracellular domain and various cyotoplasmic domains. PCDH10 was characterized a novel tumor suppressive gene in and was epigenetically silenced in multiple haematologic malignancies as well as some solid tumors such as gastric cancer, nasopharyngeal carcinoma and esophageal carcinoma. High-resolution melting (HRM) analysis has been used as a novel tool for analysis of promoter methylation. In our study, we used HRM analysis to detect the methylation levels of PCDH10 gene in 100 gastric cancers, 100 colorectal cancers, 70 pancreatic cancers and equal number of adjacent normal tissues. The frequency of PCDH10 methylation in all three types of cancers was significantly higher than that in normal tissues. Consistent with previous reports, expression levels of PCDH10 were inversely correlated with methylation levels. But we didn't find significant association between PCDH10 methylation status and TNM staging in all three types of cancers. In summary, application of HRM analysis to large amount of clinical samples proves to be a fast and high-throughput way to investigate the epigenetic status of PCDH10. And this is the first study to evaluate the prevalence of PCDH10 �methylation based on large amount of tumor samples, showing that epigenetic regulation of PCDH10 was associated with carcinogenesis.

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