1c). E7AS partially and completely blocked IL-32 and COX-2 expression, respectively (Fig. 1c), suggesting that factors other than COX-2 can induce IL-32. It has been reported that a single siRNA targeting the E7 coding region should inhibit the expression of both E6 and E7 proteins simultaneously31 and so E7AS could completely block COX-2 expression. Immunohistochemical staining for both COX-2 and IL-32 revealed the co-localization of these signals in invasive primary cancerous tissues (Fig. 1d). Expressed E7 induced significant increases in the activities of both the IL-32 and COX-2 promoters. As shown in Fig. 2, the HPV-16 E7 oncogene stimulated the promoter activities of DMXAA order both IL-32 (Fig. 2a) and COX-2 (Fig. 2b)

in a variety of cervical cancer cell lines, and E7AS specifically neutralized the E7-mediated activation of both the IL-32 (−746/+25) and COX-2 (−880/+9) promoters. In Fig. 2(a), there was no significant increase of IL-32 promoter activity induced by the control IL-32p itself (without E7) in the C33A/pOPI3 control cells (data not shown). Nor was there a significant increase of COX-2 promoter activity induced by the control E7 itself (without COX-2p) in the C33A/pOPI3 cells (Fig. 2b, data not shown). To determine the mechanism underlying the HPV-16 E7-mediated stimulation of COX-2 and IL-32, COX-2 was over-expressed in SiHa and CaSki cells and IL-32

expression was evaluated with RT-PCR and Western blot analyses. The IL-32 mRNA and protein expression levels were increased by COX-2 over-expression (Fig. 3a). In addition, IL-32 and E7 expressions were reduced in a check details dose-dependent manner by treatment with the COX-2-specific inhibitor NS398 in SiHa and CaSki cells (Fig. 3b). The levels of COX-2-derived PGE2 were reduced in the culture media from the NS398-treated SiHa and CaSki cells. Interleukin-32 levels were determined in the supernatants of COX-2 over-expressing and NS398-treated SiHa and CaSki cells using a sandwich IL-32 ELISA as reported Abiraterone previously,30 and significant expression levels of IL-32 were not detected in the culture media (data not shown) compared with the intracellular expression levels of IL-32. This result

supports the notion that IL-32 would not be secreted from cells as reported previously.12,26 Collectively, these data show that COX-2 is an upstream regulatory factor of HPV-16 E7-mediated IL-32 stimulation. To assess the regulatory effects of IL-32 on the expression of COX-2 mediated by the HPV-16 E7 oncogene in cervical cancer cells, SiHa and CaSki cells were transfected with IL-32γ and IL-32 siRNA, respectively, in independent experiments. The results of RT-PCR and Western blot analyses demonstrated that E7 and COX-2 were down-regulated in cells (over-expressed with IL-32γ) over-expressing IL-32γ and recovered by IL-32 siRNA (Figs 4a and 5a). The broad band of IL-32 proteins detected by Western blotting as shown in Fig. 3(b), suggested the various expressed forms of IL-32 proteins.