29 The levels of E7/COX-2 transcript and protein vary widely for a given cell line under control conditions in the independent experiments – i.e. in Fig. 4(a), Nontreated control SiHa is high for the expression of both gene products, whereas in Fig. 4(b), the same control is low for both markers. Furthermore, PGE2

production in the culture media was suppressed by IL-32γ over-expression (Fig. 4c) and Wnt tumor enhanced by IL-32 knock-down (Fig. 4d). Production of PGE2 in the culture supernatants of the SiHa and CaSki cells was also measured using a specific ELISA kit in the independent experiments, as described in the Materials and methods section. Similarly, with regard to PGE2 production as shown in the independent experiments, the control conditions for both cell lines, specifically SiHa cells, in each experiment are disparate, i.e. high in Fig. 4(c) and low in Fig. 4(d). The differences are considerable, suggesting that the cells are at different stages of development and the dynamic

of induction/inhibition may change with initial levels of production. Moreover, the endogenous levels of IL-32 at the onset of the assays would provide some relevance to the observed differences in basal levels. Collectively, these results indicate that E7 and COX-2 were feedback-inhibited by IL-32γ in cervical cancer cells. A variety of pro-inflammatory cytokines, learn more including IL-1β, TNF-α and IL-18, are induced by IL-32 in inflammatory

autoimmune disease.27,32 To evaluate the regulatory effects of IL-32 induced by E7-mediated COX-2 activation on the expression of other pro-inflammatory cytokines, we determined the levels of IL-1β, TNF-α and IL-18 expression after IL-32 over-expression and knock-down in SiHa and CaSki cells. Over-expression of IL-32 induced IL-1β, TNF-α and IL-18 expression (Fig. 5a), whereas IL-32 knock-down down-regulated cytokine expression in SiHa and CaSki cells (Fig. 5b). In Fig. 5 (a), various pro-inflammatory cytokines are barely detectable in SiHa (negative control) and IL-32 induced various pro-inflammatory cytokines. However, to see whether the pro-inflammatory cytokines would Etomidate be down-regulated by siRNA IL-32, PCR was optimized to show strong bands of negative control in the same lane and same cell line in Fig. 5(b). Interleukin-32 over-expression in HPV-expressing SiHa and CaSki cells feedback-inhibited the E7-mediated COX-2 activation pathway and induced other pro-inflammatory cytokines in the inflammatory/immune response. Significant variability in signals was noted in the control cohorts in independent experiments, as shown in Fig. 5(a,b). To determine whether the expression levels of IL-32-induced inflammatory cytokines would be inhibited by IL-32-specific siRNA, an optimized RT-PCR procedure was conducted to determine the expressed levels of these cytokines in the controls (Fig. 5b).