Activation of the interleukin-32 pro-inflammatory pathway in response to human papillomavirus infection and over-expression of interleukin-32 controls the expression of the human papillomavirus oncogene

Abstract

High-risk variants of human papillomavirus (HPV) induce cervical cancer by persistent infection, and are regarded as the principal aetiological factor in this malignancy. The pro-inflammatory cytokine interleukin-32 (IL-32) is present at substantial levels in cervical cancer tissues and in HPV-positive cervical cancer cells. In this study, we identified the mechanism by which the high-risk HPV-16 E7 oncogene induces IL-32 expression in cervical cancer cells. We used antisense transfection, over-expression, or knock-down of IL-32 to assess the effects of the HPV-16 E7 oncogene on IL-32 expression in cervical cancer cells. Cyclo-oxygenase 2 (COX-2) inhibitor treatment was conducted, and the expression levels, as well as the promoter activities, of IL-32 and COX-2 were evaluated in human HPV-positive cervical cancer cell lines. E7 antisense treatment reduced the expression levels and promoter activities of COX-2, which is constitutively expressed in HPV-infected cells. Constitutively expressed IL-32 was also inhibited by E7 antisense treatment. Moreover, IL-32 expression was blocked by the application of the selective COX-2 inhibitor, NS398, whereas COX-2 over-expression resulted in increased IL-32 levels. These results show that the high-risk variant of HPV induces IL-32 expression via E7-mediated COX-2 stimulation. However, E7 and COX-2 were down-regulated in the IL-32γ over-expressing cells and recovered by IL-32 small interfering RNA, indicating that E7 and COX-2 were feedback-inhibited by IL-32γ in cervical cancer cells.

Figure 1

The effect of the E7 oncogene on interleukin-32 (IL-32) expression in cervical cancer. IL-32 is induced in cervical cancer cells by the human papillomavirus (HPV) E7 oncogene. (a) IL-32 expression in cervical cancer tissues. Immunohistochemistry was conducted as described in the Materials and methods section. IL-32 expression is noted in the dysplastic epithelium of cancerous regions of the cervix (CIN 3) in contrast to the negative expression detected in normal squamous cells. (b) The effect of the E7 oncogene on IL-32 expression in C33A, C33A/pOPI3, and C33A/E7 cervical cancer cells. (c) The effect of E7 antisense (E7AS) on IL-32 and COX-2 expression in HPV⁺ cervical cancer C33A/E7, SiHa and CaSki cells. IL-32 and COX-2 were detected via Western blotting using specific antibodies as described in the Materials and methods section. (d) In the serial section, immunohistochemical staining for cyclo-oxygenase 2 (COX-2) was also conducted.

Figure 2

The effects of the E7 oncogene on interleukin-32 (IL-32) and cyclo-oxygenase-2 (COX-2) promoter activities in cervical cancer cell lines. The human papillomavirus (HPV) E7 oncogene up-regulates the promoter activities of IL-32 and COX-2 in cervical cancer cell lines. E7AS specifically neutralized the E7-mediated activation of the IL-32 promoter (−746/+25) (a) and COX-2 promoter (−880/+9) (b) in C33A/pOPI3, C33A/E7, SiHa and CaSki cervical cancer cells. Data are expressed as the means ± SD and are representative of three independent assays (*P<0·05, and **P<0·01).

Figure 3

The effect of cyclo-oxygenase-2 (COX-2) over-expression on interleukin-32 (IL-32) expression in cervical cancer cells. (a) IL-32 induction by COX-2 over-expression in SiHa and CaSki cells. (b) The inhibitory effect of the COX-2 inhibitor, NS398, on IL-32 expression in SiHa and CaSki cells. Cells transfected with pCDNA3.1 and pCDNA3.1/COX-2 (1·5 μg/μl) were incubated for 24 hr. Western blotting for IL-32 in the cell lysates was conducted using a monoclonal anti-IL-32 antibody (KU32-52).

Figure 4

Feedback inhibitory effect of IL-32 on the human papillomavirus (HPV) type 16 E7-mediated cyclo-oxygenase-2 (COX-2) activation pathway. (a) The feedback inhibition of IL-32 on E7 and COX-2 expression in SiHa and CaSki cells. COX-2 and E7 mRNAs were detected via reverse transcription-PCR using specific primer sets, and COX-2 expression was probed using a specific antibody as described in the Methods section. (b) The effect of interleukin-32 (IL-32) small-interfering (si) RNA on the HPV-16 E7-mediated cyclooxygenase-2 (COX-2) activation pathway in SiHa and CaSki cells. The effects of IL-32 siRNA on E7 and COX-2 expressions were elucidated in the SiHa and CaSki cells, respectively, in the independent experiments (n = 3). The effects of overexpressed IL-32 (c) and IL-32 siRNA (d) on PGE₂ production in cervical cancer cells. PGE₂ production in the culture supernatants of SiHa [open bar, PGE₂ (pg/ml): left axis] and CaSki [closed bar, PGE₂ (pg/ml): right axis] cells was also measured using specific ELISA kits, in the independent experiments (n = 3), as described in the Methods section. The control conditions for both cell lines, specifically SiHa cell, in each experiment are extremely disparate, i.e. high in (c) and low in (d).

Figure 5

The effects of over-expressed IL-32 on the expressions of proinflammatory cytokines and factors associated with cell cycle and apoptosis in cervical cancer cells. The expressions of proinflammatory cytokines were measured after 24 hr of over-expression and knockdown of IL-32 in SiHa and CaSki cells. (a) Induction of IL-1β, TNF-α and IL-18 by IL-32γ overexpression. (b) Down-regulation of IL-1β, TNF-α and IL-18 by IL-32 siRNA on IL-32 pro-inflammatory pathway in response to the HPV E7 oncogene. (a) and (b) were performed in independent experiments. (c) Effects of over-expressed IL-32 on expression levels of factors associated with cell cycle and apoptosis. IL-32γ over-expression induced PARP cleavage and upregulated p21 protein expression, whereas the expression levels of cyclin E and cyclin A were suppressed. Western blots were conducted using specific antibodies as described in the Methods section.

Figure 6

Feedback mechanism between cyclo-oxygenase-2 (COX-2) and interleukin-32 (IL-32) by human papillomavirus (HPV) infection. The HPV E7 oncogene triggers COX-2/PGE₂ and IL-32 production and this culminates in host inflammatory responses and this may lead to cancer. However, IL-32 over-expression in cervical cells controls HPV oncogene and COX-2 expression via a feedback inhibition mechanism for early infection. E7 expression is directly coupled to IL-32 expression. The COX2 inhibitor decreases E7 expression. Taken these results, IL-32 expression levels were enhanced in COX-2-over-expressing SiHa and CaSki cells, and treatment with the COX-2 selective inhibitor blocks E7-mediated IL-32 stimulation.