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Immunology. Mar 2011; 132(3): 410–420.
PMCID: PMC3044907

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

Keywords: cervical cancer, cyclo-oxygenase-2, feedback inhibition, human papillomavirus E7, interleukin-32

Introduction

Cervical cancer is the second most frequent cause of cancer death in women worldwide, and molecular epidemiological studies have demonstrated clearly that human papillomavirus (HPV) is a prerequisite for the development of cervical carcinoma.1,2 Approximately 200 different HPV types have been characterized, and the two most frequent high-risk HPV genotypes, HPV-16 and HPV-18, account for at least 50% of cervical cancers worldwide.3,4 Several HPV-16 type oncoproteins expressed during the early stage of infection have been associated with oncogenicity; specifically, E5, E6 and E7 have been demonstrated to contribute to the maintenance of malignant cervical cancer phenotypes.5 The function of the E5 oncoprotein-activating epidermal growth factor receptor remains to be clearly elucidated, and E6 promotes the degradation of p53 via its interaction with E6AP.6 The E7 oncoprotein binds to the pRb retinoblastoma protein, and disrupts its formation of a complex with the E2F transcription factor in the G1 phase of the cell cycle. E7 also binds to and activates cyclin complexes such as cyclin-dependent kinase cdk2 and cyclin A, which control cell cycle progression.7 The viral genes E6 and E7 found in a specific subset of HPVs are invariably expressed in HPV-positive cervical cancer cells.8 It has also been previously reported that the E7 gene of HPV-16 triggers a cellular immunosuppression and profoundly enhances the release of angiogenic cytokines by macrophages or dendritic cells.9 The E6 and E7 oncogenes also inhibit the IL-18-mediated immune response, which carries out crucial functions in host defence mechanisms against infection and cancer.10

Interleukin-32 (IL-32) is a new cytokine that has been identified as a pro-inflammatory cytokine that induces other pro-inflammatory cytokines, including IL-1β, IL-6, tumour necrosis factor-α (TNF-α) and chemokines.11 Interleukin-32 is selectively expressed in activated natural killer cells, T cells, epithelial cells, endothelial cells and blood monocytes.11,12 The IL-32 induced by IL-18 has a number of splice variants, namely, IL-32α, -β, -γ, -δ, -ε and -ζ. Their receptors have yet to be identified, although proteinase 3 has been recently identified as a specific IL-32-binding protein. Interleukin-32 has emerged as an important player in innate and adaptive immune responses13 and IL-32 associated with TNF-α appears to exacerbate TNF-α-related inflammatory arthritis and colitis.14 Expression of IL-32 may be a cancer biomarker, and high levels of IL-32 expression have been detected in several cancer cell types.15,16 Interleukin-32 knock-down was also shown to suppress anti-apoptotic proteins such as bcl-2, and induced apoptosis.15,17 Recent studies have demonstrated that viral infection stimulates IL-32 expression; IL-32 suppressed replication of HIV18 during HIV infection, thereby reducing the levels of T helper type 1 and pro-inflammatory cytokines,19,20 and was induced by infection with the influenza A virus.21 However, the role of HPV in IL-32 expression remains unclear. We detected IL-32 expression in tissues and cells obtained from patients with cervical cancer. Furthermore, as HPV plays a critical role in cervical cancer, we attempted to assess the possible role of IL-32 as an inducer of cancer and inflammation in response to HPV infection. Cyclo-oxygenase-2 (COX-2) is over-expressed in HPV-induced diseases, including cervical cancer,22,23 and is stimulated by HPV-16 E6 and E7 oncoproteins via the epidermal growth factor receptor/Ras/mitogen-activated protein kinase pathway.24 As COX-2 and IL-32 are associated with inflammatory processes, we attempted to characterize the relationship between COX-2 and IL-32 in the context of HPV infection. In this study, we evaluated the role of HPV in cervical cancer associated-IL-32 regulation as well as the feedback mechanisms between COX-2 and IL-32 occurring in response to the E7 oncogene.

Materials and methods

Cell lines and reagents

Human cervical cancer cells (C33A, SiHa and CaSki) were obtained from the American Type Culture Collection (Rockville, MD). An HPV-negative cervical cancer cell line (C33A) was prepared to establish stable cell lines expressing the E7 oncogene, and two stable cell lines (C33A/pOPI3, vector control C33A and C33A/E7, E7 expressing C33A) were established as previously described.25,26 All cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and were cultured at 37° in a humidified atmosphere of 5% CO2. N-(2-cyclohexylosyl-4-nitrophenyl)-methane sulphonamide (NS-398) was purchased from Alexis Biochemicals (San Diego, CA), dissolved in DMSO, and used at final concentrations of 50 μm and 100 μm.

Immunohistochemical detection of IL-32 in cervical cancer tissues

The following samples were analysed: 40 paraffin-embedded specimens from patients with cervical squamous cell carcinoma treated with radiation therapy or post-operative radiation therapy at the Chungnam National University Hospital (Daejeon, South Korea). The patients were grouped into the following categories: International Federation of Gynecology and Obstetrics (FIGO) stage IB (n = 16) and stage IIA–IIIB (n = 24). All tissues were subjected to immunohistochemical staining for IL-32 as described previously27 and clinically correlated with FIGO stage and survival, and the following results were obtained. In the serial section, immunohistochemical staining for COX-2 was also conducted to determine whether IL-32 and COX-2 are co-localized in cervical cancer cells. This study was approved by the Chungnam National University Hospital.

Plasmids

The IL-32γ and COX-2 were amplified from the genomic DNA of human CaSki cells via PCR, using the following primers, respectively: IL-32γ: 5′-CTGGAATTCATGTGCTTCCCGAAG-3′ (forward), 5′-GAAGGTCCTCTCTGATGACA-3′ (reverse); COX-2: 5′-CCCAAGCTTGGGCTCAGACAGCAAAGC CTA-3′ (forward), 5′-CTAGTCTAGACTAGCTACAGTTCAGTCGAACGTTCTTT-3′ (reverse). Interleukin-32γ was cloned into the EcoRI and XhoI sites of pCDNA3.1 using EcoRI and SalI, and COX-2 was ligated with pCDNA3.1 vector using the HindIII and XbaI sites. The promoters of IL-32 and COX-2 were amplified via PCR from human genomic DNA. The IL-32 promoter (−746/+25) was constructed as previously reported.21 The COX-2 promoter (−880/+9) used the following primers: 5′-CGGGATCCAAATTCTGGCCATCGCCGCTT-3′ (forward), 5′-CCAAGCTTTGACAATTGGTCGCTAA CCGAG-3′ (reverse) cloned into the MluI and HindIII sites of the pGL3-basic vector, and the inserted sequences were confirmed via DNA sequencing. Both pTarget/E7 and pTarget/E7 antisense (E7AS) were described in a previous report.25,28

Transient transfection

C33A/pOPI3, C33A/E7, SiHa and CaSki cells were seeded on six-well plates at a density of 3 × 105 cells per well, then grown to confluence, reaching approximately 80% at the time of transfection. For each well, plasmid DNA (1 μg) was introduced into the cells using an identical volume of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. The pTarget and pTarget/E7AS plasmid were transfected into C33A/E7, SiHa and CaSki cells to confirm the E7 oncogene-specific effect on IL-32 and COX-2 expression in HPV-expressing cervical cancer cells. The pGL3 basic, pGL3b/IL-32 promoter, and pGL3b/COX-2 promoter were respectively co-transfected with pTarget, pTarget/E7 and pTarget/E7AS into C33A/pOPI3, C33A/E7, SiHa and CaSki cells to determine the specific effects of E7 on the transcriptional activities of IL-32 and COX-2. Additionally, pCDNA3.1, pCDNA3.1/COX-2, pCDNA3.1/IL-32γ, siCONTROL and siIL-32 (Dharmacon, Lafayette, CO) were respectively transfected into SiHa and CaSki cells to evaluate expression between COX-2 and IL-32 by the HPV E7 oncogene. Interleukin-32γ is the most active form of IL-32 isoforms.29 All transfection plasmids were employed at a concentration of 1 μg and the small interfering (si) RNAs were used at a final concentration of 200 nm. After 6 hr the medium was replaced with basal medium and the transfected cells were incubated for 24 hr.

Analysis of promoter activities of IL-32 and COX-2

After 24 hr of incubation, the transfected cells were harvested and the cell lysates were prepared with 1 × lysis buffer (Promega, Madison, WI) containing 10 μg/ml aprotinin and 0·5 μm PMSF. Twenty microlitres of luciferase assay reagent (Promega) was added to each 50-μg protein sample, and the luciferase activities were evaluated at least in triplicate. The assay results were expressed in relative luciferase activity units. The results are expressed as the average of three independent experiments ± SD.

Reverse transcription PCR and Western blot analysis

A total of 5 μg RNAs were isolated from SiHa and CaSki cells transfected with mock, E7AS, IL-32, COX-2, siCONTROL and siIL-32 using an easy-BLUE total RNA extraction kit (iNtRon Biotechnology, Sungnam, South Korea), and the cDNA products were prepared with Moloney murine leukaemia virus reverse transcriptase (New England Biolabs, Beverly, MA). Reverse transcription–PCR (RT-PCR) analysis was performed using a Dice PCR thermal cycler (TaKaRa, Shiga, Japan) with the following primer sets: HPV E7: 5′-ATGCATGGAGATACACCTACATTGC-3′ (forward), 5′-TTATGGTTTCTGAGAACAGATGGGGC-3′ (reverse); IL-32: 5′-ATGTGCTTCCCGAAGGTCCTC-3′ (forward), 5′-TCATTTTGAGGAT TGGGGTTC-3′ (reverse); COX-2: 5′-GAAACCCACTCCAAACACAG-3′ (forward), 5′- CCCTCGCTTATGATCTGTCT-3′ (reverse); IL-1β: 5′-ATGGCAGAAGTACCTAAGCTCGC-3′ (forward), 5′-TTGACTGAAGTGGTACGTTAAACACA-3′ (reverse); TNF-α: 5′-GTCAGATCATCTTC TCGAACC-3′ (forward), 5′-AAAGTAGACCTGCCCAGACTC-3′ (reverse); IL-18: 5′-ATAGGATCCATGGCTGCTGAACCAGTA-3′ (forward), 5′-GACAGATCTGTCTTCGTTTTGAACAG T-3′ (reverse); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. Expression of proteins was analysed using Western blotting with specific antibodies. The cell lysates were prepared by treating cells with a lysis buffer [0·1% SDS, 0·1% sodium deoxycholate, 1% Triton-X-100, 1 mm EDTA, 0·5 mm EGTA, 140 mm NaCl, 10 mm Tris–HCl (pH 8·0), 10 μg/ml aprotinin and 0·5 mm PMSF] on ice and centrifuged for 30 min at 11 269 g. The protein concentration of the supernatant was measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA) and 50 μg proteins were resolved on 12% SDS–PAGE. The proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA) and blocked overnight with 5% skimmed milk. The antibodies used were specific to COX-2, GAPDH, p21 (Santa Cruz Biotechnology, Santa Cruz, CA), poly-ADP-ribose-polymerase (PARP; Cell Signaling Technology, Beverly, MA), cyclin E and cyclin A (BD Biosciences Pharmingen, San Diego, CA), and IL-32 (KU32-52).30 The blots were probed with enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK) or WEST-ZOL Plus (iNtRon Biotechnology) Western blot detection systems according to the respective manufacturers’ instructions.

Measurement of prostaglandin E2 concentrations

Culture media were collected after incubating the transfected cells for 24 hr. The levels of COX-2-derived prostaglandin E2 (PGE2) in culture media were measured with a Kookaburra Prostaglandin E2 Enzyme Immunoassay kit (Sapphire Bioscience, Crows Nest, NSW, Australia) in accordance with the manufacturer's instructions. Each assay was performed in triplicate.

Statistical analysis

All experiments were conducted either in duplicate or triplicate, and independent experiments were repeated at least three times with similar results. Comparisons between groups were conducted using Student's t-test. The differences between groups for P values < 0·05 and < 0·01 were considered significant.

Results

IL-32 expression is increased in human cervical cancer

Interleukin-32 expression was detected in 55% (n = 22) of all tumour tissues and was particularly strong in the tumour invasion site. This expression was located principally in the cytoplasm as well as in the nuclei of some tumour cells. IL-32 expression was negative in all normal epithelium but was statistically up-regulated in the dysplastic epithelium of cancerous regions of the cervix (cervical intraepithelial neoplasias) and advanced squamous cell carcinomas (Fig. 1a). In general, IL-32 expression was found in most cases exhibiting classical morphological features of HPV infection, including koilocytosis, acanthosis and papillomatosis. In contrast, IL-32 expression was usually not detected in cases that exhibited evidence of maturation arrest but lacked HPV-associated nuclear atypia. Interleukin-32 expression was detected in five of 16 sections (31%) of FIGO stage IB squamous cell carcinomas and in 17 of 24 FIGO stage IIA–IIIB squamous cell carcinomas (71%) (Table 1, P = 0·014 compared with the stage IB group). The up-regulation of IL-32 was definitively associated with transformation and progression of cervical squamous lesions. As shown in Table 1, negative cases were mainly from FIGO stage IB (67%). To obtain cytologically normal control subjects, five normal uterine cervical epithelia were obtained from age-matched (36–68 years) patients undergoing hysterectomy for various non-malignant diseases. The staining intensity exhibited borderline significance with advanced stage (P = 0·064). However, IL-32 expression was not correlated with patient survival (P = 0·79 and P = 0·90 in stage IB and IIA–IIIB, respectively, data not shown) (Fig. 1a and Table 1). To determine the effects of the HPV E7 oncogene on IL-32 expression in human cervical cancer, we confirmed IL-32 levels by the E7 oncogene in an HPV-negative C33A- and E7-stably expressing cell line (C33A/pOPI3 and C33A/E7). Interleukin-32 was induced by the HPV E7 oncogene in the C33A/E7 cells (Fig. 1b) whereas the constitutive expression of IL-32 was inhibited by E7 antisense treatment (E7AS) in the HPV-expressing C33A/E7, SiHa and CaSki cervical cancer cells. Because the IL-32 was expressed, as very low in the HPV-negative C33A cells (Fig. 1b), the change in IL-32 expression by E7AS was not confirmed in C33A cells (data not shown). As previously reported,24 COX-2 expression was also reduced in the E7AS-transfected SiHa and CaSki cells (Fig. 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).

Table 1
Correlation between interleukin-32 (IL-32) staining and stages in cervical cancer
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 ...

HPV-16 E7 oncogene induces transcriptions of IL-32 and COX-2

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 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).

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 ...

COX-2 stimulates IL-32 in response to the E7 oncogene in cervical cancer cells

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 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 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.

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 ...

IL-32 feedback inhibits the HPV-16 E7-mediated COX-2 activation pathway

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 and5a).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. We transfected IL-32γ because IL-32γ is the most active form among many other isoforms.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 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.

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 ...
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 ...

IL-32 over-expression induces pro-inflammatory cytokines and inhibits cancer development in HPV-expressing cervical cancer cells

A variety of pro-inflammatory cytokines, 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 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). We confirmed the apoptotic effects of the cells by IL-32 over-expression in HPV-expressing cervical cancer cells. To determine the effects of IL-32 over-expression on the expression of PARP, p21, cyclin E and cyclin A related to apoptosis and the cell cycle, we conducted Western blot analysis, demonstrating that the protein levels of p21 and cleaved-PARP were increased in the IL-32γ-transfected cells compared with the mock-control cells. However, the expressions of cyclin E and cyclin A were reduced in the IL-32-over-expressing SiHa and CaSki cells (Fig. 5c). These results suggested that IL-32 over-expression inhibits cancer development in cervical cancer cells, via down-regulation of the expressions of E7 and COX-2.

Discussion

In this study, we evaluated the feedback inhibition mechanism of IL-32 pro-inflammatory or cancer pathways in response to the high-risk E7 oncogene in cervical cancer cells. Recently, IL-32 has been associated with the regulation of inflammatory response during infection with the influenza A virus and with the regulation of HIV production.19,20 Expression of IL-32 has been detected in cervical cancer tissues, and IL-32 has been shown to be markedly induced by HPV-16 E7 in a variety of cervical cancer cells. When IL-32 expression was investigated according to the groups with regard to the FIGO stage IB and IIA–IIIB, there was a statistically significant (χ2 test) IL-32 expression frequency in the stage IIA–IIIB (71%) compared with stage IB (31%) disease (P = 0·014) (Table 1). However, IL-32 expression was not correlated with survival of the patients (P = 0·79 and P = 0·90 in stage IB and IIA–IIIB, respectively). Extensive studies using clinical samples are needed to investigate the discrepancy between advanced stage and survival of the patients. Additionally, COX-2 was over-expressed by HPV-16 E7 as reported previously.22,24 The COX-2 induced by HPV-16 oncoproteins has been reported to induce immortality, the inhibition of apoptosis,33 strong invasion ability,34 angiogenesis35 and suppression of the immune response36 in cervical cancer cells, via a number of mechanisms. The levels of COX-2-derived PGE2 were reduced in the culture media from the NS398-treated SiHa and CaSki cells. The levels of COX-2-derived PGE2 were reduced in the culture media from the NS398-treated SiHa and CaSki cells. Compared with the intracellular expression levels of IL-32, significant secretion of IL-32 was not detected in the supernatants of COX-2 over-expressing and NS398-treated SiHa and CaSki cells using a sandwich IL-32 ELISA.30 Although IL-32 is considered to be mainly intracellular,12,26 one may envisage that some is secreted and triggers pro-inflammation in neighbour cells. It is well known that high-risk HPV-16 expresses E6 and E7 proteins from a single polycitronic mRNA.37 An siRNA targeting HPV-16 E7 region degrades either E6, or truncated E6 (E6*) and E7 mRNAs and simultaneously results in knock-down of both E6 and E7 expression.38 Theoretically, a single siRNA targeting the E7 coding region should inhibit the expression of both E6 and E7 proteins simultaneously and be potentially more effective than an E6-specific siRNA.39 Collectively, an anti-sense E7 also can inhibit the expression of both E6 and E7 proteins simultaneously and can completely block COX-2 production. We attempted to determine whether IL-32, when coupled with COX-2, would function as a pro-inflammatory cytokine, exerting HPV-16 E7-mediated regulatory effects in cervical cancer cells. The significant induction of IL-32 and COX-2 promoter activities by HPV-16 E7 was inhibited by E7 knock-down in cervical cancer cells. As COX-2 is induced in response to an inflammatory factor40 and IL-32 also exerts immune/cancer effects,41 we identified the relationship between IL-32 and COX-2 induced by HPV-16 E7. As suggested by Figs 1 and 2(b), and also by Subbaramaiah and Dannenberg,22,24 the use of the COX-2 inhibitor NS398 results in lower expressions of IL-32 (RT-PCR and Western blot) and E7 genes (RT-PCR) (Fig. 3b). As shown in Figs 1 and 2(a), E7 expression is directly coupled to IL-32 expression. Hence, the results shown in Fig. 3 could also be interpreted as NS398 decreasing E7 expression for unknown reasons and therefore the expression of IL-32, without COX-2 being involved. Taken together these results indicate that 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. The E7-mediated production of PGE2 was also suppressed by NS398 in a dose-dependent fashion. These results demonstrate that IL-32 affects the regulation of COX-2 in response to HPV-16 E7 in cervical cancer cells. To determine the effects of IL-32 on the regulation of E7-mediated COX-2 and COX-2-derived PGE2 production, IL-32 was over-expressed and knocked-down in SiHa and CaSki cells. IL-32 over-expression was shown to inhibit the activation of E7-mediated COX-2 and E7 expression in a feedback-based manner. Furthermore, PGE2 levels were reduced in culture media by IL-32 over-expression, whereas those levels were increased in the IL-32 knock-down cell supernatants. We confirmed that E7-mediated IL-32 activation is profoundly correlated with the expression of other proinflammatory cytokines, such as IL-1β, TNF-α, and IL-18, in HPV-expressing cervical cancer cells, thereby indicating that they were induced by IL-32 over-expression, and down-regulated by IL-32 knock-down. It was previously demonstrated that HPV-16 E7 inhibits IL-18-induced IFN-γ production in human peripheral blood mononuclear and natural killer cells.10 Over-expression of IL-32 inhibited E7 oncogene expression, whereas IL-18 expression was enhanced. This suggests that the E7-mediated inhibition of IL-18 expression would be recovered via the suppression of E7, or that IL-18 could be directly induced by IL-32. Transient IL-32 over-expression would inhibit cancer development in HPV-expressing cervical cancer cells through the induction of PARP cleavage and p21 expression, as well as the suppression of cyclin E and cyclin A expression. Hence, IL-32 over-expression may prove to be resistant to the oncogenic effects of E7 through a down-regulation of HPV E7 expression, and the induction of other pro-inflammatory cytokines. Collectively, our results led us to conclude that IL-32 is a downstream regulatory factor of COX-2, and also that it performs a crucial role in the inflammatory response and cancer mediated by HPV-16 E7 in cervical cancer cells, thereby inhibiting COX-2 and HPV-16 E7 through a negative feedback mechanism.

Human papillomavirus is causally associated with cervical cancer,3 which develops over several decades from cervical intraepithelial neoplasias as the result of HPV infection. Moreover, HPV-mediated cellular transformation occurs during the abnormal viral life, apparently via the integration of the viral genome into the host DNA. Abnormal viral action by integration results in increased viral protein production.42,43 Two viral proteins, E6 and E7, perform major roles in cell cycle control,44 HPV-induced oncogenesis,45 and the inhibition of the innate host immune response.46 The results of our studies demonstrate that an HPV-16 E7→COX-2→IL-32 regulatory pathway is relevant to the response of high-risk HPV infection in cervical cancer cells. Although IL-32 over-expression inhibits the E7-mediated COX-2 activation pathway by way of a negative feedback mechanism during the early stages of infection in cervical cancer, the positive induction pathway activated in response to the HPV E7 oncogene appears to predominate over the negative feedback loop as the consequence of sustainable and prolonged HPV expression. We surmise that cervical cancer may develop via the COX-2/IL-32 activation cascade, which is itself mediated by the E7 oncogene.

In summary, the results of our study illustrate a novel mechanism by which the HPV-16 E7 oncogene activates the expression of the pro-inflammatory factors COX-2 and IL-32, and culminates in host inflammatory responses and cancer (Fig. 6). Transient IL-32 over-expression inhibits E7 and COX-2 in cervical cancer through a negative feedback mechanism. In this model, we propose that IL-32 may function as a therapeutic target molecule for the prevention or treatment of cervical cancer induced by high-risk HPV infection.

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 ...

Acknowledgments

This work was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (0920080) and in part from the basic programme (MEST 2010-0019306, 2009-0072028) of the National Research Foundation of Korea (NRF). S.L. is supported in part by the Seoul Scholarship Foundation, D.Y. is supported partially by the Priority Research Centres Programme (2009-0093824), Funds for J.H. (R13-2008-001-00000-00) and Y.Y (2009-0085906) were provided by the NRF funded by the Ministry of Education, Science, and Technology.

Disclosures

The author declares no conflict of interest.

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