Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway
© Adefuye et al.; licensee BioMed Central Ltd. 2014
Received: 17 March 2014
Accepted: 1 August 2014
Published: 8 August 2014
Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation.
Methods and results
Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE2 and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling.
SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.
In sub-Saharan Africa, cervical cancer is the most common cancer among women accounting for 22.2% of all cancer cases and also the leading cause of cancer related deaths in this region [1, 2]. Cervical cancer is a disease of multifactorial etiology usually presenting in sexually active women. Recent findings have shown that sexual transmission and persistent infection of the cervical epithelium with high risk HPV is the single most common risk factor for disease development, accounting for approximately 50% of cases . Other risk factors include, sexually transmitted infections (STIs) , immunosuppression, and multiple sexual partners . The hallmark of disease pathogenesis is characterized by chronic inflammatory response in the presence of underlining neoplasia [6, 7].
Characteristically regarded as response to tissue injury or pathogenic insult, chronic inflammation is typified by alterations to vascular, epithelial, and immune cell function . Over the last decade, numerous experimental studies using gene-disruption and gene over-expression systems in cell lines, laboratory animals, and tissue explants have provided evidence to support the role of inflammation and inflammatory pathways in the pathogenesis and progression of various human cancers including cervical cancer [8–12]. The inflammatory milieu of most cancer microenvironment has been shown to consist of tumor cells, surrounding stromal, immune and inflammatory cells which all interact intimately to produce cytokines/chemokines, growth factors, and adhesion molecules in a bid to promote tumorigenesis and metastasis . Of special relevance within this milieu are pro-inflammatory cytokines which are important mediators of chronic inflammatory responses, and have cardinal effects on malignant processes.
Interleukin 1α (IL-1α) is a pleotropic pro-inflammatory cytokine that belongs to the IL-1 family (IL-1α, IL-1β, and IL-1Ra) gene located on the long arm of chromosome 2 . IL-1α possesses a wide range of inflammatory, immunologic and tumorigenic properties [15–17]. IL-1α is secreted by a variety of cells including monocytes, tissue macrophages, neutrophils, fibroblasts, smooth muscle cells, dendritic cells, and cervical epithelium [15, 18, 19]. Accumulative evidence suggests that IL-1α plays a crucial role in tumorigenesis. Within the cancer microenvironment, IL-1α has been shown to induce the expression of metastatic genes such as the matrix metalloproteinases (MMPs) and stimulate the production of angiogenic proteins and growth factors such as IL-8, IL-6, vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGFβ) [16, 20].
Human Seminal plasma (SP) is a complex organic fluid comprising of secretions of the cowper’s, littre, prostate, and the seminal vesicles . Once deposited within the female reproductive tract (vagina and cervix) during unprotected coitus, SP has been shown to induce the expression of several pro-inflammatory cytokines including IL-1α [22–24]. In sexually active women, the molecular pathways and degree at which SP normally activates the expression of these pro-inflammatory components in any compartment of the female reproductive tract is poorly understood. SP has been shown to possess an abundance of pro-inflammatory prostaglandins (PG)  and we and others have shown that cervical cancer has up-regulated expression of PG receptors  which can be activated by both endogenous and SP-PG. In the present study, we investigated the role of SP in the regulation of IL-1α expression in normal and neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation.
IL-1α is up-regulated in cervical cancer
Seminal plasma induces expression of IL-1α in HeLa neoplastic cervical epithelial cells
SP has been shown to induce the expression of various inflammatory cytokines including IL-1α in normal cervical epithelial cells . We hypothesized that SP can also induce IL-1α expression in neoplastic cervical epithelial cells, which could potentially enhance cervical inflammation and tumorigenesis. We used a well-established HeLa (adenocarcinoma) cell line as a model system to investigate the regulation of IL-1α expression by SP in neoplastic cervical epithelium. HeLa S3 cells were treated with vehicle or SP (1:50 dilution) for 4, 8, 16, and 24 hours and the expression of IL-1α mRNA assessed using qPCR (Figure 2A). SP significantly induced the expression of IL-1α mRNA in HeLa S3 cells at all-time point investigated. Peak induction of IL-1α was observed after 4 hours of SP exposure and was 17.02 ± 4.43 fold increase, while other inductions were 15.40 ± 4.26, 10.07 ± 5.03, and 8.22 ± 2.68 fold increase for 8, 16 and 24 hours, respectively.
EP2 receptor antagonist and inhibitors of EGFR and PI3 kinase inhibit SP-mediated induction of IL-1α in HeLa neoplastic cervical epithelial cells
We next investigated the signal transduction pathways mediating SP induction of IL-1α mRNA expression, using PGE receptor 2 (EP2) antagonist and a panel of small molecule chemical inhibitors of signaling proteins. HeLa S3 cells were treated with vehicle or SP (1:50) alone or with EP2 receptor antagonist (AH-6809) or chemical inhibitors of EGFR (AG-1478), PI3 kinase (LY-294002), PTGS1 (SC-560), and PTGS2 (NS-398) for either 4 hours or 16 hours. The expression of IL-1α in HeLa S3 cells following SP treatment in the presence of receptor antagonist or chemical inhibitors was determined by qPCR after 4 hours (Figure 3A and B) and ELISA after 16 hours (Figure 3C). Figure 3A shows that EP2 receptor antagonist significantly reduced SP-mediated induction of IL-1α mRNA in HeLa S3 cells (P < 0.05). SP-mediated induction of IL-1α was also found to be markedly reduced in the presence of chemical inhibitors of EGFR (AG-1478) and PI3 kinase (LY-294002), respectively (Figure 3B; P < 0.001). Co-incubation of SP with selective PTGS1 (SC-560) and PTGS2 (NS-398) inhibitors had no inhibitory effect on SP-induced IL-1α mRNA expression (Figure 3B; P = 0.26 and 0.16, respectively). This indicates that SP-mediated induction of IL-1α mRNA expression is via EP2 and EGF receptors and not via the endogenous PTGS-PG pathway. In addition, co-treatment with EGTA [calcium chelator; 1.5 mM] and PD-98059 [ERK inhibitor; 50 μM] did not inhibit SP-mediated induction of IL-1α (data not shown).
PGE2 and EGF induces IL-1α expression in HeLa neoplastic cervical epithelial cells via their cognate receptors
Human SP is a complex organic fluid comprising of a vast diversity of antigenically distinct molecules that include prostaglandins and growth factors [26, 27]. We therefore hypothesized that PGE2 and EGF can induce IL-1α expression. To investigate the role of PGE2 and EGF on IL-1α expression, we treated HeLa cells with vehicle or PGE2 [300 nM] or human recombinant EGF [10 ng/mL] alone or together for 4, 8, 16, and 24 hours and determined IL-1α mRNA expression using qPCR. Treatment of HeLa cells with PGE2 (Figure 4A) and EGF (Figure 4B) resulted in a 2.49 ± 0.66 and 5.76 ± 0.80 maximum fold increase after 8 and 4 hours, respectively. Treatment of HeLa cells with both PGE2 and EGF together (Figure 4C) resulted in 2.39 ± 0.52, 6.60 ± 0.63, 16.31 ± 1.23 and 10.88 ± 1.52 fold increase after 4, 8, 16, and 24 hours, respectively. With peak IL-1α mRNA induction observed after 16 hours treatment (16.31 ± 1.23 fold increase). Furthermore, the inductions of IL-1α mRNA at 8, 16, and 24 hours by both ligands together was greater than by each ligand on its own and suggesting that PGE2 and EGF act synergistically in inducing the increase of IL-1α production by HeLa cells.
EP2 receptor antagonist, EGFR and PI3 kinase inhibitors inhibit PGE2 and EGF mediated induction of IL-1α in HeLa neoplastic cervical epithelial cells
Seminal plasma phosphorylates AKT via EP2/EGFR/PI3 kinase signaling to induce IL-1α expression in HeLa neoplastic cervical epithelial cells
SP induces the expression of IL-1α in the cervix via EP2/EGFR/PI3 kinase activation
Inflammation is a characterized biological response of vascularized tissues to harmful stimuli, including chemical irritants or microbial pathogens . Over the years, the role of inflammation as an etiological factor for cancer has been supported by findings that show that regular use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced incidence of certain cancers . Hence, inflammatory responses within tumor microenvironment are now recognized as a critical component for tumor progression and one of the major hall-marks of cancer [31, 32]. Despite the numerous experimental and epidemiological evidence that supports the causal relationship between inflammation and cancer, the molecular mechanisms and pathways linking inflammation and cancer remain poorly understood . The inflammatory milieu of most cancer microenvironments consist of various cells (tumor, surrounding stromal, immune and inflammatory) which all interact intimately to produce cytokines/chemokines, growth factors, and adhesion molecules in a bid to promote inflammation, tumorigenesis and metastasis . Of special relevance within this milieu are pro-inflammatory cytokines which are important mediators of chronic inflammatory responses, and have cardinal effects on malignant processes  as a result of their direct involvement in carcinogenesis, malignant transformation, tumor growth, invasion, and metastasis .
Cervical cancer is a chronic inflammatory disease and one of the leading causes of cancer-related death worldwide with a higher incidence rate reported in underdeveloped countries . It is well established that persistent infection with high-risk HPV is crucial to disease pathogenesis . However, only a subset of women infected with high-risk HPV will proceed to develop invasive cervical cancer, thus suggesting that other co-factors must be present for the development of malignancy . Studies have reported an association between the level of cervical inflammation and the development of high grade cervical neoplasia  or invasive cervical cancer . It has been reported that cervical inflammation, but not the actual diagnosis of a specific sexually transmitted infection is associated with the development of squamous intraepithelial lesions within the cervix . Direct links between increased pro-inflammatory cytokine levels in patients and increasing grade of cervical intraepithelial neoplasia and invasive cervical cancer have been established .
Using immunohistochemistry and qPCR, this present study investigated the expression of pro-inflammatory cytokine IL-1α in normal and neoplastic cervical tissue. Data presented confirmed the elevated expression of IL-1α in cervical cancer. This is in agreement with a similar study by Pao et al. (1995), where it was reported that neoplastic cervical tissue overtly expresses IL-1α . In addition, these data suggests a similar pattern of IL-1α expression in cervical cancer as demonstrated in other malignancies [40–42]. IL-1α is a pleiotropic pro-inflammatory cytokine and a member of the IL-1 family. Within the human body, IL-1α mediates normal physiological functions ranging from induction of vascular permeability and fever during sepsis to increased secretion of additional cytokines in autoimmune diseases . The production and level of IL-1α expression is elevated in numerous cancers including head and neck , breast , pancreatic , and gastric cancer  and has been associated with virulent tumor phenotype and poorer prognosis via the regulation of inflammatory genes and growth factors to enhance tumor growth and differentiation [46–48] and metastatic potential of cells . Animal studies have further confirmed the role of IL-1α in tumor development and blood vessel growth . Similarly, studies by Woodworth et al.  and Castrilli et al.  showed that IL-1α promotes in vitro growth and proliferation of both normal and human papillomavirus-immortalized and carcinoma-derived cervical epithelial cells. It is therefore probable that expression of IL-1α in cervical cancers can act via similar manner to confer virulent tumor phenotype and poorer prognosis in these patients.
IL-1α can be regulated by a host of inflammatory stimuli and recent studies have shown that seminal plasma can regulate IL-1α expression in the human cervix, post coitus [24, 52]. Conventionally, human seminal plasma was regarded primarily as a transport and survival medium for the mammalian spermatozoa traversing the cervix and the uterus during and post coitus [26, 53]. However, experimental studies using animal models shows that in addition to its role as a primary transport medium for the spermatozoa, seminal plasma also introduce to the female reproductive tract an array of antigenically distinct signaling molecules including prostaglandins, several cytokines and growth factors [26, 53–55]. These molecules interact with cognate receptors on the epithelial lining of the female reproductive tract to initiate local cellular and molecular changes reminiscent of an inflammatory response . These changes are required for maternal immune adaptation to pregnancy and for the generation of immune tolerance against fetal antigens [26, 56]. However the molecular pathway by which seminal plasma mediates the expression of IL-1α and other cytokines is yet to be fully elucidated. Hence, using HeLa (adenocarcinoma) cells, normal and neoplastic cervical tissue explants this study investigated the role of SP in the regulation of IL-1α expression in the cervix and transduction pathways by which SP induces the expression of IL-1α in neoplastic and normal cervical epithelium. In addition this study investigated PGE2 and EGF as possible ligands mediating SP induction of IL-1α in neoplastic cervical cells.
In the present study we show that exposure of HeLa neoplastic cervical epithelial cells to SP in vitro increases the expression of IL-1α. In a similar manner, SP was found to increase the expression of IL-1α in both normal and neoplastic cervical tissue explants. This in agreement with studies by Sharkey et al. (2007 and 2012) where it was reported that SP after deposition into the female reproductive tract at coitus induces the expression of pro-inflammatory cytokines including IL-1α in the cervix [24, 52] and initiates an inflammatory response. Hence it is likely that in sexually active women with underlying cervical pathology, recurrent inflammation consequent of SP-mediated IL-1α expression may enhance disease progression. Having shown that SP regulates IL-1α expression in the normal and neoplastic cervical tissue and epithelial cells, we next investigated possible signal transduction pathways by which SP mediates this role.
Employing HeLa cell line as a model, we discovered that SP induced the expression of IL-1α via the EP2 receptor, EGFR and PI3 kinase pathways since EP2 receptor antagonist (AH6809) and the inhibitors of EGFR kinase (AG1478) and PI3 kinase (LY294002) inhibited SP mediated induction of IL-1α in these neoplastic cells. In contrast, PTGS1 and PTGS2 were not shown to have a role in the induction of IL-1α by SP, since addition of their inhibitors (SC-560 and NS-398, respectively) did not reduce the induction of IL-1α. In addition, we found that SP-mediated induction of IL-1α in normal cervical tissue explant was also inhibited in the presence of the antagonist and these inhibitors. Similar in vitro studies by Battersby et al. (2006), Muller et al. (2006) and Sales et al. (2012) have shown that SP-mediated expression of pro-inflammatory and angiogenic genes in endometrial and cervical adenocarcinoma cells was significantly inhibited in the presence of AH6809  and AG-1478 (EGFR kinase inhibitor) [58, 59]. The inhibition of SP-mediated IL-1α by EP2 antagonist and EGFR kinase inhibitors suggested that the effects we observed were mediated by PGE2 and EGF present in the SP.
PGE2 has been established as the predominant PG found in SP . In the present study, we show that PGE2-mediated activation of IL-1α occurs via activation of the EP2, EGFR and Akt pathways. The role of the EP2 receptor in mediating these effects was further confirmed using the selective EP2 agonist butaprost. This is consistent with similar study by Shao et al. (2007) where it was shown that PGE2, acting via EP2 receptor activate cAMP/PKA pathway to mediate the expression of IL-1α in colon cancer cells in an autocrine/paracrine mechanism . EP2 and its signaling have been found to be up-regulated in cervical cancer  and its role in the induction of IL-1α in cervical cancer could explain the greater expression of IL-1α in cervical cancer tissue explant relative to normal cervical tissue. These data suggest that PGE2 in SP [26, 60, 61] can act via its E-series PGs receptor EP2 receptor to directly transactivate EGFR via an intracellular signaling mechanism, either by phosphorylation of cSRC or by the MMP-mediated release of heparin-bound EGF tethered to the cell membrane, [57, 59] leading to IL-1α induction. In addition to PGE2, SP has been shown to be rich in growth factors including epidermal growth factor (EGF) [62–64]. Data presented herein showed that EGF can induce IL-1α in neoplastic cervical HeLa cells. This in agreement with similar in vitro study by Hamilton et al. (2003) where it was shown that EGF induces the expression of pro-inflammatory cytokine in lung cancer cells, and the expression of this cytokine was suppressed in the presence of EGFR inhibitor (AG-1478) . It is therefore very feasible that the EGFR expressed on the membrane of these neoplastic cells can be directly activated by EGF in SP to induce IL-1α expression. The evidence that IL-1α induction by SP is due to PGE2 and EGF present in SP is in agreement with findings of Sharkey et al. (2012) who demonstrated that SP-mediated IL-1α induction in Ect1 cells occurred independently of TGF-β1, TGF-β2, and TGF-β3 which are abundant in SP .
Furthermore, our data show that concurrent treatment of HeLa S3 cells with PGE2 and EGF directed a sustained and elevated increase in IL-1α expression compared to either ligand alone or butaprost and EGF. Prior study by Sales et al. (2002) showed that PGE2 significantly induces the expression of EP4 receptor in HeLa cells , increased expression of EP4 receptor in the presence of EGF could explain the sustained and elevated increase in IL-1α expression mediated by PGE2 and EGF in these cells. Furthermore, transactivation of EGFR via PGE2-EP2 and PGE2-EP4 signaling could also augment EGF induction. In addition, PGE2 has been shown to induce the expression of amphiregulin , a ligand for EGFR and activates EGFR signaling. Taken together, it is likely that the effects of SP on IL-1α induction may be mediated by a combination of PGE2 and EGF working in synergy. Once released, IL-1α can act in an autocrine/paracrine manner within the site of production to regulate inflammation and tumorigenesis. Indeed, Shao and colleagues showed in their study that IL-1α stimulates the migration of colon cancer cells . It is therefore plausible that in sexually active women with underlying pre-invasive or invasive cervical condition, repeated exposure of the elevated EP2 receptor expressed on the neoplastic cervical epithelial cells  to PGE2 present in seminal plasma could enhance tumorigenesis following ligand-receptor binding and activation of similar intracellular signaling pathway to induce IL-1α expression. Expressed IL-1α can then stimulate cervical cancer cell migration to adjacent structures within the pelvis and perineum, hence conferring poor prognosis.
Several studies have shown that PI3 kinase-Akt signaling is deregulated in many cancers including cervical cancer where amplification of the p110α catalytic subunit has been reported [69–72]. Interestingly in this present study, SP and its constituents (PGE2 and EGF) have been shown to mediate IL-1α expression in neoplastic cervical epithelial cells via the activation of PGE2-EP2-EGFR-PI3 kinase pathways. Once activated Akt phosphorylates proteins on serine and threonine residues resulting in the modulation of multiplicity of downstream substrates, including NF-κB involved in the regulation of cell proliferation and survival [73–75]. The SP-mediated Akt phosphorylation seen in this study may act via similar mechanism to induce IL-1α expression. The SP-mediated induction of a pleotropic pro-inflammatory cytokine IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women . Furthermore, this present study is the first to demonstrate that SP regulates pro-inflammatory cytokine IL-1α expression in normal and neoplastic cervical cells via the induction of the EP2-EGFR-PI3 kinase-Akt pathways.
Materials and methods
Ethic approval for this study was obtained from the University of Cape Town Human Research Ethics Committee (HREC/REF: 067/2011). Written informed consent was obtained from all individuals before sample collection.
Chemicals and reagents
Cell culture media, penicillin-streptomycin, and fetal-calf serum were purchased from Highveld Biological (PTY) Limited (Cape Town, South Africa). Bovine serum albumin (BSA), phosphate buffered saline (PBS), and Trizol® were all purchased from Sigma Chemical Company (Cape Town, South Africa). The chemical inhibitors: AG1478, epidermal growth factor receptor (EGFR) kinase inhibitor; LY294002, phosphoinositide-3-kinase (PI3K) inhibitor; SC560, prostaglandin synthase-1 (PTGS-1) inhibitor; NS398, prostaglandin synthase-2 (PTGS-2) inhibitor; and PD98059, extracellular signal-regulated kinases 1/2 kinase (ERK1/2) inhibitor were purchased from Calbiochem (Merck, Darmstadt, Germany). Prostaglandin E2, Butaprost, PGE2 receptor antagonist (AH6809), and human recombinant epidermal growth factor (EGF) were purchased from Sigma Chemical Company (Cape Town, South Africa). Quantikine® Human IL-1α ELISA kit was purchased from R&D Systems, Minneapolis, USA. Polyclonal goat anti-IL-1α (sc-1253) and biotin conjugated secondary donkey anti-goat IgG antibody (sc-2042) were purchased from Santa Cruz Biotechnology. Streptavidin-biotin peroxidase complex and 3.3’-diaminobenzidine were all purchased from Dako North America Incorporation, USA. Total PKB (Akt) (9272 s) and phosphorylated PKB (S473) antibodies were purchased from Cell Signaling Technology. Pierce® BCA Protein Assay Kit and SuperSignal® West Pico Chemiluminescent Substrates were purchased from ThermoScientific, Rockford, USA and PVDF membrane was purchased from GE Healthcare, Amersham, United Kingdom.
Semen donors and preparation
Semen was collected from 10 healthy male volunteers attending the Andrology Laboratory of the Reproductive Medicine unit at Groote Schuur Hospital, Cape Town, South Africa. All donors had at least 72 hours of total sexual abstinence (self-reported) prior to ejaculation. Ejaculates were collected in sterile specimen jars following a voluntary self-masturbation. Parameters such as sample volume, sperm number, sperm concentration, peroxidase-positive leukocytes, pH and viscosity were noted and compared with the 2010 WHO (World Health Organization) reference values for human semen characteristics . Samples with below average parameters were excluded from the study. All samples were processed within 30 minutes of collection. The individual ejaculates were transported to the laboratory and were pooled. Seminal plasma (SP) was isolated from the pooled ejaculate by centrifugation at 15000 g for 20 minutes. Seminal plasma was then aliquoted (200 μL) and stored at −80°C until required. Prior to use seminal plasma was thawed on ice and diluted in sterile filtered serum free medium to use at a concentration of 1:50. SP has been shown to exert no toxic effect on HeLa cell viability up to and including concentration used in this study [4, 77].
Cervical tissue collection and processing
Histological typing, extent of invasiveness, and FIGO staging of carcinoma biopsies
0; carcinoma in-situ
IA; moderately differentiated
IA1; moderately differentiated
IB1; well differentiated
IB1; moderately differentiated
IIIB; moderately differentiated
Cell culture and treatments
HeLa-S3 (HeLa) authenticated and verified as cervical adenocarcinoma cells positive for HPV-18 sequence with normal levels of pRB (retinoblastoma) and low levels of p53 tumour suppressor, were purchased from BioWhittaker (Berkshire, UK). Cells were routinely maintained in DMEM nutrient mixture F-12 with Glutamax-1 and pyroxidine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (500 IU/ml penicillin and 500 μg/ml streptomycin) at 37°C and 5% CO2 (v/v). For experiments, HeLa-S3 cells were seeded in medium supplemented with 10% FBS at density of 2 × 105 cells in 3 cm diameter tissue culture dishes and allowed to attach and grow overnight after which cells were serum starved by incubating in serum free medium for 24 hours. Cells were then treated with vehicle (PBS) or SP at a dilution of 1:50 or butaprost [5 μM] or PGE2 [300 nM] or EGF [10 ng/ML] for 4, 8, 16, and 24 hours. For receptor blockade and inhibitor experiments, serum starved cells were treated with receptor antagonist/inhibitors alone or SP (1:50 dilution) alone or together for 4 or 16 hours. The antagonist and inhibitors used and their final concentrations were: EP2 receptor antagonist [AH-6809; 20 μM], inhibitors of EGFR [AG-1478; 100 nM], PI3 kinase [LY-294002; 25 μM], PTGS1 [SC-560; 15 μM], and PTGS2 [NS-398 8 μM]. The concentrations of chemical inhibitors used in this study were determined empirically by titration as described [80–83]. At the concentration and time used, the antagonist or inhibitors showed no adverse effects on cell viability when stained with 0.4% trypan blue dye. Fold increases was calculated by dividing the values obtained from the SP/SP-inhibitor treatments by the vehicle/vehicle-inhibitor treatments.
Real –Time quantitative RT-PCR
List of real-time quantitative RT-PCR primers
Enzyme linked immunosorbent assay
Quantikine® Human IL-1α ELISA kit was used to assess IL-1α protein expression. The experiment was done on HeLa S3 cells seeded at a density of 5 × 105 in 3 cm tissue culture dishes and serum starved overnight. The cells were then treated with SP (1:50) or PBS (control) for 16 and 24 hrs. Cells were lysed as described previously  and total protein quantified using Pierce® BCA Protein Assay Kit. Expressed cellular IL-1α protein was determined from the total protein in the lysate. Data are presented as fold change over control treated, which was calculated by dividing the amount of IL-1α measured in SP treated cells at the different time points by the amount measured in their respective controls. Data are presented as mean ± SEM of six independent experiments.
SDS-PAGE and Western blot analysis
Immunoblot analysis was performed on solubilized cell lysates of cultured HeLa cells that were initially separated on a 10% SDS-PAGE gel that was run at 100 volts. Thereafter, separated proteins in the gel were transferred onto PVDF membrane and subjected to immunoblot analysis. Membranes were incubated in 10 mL of blocking buffer (PBS, 0.1% Tween-20 with 5% w/v nonfat dry milk) on a shaker for 1 hour at room temperature (RT), after which membranes were washed three times for 10 minutes each with 15 mL PBS, 0.1% Tween-20 followed by an overnight incubation with rabbit anti-PKB (AKT) and anti-P-PKB (AKT) (1:1000 dilution) at 4°C with gentle shaking. Thereafter, membranes were washed three times for 10 minutes each with 15 mL of PBS, 0.1% Tween-20, incubated for 1 hour with anti-Rabbit HRP-conjugated secondary antibody (1:5000 dilution) at RT with gentle shaking and washed again three times with 15 mL PBS, 0.1% Tween-20. Protein detection was done by chemiluminescence; membranes were incubated with SuperSignal® West Pico Chemiluminescent Substrate (1 mL of SuperSignal West Pico Luminol/Enhancer Solution and 1 mL SuperSignal West Pico Stable Peroxide Solution) for 5 minutes at RT and proteins viewed with BioSpectrum™ 500 Imaging System (Ultra-Violet Products [UVP] Limited, Cambridge, UK). Densitometry on visualized protein bands was done using ImageJ version IJ 1.46r (http://www.imagej.nih.gov/ij/). Akt phosphorylation was calculated by dividing the value obtained from phosphorylated Akt channel by the value obtained from total Akt channel and expressed as fold above vehicle controls. Data are presented as mean ± SEM of three independent experiments.
Immunohistochemistry was done on archival cervical blocks (Normal n = 5, squamous cell carcinoma n = 4 and adenocarcinoma n = 4) obtained from the Department of Anatomical Pathology, University of Cape Town. Briefly, sections were deparaffinized and rehydrated by immersing in xylene twice for 5 minutes, 100% ethanol twice for 5 minute, 95% ethanol for 5 minutes, 70% ethanol for 5 minutes, 50% ethanol for 5 minutes and rinsed with water. Antigen retrieval was done by pressure cooking for 2 minutes in 0.01 M sodium citrate pH 6. Sections were blocked for endogenous peroxidase by incubating with 3% Hydrogen peroxide in methanol on a rocker at RT for 30 minutes and then rinsed with water followed by 1× TBS (50 mM Tris–HCl, 150 mM NaCl at pH 7.4). Sections were blocked using 5% normal donkey/goat serum diluted in TBS after which tissue sections were incubated with polyclonal goat anti-IL-1α (1:200) antibodies at 4°C for 18 hours. After incubation, tissue sections were then washed in TBS twice for 5 minutes each followed by incubation with biotinylated donkey anti-goat secondary IgG antibody at dilution of 1:500 at RT for 30 minutes. Tissue sections were then further incubated with streptavidin-biotin peroxidase complex (1:50) at RT for 30 minutes. Controls were incubated with biotinylated IgG secondary antibody only. Color reaction was developed by incubating with 3.3’-diaminobenzidine. Tissue sections were counterstained in aqueous hematoxylin, before mounting and coverslipping. Images were visualized and photographed using a Carl Zeiss laser scanning microscope LSM 510 (Jena, Germany).
All data in this study were analyzed by t-test or one-way ANOVA using Graph Pad Prism 5.0 software (GraphPad Software Inc., San Diego, CA). Paired T-tests were conducted on the untransformed means of the replicates between SP and control. Unpaired T-tests were performed on SP versus SP and inhibitor after conversion to fold increases. One-way ANOVA was used as an additional tool to determine the significant difference between various time points for IL-1α by real-time PCR in response to SP.
This study was conducted by a grant from Medical Research Council of South Africa (MRC) to the MRC/UCT Receptor Biology Unit and by the following grants to KJS: Poliomyelitis Research Foundation of South Africa (PRF), Cancer Association of South Africa (CANSA), National Research Foundation of South Africa (NRF) and University of Cape Town Research Committee (URC). The funders played no role in the conception, design or interpretation of the results or decision to publish. Our sincere gratitude also goes to all the members of staff of Colposcopy and Gynae-oncology clinic of Groote Schuur Hospital Cape Town for their assistance in patient recruitment.
- Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA Cancer J Clin 2005,55(2):74–108. 10.3322/canjclin.55.2.74View ArticlePubMedGoogle Scholar
- Anorlu RI: Cervical cancer: the sub-Saharan African perspective. Reprod Health Matters 2008,16(32):41–49. 10.1016/S0968-8080(08)32415-XView ArticlePubMedGoogle Scholar
- Arbyn M, Castellsague X, de Sanjose S, Bruni L, Saraiya M, Bray F, Ferlay J: Worldwide burden of cervical cancer in 2008. Ann Oncol 2011,22(12):2675–2686. 10.1093/annonc/mdr015View ArticlePubMedGoogle Scholar
- Sutherland JR, Sales KJ, Jabbour HN, Katz AA: Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo. PLoS One 2012,7(3):e33848. 10.1371/journal.pone.0033848View ArticlePubMed CentralPubMedGoogle Scholar
- Al-Daraji WI, Smith JH: Infection and cervical neoplasia: facts and fiction. Int J Clin Exp Pathol 2009,2(1):48–64.PubMed CentralPubMedGoogle Scholar
- Coussens LM, Werb Z: Inflammation and cancer. Nature 2002,420(6917):860–867. 10.1038/nature01322View ArticlePubMed CentralPubMedGoogle Scholar
- Goswami B, Rajappa M, Sharma M, Sharma A: Inflammation: its role and interplay in the development of cancer, with special focus on gynecological malignancies. Int J Gynecol Cancer 2008,18(4):591–599. 10.1111/j.1525-1438.2007.01089.xView ArticlePubMedGoogle Scholar
- Ryu HS, Chang KH, Yang HW, Kim MS, Kwon HC, Oh KS: High cyclooxygenase-2 expression in stage IB cervical cancer with lymph node metastasis or parametrial invasion. Gynecol Oncol 2000,76(3):320–325. 10.1006/gyno.1999.5690View ArticlePubMedGoogle Scholar
- Sales KJ, Katz AA, Davis M, Hinz S, Soeters RP, Hofmeyr MD, Millar RP, Jabbour HN: Cyclooxygenase-2 expression and prostaglandin E(2) synthesis are up-regulated in carcinomas of the cervix: a possible autocrine/paracrine regulation of neoplastic cell function via EP2/EP4 receptors. J Clin Endocrinol Metab 2001,86(5):2243–2249. 10.1210/jcem.86.5.7442View ArticlePubMed CentralPubMedGoogle Scholar
- Sales KJ, Katz AA, Howard B, Soeters RP, Millar RP, Jabbour HN: Cyclooxygenase-1 is up-regulated in cervical carcinomas: autocrine/paracrine regulation of cyclooxygenase-2, prostaglandin e receptors, and angiogenic factors by cyclooxygenase-1. Cancer Res 2002,62(2):424–432.PubMed CentralPubMedGoogle Scholar
- Chulada PC, Thompson MB, Mahler JF, Doyle CM, Gaul BW, Lee C, Tiano HF, Morham SG, Smithies O, Langenbach R: Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice. Cancer Res 2000,60(17):4705–4708.PubMedGoogle Scholar
- Jabbour HN, Sales KJ: Prostaglandin receptor signalling and function in human endometrial pathology. Trends Endocrinol Metab 2004,15(8):398–404. 10.1016/j.tem.2004.08.006View ArticlePubMedGoogle Scholar
- Lewis AM, Varghese S, Xu H, Alexander HR: Interleukin-1 and cancer progression: the emerging role of interleukin-1 receptor antagonist as a novel therapeutic agent in cancer treatment. J Transl Med 2006, 4:48. 10.1186/1479-5876-4-48View ArticlePubMed CentralPubMedGoogle Scholar
- Apte RN, Voronov E: Interleukin-1–a major pleiotropic cytokine in tumor-host interactions. Semin Cancer Biol 2002,12(4):277–290. 10.1016/S1044-579X(02)00014-7View ArticlePubMedGoogle Scholar
- Dinarello CA: Interleukin-1 and interleukin-1 antagonism. Blood 1991,77(8):1627–1652.PubMedGoogle Scholar
- Dinarello CA: Biologic basis for interleukin-1 in disease. Blood 1996,87(6):2095–2147.PubMedGoogle Scholar
- Auron PE: The interleukin 1 receptor: ligand interactions and signal transduction. Cytokine Growth Factor Rev 1998,9(3–4):221–237.View ArticlePubMedGoogle Scholar
- Shao J, Sheng H: Prostaglandin E2 induces the expression of IL-1alpha in colon cancer cells. J Immunol 2007,178(7):4097–4103. 10.4049/jimmunol.178.7.4097View ArticlePubMedGoogle Scholar
- Arend WP, Palmer G, Gabay C: IL-1, IL-18, and IL-33 families of cytokines. Immunol Rev 2008, 223:20–38. 10.1111/j.1600-065X.2008.00624.xView ArticlePubMedGoogle Scholar
- Konishi N, Miki C, Yoshida T, Tanaka K, Toiyama Y, Kusunoki M: Interleukin-1 receptor antagonist inhibits the expression of vascular endothelial growth factor in colorectal carcinoma. Oncology 2005,68(2–3):138–145.View ArticlePubMedGoogle Scholar
- Owen DH, Katz DF: A review of the physical and chemical properties of human semen and the formulation of a semen simulant. J Androl 2005,26(4):459–469. 10.2164/jandrol.04104View ArticlePubMedGoogle Scholar
- Robertson SA, Mayrhofer G, Seamark RF: Uterine epithelial cells synthesize granulocyte-macrophage colony-stimulating factor and interleukin-6 in pregnant and nonpregnant mice. Biol Reprod 1992,46(6):1069–1079. 10.1095/biolreprod46.6.1069View ArticlePubMedGoogle Scholar
- O'Leary S, Jasper MJ, Warnes GM, Armstrong DT, Robertson SA: Seminal plasma regulates endometrial cytokine expression, leukocyte recruitment and embryo development in the pig. Reproduction 2004,128(2):237–247. 10.1530/rep.1.00160View ArticlePubMedGoogle Scholar
- Sharkey DJ, Macpherson AM, Tremellen KP, Robertson SA: Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells. Mol Hum Reprod 2007,13(7):491–501. 10.1093/molehr/gam028View ArticlePubMedGoogle Scholar
- Templeton AA, Cooper I, Kelly RW: Prostaglandin concentrations in the semen of fertile men. J Reprod Fertil 1978,52(1):147–150. 10.1530/jrf.0.0520147View ArticlePubMedGoogle Scholar
- Robertson SA: Seminal plasma and male factor signalling in the female reproductive tract. Cell Tissue Res 2005,322(1):43–52. 10.1007/s00441-005-1127-3View ArticlePubMedGoogle Scholar
- Ness RB, Grainger DA: Male reproductive proteins and reproductive outcomes. Am J Obstet Gynecol 2008,198(6):620.e621–624.Google Scholar
- Jiang BH, Liu LZ: PI3K/PTEN signaling in tumorigenesis and angiogenesis. Biochim Biophys Acta 2008,1784(1):150–158. 10.1016/j.bbapap.2007.09.008View ArticlePubMedGoogle Scholar
- Greene ER, Huang S, Serhan CN, Panigrahy D: Regulation of inflammation in cancer by eicosanoids. Prostaglandins Other Lipid Mediat 2011,96(1–4):27–36.View ArticlePubMed CentralPubMedGoogle Scholar
- Apte RN, Dotan S, Elkabets M, White MR, Reich E, Carmi Y, Song X, Dvozkin T, Krelin Y, Voronov E: The involvement of IL-1 in tumorigenesis, tumor invasiveness, metastasis and tumor-host interactions. Cancer Metastasis Rev 2006,25(3):387–408. 10.1007/s10555-006-9004-4View ArticlePubMedGoogle Scholar
- Mantovani A: Cancer: Inflaming metastasis. Nature 2009,457(7225):36–37.View ArticlePubMedGoogle Scholar
- Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 2011,144(5):646–674. 10.1016/j.cell.2011.02.013View ArticlePubMedGoogle Scholar
- Beral V, Hermon C, Munoz N, Devesa SS: Cervical cancer. Cancer Surv 1994, 19–20:265–285.PubMedGoogle Scholar
- Smith JS, Herrero R, Bosetti C, Munoz N, Bosch FX, Eluf-Neto J, Castellsague X, Meijer CJ, Van den Brule AJ, Franceschi S, Ashley R: Herpes simplex virus-2 as a human papillomavirus cofactor in the etiology of invasive cervical cancer. J Natl Cancer Inst 2002,94(21):1604–1613. 10.1093/jnci/94.21.1604View ArticlePubMedGoogle Scholar
- Castle PE, Hillier SL, Rabe LK, Hildesheim A, Herrero R, Bratti MC, Sherman ME, Burk RD, Rodriguez AC, Alfaro M, Hutchinson ML, Morales J, Schiffman M: An association of cervical inflammation with high-grade cervical neoplasia in women infected with oncogenic human papillomavirus (HPV). Cancer Epidemiol Biomarkers Prev 2001,10(10):1021–1027.PubMedGoogle Scholar
- Yang YC, Chang CL, Huang YW, Wang DY: Possible cofactor in cervical carcinogenesis: proliferation index of the transformation zone in cervicitis. Chang Gung Med J 2001,24(10):615–620.PubMedGoogle Scholar
- Schwebke JR, Zajackowski ME: Effect of concurrent lower genital tract infections on cervical cancer screening. Genitourin Med 1997,73(5):383–386.PubMed CentralPubMedGoogle Scholar
- Sharma A, Rajappa M, Saxena A, Sharma M: Cytokine profile in Indian women with cervical intraepithelial neoplasia and cancer cervix. Int J Gynecol Cancer 2007,17(4):879–885. 10.1111/j.1525-1438.2007.00883.xView ArticlePubMedGoogle Scholar
- Pao CC, Lin CY, Yao DS, Tseng CJ: Differential expression of cytokine genes in cervical cancer tissues. Biochem Biophys Res Commun 1995,214(3):1146–1151. 10.1006/bbrc.1995.2405View ArticlePubMedGoogle Scholar
- Kurtzman SH, Anderson KH, Wang Y, Miller LJ, Renna M, Stankus M, Lindquist RR, Barrows G, Kreutzer DL: Cytokines in human breast cancer: IL-1alpha and IL-1beta expression. Oncol Rep 1999,6(1):65–70.PubMedGoogle Scholar
- Chen Z, Colon I, Ortiz N, Callister M, Dong G, Pegram MY, Arosarena O, Strome S, Nicholson JC, Van Waes C: Effects of interleukin-1alpha, interleukin-1 receptor antagonist, and neutralizing antibody on proinflammatory cytokine expression by human squamous cell carcinoma lines. Cancer Res 1998,58(16):3668–3676.PubMedGoogle Scholar
- Elaraj DM, Weinreich DM, Varghese S, Puhlmann M, Hewitt SM, Carroll NM, Feldman ED, Turner EM, Alexander HR: The role of interleukin 1 in growth and metastasis of human cancer xenografts. Clin Cancer Res 2006,12(4):1088–1096. 10.1158/1078-0432.CCR-05-1603View ArticlePubMedGoogle Scholar
- Wolf JS, Chen Z, Dong G, Sunwoo JB, Bancroft CC, Capo DE, Yeh NT, Mukaida N, Van Waes C: IL (interleukin)-1alpha promotes nuclear factor-kappaB and AP-1-induced IL-8 expression, cell survival, and proliferation in head and neck squamous cell carcinomas. Clin Cancer Res 2001,7(6):1812–1820.PubMedGoogle Scholar
- Tang RF, Wang SX, Zhang FR, Peng L, Xiao Y, Zhang M: Interleukin-1alpha, 6 regulate the secretion of vascular endothelial growth factor A, C in pancreatic cancer. Hepatobiliary Pancreat Dis Int 2005,4(3):460–463.PubMedGoogle Scholar
- Tomimatsu S, Ichikura T, Mochizuki H: Significant correlation between expression of interleukin-1alpha and liver metastasis in gastric carcinoma. Cancer 2001,91(7):1272–1276. View ArticlePubMedGoogle Scholar
- Dinarello CA, Wolff SM: The role of interleukin-1 in disease. N Engl J Med 1993,328(2):106–113. 10.1056/NEJM199301143280207View ArticlePubMedGoogle Scholar
- Tracey KJ, Cerami A: Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol 1993, 9:317–343. 10.1146/annurev.cb.09.110193.001533View ArticlePubMedGoogle Scholar
- Castrilli G, Tatone D, Diodoro MG, Rosini S, Piantelli M, Musiani P: Interleukin 1alpha and interleukin 6 promote the in vitro growth of both normal and neoplastic human cervical epithelial cells. Br J Cancer 1997,75(6):855–859. 10.1038/bjc.1997.152View ArticlePubMed CentralPubMedGoogle Scholar
- Sawai H, Funahashi H, Yamamoto M, Okada Y, Hayakawa T, Tanaka M, Takeyama H, Manabe T: Interleukin-1alpha enhances integrin alpha(6)beta(1) expression and metastatic capability of human pancreatic cancer. Oncology 2003,65(2):167–173. 10.1159/000072343View ArticlePubMedGoogle Scholar
- Voronov E, Shouval DS, Krelin Y, Cagnano E, Benharroch D, Iwakura Y, Dinarello CA, Apte RN: IL-1 is required for tumor invasiveness and angiogenesis. Proc Natl Acad Sci U S A 2003,100(5):2645–2650. 10.1073/pnas.0437939100View ArticlePubMed CentralPubMedGoogle Scholar
- Woodworth CD, McMullin E, Iglesias M, Plowman GD: Interleukin 1 alpha and tumor necrosis factor alpha stimulate autocrine amphiregulin expression and proliferation of human papillomavirus-immortalized and carcinoma-derived cervical epithelial cells. Proc Natl Acad Sci U S A 1995,92(7):2840–2844. 10.1073/pnas.92.7.2840View ArticlePubMed CentralPubMedGoogle Scholar
- Sharkey DJ, Tremellen KP, Jasper MJ, Gemzell-Danielsson K, Robertson SA: Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus. J Immunol 2012,188(5):2445–2454. 10.4049/jimmunol.1102736View ArticlePubMedGoogle Scholar
- Aumuller G, Riva A: Morphology and functions of the human seminal vesicle. Andrologia 1992,24(4):183–196.View ArticlePubMedGoogle Scholar
- Maegawa M, Kamada M, Irahara M, Yamamoto S, Yoshikawa S, Kasai Y, Ohmoto Y, Gima H, Thaler CJ, Aono T: A repertoire of cytokines in human seminal plasma. J Reprod Immunol 2002,54(1–2):33–42.View ArticlePubMedGoogle Scholar
- Fung KY, Glode LM, Green S, Duncan MW: A comprehensive characterization of the peptide and protein constituents of human seminal fluid. Prostate 2004,61(2):171–181. 10.1002/pros.20089View ArticlePubMedGoogle Scholar
- Robertson SA, Guerin LR, Moldenhauer LM, Hayball JD: Activating T regulatory cells for tolerance in early pregnancy - the contribution of seminal fluid. J Reprod Immunol 2009,83(1–2):109–116.View ArticlePubMedGoogle Scholar
- Battersby S, Sales KJ, Williams AR, Anderson RA, Gardner S, Jabbour HN: Seminal plasma and prostaglandin E2 up-regulate fibroblast growth factor 2 expression in endometrial adenocarcinoma cells via E-series prostanoid-2 receptor-mediated transactivation of the epidermal growth factor receptor and extracellular signal-regulated kinase pathway. Hum Reprod 2007,22(1):36–44.View ArticlePubMed CentralPubMedGoogle Scholar
- Sales KJ, Sutherland JR, Jabbour HN, Katz AA: Seminal plasma induces angiogenic chemokine expression in cervical cancer cells and regulates vascular function. Biochim Biophys Acta 2012,1823(10):1789–1795. 10.1016/j.bbamcr.2012.06.021View ArticlePubMedGoogle Scholar
- Muller M, Sales KJ, Katz AA, Jabbour HN: Seminal plasma promotes the expression of tumorigenic and angiogenic genes in cervical adenocarcinoma cells via the E-series prostanoid 4 receptor. Endocrinology 2006,147(7):3356–3365. 10.1210/en.2005-1429View ArticlePubMedGoogle Scholar
- Politch JA, Tucker L, Bowman FP, Anderson DJ: Concentrations and significance of cytokines and other immunologic factors in semen of healthy fertile men. Hum Reprod 2007,22(11):2928–2935. 10.1093/humrep/dem281View ArticlePubMedGoogle Scholar
- Kelly RW: Immunosuppressive mechanisms in semen: implications for contraception. Hum Reprod 1995,10(7):1686–1693.PubMedGoogle Scholar
- Elson SD, Browne CA, Thorburn GD: Identification of epidermal growth factor-like activity in human male reproductive tissues and fluids. J Clin Endocrinol Metab 1984,58(4):589–594. 10.1210/jcem-58-4-589View ArticlePubMedGoogle Scholar
- Fuse H, Sakamoto M, Okumura M, Katayama T: Epidermal growth factor contents in seminal plasma as a marker of prostatic function. Arch Androl 1992,29(1):79–85. 10.3109/01485019208987712View ArticlePubMedGoogle Scholar
- Hirata Y, Uchihashi M, Hazama M, Fujita T: Epidermal growth factor in human seminal plasma. Horm Metab Res 1987,19(1):35–37. 10.1055/s-2007-1011730View ArticlePubMedGoogle Scholar
- Hamilton LM, Torres-Lozano C, Puddicombe SM, Richter A, Kimber I, Dearman RJ, Vrugt B, Aalbers R, Holgate ST, Djukanovic R, Wilson SJ, Davies DE: The role of the epidermal growth factor receptor in sustaining neutrophil inflammation in severe asthma. Clin Exp Allergy 2003,33(2):233–240. 10.1046/j.1365-2222.2003.01593.xView ArticlePubMedGoogle Scholar
- Sharkey DJ, Macpherson AM, Tremellen KP, Mottershead DG, Gilchrist RB, Robertson SA: TGF-beta mediates proinflammatory seminal fluid signaling in human cervical epithelial cells. J Immunol 2012,189(2):1024–1035. 10.4049/jimmunol.1200005View ArticlePubMedGoogle Scholar
- Sales KJ, Katz AA, Millar RP, Jabbour HN: Seminal plasma activates cyclooxygenase-2 and prostaglandin E2 receptor expression and signalling in cervical adenocarcinoma cells. Mol Hum Reprod 2002,8(12):1065–1070. 10.1093/molehr/8.12.1065View ArticlePubMed CentralPubMedGoogle Scholar
- Dannenberg AJ, Subbaramaiah K: Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell 2003,4(6):431–436. 10.1016/S1535-6108(03)00310-6View ArticlePubMedGoogle Scholar
- Ma YY, Wei SJ, Lin YC, Lung JC, Chang TC, Whang-Peng J, Liu JM, Yang DM, Yang WK, Shen CY: PIK3CA as an oncogene in cervical cancer. Oncogene 2000,19(23):2739–2744. 10.1038/sj.onc.1203597View ArticlePubMedGoogle Scholar
- Phillips WA, St Clair F, Munday AD, Thomas RJ, Mitchell CA: Increased levels of phosphatidylinositol 3-kinase activity in colorectal tumors. Cancer 1998,83(1):41–47.View ArticlePubMedGoogle Scholar
- Sun M, Wang G, Paciga JE, Feldman RI, Yuan ZQ, Ma XL, Shelley SA, Jove R, Tsichlis PN, Nicosia SV, Cheng JQ: AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol 2001,159(2):431–437. 10.1016/S0002-9440(10)61714-2View ArticlePubMed CentralPubMedGoogle Scholar
- Shayesteh L, Lu Y, Kuo WL, Baldocchi R, Godfrey T, Collins C, Pinkel D, Powell B, Mills GB, Gray JW: PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet 1999,21(1):99–102. 10.1038/5042View ArticlePubMedGoogle Scholar
- Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA: Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995,378(6559):785–789. 10.1038/378785a0View ArticlePubMedGoogle Scholar
- Lawlor MA, Alessi DR: PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci 2001,114(Pt 16):2903–2910.PubMedGoogle Scholar
- Sekulic A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM, Abraham RT: A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 2000,60(13):3504–3513.PubMedGoogle Scholar
- Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, Behre HM, Haugen TB, Kruger T, Wang C, Mbizvo MT, Vogelsong KM: World Health Organization reference values for human semen characteristics. Hum Reprod Update 2010,16(3):231–245. 10.1093/humupd/dmp048View ArticlePubMedGoogle Scholar
- Jeremias J, David SS, Toth M, Witkin SS: Induction of messenger RNA for the 70 kDa heat shock protein in HeLa cells and the human endocervix following exposure to semen: implications for antisperm antibody production and susceptibility to sexually transmitted infections. Hum Reprod 1997,12(9):1915–1919. 10.1093/humrep/12.9.1915View ArticlePubMedGoogle Scholar
- Koss LG: The new Bethesda System for reporting results of smears of the uterine cervix. J Natl Cancer Inst 1990,82(12):988–991. 10.1093/jnci/82.12.988View ArticlePubMedGoogle Scholar
- Pecorelli S, Zigliani L, Odicino F: Revised FIGO staging for carcinoma of the cervix. Int J Gynaecol Obstet 2009,105(2):107–108. 10.1016/j.ijgo.2009.02.009View ArticlePubMedGoogle Scholar
- Sales KJ, Maldonado-Perez D, Grant V, Catalano RD, Wilson MR, Brown P, Williams AR, Anderson RA, Thompson EA, Jabbour HN: Prostaglandin F(2alpha)-F-prostanoid receptor regulates CXCL8 expression in endometrial adenocarcinoma cells via the calcium-calcineurin-NFAT pathway. Biochim Biophys Acta 2009,1793(12):1917–1928. 10.1016/j.bbamcr.2009.09.018View ArticlePubMed CentralPubMedGoogle Scholar
- Sales KJ, Grant V, Jabbour HN: Prostaglandin E2 and F2alpha activate the FP receptor and up-regulate cyclooxygenase-2 expression via the cyclic AMP response element. Mol Cell Endocrinol 2008,285(1–2):51–61.View ArticlePubMed CentralPubMedGoogle Scholar
- Sales KJ, Grant V, Cook IH, Maldonado-Perez D, Anderson RA, Williams AR, Jabbour HN: Interleukin-11 in endometrial adenocarcinoma is regulated by prostaglandin F2alpha-F-prostanoid receptor interaction via the calcium-calcineurin-nuclear factor of activated T cells pathway and negatively regulated by the regulator of calcineurin-1. Am J Pathol 2010,176(1):435–445. 10.2353/ajpath.2010.090403View ArticlePubMed CentralPubMedGoogle Scholar
- Sales KJ, Battersby S, Williams AR, Anderson RA, Jabbour HN: Prostaglandin E2 mediates phosphorylation and down-regulation of the tuberous sclerosis-2 tumor suppressor (tuberin) in human endometrial adenocarcinoma cells via the Akt signaling pathway. J Clin Endocrinol Metab 2004,89(12):6112–6118. 10.1210/jc.2004-0892View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.