Seminal plasma induces angiogenic chemokine expression in cervical cancer cells and regulates vascular function

MRC/UCT Research Group for Receptor Biology, Institute of Infectious Disease and Molecular Medicine and Division of Medical Biochemistry, University of Cape Town, South Africa.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 06/2012; 1823(10):1789-95. DOI: 10.1016/j.bbamcr.2012.06.021
Source: PubMed


Cervical cancer is one of the leading gynecological malignancies in women. We have recently shown that seminal plasma (SP) can regulate the inflammatory cyclooxygenase-prostaglandin pathway and enhance the growth of cervical epithelial tumours in vivo by promoting cellular proliferation and alteration of vascular function. This study investigated the molecular mechanism whereby SP regulates vascular function using an in vitro model system of HeLa cervical adenocarcinoma cells and human umbilical vein endothelial cells (HUVECs). We found that SP rapidly enhanced the expression of the angiogenic chemokines, interleukin (IL)-8 and growth regulated oncogene alpha (GRO) in HeLa cells in a time-dependent manner. We investigated the molecular mechanism of SP-mediated regulation of IL-8 and GRO using a panel of chemical inhibitors of cell signalling. We found that treatment of HeLa cells with SP elevated expression of IL-8 and GRO by transactivation of the epidermal growth factor receptor, activation of extracellular signal-regulated kinase and induction of cyclooxygenase enzymes and nuclear factor kappa B. We investigated the impact of IL-8 and GRO, released from HeLa cells after treatment with SP, on vascular function using a co-culture model system of conditioned medium (CM) from HeLa cells, treated with or without SP, and HUVECs. We found that CM from HeLa cells induced the arrangement of endothelial cells into a network of tube-like structures via the CXCR2 receptor on HUVECs. Taken together our data outline a molecular mechanism whereby SP can alter vascular function in cervical cancers via the pro-angiogenic chemokines, IL-8 and GRO.

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    • "These hallmarks of cancer in cervical cancer cells have now all been shown to be driven by HPV oncogenes via the induction of potent proinflammatory pathways—in particular , by inducing expression of the immediate early oncogene COX-2 and expression of the E-series prostaglandin receptors (PTGER) such as PTGER2 and PTGER4 [14] [15] [16]. In addition to the regulation of tumorigenic pathways by endogenous prostaglandins, produced in cervical cells after HPV infection and neoplastic transformation, the cervix (and vagina) can also be regulated by seminal fluid and in particular seminal plasma prostaglandins, like PGE 2 [25] [26] [27] "
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    ABSTRACT: Theconnection between human papillomavirus (HPV) infection and the consequent sequelae which establishes cervical neoplastic transformation and invasive cervical cancer has redefined many aspects of cervical cancer research. However there is still much that we do not know. In particular, the impact of external factors, like seminal fluid in sexually active women, on pathways that regulate cervical inflammation and tumorigenesis, have yet to be fully understood. HPV infection is regarded as the initiating noninflammatory cause of the disease; however emerging evidence points to resident HPV infections as drivers of inflammatory pathways that play important roles in tumorigenesis as well as in the susceptibility to other infections such as human immunodeficiency virus (HIV) infection.Moreover there is emerging evidence to support a role for seminal fluid, in particular, the inflammatory bioactive lipids, and prostaglandins which are present in vast quantities in seminal fluid in regulating pathways that can exacerbate inflammation of the cervix, speed up tumorigenesis, and enhance susceptibility to HIV infection.This review will highlight some of our current knowledge of the role of seminal fluid as a potent driver of inflammatory and tumorigenic pathways in the cervix and will provide some evidence to propose a role for seminal plasma prostaglandins in HIV infection and AIDS-related cancer.
    Full-text · Article · Aug 2014
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    • "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 [58]. 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. "
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    ABSTRACT: 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-1alpha 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-1alpha in cervical cancer, the role of SP in the regulation of IL-1alpha 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-1alpha mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1alpha 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-alpha 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-1alpha in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1alpha mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1alpha by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling. Conclusion SP-mediated induction of IL-1alpha 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.
    Full-text · Article · Aug 2014 · Journal of Molecular Signaling
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    • "In line with this hypothesis, pro-inflammatory cytokines including IL1b, IL6 and leukemia inhibitory factor (LIF) were induced in endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) following exposure to SP (Gutsche et al., 2003). SP can additionally stimulate potent angiogenic and proliferative effects in both lower and upper FRT tissues (Sales et al., 2012; Sutherland et al., 2012; Kaczmarek et al., 2013). Together , these studies suggest that SP-derived bioactive factors can signal a variety of responses in cells of both the lower and upper FRT. "
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    ABSTRACT: How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced similar sets of pathways, suggesting that SP can model the signaling effects of semen in the endometrium. SP also induced secretion of pro-inflammatory and pro-chemotactic cytokines, as well as pro-angiogenic and proliferative growth factors (P < 0.05) in both eEC and eSF. Finally, functional assays revealed that conditioned media from SP-treated eEC and eSF significantly increased (P < 0.05) chemotaxis of CD14+ monocytes and CD4+ T cells. This study is limited to in vitro analyses of the effects of SP on endometrial cells. In addition, the measured response to SP was conducted in the absence of the ovarian hormones estradiol and progesterone, as well as epithelial-stromal paracrine signaling. While this study focused on establishing the baseline cellular response of endometrial cells to SP, future work should assess how hormone signaling in the presence of appropriate paracrine interactions affects SP-induced genes in these cells. The results of this study support previous findings that SP and semen contain bioactive factors capable of eliciting chemotactic responses in the uterus, which can lead to recruitment of leukocytes to the endometrium. Future directions will explore if similar changes in gene expression do indeed occur after coitus in vivo, and how the signaling cascades initiated by SP in the endometrium can affect reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure. This project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11-1-0562 (W.C.G.); NIH 5K12-DK083021-04, NIH 1K99AI104262-01A1, The UCSF Hellman Award (N.R.R.). The authors have nothing to disclose.
    Full-text · Article · Mar 2014 · Human Reproduction
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