Mechanism And Function Of Osteopontin Via Toll-like Receptors Mediated In Ovarian Cancer Cell Metastatic Phenotype And Invasion

Osteopontin (OPN) is a phosphorylated glycoprotein that binds to the metastasis-associated cell surface receptorsαv-containing integrin as well as CD44, mediating malignant cell attachment, migration and invasion.The osteopontin gene is subject to alternative splicing, which yields three forms of message RNA, the full-length form of osteopontin (called OPN-a) and two splice variants with deletion of exon 4(referred to as OPN-c) or exon 5( named OPN-b). OPN is expressed at high levels by various cancers and has been proposed to utilize as a malignant tumor prognostic marker. Accumulating studies suggest that the OPN expressed in many types of invasive cancer and generates anchorage independent growth. Hence, the OPN may be serves as a diagnostic and prognostic marker that has value in a diagnostic panel together with other conventional tumor markers. The mechanism by which OPN leads to cancer metastasis have been well described, whereas what initial signalings mediate the production of OPN awaits further research.Toll-like receptors are a family of transmembrane receptors which contribute to the processes of host defense, tissue repair and tissue injury-induced inflammation by recognizing conserved Pathogen-associated molecular patterns (PAMPs). It is previously thought that TLRs mainly expressed in human immune-related cells and epithelial cells, but substantial data proposed the concept that the expression or up-regulation of TLRs on tumor cells promotes carcinogenesis and cancer progression by multiple mechanisms. Most researches on the mechanisms of inflammatory response initiated by TLRs have focused on the formation of primary tumor, but inflammatory mediators are also involved in the migration, invasion and metastasis of malignant cells. However, the underlying mechanism of how TLRs signaling affect the inflammatory environment to develop advanced cancer remains to be fully understood. Activation of TLRs signaling as described above has been identified as an early molecular event, regulating the expression of various cellular effectors which contributed to pathogenesis and progression of cancer. Therefore we are interested with whether TLRs signaling affect metastatic capability of advanced cancer cells by mediating the expression of OPN and investigate what the potential correlation between TLRs and OPN.In this regard, we used human low metastatic ovarian cancer cell line HO-8910 and high metastatic ovarian cancer cell line HO-8910PM (derived from the parental cell HO-8910) to explore the correlation between TLRs signaling and OPN-mediated metastasis. According to RT-PCR analysis of two kinds of cell lines, 6 members among 9 ones within the TLRs family were detected in mRNA level and TLR4 was selected for further study as steadily expressed in two cell lines. Real-time PCR data showed that Activation of TLR4 signaling in tumor cells by lipopolysaccharide can up-regulate the production of osteopontin and some metastasis-related genes including Il-8, vegf-αand tgf-β, contributing to the inflammatory tumor environment. Consistent with the mRNA level, western blotting revealed that the synthesis of three forms of osteopontin was promoted by activated TLR4 signaling. It is noted that the inductive expression level of OPN fragment (OPN-32kDa) more high in HO-8910PM, comparing with HO-8910. In addition, stimulation of LPS enhanced malignant cells proliferation, migration and invasion in vitro. Blockade of TLR4 pathway by a cellular permeable TLR4 inhibitor TAK-242 delayed cancer cell growth and reverses the enhancement of metastasis-related abilities in vitro. Furthermore, the malignant transformation of two kind cell lines, which induced by TLR4 signaling, has been evidently alleviated by neutralized antibody of osteopontin.Epigallocatechin gallate EGCG as the main component of green tea, have a wider anti-tumor and anti-inflammatory activity. The low-metastatic ovarian carcinoma cell lines HO-8910 and the highly-metastatic ovarian carcinoma cell linesHO-8910PM differentiated from HO-8910 as materials were selected as the material, and treated with different doses of EGCG.50μM EGCG which inhibited the growth of cancer cell significantly as a final working concentration by MTT detection. Then, the ovarian cancer cells were detected on the phenotypic changes in the control and experimental group of the two ovarian cancer cells by the experiment of tumor cell colony formation in soft agar, wound healing and matrigel infection assays. Finally, the ovarian cancer cells were detected on the expression changes of IL-8 and TNF-αby real-time quantitative PCR and ELISA on mRNA and protein level respectively. The results showed that HO-8910PM showed greater proliferation and transfer activity compared with HO-8910; 50μM EGCG could significantly inhibit the growth capacity of non-anchor, migration, invasion and metastasis of two ovarian cancer cells; the effect on the high transfer cell lines HO-8910PM was particularly significant; 50μM EGCG reduced the expression of inflammatory IL-8 and TNF-αand OPN-c in HO-8910PM significantly, while the down regulation effect on HO-8910 was not significant.Taken together, In this paper we studied the function mechanism of osteopontin via Toll-like receptors mediated in ovarian cancer cell metastatic phenotype and invasion. The findings of this study would imply a new mechanism between TLRs-induced inflammation and osteopontin(OPN) of cellular effectors which play an important role in the process of metastasis.