| Background and ObjectiveOvarian cancer is malignant tumor which seriously imperil the health of women, the death rate of which hold primacy in gynecologic tumor. About 70 percent of patients have local or distant metastases before they seek medical attention. Although new chemotherapy agents and advanced operation are discovered, the five-year survival rate of patients with an advanced disease is still nearly 30% during the last decade. Relapse and metastasis always result in poor clinical outcome. Recent studies indicate that there is a small subset of cancer cells in malignant tumor ,with stem cell-like characteristics, which have the capacity to both divide and expand the cancer stem cell pool and to differentiate into the heterogeneous non-tumorigenic cancer cell type and to form tumor metastasis, which lead to relapse and metastasis of tumor. If the molecular mechanism of tumor cell metastasis is clear, strategies aimed at inhibiting metastasis of cancer cells. It has been reported that tumor cell migration and metastasis share many similarities to leukocyte trafficking,which is critically regulated by chemokines and their receptors. Many cancers also express an extensive network of chemokines and chemokine receptors. So the mechanisms utilized inhoming of leukocyte directed by chemokines may also be applicable in the process of tumor cells metasatsis .Meanwhile, more and more specialists pay attention to the relationship between the chemokine SDF-1 and its receptor CXCR4.To explore the relation between the SDF-1/CXCR4 biological axis and invasiveness of ovarian cancer stem cells, we detect the expression of CXCR4 in side population cells with stem cell-like characteristics of human ovarian epithelial cancer OVCAR3 cells ,and preliminary study how SDF-1/CXCR4 biological axis work on the invasiveness of side population cells. Strategies aimed at modulating the SDF-1/CXCR4 axis may have important clinical applications to inhibit metastasis of ovarian cancer stem cells. MethodsPart.I Identification and Characterization of side population cells with stem cell-like characteristics of human ovarian epithelial cancer OVCAR3 cellsFlow cytometry and Hoechst 33342 dye efflux assay were used to isolate SP cells and non-SP cells from OVCAR3 cell line of human ovarian epithelial carcinoma.Then we observed the tumorigenicity of two subpopulations by soft agar cloning method and sphere-forming assay.Invasiveness was measured by Transwell experiment. Part.II Explore the influence of exogenous chemokine SDF-1 and AMD3100(specific antagonist of CXCR4)on side population cells'invasive ability .The expression of CXCR4 In both SP and non-SP cells was detected by immunofluorescence technique. Observe the influence of exogenous chemokine SDF-1 and AMD3100(specific antagonist of CXCR4)on side population cells'invasive ability by Transwell experiment.ResultsI. Identification and characterization of side population cells with stem cell-like characteristics of human ovarian epithelial cancer OVCAR3 cells1.The proportion of SP cells excluding Hoechst 33342 dye in OVCAR3 cells was 13.1%, by addition of verapamil, its was 1.8%,greatly decreased.2.CFE of both SP and non-SP was 46.20%±23.30% and 6.20%±3.06% ,respectively(P<0.05).3.Through 2 weeks of culture,SP cells isolated from OVCAR3 could finally generate a group of cells with a SP proportion of 28.1%,While the non- SP cells isolated synchronously generated a group of SP cells with a fewer proportion of 0.7%.4.In Transwell experiment ,the cell number that infiltrated Transwell membrane in SP group(215.9±30.9) was significantly higher than that in non-SP group (60.9±10.5). II.Explore the influence of exogenous chemokine SDF-1 and AMD3100 (specific antagonist of CXCR4) on side population cells'invasive ability .5.Immunostaining showed CXCR4 was expressed in side population cells,but non-SP cells not.6. In Transwell experiment,exogenous chemokine SDF-1 greatly provoted side population cells'invasion , while AMD3100 greatly inhibited side population cells'invasive ability,the differences of three groups were statistically significant(P<0.05).Conclusion:1. Human epithelial ovarian carcinoma cell line OVCAR3 contains a small subpopulation of cells excluding Hoechst 33342 dye . The SP,with the ability of dissymmetry fission,has better proliferative capacity and invading potential in vitro than the non-SP. So it can be applied to the ovarian cancer stem cell research.2. CXCR4 was expressed in side population cells.3. SDF-1 could induce invasion of CXCR4 side population cells of ovarian cancer and AMD3100 (specific antagonist of CXCR4) could block the effect.4. SDF-1 and CXCR4 may play an important role in the invasiveness and metastasis of side population cells of ovarian cancer, and intervention with the interaction of SDF-1 and CXCR4 could be a meaningful therapeutic target and strategy. |
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