| Background:Despite significant effort and research funds, epithelial ovarian cancer remains a very deadly disease. There are no effective screening methods that discover early stage disease; the majority of patients are diagnosed with advanced disease. Treatment modalities consist primarily of radical debulking surgery followed by taxane and platinum-based chemotherapy. Newer therapies including limited targeted agents and intraperitoneal delivery of chemotherapeutic drugs have improved disease-free intervals, but failed to yield long-lasting cures in most patients. Chemotherapeutic resistance, particularly in the recurrent setting, plagues the disease. Targeting the pathways and mechanisms behind the development of chemoresistance in ovarian cancer could lead to significant improvement in patient outcomes. In many malignancies, including blood and other solid tumors, there is a subgroup of tumor cells, separate from the bulk population, called cancer stem cells (CSCs). These CSCs are thought to be the cause of metastasis, recurrence and resistance. Previous studies have indicated that chemokines, through interaction with their specific receptors, are involved in cancer proliferation and metastasis. Recently, increasing evidence has shown that the interaction of chemokines with their receptors plays an important role in maintaining the metastastic capacity of CSCs. This includes a role for CXCR1/CXCL8 in promoting breast CSCs self-renewal and invasion; a subpopulation of migratory CD133+CXCR4+CSCs is essential for tumor metastasis in human pancreatic cancer; CXCR4+glioma stem cells involved in the metastasis of gliomas. Collectively, these findings suggest that CSCs may indeed be involved in metastatic tumor formation, and, furthermore, the metastatic potential of CSCs may be controlled, at least partially, by chemokine-mediated signaling. So far, scientists have successfully isolated and identified CD133+ovarian cancer stem cells (OCSCs). But the key factor which maintains OCSCs self-renewal and invasion is not clear.We successfully isolated OCSCs from ovarian cancer cell lines A2780.And we found that OCSCs express more chemokine CXCL16 and its receptor CXCR6 with respect to CD133-non-ovarian cancer stem cells (non ovarian cancer stem cells, nOCSCs), detected by PCR-array. Based on these studies, we may assume OCSCs autocrine CXCL16 binding to CXCR6 for maintaining its biological behavior, this study will provide the theoretical basis in finding a therapeutic target in ovarian CSCs.Objectives:1. To explore expression of CXCL16 and CXCR6 between CD133+OCSCs and CD133-nOCSCs2. To explore the effects of CXCL16/CXCR6 axis on the proliferation and invasion of OCSCsMethods:1. To determine the difference of CXCL16 and CXCR6 mRNA expression between OCSCs and nOCSCs by Real-time PCR2. To determine the difference of soluble CXCL16 between OCSCs and nOCSCs by ELISA3. To determine the difference of CXCR6 expression between OCSCs and nOCSCs by Flow Cytometry4. To assess the effect of CXCL16 on proliferation of OCSC by CCK-8 assay5. To determine the ability of invasion and migration of OCSCs after blocking of CXCL16 by neutralizing antibody or shRNA with Transwell chamber assay and matrigel invasion assay6. To determine the ability of invasion and migration of OCSCs after blocking of CXCR6 by neutralizing antibody with Transwell chamber assay and matrigel invasion assayResults:1. Compared with nOCSCs, CXCL16 and CXCR6 mRNA showed increased expression in OCSCs (P<0.01)2. OCSCs secreted much more CXCL16 (2434.00±204.65 ng/mL) than nOCSCs (129.33±31.64ng/mL) (P<0.01)3. CXCR6 expression in OCSCswas higher compared to nOCSCs (60.6±5.3% vs6.4±1.6%) (P<0.01)4. CCK-8 assay showed that the proliferation of OCSCs was not affected by CXCL16 antibody treated (P>0.05)5. The migration and invasion of OCSCs could be blocked by anti-CXCL16, migration cells number decreased from(43.00±4.00) cells/field to (21.33±3.06) cells/field (P<0.01). Invasion cells number decreased from (44.58±4.51) cells/field to (18.00±4.00) cells/field (P<0.01). The migration and invasion of OCSCs could be blocked by sh-CXCL16, (59.33±2.52) cells/field migrated in control group, but only (28.00±3.00) cells/field in sh-CXCL16 group (P<0.01). (63.67±3.51) cell cells/field invaded in control group, but (28.67±4.04) cells/field in sh-CXCL16 group (P<0.01)6. The invasion and migration of OCSCs could be blocked by anti-CXCR6, migration cells number decreased from(64.67±4.16)cells/field to(33.17±5.03)cells/field (P<0.01). Invasion cells number decreased from (55.67±3.51) cells/field to (24.33±4.04) cells/field (P<0.01)Conclusions:1. Compared with nOCSCs, OCSCs express more CXCL16 and CXCR6 in mRNA level and protein level.2. CXCL16 can not affect the proliferation of OCSCs.3. Autocrine CCL5 binding to CXCR6 maintain high ability of Invasion and Migration of CD 133+ Ovarian Cancer Stem Cells... |
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