Experimental Study On The Relevance Chemotaxis Of SDF-1/CXCR4 And Effects Of Prostate Cancer Bone Metastases

Backgroud and objectiveProstate cancer(PCa)is the most common malignant tumors in eld men, it occurred more in Europe and the United States, with the trend of aging and popular of detection serological PSA in China. Incidences of PCa appear upward trend, As a result of PCa tend to distant metastases, especially bone metastases, all kinds of treatment result is not satisfactory, Unbearable pain troubled Patients with PCa,and result in higher mortality. Therefore it is important to clarify the molecular mechanism of bone metastasis of PCa for preventing and treatment of PCa metastases.Chemokine SDF-1, as a member of the CXC chemokine subfamily, mainly expressed in bone marrow stromal and endothelial cells, CXCR4, the specific receptor of SDF-1, mainly expressed in the nerves, blood vessels and blood components. SDF-1 binding with CXCR4 is important to chemotactic effect on inducting hematopoietic cells homing and redistribution for hematopoietic stem / progenitor cells, hepatic stellate cells in vivo. Recent research has shown that SDF-1/CXCR4 biological axis related closely with invasion and metastasis in a variety of tumor. They assumed, Base on theory model of tumor cells depending on chemokines to achieve organ-specific metastasis, that high expressing of chemokine receptors in the tumor cells metastasis to organs of high expression of the corresponding chemokine ligand. But the mechanism is still unclear.ICAM-1, VCAM-1 as inflammatory adhesion molecules, mainly mediated adhesion of inflammatory cells to vascular. Ligand of lymphocyte function associated antigen -1 (LFA-1) for ICAM-1, a members theβ2 integrin subfamily。VLA-4(CD49d), as an important member of the integrinβ1subfamily, is receptor of VCAM-1. SDF-1 can improve adhesion ability of LFA-1 to promote inflammation and vascular endothelial cell adhesion by promoting LFA-1 redistribution at membrane surface of lymphocyte. And ICAM-1 binding to LFA-1 mediated migration of immune cell to the organization, cells adhesion to extracellular matrix, and so on. In recent years, ICAM-1, VCAM-1 are found higher expression significantly in many tumors than the corresponding normal tissue, the adhesions of cells to vascular mediated by inflammation molecules are obviously similar to invasion and metastasis of tumor, it suggest that inflammatory molecules may be plays an important role to promot tumor invasion and metastasis as the way of inflammation.Based on research overseas, in this study, We transfect CXCR4 gene to PCa cells by retroviral vectors, enhance the expression level of CXCR4, analysis effect of PCa cells migration to bone marrow endothelial cell by co-culture PCa cells line PC-3 and bone marrow endothelial cells BMEC-1, which SDF-1αexpressed in high level, in Transwell chamber in vitro. Cultivate PCa cells line, which Transfected with CXCR4 gene, into capsule of the prostate in SCID mice, observate effects of bone metastasis of prostate cancer, analysis the role and molecular mechanism of SDF-1 / CXCR4 signaling pathway in prostate cancer adhesion and metastasis, to provide new experimental evidence for the mechanism of bone metastasis of prostate cancer.Method1. Collectiing 148 cases archived wax blocks of surgical resection and puncture, which diagnosised prostate cancer. Detect expression of CXCR4 protein in prostate cancer tissue and benign prostatic hyperplasia tissues by immunohistochemistry, analysis relations of CXCR4 expression , clinical characteristics, the age difference, Androgen receptor (AR), pathological grade and metastasis. Further count microvessel density (MVD) of CD34 marked, observe relations prostate cancer metastasis-associated Molecules VEGF, MMP-9 with microvessel density in prostate cancer tissues.2. extracte of total RNA in mononuclear cells Separated from healthy human peripheral blood, RT-PCR amplify CXCR4 as RNA extracted a template, to obtain full-length CXCR4 gene sequence, restructur it into retrovirus vectors pLEGFP-N1,which with a green fluorescent protein (GFP), then transfected to PC-3 cells with lipofectamine, observe the expression of CXCR4 by real-time quantitative PCR and Western blotting, and detecte the invasive ability of tumor cells through cell - matrix adhesion by Transwells chamber testing in vitro. 3. Prostate cancer cells line PC-3 transfected CXCR4 gene stably treated with Chemokine SDF-1 for 24 hr, Detect mRNA and protein expression adhesion molecules LFA-1α/ ICAM-1, VLA-4/ VCAM-1 and Kinase P38MAPK by real-time quantitative PCR and Western blot; detect mRNA level and DNA binding activity of HIF-1αand P65NF-κB by real-time quantitative PCR and EMSA. Investigate affect enhanced functions of SDF-1/CXCR4 biological axis in prostate cancer cells on the expression of Moleculars appeal.4. Inoculated human prostate cancer cells line PC-3, PC-3 cells transfected CXCR4 gene and LNcap cells in subcutaneously of male SCID mice, establish transplanted human prostate cancer model of SCID mice And in situ model of tumor metastasis, observe the activities, the rate of tumor incidence, tumor growth and morphological characteristics. Detect expression of CXCR4, prostate cancer-specific antigen (PSA), as well as metastasis-associated factors VEGF and MMP-9 by Immunohistochemistry. Observe impact of cancer cells metastasis and the bone micro-environment in tumor-bearing SCID mice after CXCR4 gene transfection.Result1. Expressing of CXCR4 in Prostate cancer tissues was significantly higher than that in normal prostate tissues adjacent to cancer and benign prostatic hyperplasia (P<0.01) in 148 cases. Expression of CXCR4 protein relate with histological grade and distant metastasis in human Prostate cancer tissues. Values of MVD in Prostate cancer interstitial increased with reduce the degree of tumor differentiation. unrelated with the patient's age, the metastasis group have higher MVD values than in non-metastasis group, the values of MVD in AR-negative group are increased than in the value of AR-positive group, values of MVD increased as CXCR4, VEGF and MMP -9 protein expression gradually upregulated.2. pLEGFP-CXCR4 vector constructed successfully, which transfected into human prostate cancer cells line PC-3 after 72h, levels of CXCR4 mRNA and protein were significantly up-regulated, invasiveness and Migration of cells increased markedly(Compared with the control group, P<0.01, 89.33±11.67 vs 59.33±7.27)in vitro.3. Prostate cancer cells line PC-3 transfected CXCR4 gene stably treated with Chemokine SDF-1 for 24 hr significantly increase the expression P38MAPK mRNA and protein significantly (P<0.01, n=3), expression of phospho-P38 protein significantly increased (P<0.01, n=3). And Ratio of phospho-P38 and P38 increased, suggeste that CXCR4 gene transfection promote activity of P38 protein; Compared with the control group, expression of ICAM-1 protein in PC-3 cells increased Significantly(p<0.01, n=3), levels of ICAM-1 mRNA increased significantly (P<0.05, n=3); levels of VCAM-1 mRNA increased significantly (P<0.01, n=3), expression of VCAM-1 protein increased also (p<0.01, n=3) in PC-3 cells transfected CXCR4 gene ; levels of LFA-1αmRNA and protein increased significantly (P <0.01,n=3); levels of VLA-4 mRNA have no significant changes (P>0.05, n=3), levels of VLA-4 protein increased significantly (P<0.01, n=3).Compared with the control group, evels of P65NF-κB and HIF-1αmRNA in PC-3 cells transfected CXCR4 gene increased significantly (P<0.01, n=3), DNA binding activity of HIF-1αand P65NF-κB protein increased significantly detected by EMSA (P <0.01, n=3).4. Compared with PC-3/PEGFP-CXCR4 cells and PC-3, LNcap cells groups, significant Differences has not been found in prostate cancer lines in morphology of transplant tumor tissues, growth characteristics and the levels of PSA. Expression of VEGF and MMP-9 in groups of metastatic tumors tissues without CXCR4 transfected and PC-3/PEGFP-CXCR4 groups was significantly higher then two other groups (P<0.05). To simulate human In situ prostate cancer bone metastasis animal model by embedding tumor nodules in the subcutaneous urethra of SCID mice, that each group had not found invasion of the prostate tissues and gonad organizations. Observe SCID mice transplanted tumor transfected CXCR4 gene experiments, Compared with the control group, group of tumor transfected CXCR4 gene SCID mouse generate into a tumor need shorter time, tumor sizes biger, number of metastases more. Sites of cancer bone metastases are mainly in the lumbar vertebral. Dochondral osteogenesis Cartilage and calcification in CXCR4 transfection groups increased, and trabecular bone apparent increased, angiogenesis increased, medullary cavity mononucleosis increased.Conclusion1. Increased expression of CXCR4 in PCa can coordinated chemokine SDF-1 which from Stromal of tumor to promotes invasion and metastasis. high expression of CXCR4 in PCa cells make tumor has a higher capacity of invasion and metastasis. CXCR4 protein expression, MVD in stromal, VEGF and MMP-9 protein expression was positively correlated. 2. CXCR4 transfection can significantly increase the levels of CXCR4 mRNA and protein in prostate cancer cells line, enhance capacity of human prostate cancer cell invasion induced by SDF-1 in vitro.3. CXCR4 transfection PCa cells line PC-3 can increase levels of LFA-1α/ICAM-1, VLA-4/ VCAM-1 and P38MAPK the mRNA and protein in varying degrees, and promote the P38MAPK phosphorylation, increase levels of HIF-1αand the P65NF-κB mRNA, increased DNA binding activity of HIF-1αand P65NF-κB protein significantly.4. Human Prostate cancer SCID mouse transplantation model simulate biological behavior of metastasis for human prostate cancer, maintain a better biological characteristics of human prostate cancer, it is a excellent animal model to study metastasis mechanism and the biological treatment of prostate cancer. Transfection CXCR4 gene into prostate cancer cells promote growth and metastasis of cancer cells in SCID mice, and bone remodeled more significantly, when bone metastasis occur.