| Background and Objectives:Cancer metastasis is responsible for more than90%of the mortality in patientssuffered from cancer. Bone is the frequent site of metastasis in patients with cancer,including prostate cancer and breast cancer. Other types of cancer, such as lung, kidney andthyroid carcinoma also display osteotropism. Cancer metastasis to bone in advanced-stage isusually silent and causes the occurrence of severe bone pain, which could be refractory,spinal-cord compression, pathological skeletal fracture and hypercalcaemia. Approximately90%of patients with prostate cancer die of Metastatic Bone Disease (MBD), which is themost commonly characterized by radiography as predominance of osteoblastic bone damageas opposed to osteolytic lesion with dramatically reduced bone mineral density. Prostatecancer cells invade into the microenvironment of bone marrow, then influence the balanceof bone formation and resorption and induce the relative excess of osteoblastic activity.It has been reported that metastatic prostate cancer cells have the capability to promotethe osteoblastic differentiation via pro-osteoblastic factors. Osteoprogenitors, likeMC3T3-E1(murine calvaria clonal pre-osteoblastic cell line) cells and bone marrowmesenchymal stem cells (BMSCs), orientate along the osteoblastic lineage via theregulation of local molecules, such as WNT proteins, transforming growth factor-β (TGF-β)and bone morphogenetic proteins(BMPs) molecules. WNTs proteins of secretedglycolipoproteins mediate the processes of osteoblastic maturity through the transcriptionalactivator β-catenin. β-catenin was identified originally as a part of the adherens junctioncomplex with α-catenin and cadherin, but its function as WNTs transcriptional coactivatorhasn’t been well known until the last ten years. Binding of canonical WNT ligands to itsreceptor complex comprising one of the seven tranmembrane receptors of the FZD familyand a co-receptor LRP5or LRP6triggers WNT/β-catenin signal pathway, inhibitesβ-catenin degradation and facilitates the stabilized β-catenin translocation into nucleus and binding to TCF/Lef molecules, ultimately activates WNTs target gene transcription. TheWNT ligands also could promote bone morphogenetic protein2(BMP2) expression inosteoblasts, so the WNT/β-catenin signaling is crucial to osteogenic differentiation. Butwhether prostate cancer cells stimulates osteoblastic differentiation through WNT/β-catenin,and which signaling molecules produced by prostate cancer cells could stimulate theosteoblastic differentiation are poorly understood.Recent studies have shown that semaphorin3A (Sema3A), a member of semaphorinsfamily, exertes an osteo-protective effects by stimulating the osteoblastic bonemineralization. The functional exertion of Sema3A needes its binding to NRP1(the specificreceptor of Sema3A) and activation of wnt/β-catenin signaling. However, the role of Sema3A in prostate cancer-induced bone formation and its relationship with WNT/β-catenin hasnot been investigated.Methods:The abilities of osteostimulation among diverse PCa cell lines including C4-2(drivedfrom prostate cancer LNCaP cells), LNCaP and PC-3prostate cancer cells were evaluated,and the expression of Sema3A in different prostate cancer cells was detected. Then wefurther studied on the effect of Sema3A in C4-2induced osteoblastic differentiation byco-culture system, in which MC3T3-E1cells were cultured in osteogenic medium plus C4-2conditioned medium(CM) with or without the Sema3A RNAi. We also studied the functionof Sema3A by using neutralizing antibody for Sema3A. We detected the activation ofβ-catenin in osteoprogenitors by stimulating of C4-2CM, and the influence of Sema3A onstabilization of β-catenin.Establishment and characterization of the in vitro prostate cancer-osteoprogenitco-culture model, and the detection of Sema3A expressionThe CM was collected from each PCa cell lines, then the osteoprogenitor MC3T3-E1cells were co-cultured with diverse CM. Within3days or14days and21days co-culture,the alkaline phosphatase activity was detected by Alikaline Phosphatase Assay Kit andBCIP/NBT Alkaline Phosphatase staining, the mineralization was investigated by AlizarinRed S staining. Then the relative mRNA expression of osteoblastic marker gene, includingCol1α1, OCN and RUNX2/Cbfa1were measured by qPCR.Subsequently, the production of Sema3A in diverse prostate cancer cell lines was measured by immunofluorescence and Western-blot analysis.Mechanism of Sema3A/NRP1in PCa-induced osteoblastic differentiationMC3T3-E1cells were treated with four diverse CM, including C4-2CM, C4-2-Sema3ACM, C4-2NCCM and OM, or treated with other four different CM, including C4-2CM,C4-2Sema3A AbCM, C4-2IgGCM and OM. During cultivation for3or14d, cells wereharvested for the following tests:1. ALP activity and ALP staining of all co-cultured cells were determined by AlikalinePhosphatase Assay Kit and BCIP/NBT Alkaline Phosphatase stain Kit respectively in thethird day.2. The bone nodule formation of these cells in fourteenth day was detected by AlizarinRed S staining.3. The relative mRNA expression of Col1α1, OCN and RUNX2/Cbfa1of allco-cultured cells were measured by qPCR.4. The protein expressional levels of NRP1and β-catenin of the co-culturedosteoprogenitor cells were determined by Western-blot analysis.Results:Establishment and characterization of co-culture system1. C4-2CM and LNCaP CM stimulated alkaline phosphatase activity and minerali-zation of MC3T3-E1cells significantly(P<0.05), but PC-3CM suppressed these processesin the contrast(P<0.05).2. The relative mRNA expression levels of Col1α1, OCN and RUNX2/Cbfa1inMC3T3-E1were dramatically increased by using OM supplied C4-2CM(P<0.05).Expression of Sema3A1. Immunofluorescence analysis:Fluorescence degree by detecting with primary antibody for Sema3A and520-nmfluorescent-conjugated second-antibody in C4-2PCa cells was significantly greatercompared with fluorescence intensity of Sema3A in LNCaP and PC-3.2. Western-blot analysis:The gray value of Sema3A in C4-2cells was dramatically higher than LNCaP andPC-3cell.Mechanism of Sema3A/NRP1in PCa-induced osteoblastic differentiation 1. Osteoblastic differentiation:Activation of alkaline phosphatase: Silencing Sema3A in C4-2cells reduced the ALPactivity in MC3T3-E1cells by using lentiviral vector expressing shRNA target to block theexpressiong of Sema3A in C4-2cells(P<0.05).Mineralization: The area of bone nodule formation of MC3T3-E1by using C4-2-Sema3ACM was significantly diminished compared with C4-2CM treated group or control group.Expressiong of osteoblastic gene: The mRNA expression levels of Col1α1, OCN andRUNX2/Cbfa1in MC3T3-E1were dramatically reduced by shRNA for Sema3A(P<0.05).2. Expression of NRP1on the membrane of MC3T3-E1: Sema3A silencingdiminished the activation of NRP1dramaticlly(P<0.05) by using Western-blot analysis.3. Activation of β-catenin in the cytoplasm of MC3T3-E1: The stabilization ofβ-catenin in MC3T3-E1during osteoblastic differentiation was reduced obviously by usingshRNA traget for Sema3A(P<0.05).4. We used neutralizing antibody for Sema3A to determine whether Sema3A mediatesprostate cancer-induced osteodifferentiation directly, and the data were similar with usingshRNA for Sema3A.Conclusion:1. High amounts of Sema3A are expressed in C4-2cells, which are considered as highaggressive, dramatically pro-osteoblastic prostate cancer cell lines.2. Sema3A involves in the prostate cancer-induced pro-osteoblastic differentiation.3. Sema3A stimulates the activation of β-catenin, that indicates prostate cancer cellsmay induce the stabilization of β-catenin to promote the osteoblastic differentiation viaexpressing Sema3A. |
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