The Potential Role Of Connective Tissue Growth Factor On Human Osteosarcoma 143B Cells In Simulated Microenvironment

Objective To investigate the effects of CTGF (connective tissue growth factor) on the biological behavior of human osteosarcoma 143B cells in simulated microenvironment and their underlying mechanisms.Methods The high metastatic osteosarcoma cell line 143B was chosen as the experimental model. A co-culture system which composed of osteosarcoma 143B cells and bone marrow stromal cells HS-5 was constructed. We tried to elucidate the effects of CTGF on 143B cells with forced CTGF overexpression. Three groups in our experiment, including Co-Blank (143B+HS-5), Co-GFP (143B/AdGFP+HS-5), Co-CTGF (143B/AdCTGF+HS-5) were set up.MTT assay was used to investigate the cell viability of 143B cells in different groups, Flow cytometry (FCM) was carried out to detect the cell cycles of 143B in each group. Wound healing assay and transwell assay were carried out to detect 143B cell ability of migration and invasion, respectively, in the co-culture system. To identify the pivotal factors that may be mediated by CTGF in osteosarcoma cells, we used RT-PCR and Western blot to screen chemokines and integrin in simulated microenvironment. Western blot was carried out to detect the activated state of PI3K/AKT and Wnt/β-catenin signaling pathway.Cell suspension of HS-5 cells and 143B cells from each group at a ratio of 1:1 was mixed, and then implanted into nude mice subcutaneously. After 3 weeks, when tumors were observable, the tumor diameters were recorded every 10 days. After 40 days, tumors were harvested and fixed in buffered formaldehyde, embedded in paraffin, sectioned for further histological analysis.Results We utilized CTGF overexpression (AdCTGF) technology to regulate CTGF high expression in osteosarcoma 143B cells, which co-cultured with bone marrow stromal cells HS-5.① The results of MTT showed that Co-CTGF group cells proliferation rate increased compared to that of Co-GFP cells;②The cell cycles of 143B cells in Co-CTGF group (42.74%) was significantly higher than that of Co-GFP groups (35.96%) (p <0.05).③ The result of wound healing showed that the healing rate of Co-CTGF 143B cells (100%) were markedly higher than those of the Co-GFP cells (84.43±.24)%(p<0.01); The result of transwell without matrigel showed that the number of Co-CTGF 143B cells (68±5.1) were significantly higher than those of Co-GFP cells (34±4.2); The number of invading 143B cells which moved across the matrix barrier increased from Co-GFP group (21±4.4) to Co-CTGF (46±3.6) (p<0.05). ④Mice xenograft model showed that there was significant difference in tumor volume of Co-CTGF group vs Co-GFP group. Histological analysis showed no significant difference in heterogeneity between the two groups. ⑤ Analyzing pivotal factors results showed that the expression of αvβ3 and CXCR4 up-regulated in 143B cells, and CXCL12 up-regulated in HS-5 cells at mRNA level and protein level. ⑥ RT-PCR and Western blot analysis showed that CTGF activated the state of PI3K/AKT and Wnt/β-catenin signaling pathway.Conclusion CTGF could regulate the interaction between 143B cells and HS-5 cells, promoted the 143B proliferation, migration and invasion abilities of 143B cells in simulated microenvironment. The possible mechanism may include:①Integrin αvβ3 was stimulated in 143B cells;② CTGF promoted SDF-1 expression and then SDF-1/CXCR4 pathway and PI3K/AKT and Wnt/β-catenin signaling pathway were activated.