Osteosarcoma is the most common primary malignant bone tumor in childhood,and it maintains a high level of recurrence.Matrix metalloproteinase-1(MMP-1)was found to contribute to cancer progression.The present study wa...Osteosarcoma is the most common primary malignant bone tumor in childhood,and it maintains a high level of recurrence.Matrix metalloproteinase-1(MMP-1)was found to contribute to cancer progression.The present study was to investigate the in vitro effects of MMP-1 over-expression on the proliferation,invasion,metastasis and stem-like properties of osteosarcoma MG-63 cells.The MG-63 cells were cultured and had a full length MMP-1 cDNA inserted by the tentiviral vector (MG-63^MMP-1+).MG-63 negative control and MG-63 blank control groups were established as well.MMP-1 expression was detected in MG-63^MMP-1+,MG-63 negative control and MG-63 blank control cells using qPCR,Western blotting and immunofluorescence after 24h of culture. The cell proliferation assay was performed with a camera attached to a bioreactor,which was programmed to photograph five regions of each well every 10 min over a period of 48 h.The cell invasion assay was conducted with Matrigel to assess the invasive potential,of MG-63 cells over 24h,the qPCR analysis to measure stem cell markers,including Oct4, Sox-2,Nanog,and Pax-7,and Western blot analysis to detect invasive and metastatic potential markers TIMP-1,VEGF and BMP2/4,after 24h of culture.Immunofluorescence was used to investigate the presence of the stem cell marker Pax-7 after 24-h culture. The results showed that over-expression of MMP-1 after transfection could significantly increase minor cell proliferation and invasion (P<0.05,MG-63^MMP-1+ versus controls).Pax-7 was highly expressed in MG-63^MMP-1+ cells,with no significant changes of Oct-4,Sox-2, and Nanog observed (P<0.05).MG-63^MMP-1+ cells showed higher expression of VEGF and BMP 2/4 proteins and lower expression of TIMP-1 protein than controls (P<0.05).It was concluded that MMP-1 over-expression in MG-63 cells contributed to the proliferation, invasion,metastasis and stem-like properties of osteosarcoma cells.Future studies should focus on in vivo effects of MMP-1 over-expression and the application of MMP-1 and Pax-7 inhibition in vivo to osteosarcoma theraoies.展开更多
Objective： This study investigated the effect of NS398 on anti-proliferation and the mechanism of inducing apoptosis of human osteosarcoma cell MG-63 line in vitro. Methods： Growth suppression was detected by MTT me...Objective： This study investigated the effect of NS398 on anti-proliferation and the mechanism of inducing apoptosis of human osteosarcoma cell MG-63 line in vitro. Methods： Growth suppression was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rates were quantified by flow cytometry （FCM）. And Bcl-2 protein level were detected by Western Blotting assay. Results： Different concentration NS398 inhibited the cell growth. Through TEM and DNA electrophoresis, the special morphological changes were observed after 24, 48 and 72 h treatment with NS398. The apoptotic rates of MG-63 cells treated with NS398 were respectively, significantly higher than that of the control group（ P 〈0.01 ）. Western blot assay showed that NS398 reduced Bcl-2 protein expression. Conclusion： NS398 suppressed the proliferation of human osteosarcoma cell MG-63 line and induced apoptosis. The mechanism may be associated with down-regulation expression level of bcl-2 protein.展开更多
文摘Osteosarcoma is the most common primary malignant bone tumor in childhood,and it maintains a high level of recurrence.Matrix metalloproteinase-1(MMP-1)was found to contribute to cancer progression.The present study was to investigate the in vitro effects of MMP-1 over-expression on the proliferation,invasion,metastasis and stem-like properties of osteosarcoma MG-63 cells.The MG-63 cells were cultured and had a full length MMP-1 cDNA inserted by the tentiviral vector (MG-63^MMP-1+).MG-63 negative control and MG-63 blank control groups were established as well.MMP-1 expression was detected in MG-63^MMP-1+,MG-63 negative control and MG-63 blank control cells using qPCR,Western blotting and immunofluorescence after 24h of culture. The cell proliferation assay was performed with a camera attached to a bioreactor,which was programmed to photograph five regions of each well every 10 min over a period of 48 h.The cell invasion assay was conducted with Matrigel to assess the invasive potential,of MG-63 cells over 24h,the qPCR analysis to measure stem cell markers,including Oct4, Sox-2,Nanog,and Pax-7,and Western blot analysis to detect invasive and metastatic potential markers TIMP-1,VEGF and BMP2/4,after 24h of culture.Immunofluorescence was used to investigate the presence of the stem cell marker Pax-7 after 24-h culture. The results showed that over-expression of MMP-1 after transfection could significantly increase minor cell proliferation and invasion (P<0.05,MG-63^MMP-1+ versus controls).Pax-7 was highly expressed in MG-63^MMP-1+ cells,with no significant changes of Oct-4,Sox-2, and Nanog observed (P<0.05).MG-63^MMP-1+ cells showed higher expression of VEGF and BMP 2/4 proteins and lower expression of TIMP-1 protein than controls (P<0.05).It was concluded that MMP-1 over-expression in MG-63 cells contributed to the proliferation, invasion,metastasis and stem-like properties of osteosarcoma cells.Future studies should focus on in vivo effects of MMP-1 over-expression and the application of MMP-1 and Pax-7 inhibition in vivo to osteosarcoma theraoies.
文摘Objective： This study investigated the effect of NS398 on anti-proliferation and the mechanism of inducing apoptosis of human osteosarcoma cell MG-63 line in vitro. Methods： Growth suppression was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rates were quantified by flow cytometry （FCM）. And Bcl-2 protein level were detected by Western Blotting assay. Results： Different concentration NS398 inhibited the cell growth. Through TEM and DNA electrophoresis, the special morphological changes were observed after 24, 48 and 72 h treatment with NS398. The apoptotic rates of MG-63 cells treated with NS398 were respectively, significantly higher than that of the control group（ P 〈0.01 ）. Western blot assay showed that NS398 reduced Bcl-2 protein expression. Conclusion： NS398 suppressed the proliferation of human osteosarcoma cell MG-63 line and induced apoptosis. The mechanism may be associated with down-regulation expression level of bcl-2 protein.
