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
目的:探讨布托啡诺(BPH)对骨肉瘤(OS)细胞增殖、迁移和侵袭的影响及其相关的作用机制。方法:将MG-63细胞分为对照组、YAP抑制剂组(维替泊芬组)和BPH低、中、高浓度组,MTT法、克隆形成实验、FCM术、划痕愈合实验、Transwell实验、qPCR法...目的:探讨布托啡诺(BPH)对骨肉瘤(OS)细胞增殖、迁移和侵袭的影响及其相关的作用机制。方法:将MG-63细胞分为对照组、YAP抑制剂组(维替泊芬组)和BPH低、中、高浓度组,MTT法、克隆形成实验、FCM术、划痕愈合实验、Transwell实验、qPCR法、WB法和移植瘤实验分别检测处理后各组细胞的增殖活性、克隆形成数、细胞凋亡率、划痕愈合率,以及上皮钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)、波形蛋白(vimentin)mRNA的表达和YAP、TAZ蛋白的表达,同时观察BPH和维替泊芬对移植瘤生长的影响。结果:与对照组相比,维替泊芬组和BPH低、中、高浓度组细胞增殖活性、克隆数、划痕愈合率、侵袭细胞数,以及N-cadherin和vimentin m RNA水平、YAP和TAZ蛋白表达及移植瘤体积均显著降低(均P<0.05),细胞凋亡率、E-cadherin mRNA水平及对移植瘤的抑瘤率均升高(均P<0.05),且BPH高浓度组与维替泊芬组之间各项指标均无明显差异(均P>0.05)。结论:BPH可能通过抑制Hippo/YAP信号通路来抑制OS细胞MG-63增殖、迁移和侵袭。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.
文摘目的:探讨布托啡诺(BPH)对骨肉瘤(OS)细胞增殖、迁移和侵袭的影响及其相关的作用机制。方法:将MG-63细胞分为对照组、YAP抑制剂组(维替泊芬组)和BPH低、中、高浓度组,MTT法、克隆形成实验、FCM术、划痕愈合实验、Transwell实验、qPCR法、WB法和移植瘤实验分别检测处理后各组细胞的增殖活性、克隆形成数、细胞凋亡率、划痕愈合率,以及上皮钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)、波形蛋白(vimentin)mRNA的表达和YAP、TAZ蛋白的表达,同时观察BPH和维替泊芬对移植瘤生长的影响。结果:与对照组相比,维替泊芬组和BPH低、中、高浓度组细胞增殖活性、克隆数、划痕愈合率、侵袭细胞数,以及N-cadherin和vimentin m RNA水平、YAP和TAZ蛋白表达及移植瘤体积均显著降低(均P<0.05),细胞凋亡率、E-cadherin mRNA水平及对移植瘤的抑瘤率均升高(均P<0.05),且BPH高浓度组与维替泊芬组之间各项指标均无明显差异(均P>0.05)。结论:BPH可能通过抑制Hippo/YAP信号通路来抑制OS细胞MG-63增殖、迁移和侵袭。
