Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation,growth,and therapy resitance.The hallmarks of cancer fibroblast interactions,consisting of c...Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation,growth,and therapy resitance.The hallmarks of cancer fibroblast interactions,consisting of caveolin 1(Cav1)and mono-carboxylate ransporter 4(MCT4)(metabolic coupling markers),along with IL-6,TGFB,and lactate secretion,are considered robust biomarkers predicting recurrence and metastasis.In order to promote a novel phenotype in normal fibroblasts,we predicted that breast cancer cells could be able to cause loss of Cavl and increase of MCT4,as well as elevate IL 6 and TGF in nearby nomal fibroblasts.We created a co culture model using breast cancer(4T1)and normal fibroblast(NIH3T3)cell lines cultured under specific experimental conditions in order to directly test our theory.Moreover,we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cavl and gain of MCT4 in adjacent fibroblasts and increase lactate secretion.These results were validated using the monoculture of each group separately as a control.In this system,we show that me tformin inhibits IL-6 and TGFB secretion and re expresses Cavl in both cells.However,MCT4 and lactate stayed high after treatment with metformin.In conclusion,our work shows that co-culture with breast cancer cells may cause signifcant alterations in the phenotype and secretion of normal fibroblasts.Metformin,however,may change this state and affect fibroblasts'acquired phenotypes.Moreover,mitochondrial inhibition by metformin after 8 days of treatment,signi ficantly hinders tumor growth in mouse model of breast cancer.展开更多
In the present study, we investigated the activation of protein kinase C （PKC） family in mouse embryonic fibroblast NIH3T3 cells using gadolinium chloride as a representative lanthanide ion. With live cell imaging s...In the present study, we investigated the activation of protein kinase C （PKC） family in mouse embryonic fibroblast NIH3T3 cells using gadolinium chloride as a representative lanthanide ion. With live cell imaging system and confocal laser scanning microscopy, we found that the treatment of 50 μM GdCI3 promoted cell survival under the condition of serum-starvation. Moreover, better cell attachment and cytoskeleton reorganization were also observed. Additionally, GdC13 treatment resulted in the phosphorylation of PKC family at different time points. Furthermore, bisindolylmaleimide （a PKCpan inhibitor） could efficiently reduce the level of phosphorylated PKCpan （βIISer660）, alleviating ERK activation induced by GdC13. This finding indicated that the PKC activation was involved in GdC13-induced MAPK/ERK signaling and thus might contribute to GdClβ-indueed cell cycle progression and cell survival.展开更多
Objective： To investigate the effects of Panax notoginseng saponins （PNS） on the proliferation and differentiation in NIH3T3 cells. Methods： NIH3T3 cells were treated by various concentrations of PNS 0, 0.05, 0.10...Objective： To investigate the effects of Panax notoginseng saponins （PNS） on the proliferation and differentiation in NIH3T3 cells. Methods： NIH3T3 cells were treated by various concentrations of PNS 0, 0.05, 0.10, 0.20, and 0.40 g/L. The vitality and proliferation potential of cells were detected by 3-（4,5-dimethylthiazol- 2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay, the alkaline phosphatase （ALP） activity was measured by p-nitrophenyl phosphate （pNPP） assay, and the mineralization formation ability was tested for the cellular differentiation toward osteoblast, as well as the expression level of phosphorylated extrecellular signal-regulated kinasel/2（P-ERK1/2）, extracellular signal-regulated kinasel/2 （ERK1/2） protein kinase was analyzed by Western blot with total cell lysate of NIH3T3 cells treated by PNS. Results： Both MTT and pNPP assay showed that optical density （OD） values were increased in response to PNS treatment at a dose-dependent pattern. The mineralization formation ability was enhanced in PNS-treated NIH3T3 cells compared with untreated cells. Meanwhile, the expression level of P-ERK1/2 protein kinase was up-regulated in PNS-treated NIH3T3 cells, while, the expression level of ERK1/2 protein kinase revealed no obvious difference with or without PNS treated cells. Conclusion： PNS could pay a role to promote the proliferation and differentiation in NIH3T3 cells by means of up-regulation of P-ERK1/2 protein kinase.展开更多
Using the whole cell patch-clamp technique, we observed the outward K+ currents and studied for the first time the effects of Yb3+ on the currents and kinetics of activation and inactivation in non-excitable NIH3T3 ce...Using the whole cell patch-clamp technique, we observed the outward K+ currents and studied for the first time the effects of Yb3+ on the currents and kinetics of activation and inactivation in non-excitable NIH3T3 cell. Our results show that the outward K+ currents were promoted with increasing concentration of Ca2+ in pipette solution and saturated at the concentration of 100 μmol/L Ca2+. Yb3+ in bath solution inhibited the currents in a concentration-dependent manner. At the concentration of 1 μmol/L, Yb3+ significantly changed the Vh of activation curve and shifted the activation curve to more negative potentials, but with no effect on its slope factor k. Meanwhile, it had no effect on the inactivation curve. Potassium currents inhibition can induce a series of physiological and molecular biological functions, presumably because of its ability to depolarize the plasma membrane and enhance cell excitability resulting in increasing Ca2+ influx and cytoplast Ca2+ concentration. This process may be one of the mo- lecular mechanisms to affect cell division and proliferation of Yb3+ on NIH3T3 cells.展开更多
目的:探讨水凝胶递送碱性成纤维细胞生长因子(bFGF)对氧糖剥夺(OGD)后小鼠成纤维NIH3T3细胞功能的影响,并阐明负载bFGF的水凝胶对OGD条件下成纤维细胞氧化应激反应的改善作用。方法:制备bFGF明胶水凝胶,将对数生长期NIH3T3细胞分为空白...目的:探讨水凝胶递送碱性成纤维细胞生长因子(bFGF)对氧糖剥夺(OGD)后小鼠成纤维NIH3T3细胞功能的影响,并阐明负载bFGF的水凝胶对OGD条件下成纤维细胞氧化应激反应的改善作用。方法:制备bFGF明胶水凝胶,将对数生长期NIH3T3细胞分为空白组、20%明胶组和20%明胶+戊二醛组,采用CCK-8法检测6、12和24 h水凝胶浸出液共培养的NIH3T3细胞活性。将NIH3T3细胞分为对照组、OGD组和OGD+不同质量bFGF负载组(OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组)。采用Western blotting法检测各组细胞中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达水平。检测各组细胞中丙二醛(MDA)水平和乳酸脱氢酶(LDH)及超氧化物歧化酶(SOD)活性。采用酶联免疫吸附试验(ELISA)法检测bFGF体外释放率。结果:CCK-8法检测,与空白组比较,不同浸出时间20%明胶组和20%明胶+戊二醛组NIH3T3细胞活性差异无统计学意义(P>0.05)。Western blotting法检测,与对照组比较,OGD组NIH3T3细胞中α-SMA和Ⅰ型胶原蛋白表达水平明显升高(P<0.05或P<0.01);与OGD组比较,OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组NIH3T3细胞中α-SMA蛋白表达水平均明显降低(P<0.01),Ⅰ型胶原蛋白表达水平均明显降低(P<0.05或P<0.01),并呈剂量依赖性。与对照组比较,OGD组NIH3T3细胞中MDA水平和LDH活性升高(P<0.05),SOD活性降低(P<0.05)。与OGD组比较,OGD+1 ng bFGF组NIH3T3细胞中MDA水平和LDH活性降低(P<0.05),SOD活性升高(P<0.05)。ELISA法检测,在4 h内约10%bFGF由水凝胶中释放,在12 h内约15%bFGF由水凝胶中释放,在24 h内约21%bFGF由水凝胶中释放。结论:负载bFGF的水凝胶可以调控OGD条件下成纤维细胞的转化,抑制成纤维细胞Ⅰ型胶原蛋白表达,并改善细胞氧化应激反应,且该系统具有一定的持续释药能力。展开更多
基金the National Institute for Medical Research Development(NIMADGrant No.995813).
文摘Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation,growth,and therapy resitance.The hallmarks of cancer fibroblast interactions,consisting of caveolin 1(Cav1)and mono-carboxylate ransporter 4(MCT4)(metabolic coupling markers),along with IL-6,TGFB,and lactate secretion,are considered robust biomarkers predicting recurrence and metastasis.In order to promote a novel phenotype in normal fibroblasts,we predicted that breast cancer cells could be able to cause loss of Cavl and increase of MCT4,as well as elevate IL 6 and TGF in nearby nomal fibroblasts.We created a co culture model using breast cancer(4T1)and normal fibroblast(NIH3T3)cell lines cultured under specific experimental conditions in order to directly test our theory.Moreover,we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cavl and gain of MCT4 in adjacent fibroblasts and increase lactate secretion.These results were validated using the monoculture of each group separately as a control.In this system,we show that me tformin inhibits IL-6 and TGFB secretion and re expresses Cavl in both cells.However,MCT4 and lactate stayed high after treatment with metformin.In conclusion,our work shows that co-culture with breast cancer cells may cause signifcant alterations in the phenotype and secretion of normal fibroblasts.Metformin,however,may change this state and affect fibroblasts'acquired phenotypes.Moreover,mitochondrial inhibition by metformin after 8 days of treatment,signi ficantly hinders tumor growth in mouse model of breast cancer.
基金National Natural Science Foundation of China (Grant No.21277006 and 20637010)
文摘In the present study, we investigated the activation of protein kinase C （PKC） family in mouse embryonic fibroblast NIH3T3 cells using gadolinium chloride as a representative lanthanide ion. With live cell imaging system and confocal laser scanning microscopy, we found that the treatment of 50 μM GdCI3 promoted cell survival under the condition of serum-starvation. Moreover, better cell attachment and cytoskeleton reorganization were also observed. Additionally, GdC13 treatment resulted in the phosphorylation of PKC family at different time points. Furthermore, bisindolylmaleimide （a PKCpan inhibitor） could efficiently reduce the level of phosphorylated PKCpan （βIISer660）, alleviating ERK activation induced by GdC13. This finding indicated that the PKC activation was involved in GdC13-induced MAPK/ERK signaling and thus might contribute to GdClβ-indueed cell cycle progression and cell survival.
基金Supported by the Grant from Zhejiang Provincial Natural Science Foundation of China(No.Y207728)Traditional Chinese Medicine Adiministration Bureau of Zhejiang Province, China(No.2008YA005)
文摘Objective： To investigate the effects of Panax notoginseng saponins （PNS） on the proliferation and differentiation in NIH3T3 cells. Methods： NIH3T3 cells were treated by various concentrations of PNS 0, 0.05, 0.10, 0.20, and 0.40 g/L. The vitality and proliferation potential of cells were detected by 3-（4,5-dimethylthiazol- 2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay, the alkaline phosphatase （ALP） activity was measured by p-nitrophenyl phosphate （pNPP） assay, and the mineralization formation ability was tested for the cellular differentiation toward osteoblast, as well as the expression level of phosphorylated extrecellular signal-regulated kinasel/2（P-ERK1/2）, extracellular signal-regulated kinasel/2 （ERK1/2） protein kinase was analyzed by Western blot with total cell lysate of NIH3T3 cells treated by PNS. Results： Both MTT and pNPP assay showed that optical density （OD） values were increased in response to PNS treatment at a dose-dependent pattern. The mineralization formation ability was enhanced in PNS-treated NIH3T3 cells compared with untreated cells. Meanwhile, the expression level of P-ERK1/2 protein kinase was up-regulated in PNS-treated NIH3T3 cells, while, the expression level of ERK1/2 protein kinase revealed no obvious difference with or without PNS treated cells. Conclusion： PNS could pay a role to promote the proliferation and differentiation in NIH3T3 cells by means of up-regulation of P-ERK1/2 protein kinase.
基金supported by the National Natural Science Foundation of China (Grant No. 20637010)Shanxi Provincial Natural Science Foun-dation for Youth (Grant No. 2007021010)
文摘Using the whole cell patch-clamp technique, we observed the outward K+ currents and studied for the first time the effects of Yb3+ on the currents and kinetics of activation and inactivation in non-excitable NIH3T3 cell. Our results show that the outward K+ currents were promoted with increasing concentration of Ca2+ in pipette solution and saturated at the concentration of 100 μmol/L Ca2+. Yb3+ in bath solution inhibited the currents in a concentration-dependent manner. At the concentration of 1 μmol/L, Yb3+ significantly changed the Vh of activation curve and shifted the activation curve to more negative potentials, but with no effect on its slope factor k. Meanwhile, it had no effect on the inactivation curve. Potassium currents inhibition can induce a series of physiological and molecular biological functions, presumably because of its ability to depolarize the plasma membrane and enhance cell excitability resulting in increasing Ca2+ influx and cytoplast Ca2+ concentration. This process may be one of the mo- lecular mechanisms to affect cell division and proliferation of Yb3+ on NIH3T3 cells.
文摘目的:探讨水凝胶递送碱性成纤维细胞生长因子(bFGF)对氧糖剥夺(OGD)后小鼠成纤维NIH3T3细胞功能的影响,并阐明负载bFGF的水凝胶对OGD条件下成纤维细胞氧化应激反应的改善作用。方法:制备bFGF明胶水凝胶,将对数生长期NIH3T3细胞分为空白组、20%明胶组和20%明胶+戊二醛组,采用CCK-8法检测6、12和24 h水凝胶浸出液共培养的NIH3T3细胞活性。将NIH3T3细胞分为对照组、OGD组和OGD+不同质量bFGF负载组(OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组)。采用Western blotting法检测各组细胞中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达水平。检测各组细胞中丙二醛(MDA)水平和乳酸脱氢酶(LDH)及超氧化物歧化酶(SOD)活性。采用酶联免疫吸附试验(ELISA)法检测bFGF体外释放率。结果:CCK-8法检测,与空白组比较,不同浸出时间20%明胶组和20%明胶+戊二醛组NIH3T3细胞活性差异无统计学意义(P>0.05)。Western blotting法检测,与对照组比较,OGD组NIH3T3细胞中α-SMA和Ⅰ型胶原蛋白表达水平明显升高(P<0.05或P<0.01);与OGD组比较,OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组NIH3T3细胞中α-SMA蛋白表达水平均明显降低(P<0.01),Ⅰ型胶原蛋白表达水平均明显降低(P<0.05或P<0.01),并呈剂量依赖性。与对照组比较,OGD组NIH3T3细胞中MDA水平和LDH活性升高(P<0.05),SOD活性降低(P<0.05)。与OGD组比较,OGD+1 ng bFGF组NIH3T3细胞中MDA水平和LDH活性降低(P<0.05),SOD活性升高(P<0.05)。ELISA法检测,在4 h内约10%bFGF由水凝胶中释放,在12 h内约15%bFGF由水凝胶中释放,在24 h内约21%bFGF由水凝胶中释放。结论:负载bFGF的水凝胶可以调控OGD条件下成纤维细胞的转化,抑制成纤维细胞Ⅰ型胶原蛋白表达,并改善细胞氧化应激反应,且该系统具有一定的持续释药能力。
