目的:研究肿瘤相关钙信号传导蛋白2(tumor-associated calcium signal transducer-2,Trop-2)表达下调对食管鳞癌EC9706细胞增殖和细胞迁移的影响,并探讨其可能的分子机制.方法:将Trop-2siRNA和对照siRNA转染食管鳞癌EC9706细胞,利用实...目的:研究肿瘤相关钙信号传导蛋白2(tumor-associated calcium signal transducer-2,Trop-2)表达下调对食管鳞癌EC9706细胞增殖和细胞迁移的影响,并探讨其可能的分子机制.方法:将Trop-2siRNA和对照siRNA转染食管鳞癌EC9706细胞,利用实时荧光定量PCR和Western blotting检测转染Trop-2siRNA后食管鳞癌EC9706细胞中Trop-2mRNA和蛋白的表达.利用CCK-8试剂分析转染Trop-2siRNA后EC9706细胞的增殖变化,接着采用Boyden小室研究下调Trop-2对EC9706细胞迁移的影响,最后利用Western blotting技术分析与细胞迁移相关基因MMP-7表达的变化.结果:转染Trop-248h,Trop-2siRNA组中Trop-2mRNA和蛋白的表达显著低于未处理组和对照siRNA组(P<0.05).Trop-2表达下调能明显抑制食管鳞癌EC9706细胞的增殖(P<0.05).此外,Boyden小室结果表明,与未处理组(119.27±4.63)及对照siRNA组(112.81±5.01)相比,Trop-2siRNA组中EC9706细胞的穿膜数(65.29±4.33)明显降低.Western blotting结果表明,Trop-2siRNA能明显降低食管鳞癌EC9706细胞中MMP-7的表达.结论:Trop-2表达下调能抑制食管鳞癌细胞的增殖和迁移,其介导的迁移能力的降低可能与MMP-7表达的下调密切相关,操纵Trop-2有望成为食管鳞癌的分子治疗靶点.展开更多
P48 is a cytokine which induces monocyte differentia-tion and the induction of cytotoxic activity. In this study,the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (m...P48 is a cytokine which induces monocyte differentia-tion and the induction of cytotoxic activity. In this study,the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (mP48) were investigated. Monocyte stimulation with mP48 was found to involve the mobilization of intracellular calcium (Ca2+)and the activation and translocation of PKC from the cy-tosol to the membrane. Membane P48 induced a rapid rise of intracellular Ca2+ in a dose dependent maner. Simi-larly the stimulation of monocytes with P48 was found to involve the activation and translocation of PKC. The translocation of PKC was rapid (within 0-5 min) yet tran-sient with PKC activity returning to control levels by 8 min. The functional role of protein kineses in P48 induced TNF secretion was studied using various kinese inhibitors. The PKC inhibitors, H-7 and sphingosine, were found to inhibit P48 induced TNF secretion with 50% inhibition at 5μM HA1004, which inhibts cyclic nucleotide-dependent kinase (PKA, Ki 1.2μM), did not inhibit TNF secretion. H-8 (PKA inhibitor) was found to be an effective inhibitor of TNF secretion only at high concentrations(30μp. The Calmodulin-dependent kinase inhibitor, W7 (Ki 12μM)was found to be effective at concentration above 5μM.These findings suggest that P48-triggered TNF secretion involves transmembrane Ca2+ signaling and the subse-quent activation of at least two protein kineses, PKC and CaMK.展开更多
文摘P48 is a cytokine which induces monocyte differentia-tion and the induction of cytotoxic activity. In this study,the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (mP48) were investigated. Monocyte stimulation with mP48 was found to involve the mobilization of intracellular calcium (Ca2+)and the activation and translocation of PKC from the cy-tosol to the membrane. Membane P48 induced a rapid rise of intracellular Ca2+ in a dose dependent maner. Simi-larly the stimulation of monocytes with P48 was found to involve the activation and translocation of PKC. The translocation of PKC was rapid (within 0-5 min) yet tran-sient with PKC activity returning to control levels by 8 min. The functional role of protein kineses in P48 induced TNF secretion was studied using various kinese inhibitors. The PKC inhibitors, H-7 and sphingosine, were found to inhibit P48 induced TNF secretion with 50% inhibition at 5μM HA1004, which inhibts cyclic nucleotide-dependent kinase (PKA, Ki 1.2μM), did not inhibit TNF secretion. H-8 (PKA inhibitor) was found to be an effective inhibitor of TNF secretion only at high concentrations(30μp. The Calmodulin-dependent kinase inhibitor, W7 (Ki 12μM)was found to be effective at concentration above 5μM.These findings suggest that P48-triggered TNF secretion involves transmembrane Ca2+ signaling and the subse-quent activation of at least two protein kineses, PKC and CaMK.
