AIM: To study the effects of non-cytotoxJc concentrations of docetaxel on some important angiogenic factors of LS174T Cells.METHODS: The non-cytotoxic concentration of docetaxel and the activity of gelatinase were det...AIM: To study the effects of non-cytotoxJc concentrations of docetaxel on some important angiogenic factors of LS174T Cells.METHODS: The non-cytotoxic concentration of docetaxel and the activity of gelatinase were determined with MTT and gelatin zymography respectively, the expression of VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), MMP (matrix metalloproteinase) 2 and MMP 9 was investigated with RT-PCR and Western blot.RESULTS: The maximum non-cytotoxic concentration of docetaxel on LS174T Cells was 1.0 ng/ml. Compared with the solvent control group, 0.1, 0.5, 1.0 ng/ml of docetaxel could downregulate the expression of VEGF, bFGF, MMP 2and MMP 9 and suppress the activity of gelatinase.CONCLUSION: Our study suggests that the non-cytotoxic concentrations of docetaxel have strong antiangiogenic activity on LS174T Cells, which suggests docetaxel may be a promising antiangiogenic agent.展开更多
Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and ...Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and DNA damage-inducible protein (gadd45) family exhibit disordered expression in several types of malignant diseases and are thus a novel target for cancer therapy. However, there have been only few investigations of whether HDAC inhibitors affect the expression of gadd45 genes. We examined the effects of a HDAC inhibitor, trichostatin A (TSA), on the time-dependent expression of gadd45 genes in the human colon cancer cell line LS174T. Addition of TSA to LS174T cells induced inhibition of cell proliferation by arresting the cell cycle. We found that TSA treatment of LS174T cells induced rapid upregulation of gadd45β mRNA expression within 15 min, reaching a peak level at 3 h. Although the time-dependent expression pattern of gadd45β mRNA was similar to that of gadd45β mRNA, the peak level of gadd45β was lower than that of gadd45β. TSA treatment also upregulated the mRNA level of p21Waf1/Cip1, a prolif- eration inhibitor, after 3 h, but downregulated the mRNA levels of cyclin D1, a proliferation inducer, after 3 h, and of c-Myc after 1 h. TSA treatment induced a certain level of apoptosis, but the mRNA level of p53, a potent apoptosis inducer, was down-regulated after 3 h. These results suggest that the up-regulation of p21Waf1/Cip1 and apoptosis was independent of p53 and that the early upregulation of gadd45β gene, which precedes the upregulation of p21Waf1/Cip1 and the downregulation of cyclin D1, are important in TSA-treated LS174T cells.展开更多
Mucin 2 and occludin play a crucial role in preserving the intestinal mucosal integrity. However, the role for leucine mediating intestinal mucin 2 and occludin expression has little been investigated. The current stu...Mucin 2 and occludin play a crucial role in preserving the intestinal mucosal integrity. However, the role for leucine mediating intestinal mucin 2 and occludin expression has little been investigated. The current study was conducted to test the hypothesis that leucine treatment could increase mucin 2 and occludin levels in LS174 T cells. The LS174 T cells were incubated in the Dulbecco's Modified Eagle Medium(DMEM)supplementing 0, 0.5 and 5 mmol/L L-leucine for the various durations. Two hours after the leucine treatment, the inhibitor of mammalian target of rapamycin(mTOR) and protein kinase B(Akt) phosphorylation in LS174 T cells were significantly increased(P < 0.05), and the mucin 2 and occludin levels were also significantly enhanced(P < 0.05). However, the pretreatment of 10 nmol/L rapamycin, which was an mTOR inhibitor, or 1 μmol/L wortmanin, which was an inhibitor of phosphatidylinositol 3-kinase(PI3 K), completely inhibited leucine-induced mTOR or Akt phosphorylation(P < 0.05), and significantly reduced leucine-stimulated mucin 2 and occludin levels(P < 0.05). These results suggest that leucine treatment promotes the mucin 2 and occludin levels in LS174 T cells partially through the PI3 K-Akt-mTOR signaling pathway.展开更多
目的构建表达高度胸苷磷酸化酶(TP)活性并可以稳定传代的结肠癌细胞株。方法合成包含TP c DNA全长的真核细胞表达载体,用慢病毒包装后转染人结肠癌细胞株SW480、LOVO、HT29和LS174T,流式细胞仪检测转染效率,实时荧光定量RT-PCR方法检测T...目的构建表达高度胸苷磷酸化酶(TP)活性并可以稳定传代的结肠癌细胞株。方法合成包含TP c DNA全长的真核细胞表达载体,用慢病毒包装后转染人结肠癌细胞株SW480、LOVO、HT29和LS174T,流式细胞仪检测转染效率,实时荧光定量RT-PCR方法检测TP的m RNA表达,蛋白印迹法(Western blot)检测TP蛋白表达。结果转染TP c DNA后,SW480、LOVO、HT29与LS174T在传代5代后转染效率仍稳定在95%左右。与未转染TP基因的亲代细胞相比,SW480-TP、LOVO-TP、HT29-TP与LS174T-TP的TP m RNA的表达分别提高了(694.56±171.53)、(282.46±86.85)、(8.45±0.15)和(2 615.02±253.97)倍,差异有统计学意义(P<0.05);并且TP蛋白的表达水平也显著提高。结论慢病毒载体能够高效地将TP c DNA转染至人结肠癌细胞SW480、LOVO、HT29和LS174T中,所得克隆能够稳定传代,并能显著提高4株结肠癌细胞TP的m RNA和蛋白表达水平。展开更多
文摘AIM: To study the effects of non-cytotoxJc concentrations of docetaxel on some important angiogenic factors of LS174T Cells.METHODS: The non-cytotoxic concentration of docetaxel and the activity of gelatinase were determined with MTT and gelatin zymography respectively, the expression of VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), MMP (matrix metalloproteinase) 2 and MMP 9 was investigated with RT-PCR and Western blot.RESULTS: The maximum non-cytotoxic concentration of docetaxel on LS174T Cells was 1.0 ng/ml. Compared with the solvent control group, 0.1, 0.5, 1.0 ng/ml of docetaxel could downregulate the expression of VEGF, bFGF, MMP 2and MMP 9 and suppress the activity of gelatinase.CONCLUSION: Our study suggests that the non-cytotoxic concentrations of docetaxel have strong antiangiogenic activity on LS174T Cells, which suggests docetaxel may be a promising antiangiogenic agent.
文摘Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and DNA damage-inducible protein (gadd45) family exhibit disordered expression in several types of malignant diseases and are thus a novel target for cancer therapy. However, there have been only few investigations of whether HDAC inhibitors affect the expression of gadd45 genes. We examined the effects of a HDAC inhibitor, trichostatin A (TSA), on the time-dependent expression of gadd45 genes in the human colon cancer cell line LS174T. Addition of TSA to LS174T cells induced inhibition of cell proliferation by arresting the cell cycle. We found that TSA treatment of LS174T cells induced rapid upregulation of gadd45β mRNA expression within 15 min, reaching a peak level at 3 h. Although the time-dependent expression pattern of gadd45β mRNA was similar to that of gadd45β mRNA, the peak level of gadd45β was lower than that of gadd45β. TSA treatment also upregulated the mRNA level of p21Waf1/Cip1, a prolif- eration inhibitor, after 3 h, but downregulated the mRNA levels of cyclin D1, a proliferation inducer, after 3 h, and of c-Myc after 1 h. TSA treatment induced a certain level of apoptosis, but the mRNA level of p53, a potent apoptosis inducer, was down-regulated after 3 h. These results suggest that the up-regulation of p21Waf1/Cip1 and apoptosis was independent of p53 and that the early upregulation of gadd45β gene, which precedes the upregulation of p21Waf1/Cip1 and the downregulation of cyclin D1, are important in TSA-treated LS174T cells.
基金financially supported by the grant from the National Natural Science Foundation of China (31201812)the earmarked fund for the China Agriculture Research System(CARS-36)the grant from the Science and Technology Support Program of Sichuan Province(13ZC2237)
文摘Mucin 2 and occludin play a crucial role in preserving the intestinal mucosal integrity. However, the role for leucine mediating intestinal mucin 2 and occludin expression has little been investigated. The current study was conducted to test the hypothesis that leucine treatment could increase mucin 2 and occludin levels in LS174 T cells. The LS174 T cells were incubated in the Dulbecco's Modified Eagle Medium(DMEM)supplementing 0, 0.5 and 5 mmol/L L-leucine for the various durations. Two hours after the leucine treatment, the inhibitor of mammalian target of rapamycin(mTOR) and protein kinase B(Akt) phosphorylation in LS174 T cells were significantly increased(P < 0.05), and the mucin 2 and occludin levels were also significantly enhanced(P < 0.05). However, the pretreatment of 10 nmol/L rapamycin, which was an mTOR inhibitor, or 1 μmol/L wortmanin, which was an inhibitor of phosphatidylinositol 3-kinase(PI3 K), completely inhibited leucine-induced mTOR or Akt phosphorylation(P < 0.05), and significantly reduced leucine-stimulated mucin 2 and occludin levels(P < 0.05). These results suggest that leucine treatment promotes the mucin 2 and occludin levels in LS174 T cells partially through the PI3 K-Akt-mTOR signaling pathway.
文摘目的构建表达高度胸苷磷酸化酶(TP)活性并可以稳定传代的结肠癌细胞株。方法合成包含TP c DNA全长的真核细胞表达载体,用慢病毒包装后转染人结肠癌细胞株SW480、LOVO、HT29和LS174T,流式细胞仪检测转染效率,实时荧光定量RT-PCR方法检测TP的m RNA表达,蛋白印迹法(Western blot)检测TP蛋白表达。结果转染TP c DNA后,SW480、LOVO、HT29与LS174T在传代5代后转染效率仍稳定在95%左右。与未转染TP基因的亲代细胞相比,SW480-TP、LOVO-TP、HT29-TP与LS174T-TP的TP m RNA的表达分别提高了(694.56±171.53)、(282.46±86.85)、(8.45±0.15)和(2 615.02±253.97)倍,差异有统计学意义(P<0.05);并且TP蛋白的表达水平也显著提高。结论慢病毒载体能够高效地将TP c DNA转染至人结肠癌细胞SW480、LOVO、HT29和LS174T中,所得克隆能够稳定传代,并能显著提高4株结肠癌细胞TP的m RNA和蛋白表达水平。
