目的:探讨甲基化酶抑制剂5'-氮杂-2'-脱氧胞苷(5'-Aza-2'-deoxycytidine,5'-Aza-CdR)对结直肠癌细胞株HT-29和LoVo中CDX2基因甲基化水平、mRNA及蛋白表达的影响.方法:用不同浓度5'-Aza-CdR处理结直肠癌细胞株HT...目的:探讨甲基化酶抑制剂5'-氮杂-2'-脱氧胞苷(5'-Aza-2'-deoxycytidine,5'-Aza-CdR)对结直肠癌细胞株HT-29和LoVo中CDX2基因甲基化水平、mRNA及蛋白表达的影响.方法:用不同浓度5'-Aza-CdR处理结直肠癌细胞株HT-29和LoVo.应用TaqMan探针为基础的实时定量PCR(Methylight)方法、SYBR Green PCR方法及蛋白印迹实验(Western blot)检测药物处理前后HT-29和LoVo细胞中CDX2基因的甲基化状态、mRNA和蛋白表达.结果:Methylight检测HT-29和LoVo细胞中C D X2蛋白在药物作用异常甲基化未得到逆转;实时荧光定量PCR和Western blot检测到0.5、1.0、1.5μmol/L 5'-Aza-CdR处理后C D X2基因m R N A和蛋白重新表达,实时荧光定量PCR检测对照组和不同浓度实验组在HT-29细胞株mRNA相对表达量分别为1.000±0.000、0.973±0.024、1.014±0.019和1.094±0.020;在LoVo细胞株相对表达量分别为1.000±0.000、0.966±0.038、1.050±0.029和1.007±0.019.Western blot检测CDX2蛋白在对照组和不同浓度实验组中HT-29细胞株相对表达量分别为0.454±0.049、0.501±0.041、0.340±0.050和0.531±0.046;LoVo细胞株相对表达量分别为0.527±0.037、0.415±0.037、0.432±0.040和0.626±0.046.以上作用无时间、剂量依赖性,但CDX2基因mRNA在HT-29细胞株表达(F=25.146,P=0.000)和LoVo细胞株表达(F=5.470,P=0.024)具有统计学差异,C D X2蛋白表达在HT-29细胞株表达(F=9.700,P=0.005)和LoVo细胞株表达(F=17.701,P=0.001)亦具有统计学差异.结论:结直肠癌细胞株HT-29和LoVo中CDX2基因mRNA和蛋白表达不受中DNA甲基化的表观遗传修饰的调控.展开更多
Chardonnay and Tinta Cao grape pomaces were generated by the winemaking process. The pomaces were extracted with 50% acetone and tested for antioxidant capacities using the oxygen radical absorbance capacity (ORAC) as...Chardonnay and Tinta Cao grape pomaces were generated by the winemaking process. The pomaces were extracted with 50% acetone and tested for antioxidant capacities using the oxygen radical absorbance capacity (ORAC) assay, DPPH□EC50 , and ABTS.+ scavenging capacity tests. Cytotoxicity and antiproliferative activities against Caco-2 and HT-29 human colon cancer cell lines were also analyzed. The quantitative detection of caspase-3 activity during the early apoptotic process was evaluated by fluorometric immunosorbent enzyme assay (FIENA). Induction of late stage apoptosis was analyzed by DNA fragmentation. Total phenolic content (TPC), individual phenolic acids, total oil, and fatty acid profile were also analyzed. The Tinta Cao pomace had the highest antioxidant capacity in all tests with an ORAC value of 386.5 μmol trolox equivalents (TE) per g pomace (μmol TE/g), its DPPH□EC50 value was 94.6 mg equivalents/L, and its ABTS.+ value was 806.7 μmol TE/g, which was more than twice as high as the Chardonnay. Both Tinta Cao and Chardonnay grape pomace extracts appear to contain components that inhibit the proliferation of Caco-2 and HT-29 cancer cells, at least in part, by triggering apoptosis. Expression of caspase-3 was induced by Tinta Cao and Chardonnay pomace extracts at 3 g/L after 4 hours of treatment with a 308% and 229% increase compared to control, respectively. An increase in DNA fragmentation was also observed with both grape pomace treatments. This study demonstrated that these tested grape pomaces were potent scavengers of free radicals and may provide some level of protection against certain cancers.展开更多
文摘目的:探讨甲基化酶抑制剂5'-氮杂-2'-脱氧胞苷(5'-Aza-2'-deoxycytidine,5'-Aza-CdR)对结直肠癌细胞株HT-29和LoVo中CDX2基因甲基化水平、mRNA及蛋白表达的影响.方法:用不同浓度5'-Aza-CdR处理结直肠癌细胞株HT-29和LoVo.应用TaqMan探针为基础的实时定量PCR(Methylight)方法、SYBR Green PCR方法及蛋白印迹实验(Western blot)检测药物处理前后HT-29和LoVo细胞中CDX2基因的甲基化状态、mRNA和蛋白表达.结果:Methylight检测HT-29和LoVo细胞中C D X2蛋白在药物作用异常甲基化未得到逆转;实时荧光定量PCR和Western blot检测到0.5、1.0、1.5μmol/L 5'-Aza-CdR处理后C D X2基因m R N A和蛋白重新表达,实时荧光定量PCR检测对照组和不同浓度实验组在HT-29细胞株mRNA相对表达量分别为1.000±0.000、0.973±0.024、1.014±0.019和1.094±0.020;在LoVo细胞株相对表达量分别为1.000±0.000、0.966±0.038、1.050±0.029和1.007±0.019.Western blot检测CDX2蛋白在对照组和不同浓度实验组中HT-29细胞株相对表达量分别为0.454±0.049、0.501±0.041、0.340±0.050和0.531±0.046;LoVo细胞株相对表达量分别为0.527±0.037、0.415±0.037、0.432±0.040和0.626±0.046.以上作用无时间、剂量依赖性,但CDX2基因mRNA在HT-29细胞株表达(F=25.146,P=0.000)和LoVo细胞株表达(F=5.470,P=0.024)具有统计学差异,C D X2蛋白表达在HT-29细胞株表达(F=9.700,P=0.005)和LoVo细胞株表达(F=17.701,P=0.001)亦具有统计学差异.结论:结直肠癌细胞株HT-29和LoVo中CDX2基因mRNA和蛋白表达不受中DNA甲基化的表观遗传修饰的调控.
文摘Chardonnay and Tinta Cao grape pomaces were generated by the winemaking process. The pomaces were extracted with 50% acetone and tested for antioxidant capacities using the oxygen radical absorbance capacity (ORAC) assay, DPPH□EC50 , and ABTS.+ scavenging capacity tests. Cytotoxicity and antiproliferative activities against Caco-2 and HT-29 human colon cancer cell lines were also analyzed. The quantitative detection of caspase-3 activity during the early apoptotic process was evaluated by fluorometric immunosorbent enzyme assay (FIENA). Induction of late stage apoptosis was analyzed by DNA fragmentation. Total phenolic content (TPC), individual phenolic acids, total oil, and fatty acid profile were also analyzed. The Tinta Cao pomace had the highest antioxidant capacity in all tests with an ORAC value of 386.5 μmol trolox equivalents (TE) per g pomace (μmol TE/g), its DPPH□EC50 value was 94.6 mg equivalents/L, and its ABTS.+ value was 806.7 μmol TE/g, which was more than twice as high as the Chardonnay. Both Tinta Cao and Chardonnay grape pomace extracts appear to contain components that inhibit the proliferation of Caco-2 and HT-29 cancer cells, at least in part, by triggering apoptosis. Expression of caspase-3 was induced by Tinta Cao and Chardonnay pomace extracts at 3 g/L after 4 hours of treatment with a 308% and 229% increase compared to control, respectively. An increase in DNA fragmentation was also observed with both grape pomace treatments. This study demonstrated that these tested grape pomaces were potent scavengers of free radicals and may provide some level of protection against certain cancers.
