The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression leve...The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIFl-ct, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 ~unol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIFI-a, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the ex- pression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, $6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib （IC50：1.385 vs. 15.09 ~mol/L respec- tively）, whereas combination of NVP-BKM120 and gefitinib （1 ~trnol/L） did not show more obvious ef- fect than NVP-BKM120 used alone on inhibition of cell growth （P〉0.05）. NVP-BKM120 （1 ~unol/L） increased the proportion ofH1975 cells in G0~G1 phase and the effect was concentration-dependent, and 2 ~maol/L NVP-BKM120 promoted apoptosis ofH1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib （1 ~unol/L）-treated group or between DMSO-treated control group and gefitinib （1 Ixmol/L）-treated alone group （P〉0.05 for all）. It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 （1 ~展开更多
[目的]探索信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)新型抑制剂苦蘵内酯J诱导肺腺癌细胞凋亡的分子机制。[方法]以5、10、20、25、30、40μmol·L^(-1)苦蘵内酯J处理表皮细胞生长因子受...[目的]探索信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)新型抑制剂苦蘵内酯J诱导肺腺癌细胞凋亡的分子机制。[方法]以5、10、20、25、30、40μmol·L^(-1)苦蘵内酯J处理表皮细胞生长因子受体(epidermal growth factor receptor,EGFR)T790M继发突变的肺腺癌细胞株H1975细胞24h或48h后,CCK-8法检测细胞存活率;5、10、15、20μmol·L^(-1)苦蘵内酯J处理H1975细胞24h后,Annexin V-PI染色及流式细胞术检测细胞凋亡率,Western blot检测凋亡相关蛋白的表达;5、10、15、20μmol·L^(-1)苦蘵内酯J处理H1975细胞4h或20μmol·L^(-1)苦蘵内酯J处理H1975细胞0.5、1、2、4h后,Western blot检测STAT3蛋白表达及其磷酸化水平;以20μmol·L^(-1)蛋白酶体抑制剂MG132及10μmol·L^(-1)苦蘵内酯J联合处理或单独处理H1975细胞,阐明苦蘵内酯J对STAT3蛋白的降解作用;20μmol·L^(-1)苦蘵内酯J处理H1975细胞4h后,实时荧光定量PCR检测STAT3 m RNA表达情况。[结果]H1975细胞存活率随苦蘵内酯J作用时间延长和作用浓度增加而不断降低,20μmol·L^(-1)苦蘵内酯J处理24h和48h,H1975细胞存活率分别为65.9%和44.2%;20μmol·L^(-1)苦蘵内酯J处理24h后,H1975细胞凋亡率为51.1%;Western blot检测显示20μmol·L^(-1)苦蘵内酯J处理24h后,H1975细胞内cleaved-caspase 9、cleaved-caspase 3和cleaved-PARP蛋白表达水平显著高于未处理组(P<0.05,P<0.01,P<0.001);20μmol·L^(-1)苦蘵内酯J处理4h后,H1975细胞内STAT3总蛋白和STAT3-Tyr表达水平显著低于与未处理组(P<0.001);蛋白酶体抑制剂MG132可恢复STAT3的总蛋白水平;实时荧光定量PCR结果显示,苦蘵内酯J对STAT3 m RNA表达无影响(P>0.05)。[结论]苦蘵内酯J能够抑制H1975细胞增殖并诱导细胞凋亡,其可能的机制是引起STAT3泛素化降解并抑制STAT3磷酸化。展开更多
基金supported by grants from the Wu Jieping Medical Foundation of China(No.320-6700-09069)the Hubei Provincial Natural Science Foundation of China(No.2010CDB07702)Novartis International AG
文摘The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIFl-ct, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 ~unol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIFI-a, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the ex- pression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, $6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib （IC50：1.385 vs. 15.09 ~mol/L respec- tively）, whereas combination of NVP-BKM120 and gefitinib （1 ~trnol/L） did not show more obvious ef- fect than NVP-BKM120 used alone on inhibition of cell growth （P〉0.05）. NVP-BKM120 （1 ~unol/L） increased the proportion ofH1975 cells in G0~G1 phase and the effect was concentration-dependent, and 2 ~maol/L NVP-BKM120 promoted apoptosis ofH1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib （1 ~unol/L）-treated group or between DMSO-treated control group and gefitinib （1 Ixmol/L）-treated alone group （P〉0.05 for all）. It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 （1 ~
文摘[目的]探索信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)新型抑制剂苦蘵内酯J诱导肺腺癌细胞凋亡的分子机制。[方法]以5、10、20、25、30、40μmol·L^(-1)苦蘵内酯J处理表皮细胞生长因子受体(epidermal growth factor receptor,EGFR)T790M继发突变的肺腺癌细胞株H1975细胞24h或48h后,CCK-8法检测细胞存活率;5、10、15、20μmol·L^(-1)苦蘵内酯J处理H1975细胞24h后,Annexin V-PI染色及流式细胞术检测细胞凋亡率,Western blot检测凋亡相关蛋白的表达;5、10、15、20μmol·L^(-1)苦蘵内酯J处理H1975细胞4h或20μmol·L^(-1)苦蘵内酯J处理H1975细胞0.5、1、2、4h后,Western blot检测STAT3蛋白表达及其磷酸化水平;以20μmol·L^(-1)蛋白酶体抑制剂MG132及10μmol·L^(-1)苦蘵内酯J联合处理或单独处理H1975细胞,阐明苦蘵内酯J对STAT3蛋白的降解作用;20μmol·L^(-1)苦蘵内酯J处理H1975细胞4h后,实时荧光定量PCR检测STAT3 m RNA表达情况。[结果]H1975细胞存活率随苦蘵内酯J作用时间延长和作用浓度增加而不断降低,20μmol·L^(-1)苦蘵内酯J处理24h和48h,H1975细胞存活率分别为65.9%和44.2%;20μmol·L^(-1)苦蘵内酯J处理24h后,H1975细胞凋亡率为51.1%;Western blot检测显示20μmol·L^(-1)苦蘵内酯J处理24h后,H1975细胞内cleaved-caspase 9、cleaved-caspase 3和cleaved-PARP蛋白表达水平显著高于未处理组(P<0.05,P<0.01,P<0.001);20μmol·L^(-1)苦蘵内酯J处理4h后,H1975细胞内STAT3总蛋白和STAT3-Tyr表达水平显著低于与未处理组(P<0.001);蛋白酶体抑制剂MG132可恢复STAT3的总蛋白水平;实时荧光定量PCR结果显示,苦蘵内酯J对STAT3 m RNA表达无影响(P>0.05)。[结论]苦蘵内酯J能够抑制H1975细胞增殖并诱导细胞凋亡,其可能的机制是引起STAT3泛素化降解并抑制STAT3磷酸化。
