甲基莲心碱增强STI571对K562/G01细胞敏感性及其机制研究
摘要
目的
探讨甲基莲心碱增强STI571对K562／G01细胞敏感性,对细胞增殖、凋亡、bcr/abl融合基因和p210Bcr-abl融合蛋白表达的影响,为临床应用提供理论及实验依据。
方法
采用改良四氮唑盐法(MTT)检测细胞毒性；流式细胞仪测定经药物处理的K562／G01细胞凋亡率；分光光度法检测定Caspase-3活性；RT-PCR法和蛋白免疫印迹(Western-blot)法检测Nef对bcr/abl融合基因及p210Bcr-abl融合蛋白表达的影响。
结果
(1)①Nef2～64μmol/L、As2O310～160μmol/L对K562／G01细胞增殖均有抑制作用,Nef、As203对K562／G01细胞的IC10为8.1±0.16μmol/L.10.42±0.64.②STI 571在K562细胞的IC50为0.63±0.04μmol/L,对K562／G01细胞的IC50为26.52±1.35μmol/L,K562细胞对STI 571的耐药倍数是42.1倍。③Nef4μmol/L、Nef 8μmol/L.As20310μmol/L与STI 571(0.01～100μmol/L)联合用药组与STI 571(0.01～100μmol/L)单独用药组相比均不同程度的降低了K562／G01细胞的IC50值,IC50由29.41±0.48μmol／L降至14.39±0.48、8.80±0.25和12.75±1.50μmol/L.
(2)①PI染色流式细胞术检测各给药组24、48、72和96h凋亡率,与阴性对照组相比Nef 4μmol/L和8μmol/L组细胞凋亡率无显著增高(p>0.05), STI 571 10μmol/L联合Nef 8μmol/L组与STI 571组比的凋亡率显著增高(p<0.05)。②K562／G01细胞经药物处理培养48h、96h后,采用分光光度法检测经药物处理48h后细胞Caspase-3活性,与阴性对照相比,Nef 4μmol/L、8μmol/L组Caspase-3活性无显著增高(p>0.05),联合用药组Caspase-3活性显著高于STI 571(p<0.05)组和As203组(p<0.05)；而经药物处理96h后,Nef4μmol/L、8μmol/L组Caspase-3活性显著增高(p>0.05)。
(3)①Western-blotting结果显示：与阴性对照组比较,Nef 4μmol/L、8μmol/L组p210Bcr-abl蛋白表达无显著变化(p>0.05)；与STI 571 10μmol/L组相比,STI 571 10μmol/L联合Nef 8μmol/L用药组p210Bcr-abl蛋白表达显著下降(p<0.05)。②RT-PCR结果显示：与阴性对照组相比,Nef 4、8μmol/L组bcr/abl融合基因表达无显著变化(p>0.05)；与STI 571组相比,STI 571 10μmol/L联合Nef 8μmol/L用药组bcr／abl融合基因表达显著下降(p<0.01)。
结论
Nef能提高K562／G01细胞对STI 571敏感性；通过增强STI 571对K562／G01细胞的促进凋亡及Caspase-3活化作用；同时Nef能增强STI571下调bcr/abl融合基因及p210Bcr-abl融合蛋白表达。
OBJECTIVE
To observe neferine(nef) neferine enhances the sensitivity of STI571 and the molecular mechanism of reversal multidrug resistance on K562/G01 cells.
METHODS
Cytotoxicity was determined by improved MTT assay.The influence of nef to affect STI571 to induce K562/G01 cells apoptosis was detected by PI staining flow cytometry.The activity of caspase-3 was detected by Caspase 3 Activity Assay Kit.The influence on bcr-abl mRNA and p210Bcr-abl expression was determined by RT-PCR and protein immunoblotting (Western blotting).
RESULTS:
(1)①Nef 2～64μmol/L、As2O3 10～160μmol/L exhibited an inhibition activity to roliferation in K562/G01 cells. The IC10 of Nef was 8.1±0.16μmol/L and the IC10 of ATO was 10.42±0.64μmol/L.②The IC50 of STI 571 was 0.63±0.04μmol/L in K562 cells and 26.52±1.35μmol/L in K562/G01 cells. Drug resistance activity of K562/G01 cells to STI 571 was 42.1-fold greater than that of K562 cells.③Nef (4μmol/L)、Nef (8μmol/L)、As2O3 (10μmol/L) decreased IC50 of STI 571 in K562/G01 cells from 29.41±0.48μmol/L to 14.39±0.48μmol/L、8.80±0.25μmol/L和12.75±1.50μmol/L.
(2)①The application of single drug of Nef has no effect to induce K562/G01 cells apoptosis (p>0.05). Nef in combination with STI 571 significantly degraded apoptosis than treated by STI571 (p<0.05).②The application of single drug of Nef after 48 h has no effect to inhence caspase-3 activity in K562/G01 cells (p>0.05).Nef in combination with STI571 significantly inhence caspase-3 activity than treated by STI571 (p <0.05). The application of single drug of Nef after 96 h has inhenced caspase-3 activity in K562/G01 cells (p<0.01).
(3)①The application of single drug of Nef has no effect on p210Bcr-abl expression in K562/G01 cells (p>0.05). Nef in combination with STI571 significantly degraded p210Bcr-abl expression in K562/G01 cells than treated by STI571 (p<0.05).②The application of single drug of Nef has no effect on bcr/abl mRNA expression in K562/G01 cells (p>0.05). Nef in combination with STI571 significantly degraded bcr-abl mRNA expression in K562/G01 cells than treated by STI571 (p< 0.05).
CONCLUSIONS
Nef has a potency of anticancer to K562/G01 cells. (2) Nef has no effect on bcr/abl mRNA and p210Bcr-abl protein expression but can enhanced the effects of STI571 on down-regulate bcr-abl mRNA and p210Bcr-abl protein expression.Nef is able to potentiate the anticancer function of STI 571.
探讨甲基莲心碱增强STI571对K562／G01细胞敏感性,对细胞增殖、凋亡、bcr/abl融合基因和p210Bcr-abl融合蛋白表达的影响,为临床应用提供理论及实验依据。
方法
采用改良四氮唑盐法(MTT)检测细胞毒性；流式细胞仪测定经药物处理的K562／G01细胞凋亡率；分光光度法检测定Caspase-3活性；RT-PCR法和蛋白免疫印迹(Western-blot)法检测Nef对bcr/abl融合基因及p210Bcr-abl融合蛋白表达的影响。
结果
(1)①Nef2～64μmol/L、As2O310～160μmol/L对K562／G01细胞增殖均有抑制作用,Nef、As203对K562／G01细胞的IC10为8.1±0.16μmol/L.10.42±0.64.②STI 571在K562细胞的IC50为0.63±0.04μmol/L,对K562／G01细胞的IC50为26.52±1.35μmol/L,K562细胞对STI 571的耐药倍数是42.1倍。③Nef4μmol/L、Nef 8μmol/L.As20310μmol/L与STI 571(0.01～100μmol/L)联合用药组与STI 571(0.01～100μmol/L)单独用药组相比均不同程度的降低了K562／G01细胞的IC50值,IC50由29.41±0.48μmol／L降至14.39±0.48、8.80±0.25和12.75±1.50μmol/L.
(2)①PI染色流式细胞术检测各给药组24、48、72和96h凋亡率,与阴性对照组相比Nef 4μmol/L和8μmol/L组细胞凋亡率无显著增高(p>0.05), STI 571 10μmol/L联合Nef 8μmol/L组与STI 571组比的凋亡率显著增高(p<0.05)。②K562／G01细胞经药物处理培养48h、96h后,采用分光光度法检测经药物处理48h后细胞Caspase-3活性,与阴性对照相比,Nef 4μmol/L、8μmol/L组Caspase-3活性无显著增高(p>0.05),联合用药组Caspase-3活性显著高于STI 571(p<0.05)组和As203组(p<0.05)；而经药物处理96h后,Nef4μmol/L、8μmol/L组Caspase-3活性显著增高(p>0.05)。
(3)①Western-blotting结果显示：与阴性对照组比较,Nef 4μmol/L、8μmol/L组p210Bcr-abl蛋白表达无显著变化(p>0.05)；与STI 571 10μmol/L组相比,STI 571 10μmol/L联合Nef 8μmol/L用药组p210Bcr-abl蛋白表达显著下降(p<0.05)。②RT-PCR结果显示：与阴性对照组相比,Nef 4、8μmol/L组bcr/abl融合基因表达无显著变化(p>0.05)；与STI 571组相比,STI 571 10μmol/L联合Nef 8μmol/L用药组bcr／abl融合基因表达显著下降(p<0.01)。
结论
Nef能提高K562／G01细胞对STI 571敏感性；通过增强STI 571对K562／G01细胞的促进凋亡及Caspase-3活化作用；同时Nef能增强STI571下调bcr/abl融合基因及p210Bcr-abl融合蛋白表达。
OBJECTIVE
To observe neferine(nef) neferine enhances the sensitivity of STI571 and the molecular mechanism of reversal multidrug resistance on K562/G01 cells.
METHODS
Cytotoxicity was determined by improved MTT assay.The influence of nef to affect STI571 to induce K562/G01 cells apoptosis was detected by PI staining flow cytometry.The activity of caspase-3 was detected by Caspase 3 Activity Assay Kit.The influence on bcr-abl mRNA and p210Bcr-abl expression was determined by RT-PCR and protein immunoblotting (Western blotting).
RESULTS:
(1)①Nef 2～64μmol/L、As2O3 10～160μmol/L exhibited an inhibition activity to roliferation in K562/G01 cells. The IC10 of Nef was 8.1±0.16μmol/L and the IC10 of ATO was 10.42±0.64μmol/L.②The IC50 of STI 571 was 0.63±0.04μmol/L in K562 cells and 26.52±1.35μmol/L in K562/G01 cells. Drug resistance activity of K562/G01 cells to STI 571 was 42.1-fold greater than that of K562 cells.③Nef (4μmol/L)、Nef (8μmol/L)、As2O3 (10μmol/L) decreased IC50 of STI 571 in K562/G01 cells from 29.41±0.48μmol/L to 14.39±0.48μmol/L、8.80±0.25μmol/L和12.75±1.50μmol/L.
(2)①The application of single drug of Nef has no effect to induce K562/G01 cells apoptosis (p>0.05). Nef in combination with STI 571 significantly degraded apoptosis than treated by STI571 (p<0.05).②The application of single drug of Nef after 48 h has no effect to inhence caspase-3 activity in K562/G01 cells (p>0.05).Nef in combination with STI571 significantly inhence caspase-3 activity than treated by STI571 (p <0.05). The application of single drug of Nef after 96 h has inhenced caspase-3 activity in K562/G01 cells (p<0.01).
(3)①The application of single drug of Nef has no effect on p210Bcr-abl expression in K562/G01 cells (p>0.05). Nef in combination with STI571 significantly degraded p210Bcr-abl expression in K562/G01 cells than treated by STI571 (p<0.05).②The application of single drug of Nef has no effect on bcr/abl mRNA expression in K562/G01 cells (p>0.05). Nef in combination with STI571 significantly degraded bcr-abl mRNA expression in K562/G01 cells than treated by STI571 (p< 0.05).
CONCLUSIONS
Nef has a potency of anticancer to K562/G01 cells. (2) Nef has no effect on bcr/abl mRNA and p210Bcr-abl protein expression but can enhanced the effects of STI571 on down-regulate bcr-abl mRNA and p210Bcr-abl protein expression.Nef is able to potentiate the anticancer function of STI 571.
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