目的:探讨萝卜硫素(Sulforaphane,SFN)对胰腺癌PANC-1细胞增殖、周期、转移和凋亡的影响及其机制。方法:将PANC-1细胞随机分为四组:对照组(sh-NC)、SFN组(sh-NC+SFN)、实验组(sh-TP53)和sh-TP53+SFN组,并制备不同浓度的SFN溶液(0、6、8...目的:探讨萝卜硫素(Sulforaphane,SFN)对胰腺癌PANC-1细胞增殖、周期、转移和凋亡的影响及其机制。方法:将PANC-1细胞随机分为四组:对照组(sh-NC)、SFN组(sh-NC+SFN)、实验组(sh-TP53)和sh-TP53+SFN组,并制备不同浓度的SFN溶液(0、6、8、10、16和20μg/mL)。细胞增殖-毒性检测(Cell counting Kit-8,CCK-8)试剂盒检测细胞存活率,划痕实验和Transwell实验检测细胞迁移和侵袭能力,流式细胞术用于分析细胞周期和细胞凋亡能力,蛋白免疫印迹法(Western Blot,WB)检测凋亡蛋白B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma 2 associated X protein,Bax)、B淋巴细胞瘤-2蛋白(B-cell lymphoma 2,Bcl-2)、半胱氨酸天冬氨酸蛋白酶3(Cysteinyl aspartate specific proteinase-3,Caspase-3)和半胱氨酸天冬氨酸蛋白酶9(Cysteinyl aspartate specific proteinase-9,Caspase-9),周期蛋白细胞周期蛋白依赖性激酶1(cyclin-dependent protein kinase 1,CDK1)和细胞周期蛋白B(Cyclin B),转移蛋白基质金属蛋白酶-9(Matrix metallopeptidase-9,MMP-9)和E-钙粘蛋白(E-cadherin),p53信号通路中生长停滞与DNA损伤诱导蛋白45A(growth arrest and DNA damage inducible alpha,GADD45A),细胞周期依赖性蛋白激酶抑制因子1A(cyclin-dependent kinase inhibitors 1A,p21)和肿瘤蛋白p53(Tumor protein p53,TP53)的表达水平,研究SFN对细胞增殖以及p53信号通路的影响。结果:SFN以浓度依赖性的方式抑制PANC-1细胞增殖。与对照组相比,SFN处理后的PANC-1细胞增殖率显著降低(P<0.001);迁移及侵袭能力减弱(P<0.01,P<0.0001);阻滞PANC-1细胞在G2/M期(P<0.0001);同时促进细胞凋亡(P<0.0001);上调Bax、Caspase-3、Caspase-9、E-cadherin、GADD45A、p21和TP53的蛋白水平,下调Bcl-2、CDK1、Cyclin B和MMP-9的蛋白水平(P<0.05,P<0.01,P<0.001,P<0.0001)。与SFN组相比,敲低TP53能显著下调SFN抑制PANC-1细胞增殖、迁移、侵袭和阻滞在G2/M期的能力,显著下调SFN促进PANC-1细胞凋亡的能力,下调Bax、Caspase-3、Caspase-9、E-cadherin、GADD45A、p21和TP53的蛋白水平,上调Bcl-2、CDK1、Cyclin B和MMP-9的蛋白水平(P<0.05,P<0.01,P<0.001,P<0.0001)。结论:SFN抑制胰腺癌PANC-1细胞活性与激活p53信号通路相关。展开更多
Following the publication,concerns have been raised about a number of figures in this article.An unexpected area of similarity was identified in terms of the cellular data,where the results from differently performed ...Following the publication,concerns have been raised about a number of figures in this article.An unexpected area of similarity was identified in terms of the cellular data,where the results from differently performed experiments were intended to have been shown,although the areas immediately surrounding this area featured comparatively different distributions of cells.展开更多
Background:The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy.Here,we studied the efficacy of the genotoxic agent etoposide(Eto)and irradiation in indu...Background:The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy.Here,we studied the efficacy of the genotoxic agent etoposide(Eto)and irradiation in inducing senescence of Panc02 pancreatic cancer cells,and the capability of the Bcl-2 inhibitor navitoclax(ABT-263;Nav)to trigger senolysis.Methods:Panc02 cells were treated with Eto or irradiated with 5–20 Gy before exposure to Nav.Cell survival,proliferation,and senescence were assessed by trypan blue staining,quantification of DNA synthesis,and staining of senescence-associatedβ-galactosidase(SA-β-Gal)-positive cells,respectively.Levels of mRNA were determined by real-time polymerase chain reaction,and protein expression was analyzed by immunoblotting.Panc02 cells were also grown as pancreatic tumors in mice,which were subsequently treated with Eto and Nav.Results:Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav.In vivo,Eto and Nav together,but not Eto alone,significantly reduced the proportion of proliferating cells.The expression of the senescence markerγH2AX and tumor infiltration with T-cells were not affected by the treatment.In vitro,almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive.Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto.In response to triggers of senescence,cultured Panc02 cells showed increased protein levels ofγH2AX and the autophagy marker LC3B-II,and higher mRNA levels of Cdkn1a,Mdm2,and PAI-1,while the effects of Nav were variable.Conclusions:In vitro and in vivo,the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone.Our data also provide some evidence for senolytic effects of Nav in vitro.展开更多
目的研究重楼皂苷Ⅶ对人胰腺癌PANC-1细胞增殖、迁移与侵袭作用及机制。方法采用MTT法检测重楼皂苷Ⅶ对PANC-1细胞存活率的影响;观察重楼皂苷Ⅶ对PANC-1细胞形态的影响;采用划痕实验和Transwell小室法考察重楼皂苷Ⅶ对PANC-1细胞迁移和...目的研究重楼皂苷Ⅶ对人胰腺癌PANC-1细胞增殖、迁移与侵袭作用及机制。方法采用MTT法检测重楼皂苷Ⅶ对PANC-1细胞存活率的影响;观察重楼皂苷Ⅶ对PANC-1细胞形态的影响;采用划痕实验和Transwell小室法考察重楼皂苷Ⅶ对PANC-1细胞迁移和侵袭能力的影响;采用流式细胞术检测重楼皂苷Ⅶ对PANC-1细胞凋亡的影响;采用Western blotting法检测重楼皂苷Ⅶ对PANC-1细胞半胱氨酸蛋白酶-3(Caspase-3)、B细胞淋巴瘤-2(B-celllymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-associated X protein,Bax)、聚腺苷二磷酸核糖聚合酶(poly ADP-ribose polymerase,PARP)、DNA酶抑制物(inhibitor of caspase-activated deoxyribonuclease,ICAD)和程序性死亡分子配体-1(programmed death ligand 1,PD-L1)蛋白表达的影响。结果重楼皂苷Ⅶ可抑制PANC-1细胞存活率,显著抑制PANC-1细胞迁移和侵袭能力(P<0.01),显著诱导PANC-1细胞凋亡(P<0.05、0.01),显著升高PANC-1细胞中凋亡相关蛋白Cleaved Caspase-3、PARP、Bax表达水平(P<0.01),显著降低pro-PARP、ICAD、Bcl-2、PD-L1蛋白表达水平(P<0.05、0.01)。结论重楼皂苷Ⅶ能够抑制PANC-1细胞增殖、迁移和侵袭,并可能通过下调PD-L1表达诱导PANC-1细胞凋亡。展开更多
目的:建立稳定转染人宫颈癌癌基因(human cervical cancer oncogene,HCCR)siRNA真核表达质粒的人胰腺癌PANC1细胞株,探讨siRNA干扰HCCR的表达后对人胰腺癌PANC1细胞增殖、凋亡和侵袭的影响。方法:通过脂质体转染法将含HCCRsiRNA的真核...目的:建立稳定转染人宫颈癌癌基因(human cervical cancer oncogene,HCCR)siRNA真核表达质粒的人胰腺癌PANC1细胞株,探讨siRNA干扰HCCR的表达后对人胰腺癌PANC1细胞增殖、凋亡和侵袭的影响。方法:通过脂质体转染法将含HCCRsiRNA的真核表达质粒pGCsi-HCCR稳定转染至人胰腺癌细胞株PANC1,抗生素G418筛选获得稳转细胞株;Western blot检测PANC1细胞中HCCR的表达,同时检测肿瘤相关基因p53蛋白表达的变化;流式细胞仪检测PANC1细胞的细胞周期和凋亡率变化;MTT比色法检测siRNA干扰后对PANC1细胞增殖能力的影响;Transwell侵袭实验观察siRNA干扰后对PANC1细胞侵袭能力的影响。结果:Western blot证实siRNA稳转组的PANC1细胞株和空载体稳转组比较HCCR蛋白表达水平下调,稳转细胞株建立成功。siRNA稳转组p53蛋白表达下降。siRNA稳转组的S期细胞数目减少而G0/G1期细胞数目增加,细胞凋亡增加。MTT结果显示siRNA稳转组1和稳转组2细胞吸光度分别为空载体组细胞的0.65倍和0.68倍,细胞增殖能力下降。Transwell侵袭实验显示siRNA稳转组细胞和空载体组细胞穿膜数分别为24.4±9.9和49.1±15.4(P<0.01),稳转组细胞侵袭能力下降。结论:siRNA干扰HCCR的表达后能抑制胰腺癌PANC1细胞增殖和侵袭,促进其凋亡。展开更多
文摘目的:探讨萝卜硫素(Sulforaphane,SFN)对胰腺癌PANC-1细胞增殖、周期、转移和凋亡的影响及其机制。方法:将PANC-1细胞随机分为四组:对照组(sh-NC)、SFN组(sh-NC+SFN)、实验组(sh-TP53)和sh-TP53+SFN组,并制备不同浓度的SFN溶液(0、6、8、10、16和20μg/mL)。细胞增殖-毒性检测(Cell counting Kit-8,CCK-8)试剂盒检测细胞存活率,划痕实验和Transwell实验检测细胞迁移和侵袭能力,流式细胞术用于分析细胞周期和细胞凋亡能力,蛋白免疫印迹法(Western Blot,WB)检测凋亡蛋白B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma 2 associated X protein,Bax)、B淋巴细胞瘤-2蛋白(B-cell lymphoma 2,Bcl-2)、半胱氨酸天冬氨酸蛋白酶3(Cysteinyl aspartate specific proteinase-3,Caspase-3)和半胱氨酸天冬氨酸蛋白酶9(Cysteinyl aspartate specific proteinase-9,Caspase-9),周期蛋白细胞周期蛋白依赖性激酶1(cyclin-dependent protein kinase 1,CDK1)和细胞周期蛋白B(Cyclin B),转移蛋白基质金属蛋白酶-9(Matrix metallopeptidase-9,MMP-9)和E-钙粘蛋白(E-cadherin),p53信号通路中生长停滞与DNA损伤诱导蛋白45A(growth arrest and DNA damage inducible alpha,GADD45A),细胞周期依赖性蛋白激酶抑制因子1A(cyclin-dependent kinase inhibitors 1A,p21)和肿瘤蛋白p53(Tumor protein p53,TP53)的表达水平,研究SFN对细胞增殖以及p53信号通路的影响。结果:SFN以浓度依赖性的方式抑制PANC-1细胞增殖。与对照组相比,SFN处理后的PANC-1细胞增殖率显著降低(P<0.001);迁移及侵袭能力减弱(P<0.01,P<0.0001);阻滞PANC-1细胞在G2/M期(P<0.0001);同时促进细胞凋亡(P<0.0001);上调Bax、Caspase-3、Caspase-9、E-cadherin、GADD45A、p21和TP53的蛋白水平,下调Bcl-2、CDK1、Cyclin B和MMP-9的蛋白水平(P<0.05,P<0.01,P<0.001,P<0.0001)。与SFN组相比,敲低TP53能显著下调SFN抑制PANC-1细胞增殖、迁移、侵袭和阻滞在G2/M期的能力,显著下调SFN促进PANC-1细胞凋亡的能力,下调Bax、Caspase-3、Caspase-9、E-cadherin、GADD45A、p21和TP53的蛋白水平,上调Bcl-2、CDK1、Cyclin B和MMP-9的蛋白水平(P<0.05,P<0.01,P<0.001,P<0.0001)。结论:SFN抑制胰腺癌PANC-1细胞活性与激活p53信号通路相关。
文摘Following the publication,concerns have been raised about a number of figures in this article.An unexpected area of similarity was identified in terms of the cellular data,where the results from differently performed experiments were intended to have been shown,although the areas immediately surrounding this area featured comparatively different distributions of cells.
基金supported by a grant from the Bundesministerium fur Bildung und Forschung(01ZX1903A).
文摘Background:The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy.Here,we studied the efficacy of the genotoxic agent etoposide(Eto)and irradiation in inducing senescence of Panc02 pancreatic cancer cells,and the capability of the Bcl-2 inhibitor navitoclax(ABT-263;Nav)to trigger senolysis.Methods:Panc02 cells were treated with Eto or irradiated with 5–20 Gy before exposure to Nav.Cell survival,proliferation,and senescence were assessed by trypan blue staining,quantification of DNA synthesis,and staining of senescence-associatedβ-galactosidase(SA-β-Gal)-positive cells,respectively.Levels of mRNA were determined by real-time polymerase chain reaction,and protein expression was analyzed by immunoblotting.Panc02 cells were also grown as pancreatic tumors in mice,which were subsequently treated with Eto and Nav.Results:Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav.In vivo,Eto and Nav together,but not Eto alone,significantly reduced the proportion of proliferating cells.The expression of the senescence markerγH2AX and tumor infiltration with T-cells were not affected by the treatment.In vitro,almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive.Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto.In response to triggers of senescence,cultured Panc02 cells showed increased protein levels ofγH2AX and the autophagy marker LC3B-II,and higher mRNA levels of Cdkn1a,Mdm2,and PAI-1,while the effects of Nav were variable.Conclusions:In vitro and in vivo,the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone.Our data also provide some evidence for senolytic effects of Nav in vitro.
文摘目的研究重楼皂苷Ⅶ对人胰腺癌PANC-1细胞增殖、迁移与侵袭作用及机制。方法采用MTT法检测重楼皂苷Ⅶ对PANC-1细胞存活率的影响;观察重楼皂苷Ⅶ对PANC-1细胞形态的影响;采用划痕实验和Transwell小室法考察重楼皂苷Ⅶ对PANC-1细胞迁移和侵袭能力的影响;采用流式细胞术检测重楼皂苷Ⅶ对PANC-1细胞凋亡的影响;采用Western blotting法检测重楼皂苷Ⅶ对PANC-1细胞半胱氨酸蛋白酶-3(Caspase-3)、B细胞淋巴瘤-2(B-celllymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-associated X protein,Bax)、聚腺苷二磷酸核糖聚合酶(poly ADP-ribose polymerase,PARP)、DNA酶抑制物(inhibitor of caspase-activated deoxyribonuclease,ICAD)和程序性死亡分子配体-1(programmed death ligand 1,PD-L1)蛋白表达的影响。结果重楼皂苷Ⅶ可抑制PANC-1细胞存活率,显著抑制PANC-1细胞迁移和侵袭能力(P<0.01),显著诱导PANC-1细胞凋亡(P<0.05、0.01),显著升高PANC-1细胞中凋亡相关蛋白Cleaved Caspase-3、PARP、Bax表达水平(P<0.01),显著降低pro-PARP、ICAD、Bcl-2、PD-L1蛋白表达水平(P<0.05、0.01)。结论重楼皂苷Ⅶ能够抑制PANC-1细胞增殖、迁移和侵袭,并可能通过下调PD-L1表达诱导PANC-1细胞凋亡。
