OBJECTIVE TNF-related apoptosis-inducing ligand(TRAIL)is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors.However,most breast cancer cells ...OBJECTIVE TNF-related apoptosis-inducing ligand(TRAIL)is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors.However,most breast cancer cells are resistant to TRAIL-induced apoptosis.Our objectives are to investigate the underlying molecular mechanisms and to develop strategies to overcome such resistance.METHODS To identify modulators of TRAIL-induced apoptosis,we carried out a genome wide si RNA screen.To validate the screening result,we either silenced or overexpressed the identified genes in various breast cancer cells and changes in growth and TRAIL-induced cell apoptosis were determined in vitro and in an orthotopic xenograft mouse model.Finally,we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL-therapy.RESULTS We unexpectedly identified androgen receptor(AR)to be responsible for TRAIL resistance.While AR is classically viewed as the key factor in prostate cancer progression,we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple-negative breast cancers(TNBC)that are highly aggressive with poor prognosis.Importantly,breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance.AR overexpression in MDA-MB-231 and MDA-MB-436 cells suppressed the TRAIL sensitivity whereas knockdown of AR rendered MCF-7 and MDA-MB-453 cells sensitive to TRAIL-induced apoptosis.AR overexpression also induced TRAIL resistance in breast tumors in vivo.Further,we observed an upregulation of the TRAIL receptor,death receptor 5(DR5)in breast cancer cells,following the removal or inhibition of AR by its antagonists Casodex and MDV3100.Treatment with AR antagonists also enhanced TRAIL-induced breast cancer cell apoptosis.CONCLUSION AR signaling suppresses TRAIL-induced breast cancer cell apoptosis,in part,by suppressing DR5 expression,and a combination of AR antagonists together with TRAIL may be a novel and effective therapy for TNBC.展开更多
背景与目的:靶向治疗是乳腺癌等实体瘤治疗的又一有效手段,靶向药物的开发是临床治疗的需要。本文探讨哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)特异性抑制剂雷帕霉素和甲基转移酶抑制剂5-脱氧氮杂胞苷(5-aza-2’-de...背景与目的:靶向治疗是乳腺癌等实体瘤治疗的又一有效手段,靶向药物的开发是临床治疗的需要。本文探讨哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)特异性抑制剂雷帕霉素和甲基转移酶抑制剂5-脱氧氮杂胞苷(5-aza-2’-deoxycytidine,5-Aza-cdR)对雌激素受体阴性人乳腺癌细胞株MCF-7体外培养的抑制作用,为临床靶向治疗提供实验依据。方法:取对数生长期生长良好的乳腺癌MCF-7细胞株,按2.0×106细胞/孔接种于24孔板,按析因试验设置4个组:雷帕霉素组(R组)、5-Aza-cdR组(Aza组)、两药联合组和对照组。采用MTT法观察雷帕霉素、5-Aza-cdR对体外培养的MCF-7细胞系生长的抑制作用;应用流式细胞仪检测凋亡细胞和细胞周期。结果:与对照组相比,R组和Aza组均有一定的抑制乳腺癌细胞MCF-7细胞体外培养生长的作用(P<0.05),联合组对乳腺癌细胞生长的抑制作用与R组相同(P>0.05)。在干预48h时,R组和联合组即达到有效抑制率。Aza组干预120h达到有效抑制率。Aza组细胞抑制率低于联合组(P<0.05),R组与联合组细胞抑制率差异无显著性(P>0.05)。在72h时间点,R组、Aza组、联合组、对照组的早期凋亡细胞百分率分别为40.9%、22.7%、37.3%、24.1%;晚期凋亡细胞分别为17.8%、43.7%、27%、40.2%;与对照组相比,R组、联合组均能有效地诱导MCF-7早期细胞凋亡(P<0.05)。Aza组早期细胞凋亡率均较R组和联合组低(P<0.05),但晚期细胞凋亡率较R组和联合组高(P<0.05);与对照组相比差异无显著性(P>0.05)。雷帕霉素4.0×10-5mg/L与5-Aza-cdR25μmol/L干预无明显交互作用(P>0.05)。联用组与R组比较,细胞早期凋亡率下降(P<0.05)。在72h时间点,4组的G0、G1期细胞分别为60.1%、67.3%、66.8%和6%;S期细胞分别为34.2%、17.1%、22.2%和38.5%;G2期细胞分别为5.8%、15.6%、11.1%和55.4%。与对照组比较,R组、Aza组、联合组对乳腺癌MCF-7细胞均有较强的G0、G1期阻滞作用(P<0.05)。与联合组相比联合用药不增强G0、G1期周期阻滞作用(P>0.05)。雷帕霉素4.0×10-5mg/L与5-Aza-cdR25μmol/L干预在细胞G0、G1期无明显的交互作用(P>0.05)。结论:体外培养抑制试验证明,雷帕霉素、5-Aza-cdR均能抑制乳腺癌细胞株MCF-7的生长。两药无交互作用,临床无联合用药价值。雷帕霉素联合5-Aza-cdR不增加细胞凋亡。5-Aza-cdR能干预细胞周期,其可能的机制是诱导细胞成熟分化。联合用药不增强MCF-7细胞的周期阻滞和凋亡。展开更多
目的探讨Beclin 1蛋白与三阴性乳腺癌细胞对5-Fu敏感度的关系。方法应用慢病毒介导p Len O-GTP-BECN1表达载体稳定转染至BT-549细胞,嘌呤霉素筛选阳性细胞,设立对照组。使用不同浓度5-Fu处理各组细胞,MTT法比较细胞对5-Fu的敏感度;流式...目的探讨Beclin 1蛋白与三阴性乳腺癌细胞对5-Fu敏感度的关系。方法应用慢病毒介导p Len O-GTP-BECN1表达载体稳定转染至BT-549细胞,嘌呤霉素筛选阳性细胞,设立对照组。使用不同浓度5-Fu处理各组细胞,MTT法比较细胞对5-Fu的敏感度;流式细胞术和Hoechst33342实验检测细胞凋亡情况;Western blot检测细胞凋亡相关蛋白Caspase-3和Caspase-8表达情况。结果成功构建稳定表达Beclin 1蛋白的BT-549细胞株。p Len O-GTP-BECN1组对5-Fu敏感度显著高于其他两组;5-Fu作用48 h后,p Len O-GTP-BECN1组的IC50为(3.54±0.20)μg/ml,显著低于空白载体p Len O-GTP组(14.45±1.81)μg/ml和空白组(79.40±8.34)μg/ml(F=207.902;P<0.00)。5-Fu作用前,p Len O-GTP-BECN1组凋亡率为(12.14±0.76)%,同p Len O-GTP组(13.57±1.04)%和空白组(13.75±1.29)%(F=2.096;P=0.204)之间差异并无统计学意义;5-Fu作用48 h后,p Len O-GTP-BECN1组凋亡率为(51.01±3.26)%,显著高于p Len O-GTP组(42.17±4.6)%和空白组(38.42±2.93)%(F=9.32;P<0.01);接受5-Fu处理后,p Len O-GTP-BECN1组细胞核凋亡特征改变较其他两组更明显。5-Fu作用后,p Len O-GTP-BECN1组细胞Caspase-3相对表达量为(0.57±0.0028),显著高于p Len O-GTP组(0.42±0.010)和空白组(0.30±0.046)(F=46.134;P<0.01);p Len O-GTP-BECN1组细胞Caspase-8相对表达量为(0.44±0.0038),显著高于p Len O-GTP组(0.18±0.0095)和空白组(0.27±0.00060)(F=1006.757;P<0.00)。结论Beclin 1显著提高三阴性乳腺癌细胞对5-Fu的敏感度,其中促进凋亡发生可能是重要机制之一。展开更多
AIM To filtrate breast cancer resistance protein(BCRP)-mediated resistance agents and investigatethe mechanism,so as to provide valuable datum for optimization clinical chemotherapy scheme to tumor withevaluation mark...AIM To filtrate breast cancer resistance protein(BCRP)-mediated resistance agents and investigatethe mechanism,so as to provide valuable datum for optimization clinical chemotherapy scheme to tumor withevaluation marker of BCRP expression.METHODS MTT assay was used to filtrate BCRP-mediatedresistance agents with PA317/Tet-on/TRE-BCRP cell of different expression levels of BCRP after treated withdifferent concentration anticancer agents.High performance liquid chromatography(HPLC) was applied tomeasure relative dose of intracellular retention resistance agents.Nuclear DNA fluorescence dye,Hochest33258,staining and flow cytometry were adopted to detect apoptotic cells after treated with drugs.RESULTSThere were shown increasing durg-resistance to 5-fluorouracil,methotrexate,doxirubicin,pirarubicin,etoposide and mitoxantrone followed with increasing expression of BCRP on PA317/Tet-on/TRE-BCRPcells(P<0.05,n=3),but shown sensitive to paclitaxel,cisplatin,vincristine,mitomycin and vindesine.Therealso was shown significant negative correlation between the intracellular retention dose of 5-fluorouracil withdifferent expression of BCRP(r=-0.885,P<0.05,n=3).There were shown parallel results ofthat decreasingcellular apoptotic rate with increasing cellular expression of BCRP after treated with 5-fluorouracil byfluorescence dye staining and flow cytometry(P<0.05,n=3),and also shown significate rise of the apoptoticrate of BCRP expression cells after treated with Ko143 (P<0.05,n=3).Every group of cells could be differentextently blocked in phase of G_0/G_1 treated with 5-fluorouracil.CONCLUSION Resistance of 5-fluorouracilcould be especially mediated by conjugated with BCRP and acted as drug exclude-pump substrate.Cellularability resistant to 5-fluorouracil-induced apoptosis could be reinforced by BCRP expression.展开更多
基金supported by National Institutes of Health(R21CA193271 and R01HL116849)National Natural Science Foundation of China(31100595 and 31300683)
文摘OBJECTIVE TNF-related apoptosis-inducing ligand(TRAIL)is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors.However,most breast cancer cells are resistant to TRAIL-induced apoptosis.Our objectives are to investigate the underlying molecular mechanisms and to develop strategies to overcome such resistance.METHODS To identify modulators of TRAIL-induced apoptosis,we carried out a genome wide si RNA screen.To validate the screening result,we either silenced or overexpressed the identified genes in various breast cancer cells and changes in growth and TRAIL-induced cell apoptosis were determined in vitro and in an orthotopic xenograft mouse model.Finally,we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL-therapy.RESULTS We unexpectedly identified androgen receptor(AR)to be responsible for TRAIL resistance.While AR is classically viewed as the key factor in prostate cancer progression,we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple-negative breast cancers(TNBC)that are highly aggressive with poor prognosis.Importantly,breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance.AR overexpression in MDA-MB-231 and MDA-MB-436 cells suppressed the TRAIL sensitivity whereas knockdown of AR rendered MCF-7 and MDA-MB-453 cells sensitive to TRAIL-induced apoptosis.AR overexpression also induced TRAIL resistance in breast tumors in vivo.Further,we observed an upregulation of the TRAIL receptor,death receptor 5(DR5)in breast cancer cells,following the removal or inhibition of AR by its antagonists Casodex and MDV3100.Treatment with AR antagonists also enhanced TRAIL-induced breast cancer cell apoptosis.CONCLUSION AR signaling suppresses TRAIL-induced breast cancer cell apoptosis,in part,by suppressing DR5 expression,and a combination of AR antagonists together with TRAIL may be a novel and effective therapy for TNBC.
文摘背景与目的:靶向治疗是乳腺癌等实体瘤治疗的又一有效手段,靶向药物的开发是临床治疗的需要。本文探讨哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)特异性抑制剂雷帕霉素和甲基转移酶抑制剂5-脱氧氮杂胞苷(5-aza-2’-deoxycytidine,5-Aza-cdR)对雌激素受体阴性人乳腺癌细胞株MCF-7体外培养的抑制作用,为临床靶向治疗提供实验依据。方法:取对数生长期生长良好的乳腺癌MCF-7细胞株,按2.0×106细胞/孔接种于24孔板,按析因试验设置4个组:雷帕霉素组(R组)、5-Aza-cdR组(Aza组)、两药联合组和对照组。采用MTT法观察雷帕霉素、5-Aza-cdR对体外培养的MCF-7细胞系生长的抑制作用;应用流式细胞仪检测凋亡细胞和细胞周期。结果:与对照组相比,R组和Aza组均有一定的抑制乳腺癌细胞MCF-7细胞体外培养生长的作用(P<0.05),联合组对乳腺癌细胞生长的抑制作用与R组相同(P>0.05)。在干预48h时,R组和联合组即达到有效抑制率。Aza组干预120h达到有效抑制率。Aza组细胞抑制率低于联合组(P<0.05),R组与联合组细胞抑制率差异无显著性(P>0.05)。在72h时间点,R组、Aza组、联合组、对照组的早期凋亡细胞百分率分别为40.9%、22.7%、37.3%、24.1%;晚期凋亡细胞分别为17.8%、43.7%、27%、40.2%;与对照组相比,R组、联合组均能有效地诱导MCF-7早期细胞凋亡(P<0.05)。Aza组早期细胞凋亡率均较R组和联合组低(P<0.05),但晚期细胞凋亡率较R组和联合组高(P<0.05);与对照组相比差异无显著性(P>0.05)。雷帕霉素4.0×10-5mg/L与5-Aza-cdR25μmol/L干预无明显交互作用(P>0.05)。联用组与R组比较,细胞早期凋亡率下降(P<0.05)。在72h时间点,4组的G0、G1期细胞分别为60.1%、67.3%、66.8%和6%;S期细胞分别为34.2%、17.1%、22.2%和38.5%;G2期细胞分别为5.8%、15.6%、11.1%和55.4%。与对照组比较,R组、Aza组、联合组对乳腺癌MCF-7细胞均有较强的G0、G1期阻滞作用(P<0.05)。与联合组相比联合用药不增强G0、G1期周期阻滞作用(P>0.05)。雷帕霉素4.0×10-5mg/L与5-Aza-cdR25μmol/L干预在细胞G0、G1期无明显的交互作用(P>0.05)。结论:体外培养抑制试验证明,雷帕霉素、5-Aza-cdR均能抑制乳腺癌细胞株MCF-7的生长。两药无交互作用,临床无联合用药价值。雷帕霉素联合5-Aza-cdR不增加细胞凋亡。5-Aza-cdR能干预细胞周期,其可能的机制是诱导细胞成熟分化。联合用药不增强MCF-7细胞的周期阻滞和凋亡。
文摘目的探讨Beclin 1蛋白与三阴性乳腺癌细胞对5-Fu敏感度的关系。方法应用慢病毒介导p Len O-GTP-BECN1表达载体稳定转染至BT-549细胞,嘌呤霉素筛选阳性细胞,设立对照组。使用不同浓度5-Fu处理各组细胞,MTT法比较细胞对5-Fu的敏感度;流式细胞术和Hoechst33342实验检测细胞凋亡情况;Western blot检测细胞凋亡相关蛋白Caspase-3和Caspase-8表达情况。结果成功构建稳定表达Beclin 1蛋白的BT-549细胞株。p Len O-GTP-BECN1组对5-Fu敏感度显著高于其他两组;5-Fu作用48 h后,p Len O-GTP-BECN1组的IC50为(3.54±0.20)μg/ml,显著低于空白载体p Len O-GTP组(14.45±1.81)μg/ml和空白组(79.40±8.34)μg/ml(F=207.902;P<0.00)。5-Fu作用前,p Len O-GTP-BECN1组凋亡率为(12.14±0.76)%,同p Len O-GTP组(13.57±1.04)%和空白组(13.75±1.29)%(F=2.096;P=0.204)之间差异并无统计学意义;5-Fu作用48 h后,p Len O-GTP-BECN1组凋亡率为(51.01±3.26)%,显著高于p Len O-GTP组(42.17±4.6)%和空白组(38.42±2.93)%(F=9.32;P<0.01);接受5-Fu处理后,p Len O-GTP-BECN1组细胞核凋亡特征改变较其他两组更明显。5-Fu作用后,p Len O-GTP-BECN1组细胞Caspase-3相对表达量为(0.57±0.0028),显著高于p Len O-GTP组(0.42±0.010)和空白组(0.30±0.046)(F=46.134;P<0.01);p Len O-GTP-BECN1组细胞Caspase-8相对表达量为(0.44±0.0038),显著高于p Len O-GTP组(0.18±0.0095)和空白组(0.27±0.00060)(F=1006.757;P<0.00)。结论Beclin 1显著提高三阴性乳腺癌细胞对5-Fu的敏感度,其中促进凋亡发生可能是重要机制之一。
文摘AIM To filtrate breast cancer resistance protein(BCRP)-mediated resistance agents and investigatethe mechanism,so as to provide valuable datum for optimization clinical chemotherapy scheme to tumor withevaluation marker of BCRP expression.METHODS MTT assay was used to filtrate BCRP-mediatedresistance agents with PA317/Tet-on/TRE-BCRP cell of different expression levels of BCRP after treated withdifferent concentration anticancer agents.High performance liquid chromatography(HPLC) was applied tomeasure relative dose of intracellular retention resistance agents.Nuclear DNA fluorescence dye,Hochest33258,staining and flow cytometry were adopted to detect apoptotic cells after treated with drugs.RESULTSThere were shown increasing durg-resistance to 5-fluorouracil,methotrexate,doxirubicin,pirarubicin,etoposide and mitoxantrone followed with increasing expression of BCRP on PA317/Tet-on/TRE-BCRPcells(P<0.05,n=3),but shown sensitive to paclitaxel,cisplatin,vincristine,mitomycin and vindesine.Therealso was shown significant negative correlation between the intracellular retention dose of 5-fluorouracil withdifferent expression of BCRP(r=-0.885,P<0.05,n=3).There were shown parallel results ofthat decreasingcellular apoptotic rate with increasing cellular expression of BCRP after treated with 5-fluorouracil byfluorescence dye staining and flow cytometry(P<0.05,n=3),and also shown significate rise of the apoptoticrate of BCRP expression cells after treated with Ko143 (P<0.05,n=3).Every group of cells could be differentextently blocked in phase of G_0/G_1 treated with 5-fluorouracil.CONCLUSION Resistance of 5-fluorouracilcould be especially mediated by conjugated with BCRP and acted as drug exclude-pump substrate.Cellularability resistant to 5-fluorouracil-induced apoptosis could be reinforced by BCRP expression.
