p14ARF增强顺铂诱导的骨肉瘤U2OS细胞凋亡的研究

目的探讨p14ARF对顺铂诱导骨肉瘤U2OS细胞凋亡的影响及其机制,为提高骨肉瘤化疗敏感性提供依据。方法在不表达p14ARF的U2OS细胞中稳定转染pcDNA3.1-p14ARF质粒,构建稳定表达株;采用RT-PCR和Western blot法鉴定其表达;台盼蓝拒染法测定生长抑制作用;Hoechst33258荧光染色检测凋亡;Western blot法检测Caspase-3,9和PARP的表达和活化。结果 mRNA和蛋白水平,U2OS和U2OS-vec细胞未见p14ARF表达,U2OS-ARF细胞可见p14ARF的高表达;p14ARF单独对U2OS细胞的生长无影响,但顺铂(5μM)处理72小时后,U2OS-ARF细胞的生长抑制率明显高于U2OS和U2OS-vec细胞,相对于U2OS和U2OS-vec细胞,U2OS-ARF细胞不仅表现更为明显的凋亡形态学变化,还明显出现了Caspase-3,9和PARP的活化裂解。结论 p14ARF能够增强顺铂诱导的骨肉瘤U2OS细胞毒作用和凋亡,通过激活Caspase-PARP级联活化,提高骨肉瘤U2OS细胞对顺铂的敏感性。     

小分子抑制剂Spautin-1增强骨肉瘤细胞化疗敏感性

目的 观察小分子抑制剂Spautin-1对骨肉瘤细胞株(U2OS)化疗敏感性的影响.方法 培养U2OS细胞,使用有效自噬抑制浓度的Spautin-1(10 μmol/L)和顺铂(5μmol/L)处理U2OS,细胞计数试剂盒(CCK-8)法检测Spautin-1对于顺铂(Cisplatin)介导的U2OS的增殖抑制作用的影响,流式细胞术和Western blot检测Spautin-1对顺铂介导的U2OS的凋亡诱导作用的影响.结果 有效自噬抑制浓度的Spautin-1(10 μmol/L)对U2OS无明显的生长抑制及凋亡诱导作用(P＞0.05),而此浓度的Spautin-1可明显增强顺铂对U2OS的增殖抑制(P＜0.05),与顺铂处理组(16.5％)比较,Spautin-1与顺铂共处理后U2OS细胞凋亡比例明显增加(35.0％),切割后的半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)和聚腺苷二磷酸核糖聚合酶(PARP)明显升高.结论 小分子抑制剂Spautin-1可增强U2OS对顺铂的敏感性.     

TRAIL联合顺铂诱导肝癌细胞HepG2凋亡的研究

目的 探讨顺铂增加TRAIL诱导肝癌细胞HepG2凋亡的分子机制，提供TRAIL应用肝癌临床治疗的一种新的用药模式。方法 以肝癌细胞HepG2为研究对象，应用流式细胞仪分析顺铂联合应用TRAIL对细胞周期的影响，Western blot检测顺铂作用前后HepG2细胞在TRAIL诱导凋亡过程中Caspase-8、Caspase-3、DR4、DR、DcR，c—FLIP及RIP的表达变化。结果 顺铂能够增强TRAIL诱导HepG2细胞凋亡的能力，两者联合应用具有明显的协同作用，顺铂预先处理能够下调c-FLIP及RIP的表达及增加DR的表达。结论 TRAIL联合顺铂可以诱导肝癌细胞HepG2凋亡，其机制是通过下调c-FLIP及RIP的表达，解除Caspase-8受抑，恢复凋亡信号的传导来实现的。死亡受体D＆的表达上调也可能参与了这一变化过程。     

咖啡因促进顺铂诱导骨肉瘤细胞凋亡的实验研究

目的 :探讨咖啡因促进顺铂诱导骨肉瘤细胞凋亡的作用。方法 :以人骨肉瘤细胞 (OS -73 2 )为受试对象 ,设咖啡因组、顺铂组、混合组、对照组 ,从三个方面观察咖啡因是否具有促进顺铂诱导骨肉瘤细胞凋亡的作用。结果 :药物作用各组骨肉瘤细胞均可见凋亡的发生。在咖啡因与顺铂共同作用后线粒体跨膜电位下降的骨肉瘤细胞百分率明显增加 (P <0 .0 5 )。TUNEL法检测混合组骨肉瘤细胞凋亡百分率较其他各组明显增高 (P <0 .0 5 )。结论 :咖啡因有促进顺铂诱导骨肉瘤细胞凋亡的作用。     

阻断PI3K/AKT/mTOR通路增强顺铂诱导的人皮肤黑素瘤细胞株A375凋亡的机制研究

目的:本实验探讨抑制PI3K/AKT/mTOR通路对顺铂诱导人皮肤黑素瘤细胞株A375细胞凋亡作用及其机制。方法:用顺铂处理人皮肤黑素瘤细胞株A375细胞后Western blot检测细胞凋亡、PI3K/AKT/mTOR通路的活化情况以及细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)。用PI3K的抑制剂LY294002(LY)和mTOR的抑制剂雷帕霉素(Rapamycin,Rap)分别预处理以研究其对顺铂处理后人皮肤黑素瘤细胞株A375细胞凋亡的协同作用。为进一步探索阻断PI3K/AKT/mTOR通路对顺铂处理后人皮肤黑素瘤细胞株A375细胞凋亡的协同作用机制,采用Western blot检测阻断PI3K/AKT/mTOR后联用顺铂处理人皮肤黑素瘤细胞株A375细胞中Bcl-2、Bcl-xl蛋白表达。结果:顺铂处理后的A375黑素瘤细胞中PARP的活化剪切体表达量水平呈浓度和时间依赖性增加,细胞活力呈浓度和时间依赖性降低(P0.05)。顺铂处理A375黑素瘤细胞后PI3K/AKT/mTOR通路的活化。阻断PI3K/AKT/mTOR通路再用顺铂联合处理A375黑素瘤细胞后PARP的活化剪切体表达量明显上调以及细胞活力明显降低(P0.05)。阻断PI3K/AKT/mTOR通路后再用顺铂联合处理A375黑素瘤细胞发现Bcl-2蛋白和Bcl-xl表达水平下调。结论:阻断PI3K/AKT/mTOR通路对顺铂诱导的细胞凋亡具有协同作用,该协同作用可能与Bcl-2和Bcl-xl蛋白下调有关。     

不同强度恒磁场协同顺铂促进U2骨肉瘤细胞凋亡的分子机制

[目的]探讨不同强度恒磁场促进顺铂诱导的U2骨肉瘤细胞(U2-OS)细胞凋亡及p53、bcl-2、c-myc基因表达变化的情况.[方法]分别使用1 mT、10 mT、100 mT恒磁场和加入3.0 μg/ml顺铂的细胞培养液共同培养U2-OS细胞24 h.采用四甲基氮唑蓝比色法(MTT法)、流式细胞术、逆转录-聚合酶链反应(RT-PCR)检测恒磁场和顺铂联合对U2骨肉瘤细胞体外增殖、凋亡及相关基因表达.[结果]不同强度恒磁场能促进顺铂对人U2-OS细胞的抑制作用,10 Gs场强下其抑制率、凋亡率最高;凋亡率达37.66%,而p53 mRNA表达最强,bcl-2、c-myc mRNA表达最弱.[结论]不同强度恒磁场协同顺铂通过诱导细胞内p53、bcl-2、c-myc基因表达的变化是其抗肿瘤的机制之一,10 Gs场强下协同顺铂的抗肿瘤作用最强.     

华蟾酥毒基诱导骨肉瘤细胞凋亡的体外实验研究

 目的 研究华蟾酥毒基（cinobufagin）对多种骨肉瘤细胞系的增殖抑制和诱导凋亡作用,并探讨其相关机制。方法 不同浓度的华蟾素毒基分别处理骨肉瘤细胞系U2OS、MG63和SaO2后,四甲基偶氮唑蓝（MTT）法观察不同骨肉瘤细胞系的增殖活性,流式细胞仪检测细胞周期的变化,细胞核荧光染色及Annexin V/ PI染色检测细胞凋亡,Western blot检测IAPs和Bcl-2家族凋亡相关蛋白凋亡相关蛋白Bax、cleaved-PARP、xIAP、cIAP-1、survivin及p65的表达。结果 MTT检测显示,华蟾酥毒基可明显抑制骨肉瘤细胞U2OS、MG63和SaO2的增殖,其抑制细胞增殖的作用呈剂量和时间依赖性,其对U2OS、MG63和 SaO2细胞的48 h IC50值分别为（104.83±16.96） nmol/L、（47.07±7.5） nmol/L和（136.72±10.08） nmol/L。100 nmol/L 华蟾酥毒基处理骨肉瘤细胞12 h后,流式细胞仪检测可见骨肉瘤U2OS、MG63和 SaO2的G₀/G₁期细胞比例下降,G2/M 期细胞比例增加,表明华蟾素毒基主要通过将细胞阻滞在G₂/M 期,以抑制骨肉瘤细胞的增殖。进一步Hoechst 33258细胞核染色发现,华蟾素毒基可诱导骨肉瘤细胞产生典型凋亡小体,Annexin V/ PI检测100 nmol/L 华蟾酥毒基作用48 h后,U2OS、MG63和 SaO2细胞凋亡率分别为33.6%±6.4%、36.4%±7.8% 及 29.3%±5.1%。表明华蟾酥毒基的抗骨肉瘤效果是通过诱导骨肉瘤细胞凋亡来实现。在探讨其诱导骨肉瘤细胞凋亡的相关机制中,通过Western blot检测发现其诱导凋亡的作用机制是通过上调IAPs和Bcl-2家族凋亡相关蛋白Bax、cleaved-PARP,下调xIAP、cIAP-1、survivin及p65蛋白来实现。结论 华蟾酥毒基可明显抑制骨肉瘤细胞株U2OS、MG63和 SaO2细胞增殖,阻滞细胞在G₂/M期,并诱导其凋亡,其诱导凋亡的作用机制为调节IAPs和Bcl-2凋亡相关家族蛋白的活性。     

α干扰素增强骨肉瘤U2OS细胞对足叶乙甙的敏感性

目的探讨α干扰素对人骨肉瘤U20S细胞足叶乙甙敏感性的作用及其机制，为提高骨肉瘤化疗的敏感性探索新的治疗方法。方法应用MTT法测定IFNα和VPl6单用以及联用对U20S细胞的细胞毒作用；Hoeehst33258荧光染色检测药物对U20S细胞凋亡的影响；Westernblot法检测Caspase-3、8、9的表达和活化。结果IFNα在50U／ml、500U／ml、5000U／ml等剂量处理48h、72h、96h对U20S细胞无毒性作用，却明显增强了VPl6（2．5μg／m1）诱导的细胞毒作用；与单药组相比，IFNα（5000U／m1）与VPl6（2．5μg／m1）联用72h后U20S细胞出现更为明显的凋亡特征性形态变化；并且联合用药组的凋亡关键酶Caspase-3、8、9均发生明显断裂活化。结论IFNα能够增强VP16诱导的骨肉瘤U20S细胞毒作用和凋亡，且与Caspase级联活化有关，二者联合应用可能成为提高骨肉瘤化疗敏感性的有效途径。     

单启动子双表达载体pIRES-p14ARF-p53的构建及其对骨肉瘤细胞增殖的抑制作用

目的 构建双质粒表达载体pIRES-p14ARF-p53，并研究其对骨肉瘤细胞增殖生长的抑制作用。方法 利用基因工程技术，将从培养的正常人肝细胞系L02细胞中扩增出的p14cDNA（0．5kb）亚克隆至pIRES载体中，通过聚合酶链反应（PCR）、限制性内切酶酶切鉴定重组质粒pIRES-p14ARF-p53。通过脂质体介导转染入骨肉瘤MG-63细胞中，并筛选出阳性克隆，流式细胞仪测定瘤细胞DNA含量和细胞周期；逆转录（RT）-PCR和Western bolt对稳定转染后的瘤细胞p53、p14ARF蛋白的表达进行定性和半定量检测。噻唑蓝（MTT）比色法与细胞生长曲线观察细胞增殖情况。结果 成功构建出双质粒表达载体pIRES-p14ARF-p53。转染后瘤细胞形态由转染前密集排列的多角形、星形转变为排列稀疏的长梭形细胞，瘤细胞生长速度较前缓慢，群体倍增时间比转染前增加了2．3倍，且易出现脱壁并见散在瘤细胞凋亡；流式细胞仪检测发现转染后的瘤细胞多停滞于G1期；RT-PCR与Western blot检测证实p14ARF、p53基因在靶细胞mRNA和蛋白水平分别有独立表达，转染MG-63后24、48、72、96h瘤细胞生长抑制率分别为：33．43％、69．37％、66，19％、75．26％，差异有统计学意义（P〈0．01）。结论 野生型p53和p14ARF协同抑制骨肉瘤细胞的增殖促进瘤细胞的凋亡，为进一步研究p14ARF与p53在骨肉瘤基因治疗的体内实验奠定了基础。     

塞来昔布协同顺铂P13K/Akt途径诱导骨肉瘤MG-63细胞凋亡的实验研究

目的 探讨环氧化酶-2(COX-2)选择性抑制剂塞来昔布对人骨肉瘤MG-63细胞增殖和凋亡的影响及作用机制.方法 塞来昔布(50 μmol/L或100 μmol/L)和(或)顺铂(10 μg/ml)作用骨肉瘤MG-63细胞48 h后,MTT法测定细胞增殖的抑制率,电子显微镜和流式细胞仪检测细胞凋亡,RT-PCR法检测基因水平COX-2表达变化,Western blot检测COX-2及凋亡相关蛋白表达变化.P13K抑制剂Wortmannin作用MG-63细胞48 h,检测蛋白表达变化.结果 塞来昔布导致MG-63细胞阻滞在G1期,通过激活半胱氨酸天冬氨酸蛋白酶(caspase)-9的内源性凋亡途径诱导细胞凋亡;顺铂单药作用后骨肉瘤MG-63细胞凋亡率为5.93%,而联合应用塞来昔布50μmol/L或100 μmol/L后,凋亡率分别为6.66%和37.15%,与顺铂联合具有明显的协同作用;COX-2蛋白表达未降低.塞来昔布联合顺铂明显降低P13K/Akt、survivin、Bcl-2的表达,检测到caspase-9、caspase-3的激活和PARP裂解片段.Wortmannin作用MG-63细胞48 h,检测到pAkt(Thr308)、Bcl-2、survivin表达下调.结论 塞来昔布可通过非COX-2途径诱导骨肉瘤MG-63细胞凋亡,与P13K/Akt、survivin、Bcl-2蛋白相关,并且PI3K/Akt途径在survivin、Bcl-2表达调控中发挥重要作用.这可能是塞来昔布药物干预的中心环节.     

壳五糖对骨肉瘤细胞抗肿瘤作用的研究

目的探讨壳五糖对骨肉瘤细胞的抗肿瘤作用及其作用机制。方法使用不同质量浓度的壳寡糖单糖(聚合度为2~6)处理3种骨肉瘤细胞系(MNNG、MG63、U2OS),48 h后采用CCK-8法评估细胞活力及细胞增殖情况,并通过流式细胞技术分析细胞周期及细胞凋亡的变化情况。利用Transwell实验检测细胞的迁移和侵袭能力。采用蛋白质免疫印迹法检测壳五糖处理前后细胞中磷酸肌醇-3-激酶(PI3K)/丝氨酸-苏氨酸蛋白激酶(AKT)信号转导通路中关键蛋白表达水平的变化。结果壳寡糖单糖以剂量依赖性方式抑制骨肉瘤细胞的增殖,其中以壳五糖作用效果最佳,其在3种骨肉瘤细胞中的半数抑制浓度(IC50)分别为1.55 mg/mL(MNNG)、0.84 mg/mL(MG63)、0.76 mg/mL(U2OS)。壳五糖处理3种骨肉瘤细胞后显著抑制这些细胞的增殖和迁移能力,同时影响骨肉瘤细胞周期并促进细胞凋亡。蛋白质免疫印迹法检测显示,PI3K/AKT信号转导通路中的磷酸化PI3K、磷酸化AKT的表达明显下降。结论壳五糖可有效抑制骨肉瘤细胞的增殖、迁移、侵袭能力,其作用机制可能是通过调控PI3K/AKT信号转导通路产生的。     

过表达FOXO1对骨肉瘤细胞侵袭迁移能力的影响及机制研究

［摘要］ 目的 探讨FOXO1对骨肉瘤细胞侵袭迁移能力的影响及机制。方法 采用实时荧光定量PCR（qRT?PCR）的方法检测人正常成骨细胞hFOB 1.19和人骨肉瘤细胞U2OS、MNNG/HOS中FOXO1的mRNA表达水平。利用pcDNA3.1?FOXO1重组质粒建立FOXO1过表达的细胞模型,空载质粒（pcDNA3.1?vector）作为对照,qRT?PCR和蛋白质印迹法（Western blot）验证转染效率。确定成功过表达FOXO1基因后利用Cell Counting Kit?8（CCK?8）实验检测骨肉瘤细胞的增殖能力（吸光度值）,Transwell实验体外检测骨肉瘤细胞的侵袭和迁移能力,并采用qRT?PCR和western blot的方法检测基质金属蛋白酶9（MMP9）的表达变化。结果 人骨肉瘤细胞MNNG/HOS、U2OS中FOXO1的mRNA的表达水平显著低于人成骨细胞hFOB 1.19（P<0.05）,且骨肉瘤细胞U2OS的FOXO1的表达水平较低。重组质粒pcDNA3.1?FOXO1转染后,骨肉瘤细胞U2OS的FOXO1表达水平显著升高。与对照组（pcDNA3.1?vector）比较,U2OS?FOXO1过表达组的细胞的增殖能力降低（P<0.05）,侵袭迁移和迁移能力降低,MMP9较对照组表达水平明显降低（P<0.05）。结论 骨肉瘤细胞中FOXO1表达水平明显低于正常成骨细胞。过表达FOXO1可能通过下调MMP9抑制骨肉瘤细胞的侵袭和迁移能力。     

RecQ5表达与骨肉瘤组织恶性程度间的关系分析

目的分析RecQ5在不同组织和细胞上的表达差异,推断RecQ5与组织恶性程度间的关系。方法选取骨肉瘤标本共35例、癌旁组织标本20例及正常骨组织标本20例,通过免疫组织化学染色法检测不同组织中RecQ5的表达情况,并进一步分析其表达程度与骨肉瘤分期的关系;培养hFOB1.19、U2OS和MG-63细胞株,通过Real-timePCR和Westernblot法检测RecQ5的表达程度。结果RecQ5在人骨肉瘤组织中低表达。正常骨组织中,RecQ5的表达阳性率为100%(20/20),在癌旁组织中的表达阳性率为90%(18/20),而在骨肉瘤组织中,RecQ5表达阳性率为68.5%(24/35);正常骨组织、癌旁组织与骨肉瘤组织的RecQ5表达阳性率之间比较差异具有统计学意义(P=0.004),但正常骨组织与癌旁组织之间的阳性率差异无统计学意义(P>0.05)。RecQ5的表达强度随着组织恶性程度的不断增加而逐渐减弱,差异具有统计学意义(P<0.05),且在骨肉瘤组织中,其也随着肿瘤分期增加而表达强度逐渐减弱(P<0.05)。RecQ5在人骨肉瘤细胞株MG-63和U2OS中mRNA和蛋白表达水平较正常人成骨细胞hFOB1.19中低,差异有统计学意义(P<0.05)。结论RecQ5的表达水平与组织的恶性程度呈负相关,RecQ5的缺失与骨肉瘤的发生存在相关性。     

MEK inhibitor, U0126, attenuates cisplatin-induced renal injury by decreasing inflammation and apoptosis

BACKGROUND: Although inflammation and apoptosis are known to play important roles in cisplatin nephrotoxicity, the exact intracellular signaling mechanisms are not well understood. Recent reports that extracellular signal-regulated kinase (ERK1/2) pathway mediates cisplatin-induced caspase activation and apoptosis in cultured renal tubular cells led us to investigate the effect of MAPK/ERK kinase (MEK) inhibitor, an immediate upstream of ERK1/2 in cisplatin-induced acute renal failure (ARF) in mice. METHODS: The effect of MEK/ERK1/2 inhibition on kidney tumor necrosis factor-alpha (TNF-alpha (gene expression, inflammation, the activation of tissue caspases, and apoptosis were examined in addition to its effects on renal function and histology in cisplatin-induced ARF in mice. RESULTS: Pretreatment of MEK inhibitor, U0126, decreased ERK1/2 phosphorylation following cisplatin administration with significant functional and histologic protection. This beneficial effect was accompanied by decrease in TNF-alpha gene expression level and inflammation, as well as in caspase 3 activity and apoptosis. CONCLUSION: These data provide evidence that ERK1/2 pathway functions as an upstream signal for TNF-alpha-mediated inflammation and caspase 3-mediated apoptosis in cisplatin-induced ARF in mice and suggest that ERK1/2 can be a novel therapeutic target in cisplatin nephrotoxicity.     

miR-181a-5p通过HOXB4抑制骨肉瘤细胞HOS增殖和迁移

目的：研究miR-181a-5p对HOS骨肉瘤细胞增殖、周期和迁移的影响及其机制。方法 ：采用实时定量PCR检测hFOB1.19成骨细胞和HOS、U2OS、MG63骨肉瘤细胞系中miR-181a-5p及HOXB4的表达情况。利用Lipofectamine 2000将miR-181a-5p mimics和miR-181a-5p inhibitor分别转染至人骨肉瘤HOS细胞中（分别为过表达组和抑制剂组）,并设置miR阴性对照组;CCK-8法检测各组细胞的增殖能力变化,流式细胞术检测各组细胞的细胞周期变化,划痕愈合实验以及Transwell迁移实验检测各组细胞的迁移能力变化。Targetscan网站预测miR-181a-5p的靶向基因,并通过双荧光素酶报告基因系统及Western blot验证靶向关系。结果：与成骨细胞hFOB1.19相比,miR-181a-5p在骨肉瘤细胞HOS、U2OS和MG63中低表达（P<0.05）,而HOXB4在骨肉瘤中高表达（P<0.05）。与阴性对照组相比,过表达miR-181a-5p抑制骨肉瘤HOS细胞的增殖和迁移能力,并且处于细胞周期S期的细胞...     

Expression and Clinical Significance of High‐Mobility Group AT‐hook 2 (HMGA2) in Osteosarcoma

ObjectiveAlthough high‐mobility group AT‐hook 2 (HMGA2) has been shown to have crucial roles in the pathogenesis and metastasis of various malignancies, its expression and significance in osteosarcoma remain unknown. Here we evaluate the expression, clinical prognostic value, and overall function of HMGA2 in osteosarcoma.MethodsSixty‐nine osteosarcoma patient specimens within a tissue microarray (TMA) were analyzed by immunohistochemistry for HMGA2 expression. Demographics and clinicopathological information including age, gender, tumor location, metastasis, recurrence, chemotherapy response, follow‐up time, and disease status were also collected. After validation of expression, we determined whether there was a correlation between HMGA2 expression and patient clinicopathology. HMGA2 expression was also evaluated in osteosarcoma cell lines and patient tissues by Western blot, we analyzed the expression of HMGA2 in the human osteosarcoma cell lines MG63, 143B, U2OS, Saos‐2, MNNG/HOS, and KHOS. HMGA2‐specific siRNA and clonogenic assays were then used to determine the effect of HMGA2 inhibition on osteosarcoma cell proliferation, growth, and chemosensitivity.ResultsHMGA2 expression was elevated in the osteosarcoma patient specimens and human osteosarcoma cell lines. HMGA2 was differentially expressed in human osteosarcoma cell lines. Specifically, a relatively high expression of HMGA2 was present in KHOS, MNNG/HOS, 143B and a relatively low expression was in MG63, U2OS as well as Saos‐2. HMGA2 expression is correlated with metastasis and shorter overall survival. High HMGA2 expression is an independent predictor of poor osteosarcoma prognosis. There was no significant correlation between HMGA2 expression and the age, gender, or tumor site of the patient. HMGA2 expression is predominantly within the nucleus. The expression of HMGA2 also directly correlated to neoadjuvant chemoresistance. There was a significant reduction of HMGA2 expression in the siRNA transfection group. After the use of siRNA, the proliferation of osteosarcoma cells is decreased and the chemosensitivity of osteosarcoma cells is significantly increased.ConclusionOur study supports HMGA2 as a potential prognostic biomarker and therapeutic target in osteosarcoma.     

雷帕霉素对人骨肉瘤细胞增殖和凋亡的影响

目的 观察雷帕霉素(RAPA)对人骨肉瘤细胞U-2OS和Saos-2增殖和凋亡的影响并探讨其机制.方法 体外培养骨肉瘤细胞株U-2OS和Saos-2;噻唑蓝(MTT)比色法和流式细胞仪检测不同浓度雷帕霉素对骨肉瘤细胞增殖和凋亡的影响;Western blot检测雷帕霉素作用后骨肉瘤细胞Fas蛋白表达的变化.结果 MTT检测显示雷帕霉素可抑制入骨肉瘤细胞增殖,在雷帕霉素浓度达到20 nmol/L时,抑制作用显著提高(P＜0.05),增殖抑制率分别达到31.2％和28.7％.流式细胞检测显示在雷帕霉素浓度达到20 nmol/L时,其诱导骨肉瘤细胞凋亡的作用显著增强(P＜0.05).Western blot检测显示经雷帕霉素作用48 h后的骨肉瘤细胞Fas蛋白的表达明显增强.结论 雷帕霉素对入骨肉瘤细胞株U-2OS和Saos-2的增殖有抑制作用并可诱导其发生凋亡,其可能是通过上调Fas蛋白的表达而发挥作用的.     

Novel quinazoline HMJ‐30 induces U‐2 OS human osteogenic sarcoma cell apoptosis through induction of oxidative stress and up‐regulation of ATM/p53 signaling pathway

Human osteogenic sarcoma is the most common primary bone tumor. Despite of the success of frontline therapy, about 40% of patients have disease progression and further therapy is palliative and toxic. In this study, we developed a novel quinazoline HMJ‐30 to investigate the cell growth inhibition and apoptotic responses in U‐2 OS human osteogenic sarcoma cells. Our results demonstrated that HMJ‐30 significantly reduced cell viabilities of U‐2 OS, HOS, and 143B cells in a dose‐dependent manner, but it exhibited low cytotoxicity in normal hFOB cells. HMJ‐30 induced DNA damage and apoptosis in U‐2 OS cells as revealed by morphologic changes, comet assay and DAPI staining. Immuno‐staining, colorimetric assays, and Western blotting analyses indicated that activities of caspase‐8, caspase‐9, and caspase‐3 and the levels of Bcl‐2 family‐related proteins (Bcl‐2, Mcl‐1, Bax, BAD, and t‐Bid) were altered in HMJ‐30‐treated U‐2 OS cells. Pretreatment of cells with caspase‐8, ‐9, and ‐3 specific inhibitors significantly reduced the cell growth inhibition. HMJ‐30‐induced apoptosis was mediated through both death‐receptor and mitochondria‐dependent apoptotic pathways in U‐2 OS cells. HMJ‐30 induced early phosphorylation of p53^Ser18 was through the activation of ataxia telangiectasia mutated (ATM) in U‐2 OS cells. The cell growth inhibition by HMJ‐30 was substantially attenuated either by the pre‐incubation of U‐2 OS cells with N‐acetylcysteine (NAC, an antioxidant) and caffeine (an ATM kinase inhibitor) or by p53 knockdown via RNAi. In conclusion, ROS dependent‐ATM/p53 signaling pathway is involved in HMJ‐30‐induced apoptosis in U‐2 OS cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1448–1456, 2011     

Protein tyrosine phosphatase non-receptor type 12 suppresses tumor progression in osteosarcoma cells

BackgroundProtein tyrosine phosphatase non-receptor 12 (PTPN12) plays a prominent role in various cancers as a tumor suppressor. However, the expression of PTPN12 and its biological functions in osteosarcoma (OS) remains to be determined.MethodsPTPN12 expression in OS was explored in public databases and detected by immunohistochemistry and Western blot. The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay and colony formation. The cell migration and invasion were assessed by the Transwell assay. Flow cytometry analysis was applied to detect cell apoptosis and cell cycle distribution. To investigate the related mechanism, the levels of EGFR and downstream proteins were detected by Western blot.ResultsPTPN12 expression was significantly decreased in OS samples in GEO database and our hospital. OS cell lines in Cancer Cell Line Encyclopedia (CCLE) database and our cultured OS cells also demonstrated low PTPN12 expression. Lentivirus-induced overexpression of PTPN12 significantly inhibited the cell viability, migration and invasion of 143B and U2OS cells. The results of flow cytometry found that PTPN12 overexpression promoted cell apoptosis and induced cell cycle arrest at G1 phase in 143B and U2OS cells. The phosphorylation levels of EGFR and subsequent proteins of the PI3K/AKT and ERK pathways were inactivated as a result of PTPN12 overexpression in OS.ConclusionPTPN12 plays a tumor suppressive role in OS cells. Restoring of PTPN12 activity may provide new insights for the treatment of this disease.