慢病毒介导ITGB4 shRNA对H460SM细胞增殖的抑制作用

目的 体外实验研究β4整合素shRNA对非小细胞肺癌H460SM细胞增殖的抑制作用,为非小细胞肺癌早期诊断和治疗提供新的靶点.方法 实时定量RT-PCR验证β4整合素表达水平的变化,显微镜下观察细胞形态的变化.通过细胞计数和MTS实验分析抑制β4整合素表达对H460SM细胞增殖的影响;流式细胞术分析抑制β4整合素表达对H460SM细胞凋亡和细胞周期的影响.结果 与阴性对照组(H460SM-NS)比较,稳定表达β4整合素shRNA的实验组(H460SM-68、H460SM-71)细胞β4整合素表达水平明显降低(P＜0.01);与阴性对照组比较,实验组细胞的增殖受到明显抑制(P＜0.05);实验组细胞凋亡率明显高于阴性对照组,差异有统计学意义(P＜0.01);实验组细胞增殖指数PI明显低于阴性对照组(P＜0.05),G0/G1期细胞数明显高于阴性对照组(P＜0.05),提示抑制β4整合素的表达,细胞周期可能被阻滞在G1/S检测点,导致细胞增殖受到抑制.结论 抑制β4整合素的表达,可以抑制非小细胞肺癌H460SM细胞增殖,并可能诱导H460SM细胞凋亡.癌细胞增殖受到抑制可能与细胞周期调控有关.抑制β4整合素的表达,可能不影响非小细胞肺癌细胞凋亡.     

全反式维甲酸增强顺铂对A549 细胞化疗敏感性及对Survivin mRNA 和COX-2 mRNA表达的影响

目的:体外观察全反式维甲酸（all-trans retinoic acid ,ATRA）增强顺铂（cisplatin,DDP ）对人类非小细胞肺癌细胞株A549 细胞的增殖抑制及对凋亡抑制蛋白Survivin mRNA和环氧化酶-2（cyclooxygenase- 2,COX-2）mRNA 表达的影响。方法:应用不同浓度组DDP（0.5、5、50mg/L）、ATRA（0.1、1、10μ mol/L）以及联合用药组（ATRA 1 μ mol/L,DDP 5mg/L）,处理肺腺癌细胞株A549 细胞,采用四甲基偶氮唑盐（MTT）比色法观察不同浓度DDP 组、不同浓度ATRA 组及联合用药组对A549 细胞生长的影响;应用实时荧光定量聚合酶链反应（real-time polymerase chain reaction ,RT-PCR）检测DDP 组、ATRA 组及联合用药组处理前后A549 细胞中Survivin mRNA和COX-2 mRNA 表达变化;应用流式细胞术观察DDP 组、ATRA 组及联合用药组处理前后细胞凋亡率。结果:与空白对照组相比,单独应用DDP 、ATRA 处理A549 细胞均诱导细胞凋亡,且呈浓度依赖性。与单独应用DDP 的作用相比,联合用药组可更显著抑制A549 的增殖,增加细胞的凋亡率（P<0.05）,并增强对A549 细胞Survivin mRNA和COX-2 mRNA表达的抑制作用（P<0.05）;并且,流式细胞术测定结果显示联合用药组的早期凋亡率（7.37± 3.83）% 、中晚期凋亡率（34.37±2.08）% 、继发性坏死率（7.44± 0.46）% 均较单独应用DDP 组高（3.55± 0.75）% 、（6.62± 0.33）% 、（3.03± 0.05）% ,P 均<0.05。结论:全反式维甲酸能够明显提高非小细胞肺癌对顺铂的敏感性,其机制可能与抑制非小细胞肺癌细胞Survivin mRNA和COX-2 mRNA 的表达有关。      

CXCL4对非小细胞肺癌H460细胞顺铂耐药性的影响及其机制探讨

目的:探讨血小板因子4（CXCL4）对人非小细胞肺癌H460细胞顺铂耐药性的影响及其作用机制。方法:体外诱导法建立顺铂耐药的非小细胞肺癌细胞株H460/DDP,并鉴定其生物学特性。采用CCK-8法分别检测H460及H460/DDP对顺铂的耐药性。qRT-PCR及Western Blot检测亲本细胞株H460及顺铂耐药株H460/DDP中CXCL4的mRNA及蛋白表达水平。通过CCK-8法检测过表达或者敲低CXCL4后亲本细胞株H460或顺铂耐药株H460/DDP对顺铂的敏感性。最后利用Western Blot及qRT-PCR法探讨CXCL4抵抗非小细胞肺癌化疗敏感性的调控机制。结果:成功构建了非小细胞肺癌顺铂耐药株H460/DDP（H460/DDP IC50=55.005 μg/ml）,CCK-8结果提示顺铂耐药株H460/DDP与亲本细胞株H460细胞的增殖速度没有明显差异（P＞0.05）;顺铂耐药株H460/DDP中,CXCL4 mRNA及蛋白水平均显著增高（P＜0.000 1）;在亲本细胞株H460中稳定过表达CXCL4能显著降低其对顺铂的敏感性（P＜0.000 1）,而于顺铂耐药株H460/DDP中敲低CXCL4能显著增加H460/DDP对顺铂的敏感性（P＜0.000 1）;Western Blot及qRT-PCR结果显示,CXCL4能激活Wnt/β-catenin 信号通路,促进下游基因MYC、CyclinD1、MMP-7、CDK4的表达。结论:CXCL4通过调控Wnt/β-catenin 信号通路促进非小细胞肺癌对顺铂的耐药性。     

醋酸甲地孕酮联合三氧化二砷对肝癌HepG2细胞株体外抑制作用的研究

目的 探讨醋酸甲地孕酮（MA）联合三氧化二砷（As2O3）对肝癌HepG2细胞株体外增殖的影响及其可能机制。方法 将对数生长期HepG2细胞分为4组：MA单药组、As2O3单药组、MA+As2O3联合组和对照组,根据前期研究结果确定药物剂量分别为75.0 μmol/L MA、1.0 μg/ml As2O3,对照组加入等量细胞培养液。24 h后采用CCK-8方法、流式细胞学检测及Western blotting检测MA单药组、As2O3单药组及MA+As2O3联合组对HepG2细胞增殖、细胞凋亡及X连锁凋亡抑制蛋白（XIAP）表达的影响。结果CCK-8法显示MA+As2O3组较MA或As2O3单药组均能够明显抑制HepG2细胞生长;流式细胞仪结果显示MA+As2O3联合组的细胞凋亡率高于MA或As2O3单药组。MA+As2O3作用后XIAP表达较MA或As2O3单药组降低。结论MA联合As2O3能够发挥对HepG2细胞协同抑制作用,其中诱导细胞凋亡可能是其机制之一。     

抑制Bcl-2基因表达增强非小细胞肺癌NCI-H460细胞放射敏感性的研究

背景与目的:Bcl-2基因是一种重要的抑制细胞凋亡的基因,广泛存在于各种肿瘤细胞中.本研究利用短发夹RNA(shRNA)重组质粒抑制Bcl-2基因的表达,观察其对非小细胞肺癌NCI-H460的细胞凋亡和放射敏感性变化的影响.方法:将特异性抑制Bcl-2基因表达的干扰质粒Bcl-2/shRNA稳定转染NCI-H460细胞,获得H460-RNAi细胞,同时设转染含无义序列Neg-shRNA质粒的H460-Neg细胞及未转染的H460细胞为对照组,采用Western blot法,观察Bcl-2基因在蛋白表达水平的变化;X射线照射后,采用细胞形态学及克隆形成实验,检测RNA干扰对NCI-H460细胞凋亡和放射敏感性的影响.结果:H460-RNAi细胞的Bcl-2蛋白表达较对照组明显降低(P<0.05);H460-RNAi细胞的凋亡率[(10.8±0.7)%]明显高于对照组(P<0.05),4 Gy X线照射48 h后的凋亡率[(24.9±1.4)%]明显高于对照组(P<0.05)及未照射组(P<0.05):H460-RNAi细胞的存活参数D0、Dq、N和SF2值分别为0.97、0.75、2.18和0.26,均低于其余两组,而H460细胞组与H460-Neg细胞组比较差异无统计学意义(分别为1.39、1.39、2.72、0.52和1.21、1.28、2.87、0.46).结论:Bcl-2/shRNA重组质粒可以有效抑制非小细胞肺癌NCI-H460细胞的Bcl-2基因的表达,增强NCI-H460的细胞凋亡及放射敏感性.     

全反式维甲酸与顺铂联合对肺腺癌A549细胞株增殖与凋亡影响的观察

目的:体外观察全反式维甲酸(ATRA)联合顺铂(DDP)抑制肺腺癌细胞株A549增殖及诱导其凋亡的作用.方法:应用四甲基偶氮唑盐比色法(MTT)测定A549细胞生长抑制率,荧光定量RT-PCR法测定A549细胞维甲酸受体-β(RARβ)mRNA、血管内皮细胞生长因子(VEGF)mRNA的转录情况.结果:应用DDP(5、 10 和 50 mg/L)之后,与对照组相比,可以显著抑制A549细胞的增殖作用,且随着药物浓度的增加,其抑制作用增强,P＜0.05;ATRA+DDP联合用药组的细胞增殖抑制作用较单独应用DDP有更高的抑制强度,与DDP组相比,ATRA+DDP组增加了RARβ的表达,抑制了VEGF的表达,A549细胞的凋亡率增加,P＜0.05.结论:全反式维甲酸联合顺铂能更有效抑制A549细胞的增殖,增加非小细胞肺癌对顺铂化疗的敏感性.     

三氧化二砷联合氟尿嘧啶体外对人类大肠癌细胞生长及端粒酶活性的影响

目的:探讨三氧化二砷(As2O3)注射液与氟尿嘧啶(5-FU)联合应用对人类大肠癌细胞的生长及其端粒酶活性的影响。方法:采用四甲基偶氮唑蓝比色(MTT)法观察As2O3注射液和/或5-FU对人类大肠癌细胞株LoVo和CoLo-320生长的影响;应用端粒重复序列扩增法(TRAP)—聚丙烯酰胺凝胶电泳(PAGE)银染法检测As2O3注射液和/或5-FU处理前后大肠癌细胞端粒酶活性的变化。结果:与As2O3或5-FU单药作用相比,As2O3与5-FU联合应用可显著增强抑制人结肠癌细胞增殖作用,并对细胞端粒酶活性具有增强抑制作用。结论:与各单药相比较,As2O3注射液联合5-FU能够明显增强抗大肠癌作用,其机制可能与增强抑制癌细胞的端粒酶活性有关。     

氧化砷诱导人小细胞肺癌细胞凋亡的研究

目的 研究砷剂（As2O3)对人小细胞肺癌细胞凋亡诱导作用以及干扰素（IFNα－2b)对As2O3作用的影响。方法 采用DNA电泳、TUNEL原位末端标记和免疫细胞化学染色检测不同浓度As2O3作用不同时间对人小细胞肺癌细胞凋亡诱导作用及相关主相关基因蛋白的表达。结果 在一定的浓度范围内,As2O3可诱导人小细胞肺癌细胞凋亡,并且随浓度的升高和作用时间的延长,凋亡细胞数增加,相关基因蛋白Bcl-2、PCNA表达降低。联合使用IFNα-2b可增强凋亡诱导作用。结论 As2O3可诱导人小细胞肺癌细胞凋亡,且与药物浓度和作用时间有关。IFNα-2b可增强此作用。     

凋亡抑制蛋白XIAP基因对A549细胞凋亡和化疗敏感性的影响

背景与目的X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)是新发现的一个IAP家族中的主要成员,是IAP家族中最强的凋亡抑制因子,它可直接抑制caspases并可多途径调节细胞凋亡。XIAP基因在大多数的肿瘤细胞株中过表达,其表达与肿瘤的进展、复发、预后以及肿瘤化疗的耐药密切相关。研究通过RNAi方法下调非小细胞肺癌(NSCLC)细胞NIC-A549的XIAP基因表达后,研究XIAP siRNA特异序列在NSCLC细胞凋亡和化疗敏感性方面的作用。方法应用半定量RT-PCR法检测A549细胞中XIAP mRNA基因的表达,设计并构建XIAP干扰性小RNA(small interfering RNA,siRNA)序列的表达载体,转染siRNA载体至A549中。荧光纤维镜确定转染效率,MTT法测定细胞增殖率及化疗药物对细胞杀伤率,流式细胞仪测定细胞凋亡率。结果酶切和DNA测序证实XIAP siRNA构建成功。荧光纤维镜显示阳性转染组及阴性转染组的细胞转染效率无差异。半定量RT-PCR法示阳性转染组较阴性转染组、未转染组细胞XIAP mRNA表达明显降低;与对照组相比,顺铂对阳性转染组XIAP mRNA表达的抑制作用明显增强、阳性转染组细胞增殖在24h、48h、72h、96h明显受抑制;细胞杀伤率、凋亡率明显增加。结论XIAP基因在NSCLC中表达增高,可抑制NSCLC细胞凋亡,导致NSCLC化疗耐药。XIAP siRNA序列可特异性地抑制NSCLC细胞增长,下调XIAP基因的表达,促进细胞凋亡,增加NSCLC化疗敏感性。XIAP siRNA序列有可能成为NSCLC治疗的靶标。     

三氧化二砷对人胃癌细胞系MGC803凋亡及耐药基因表达影响的实验研究

目的:研究三氧化二砷(As2O3)对人胃癌细胞系MGC803的抑制和诱导凋亡作用及对MRP基因表达的影响.方法:选用人胃癌MGC803细胞系,运用体外细胞培养法,MTT法,流式细胞术检测As2O3对人其抑制和诱导凋亡作用;用逆转录-聚合酶链反应(RT-PCR)方法检测MRP mRNA的表达.结果:As2O3对人胃癌MGC803 细胞具有抑制作用,其抑制率呈时间-剂量依赖关系.不同浓度的As2O3均可诱导凋亡.1.0μmol/L、2.0μmol/L的As2O3可下调MRP mRNA的表达.结论:As2O3具有抗肿瘤作用,主要是通过诱导细胞凋亡实现的,其机制与下调MRP mRNA 表达有密切关系.     

XIAP and survivin as therapeutic targets for radiation sensitization in preclinical models of lung cancer

Survivin and XIAP are members of inhibitors of apoptosis (IAPs) family. They are upregulated in various malignancies. Inactivation of these molecules has resulted in chemosensitization. The purpose of this study was to determine whether inhibition of survivin, XIAP, or both enhances radiotherapy in a lung cancer model. Transient transfection of H460 cells with antisense oligonucleotides (ASOs) against either molecule has specifically reduced their expression, by Western analysis. Results from 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and clonogenic assays suggest that inhibition of survivin or XIAP greatly decreased cell survival following irradiation. A significantly increased number of apoptotic cells were detected when H460 cells were treated with either antisurvivin, anti-XIAP or both ASOs (P=0.03, 0.0003 and 0.01, respectively) plus irradiation. H460 xenografts that were treated with ASOs plus radiotherapy demonstrated growth delay beyond 15 days. Growth delay in the groups of combined treatment was greater than that in other groups. However, treatment with ASOs alone did not affect tumor growth delay in mice, but decreased the survival of H460 cells in culture. Antisense treatment did not cause any mortality or weight loss during the 32 days of study. These data suggest that inhibition of survivin or XIAP radiosensitizes H460 lung cancer cells by upregulating apoptosis and downregulating cell survival. Combination of radiotherapy and inhibition of survivin and XIAP through the antisense approach results in improved tumor control by radiotherapy in a mouse model of lung cancer.     

Arsenic trioxide exerts synergistic effects with cisplatin on non-small cell lung cancer cells via apoptosis induction

Background

Despite multidisciplinary treatment, lung cancer remains a highly lethal disease due to poor response to chemotherapy. The identification of therapeutic agents with synergistic effects with traditional drugs is an alternative for lung cancer therapy. In this study, the synergistic effects of arsenic trioxide (As₂O₃) with cisplatin (DDP) on A549 and H460 non-small cell lung cancer (NSCLC) cells were explored.

Methods

A549 and H460 human lung cancer cells were treated with As₂O₃ and/or DDP. Cell growth curves, cell proliferation, cell cycle, and apoptosis of human cancer cell lines were determined by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method, clonogenic assay, and flow cytometry (FCM). Apoptosis was further assessed by TUNEL staining. Cell cycle and apoptosis related protein p21, cyclin D1, Bcl-2, bax, clusterin, and caspase-3 were detected by western blot.

Results

MTT and clonogenic assay showed As₂O₃ within 10^-2 μM to 10 μM exerted inhibition on the proliferation of NSCLC cells, and 2.5 μM As₂O₃ exerted synergistic inhibition on proliferation with 3 μg/ml DDP. The combination indices (CI) for A549 and H460 were 0.5 and 0.6, respectively, as confirmed by the synergism of As₂O₃ with DDP. FCM showed As₂O₃ did not affect the cell cycle. The G0/G1 fraction ranged from 57% to 62% for controlled A549 cells and cells treated with As₂O₃ and/or DDP. The G0/G1 fraction ranged from 37% to 42% for controlled H460 cells and cells treated with As₂O₃ and/or DDP. FCM and TUNEL staining illustrated that the combination of As₂O₃ and DDP provoked synergistic effects on apoptosis induction based on the analysis of the apoptosis index. Western blotting revealed that the expression of cell cycle related protein p21 and cyclin D1 were not affected by the treatments, whereas apoptosis related protein bax, Bcl-2, and clusterin were significantly regulated by As₂O₃ and/or DDP treatments compared with controls. The expression of caspase-3 in cells treated with the combination of As₂O₃ and DDP did not differ from that in cells treated with a single agent.

Conclusion

As₂O₃ exerted synergistic effects with DDP on NSCLC cells, and the synergistic effects were partly due to the induction of caspase-independent apoptosis.     

甘露糖对6个人非小细胞肺癌细胞系放射敏感性影响

目的 探讨甘露糖对6个人非小细胞肺癌细胞系放射敏感性的影响及其可能机制。方法 采用Western blot检测磷酸甘露糖异构酶在6个肺癌细胞系中的表达。利用MTT法观察甘露糖对肺癌细胞系的增殖抑制作用;分别给予对照组、甘露糖组0、2、4、6、8、10Gy照射,采用平板克隆形成实验检测甘露糖对6个肺癌细胞放射敏感性的影响;流式细胞术检测对照组、甘露糖组、照射组及联合组细胞的凋亡情况。结果 6个肺癌细胞系中磷酸甘露糖异构酶表达量各不相同,其中A549细胞表达量最高,H460细胞表达量最低。11.1mmol/L甘露糖对A549、H460细胞系抑制作用相同,随着甘露糖浓度增加,对H460细胞系抑制作用更显著。采用11.1mmol/L的甘露糖可明显增加H460细胞系放射敏感性及细胞凋亡率,而对A549细胞系放射敏感性和凋亡率影响不大。结论 在磷酸甘露糖异构酶高表达的6个肺癌细胞系中,甘露糖可增强部分肿瘤细胞的放射敏感性。     

Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide

The inhibitor of apoptosis proteins (IAPs) plays a central role in repressing caspase-mediated cell death. However, little is known about the actual role of endogenously expressed IAPs in cancer cells. We found that the cytochrome c/apoptotic protease-activating factor-1 (apoptosome)-dependent caspase activation is deficient in human non-small cell lung cancer (NSCLC) NCI-H460 cells. This dysfunctional apoptosome activity was not correlated with any decrease of apoptosome component factors, but it was linked to an increased X-linked inhibitor of apoptosis protein (XIAP). In H460 cells, the overexpressed XIAP, but not c-IAP1, bound to the processed form of caspase-9 and suppressed the activation of downstream effector caspases. Moreover, the defect in apoptosome activity in H460 cells was dramatically restored by the IAP-targeting SmacN7 peptide, which disrupted XIAP-caspase-9 binding, indicating an essential role of the IAP in the apoptosome inhibition. However, the SmacN7 did not show any striking effect on the apoptosome activity of normal lung fibroblast cells, although these cells also expressed modest amounts of IAP. To explore the therapeutic approach, we additionally developed SmacN7(R)8, a newly designed cell permeable peptide. The SmacN7(R)8 selectively reversed the apoptosis resistance of H460 cells, and when in combination with chemotherapy, regressed the tumor growth in vivo with little toxicity to the mice. Our results indicate that IAP-dependent suppression of apoptosome predominantly occurs in IAP-overexpressing tumor, and the IAP-targeting Smac peptide is an effective molecule to increase tumor cell death induced by chemotherapy in vitro and in vivo.     

Plumbagin from Plumbago Zeylanica L Induces Apoptosis in Human Non-small Cell Lung Cancer Cell Lines through NFkB Inactivation

Objective: To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer celllines, and investigate the underlying mechanisms. Materials and Methods: Human non-small cell lung cancercell lines A549, H292 and H460 were treated with various concentrations of plumbagin. Cell proliferationrates was determined using both cell counting kit-8 (CCK-8) and clonogenic assays. Apoptosis was detected byannexin V/propidium iodide double-labeled flow cytometry and TUNEL assay. The levels of reactive oxygenspecies (ROS) were detected by flow cytometry. Activity of NF-kB was examined by electrophoretic mobilityshift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of bothNF-kB regulated apoptotic-related gene and activation of p65 and IkBk. Results: Plumbagin dose-dependentlyinhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were10.3 μmol/L, 7.3 μmol/L, and 6.1 μmol/L for 12 hours, respectively. The compound concentration-dependentlyinduced apoptosis of the three cell lines. Treatment with plumbagin increased the intracellular level of ROS, andinhibited the activation of NK-kB. In addition to inhibition of NF-kB/p65 nuclear translocation, the compoundalso suppressed the degradation of IkBk. ROS scavenger NAC highly reversed the effect of plumbagin onapoptosis and inactivation of NK-kB in H460 cell line. Treatment with plumbagin also increased the activity ofcaspase-9 and caspase-3, downregulated the expression of Bcl-2, upregulated the expression of Bax, Bak, andCytC. Conclusions: Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells throughan NF-kB-regulated mitochondrial-mediated pathway, involving activation of ROS.     

The proteasomal and apoptotic phenotype determine bortezomib sensitivity of non-small cell lung cancer cells

Bortezomib is a novel anti-cancer agent which has shown promising activity in non-small lung cancer (NSCLC) patients. However, only a subset of patients respond to this treatment. We show that NSCLC cell lines are differentially sensitive to bortezomib, IC₅₀ values ranging from 5 to 83 nM. The apoptosis-inducing potential of bortezomib in NSCLC cells was found to be dependent not only on the apoptotic phenotype but also on the proteasomal phenotype of individual cell lines. Upon effective proteasome inhibition, H460 cells were more susceptible to apoptosis induction by bortezomib than SW1573 cells, indicating a different apoptotic phenotype. However, exposure to a low dose of bortezomib did only result in SW1573 cells, and not in H460 cells, in inhibition of proteasome activity and subsequent apoptosis. This suggests a different proteasomal phenotype as well. Additionally, overexpression of anti-apoptotic protein Bcl-2 in H460 cells did not affect the proteasomal phenotype of H460 cells but did result in decreased bortezomib-induced apoptosis. In conclusion, successful proteasome-inhibitor based treatment strategies in NSCLC face the challenge of having to overcome apoptosis resistance as well as proteasomal resistance of individual lung cancer cells. Further studies in NSCLC are warranted to elucidate underlying mechanisms.