盐霉素增强吉非替尼诱导人肺腺癌细胞株A549凋亡的作用

 目的：探讨盐霉素联合吉非替尼诱导人肺腺癌细胞株A549凋亡的协同作用。方法：采用MTT的方法检测盐霉素对A549细胞生长的抑制作用;流式细胞术检测盐霉素对A549细胞凋亡和线粒体膜电位的影响;比色法检测caspase-3、-8和-9活性;Western bloting 分析细胞色素C、Bcl-2、p-EGFR、p-Akt和p-ERK蛋白水平。结果：盐霉素与吉非替尼单用均出现不同程度的细胞增殖抑制作用和诱导细胞凋亡作用;而盐霉素与吉非替尼联合作用,能更显著地抑制细胞增殖,且凋亡细胞显著增加（P<0.05）。盐霉素单独作用A549细胞,线粒体膜电位显著下降,细胞内活性氧和Ca2+在短期内显著升高,胞浆细胞色素C含量以及caspase-3、-8和-9活性均显著增加,与对照组比较差异均有统计学意义;吉非替尼单用则主要表现为对p-EGFR、p-Akt和p-ERK蛋白表达的抑制作用,而对胞浆细胞色素C含量以及caspase-3、-8和-9活性影响较少。Western blotting检测发现,联合用药组的Bcl- 2、p-EGFR、p-Akt和p-ERK蛋白表达量明显减少,但是对EGFR、Akt和ERK总蛋白水平无显著影响。结论：盐霉素与吉非替尼联用具有较好的协同作用,可能通过Bcl-2途径及线粒体凋亡途径诱导人肺腺癌A549细胞凋亡,提高A549细胞对吉非替尼的敏感性。     

吉非替尼上调NKG2D配体表达增强A549细胞对NK细胞杀伤的敏感性北大核心CSCD

目的:研究表皮生长因子受体酪氨酸激酶抑制剂吉非替尼对人肺腺癌A549细胞NKG2D配体表达及NK细胞杀伤活性的影响及其分子机制。方法:MTT法测定吉非替尼对A549细胞增殖抑制率,流式细胞仪检测吉非替尼、EGFR下游分子LY294002(PI3-K抑制剂)、SB203580(MAPK抑制剂)、STAT21(STAT3抑制剂)、Rottlerin(PKC抑制剂)作用A549细胞24小时后A549细胞NKG2D配体的表达。乳酸脱氢酶释放法检测不同效靶比时,NK细胞对吉非替尼作用前、后A549细胞的杀伤活性。结果:吉非替尼上调A549细胞MICB、ULBP1表达,增强A549细胞对NK细胞杀伤的敏感性,EGFR下游分子MAPK、STAT3抑制剂不影响A549细胞NKG2D配体的表达,PI3-K抑制剂下调A549细胞MICA表达,PKC抑制剂上调ULBP1表达。结论:吉非替尼上调NKG2D配体表达增强A549细胞对NK细胞杀伤的敏感性。     

雷帕霉素对顺铂作用下人肺腺癌A549及耐药A549/DDP细胞增殖、侵袭、黏附及自噬凋亡的影响

 目的:研究雷帕霉素（Rap）对顺铂（DDP）作用下人肺腺癌A549及耐药A549/DDP 细胞增殖、迁移、黏附及其自噬凋亡的影响。方法: 培养人肺腺癌A549及耐药A549/DDP细胞株,利用MTT方法分别检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞增殖抑制率的影响;Transwell方法检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞体外侵袭能力的影响;黏附实验检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞体外侵袭能力的影响;流式细胞术检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞凋亡的影响;Western blotting检测Rap和DDP单独与联合作用对A549及耐药A549/DDP细胞自噬标志蛋白beclin-1和LC3表达的影响。 结果: 与Rap或DDP单独作用组相比,Rap和DDP联合作用能够同时显著抑制人肺腺癌A549及耐药A549/DDP细胞增殖、体外侵袭能力及细胞黏附能力,并能够促进细胞凋亡和自噬标志蛋白beclin-1和LC3的表达（均P＜0.05）。结论: Rap能够通过促进细胞自噬而增强DDP的作用,进而抑制人肺腺癌A549及耐药A549/DDP细胞的增殖、侵袭、黏附并促进细胞凋亡作用,具有协同作用。     

厄罗替尼联合塞来昔布阻断EGFR和COX-2抑制肺癌A549细胞增殖

目的探讨厄罗替尼联合塞来昔布对肺腺癌A549细胞系凋亡、表皮生长因子受体(EGFR)和环氧合酶-2(COX-2)表达的影响。方法厄罗替尼、塞来昔布单独或联合干预细胞48 h后,倒置相差显微镜观察细胞形态;四甲基偶氮唑盐(MTT)法测定细胞抑制率;Hoechst33258法和TUNEL法检测细胞凋亡和流式细胞术检测细胞周期;免疫荧光检测EGFR和COX-2蛋白表达。结果厄罗替尼联合塞来昔布组相比单药组A549细胞明显出现大量颗粒和空泡,细胞变圆开始脱落;二者联合作用时抑制作用更强(P<0.05),厄罗替尼与塞来昔布均能诱导细胞凋亡,联合作用后细胞凋亡率更高(P<0.05),并使细胞发生明显的G1期阻滞(P<0.05),进一步下调了EGFR和COX-2蛋白的表达(P<0.05)。结论厄罗替尼与塞来昔布联合应用可协同介导细胞凋亡,阻滞细胞周期于G0/G1期及阻滞EGFR和COX-2信号途径。     

重组人IL-24联合顺铂体外抗肺癌细胞生长的实验研究

目的探讨重组人白细胞介素-24(rhIL-24)联合顺铂(DDP)对人肺腺癌A549细胞的体外抑制效应及对A549细胞凋亡、细胞周期的影响。方法 rhIL-24、DDP及rhIL-24+DDP干预A549细胞,用CCK-8法检测A549细胞增殖抑制率;用流式细胞仪检测细胞凋亡及细胞周期的变化。结果 rhIL-24联合DDP作用24 h对人肺腺癌A549细胞增殖抑制率为32.8%,与单独DDP组比较有显著性差异(P0.05);流式细胞仪检测rhIL-24联合DDP作用24 h对人肺腺癌A549细胞凋亡率为24.9%;细胞周期检测中rhIL-24作用A549细胞24 h后S期细胞增多。结论 rhIL-24能抑制A549细胞增殖,联合DDP组效果优于单独用药组,具有化疗增敏效应;rhIL-24能诱导A549细胞凋亡并能影响细胞周期。     

腺病毒介导的IL-24增强紫杉醇对A549肺腺癌细胞生长的抑制作用

目的探讨腺病毒介导的白细胞介素24(Ad-IL-24)联合紫杉醇对体外培养的人肺腺癌A549细胞生长的影响。方法用Ad-IL-24、紫杉醇、Ad-IL-24联合紫杉醇处理A549细胞,以CCK-8法测定细胞生长抑制率,流式细胞术检测细胞凋亡和周期的变化。结果 Ad-IL-24可明显抑制人肺腺癌A549细胞生长并诱导细胞凋亡,与紫杉醇联合处理后,其抑制肺癌细胞生长及诱导细胞凋亡作用明显优于单用紫杉醇组及Ad-IL-24组,并引起G2/M期阻滞。结论 Ad-IL-24感染肺癌A549细胞可增强紫杉醇对肿瘤细胞生长的抑制作用。     

阿糖胞苷诱导肺腺癌细胞凋亡与p73激活相关

目的 体外观察阿糖胞苷(1-β-D-arabinofuranosylcytosine, Ara-C)对肺腺癌A549细胞凋亡的诱导作用,探讨p53、p73基因在此凋亡过程中的调控作用.方法 Ara-C体外作用于A549细胞,TUNEL法检测A549细胞的凋亡;透射电镜观察A549细胞凋亡的典型超微结构;免疫印迹法检测A...     

PLCE1通过调控p53抑制肺腺癌A549细胞凋亡

目的:探讨磷脂酶Cε1(PLCE1)抑制肺腺癌A549细胞凋亡的作用机制。方法:选用人肺腺癌细胞株A549作为研究对象。采用real-time PCR和Western blotting法分别检测PLCE1抑制剂U-73122处理前、后肺腺癌细胞株A549中PLCE1和p53 mRNA和蛋白水平的表达;流式细胞术检测细胞凋亡。结果:肺腺癌细胞株A549高表达PLCE1,低表达p53;抑制PLCE1表达后A549细胞中p53表达上调,细胞凋亡明显增加。结论:PLCE1通过抑制肺腺癌A549细胞株中p53的表达,从而抑制A549细胞凋亡。     

HGF/c-Met信号通路在克唑替尼诱导不同肺癌细胞株凋亡中的作用

 目的: 观察克唑替尼(crizotinib)诱导不同肺癌细胞株凋亡中HGF/c-Met信号通路的变化并探讨其调控机制。方法: 采用噻唑蓝(MTT)法检测克唑替尼对H1993(c-Met扩增的肺腺癌细胞)、H2228(含有EML4-ALK融合基因的肺癌细胞)和A549细胞的活力抑制情况;采用流式细胞术检测3种细胞在克唑替尼作用后24 h、48 h和72 h的凋亡率;采用Western blot检测细胞在克唑替尼作用前后HGF/c-Met信号通路中MET蛋白及其磷酸化形式p-MET的水平,同时观察其下游通路关键蛋白AKT、ERK、p-AKT和p-ERK的变化情况。结果: MTT结果表明克唑替尼作用72 h后,H1993、H2228和A549细胞株的细胞活力抑制率均呈剂量依赖性升高。流式细胞术检测发现随着克唑替尼作用时间的延长,细胞凋亡率呈时间依赖性增加(P<0.05)。Western blot检测结果提示在H1993细胞株和H2228细胞株中,p-MET、p-AKT和p-ERK随着时间的延长蛋白水平呈现下降趋势。而在A549细胞株中p-AKT、p-ERK和p-MET在药物作用72 h后的变化趋势不明显。结论: 初步证实HGF/c-Met信号通路与克唑替尼诱导肺癌细胞株H1993和H2228凋亡相关。     

MADD在肺腺癌组织中的表达及对肺腺癌A549细胞增殖和凋亡的影响

目的:探讨MADD在肺正常组织及肺腺癌组织中的表达及其对肺腺癌细胞增殖和凋亡的影响。方法:收集肺临床病理组织标本,免疫组化法检测肺正常组织和肺癌组织MADD表达;培养人肺腺癌A549细胞,逆转录PCR检测其IG20基因表达;用携带MADD基因的质粒和能够沉默MADD表达的慢病毒载体分别转染A549细胞,Western blot、MTT分析和流式细胞术检测其MADD表达、增殖及凋亡。结果:肺腺癌组织MADD表达水平明显高于肺正常组织和肺鳞癌组织;A549细胞能够表达MADD;高表达MADD能抑制A549细胞凋亡,提高其增殖活力,而沉默MADD表达则能促进A549细胞凋亡,降低其增殖活力。结论:肺腺癌组织MADD表达明显增高;MADD可通过抑制凋亡来促进肺腺癌细胞生存。     

Relationship between epidermal growth factor receptor (EGFR) mutation and serum cyclooxygenase-2 Level,and the synergistic effect of celecoxib and gefitinib on EGFR expression in non-small cell lung cancer cells

Epidermal growth factor receptor (EGFR) mutations occur mostly in patients with lung adenocarcinoma; such patients are also more likely to express cyclooxygenase-2 (COX-2), indicating a possible relationship between EGFR mutation and COX-2. The COX-2 and EGFR pathways mutually enhance their procarcinogenic effects in different tumor types. Therefore, simultaneous EGFR and COX-2 inhibition may be a promising therapeutic approach for patients with lung adenocarcinoma. We obtained tissue and serum samples from patients with non-small cell lung cancer (NSCLC) to detect the relationship between EGFR mutation and serum COX-2 level. Subsequently, gefitinib was combined with celecoxib to investigate the efficacy of inhibition in vitro in two NSCLC cell lines: HCC827 (del E746-A750) and A549 (wild-type EGFR). The cells were treated with gefitinib or celecoxib alone or with gefitinib plus celecoxib. Cell proliferation and apoptosis were assessed and correlated with expression of COX-2 and phosphorylated (p)-EGFR. The EGFR mutation rate of the high-COX-2 patients was significantly higher than that in the low-COX-2 patients. Multivariate analysis showed that high COX-2 levels were independently associated with EGFR mutation. Celecoxib and gefitinib inhibited cell growth in both cell lines. At sufficiently high concentrations, celecoxib plus gefitinib significantly mutually enhanced their anti-proliferative and apoptotic effects in both cell lines. At low concentrations, the combination had no additional effects on A549 cells. There was increased down regulation of COX-2 and p-EGFR when both cell lines were treated with high-concentration celecoxib plus gefitinib compared to either agent alone. This study demonstrates that high serum COX-2 levels may indicate EGFR mutations and that the efficacy of combined celecoxib and gefitinib is significantly greater in NSCLC cells with EGFR mutations; at high concentrations, the combination is efficacious in wild-type NSCLC cells.     

Therapeutic effect of 188Re-MAG3-depreotide on non-small cell lung cancer in vivo and in vitro

Objective: To investigate the in vivo and in vitro therapeutic effect of 188Re-MAG3-depreotide on non-small cell lung cancer (NSCLC). Methods: MTT was done to measure the cell proliferation; flow cytometry to detect cell apoptosis; Transwell invasion assay to determine the invasiveness of NSCLC. In addition, HE staining, TUNEL staining and immunohistochemistry for CD34 were employed to investigate the influence of 188Re-MAG3-depreotide on the growth of NSCLC. Results: 1) Within 2-6 days, the inhibitory effect of 188Re-MAG3-depreotide on the proliferation of A549 cells and SPC-A1 cells increased over time. 2) At 48 h after treatment with 188Re-MAG3-depreotide, the apoptosis rate of A549 cells and SPC-A1 cells was 23.1% and 22.6%, respectively. 3) After 188Re-MAG3-depreotide treatment, the number of invasive A549 cells and SPC-A1 cells was reduced by about 3 times when compared with control group. 4) The cancer in the control group presented with unlimited growth. The cancer growth continued after treatment with 188Re or MAG3-depreotide alone, while the cancer growth was markedly inhibited after 188Re-MAG3-depreotide treatment when compared with control group. Conclusion: 188Re-MAG3-depreotide can inhibit the proliferation and invasion of A549 cells and SPC-A1 cells. Treatment with 7.4MBq 188Re-MAG3-depreotide via tail vein can significantly suppress the in vivo cancer growth and induce the apoptosis of cancer cells. These findings demonstrate that 188Re-MAG3-depreotide can induce the apoptosis of NSCLC cells and directly kill the NSCLC cells, which provide evidence for the radiotherapy of NSCLC.     

Somatic mutations of the epidermal growth factor receptor and non-small-cell lung cancer

Frequent overexpression of epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) makes EGFR a new therapeutic target. Two specific EGFR tyrosine kinase inhibitors, gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva), have been developed and approved by the US Food and Drug Administration for second-line and third-line treatment of advanced NSCLC. Clinical trials have shown considerable variability in the response rate between different patients with NSCLC, which led to the discovery of somatic EGFR-activating mutations. This brief review summarises the discovery and functional consequences of the mutations, their clinicopathological features and significant implications in the treatment and prognosis of NSCLC.     

Mig-6 overcomes gefitinib resistance by inhibiting EGFR/ERK pathway in non-small cell lung cancer cell lines

Non small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and is the most common cause of lung cancer death. Currently, the epidermal growth factor receptor inhibitor gefitinib is widely used for patients with advanced NSCLC. However, drug resistance is a major obstacle. Mig-6 is a feedback inhibitor of EGFR and its down-stream pathway; it has been shown to play a role in gefitinib sensitivity. There is neither systematical research on the relationship between Mig-6 expression and gefitinib sensitivity, nor has the contribution of up-regulated Mig-6 on the gefitinib-resistant cell lines. In the present work, four NSCLC cell lines (H1299, A549, PC-9, and PC-9/AB11) with different sensitivities to gefitinib were subjected to analysis of the expression of Mig-6. We found that Mig-6 is over-expressed in gefitinib-sensitive NSCLC cell lines, but is low in gefitinib-resistant NSCLC cell lines. Further analysis revealed that over-expression of Mig-6 increased cell apoptosis and inhibited proliferation of gefitinib-resistant NSCLC cells treated with gefitinib, whereas lowering the expression of Mig-6 decreased cell apoptosis and promoted cell proliferation after treatment with gefitinib in gefitinib-sensitive NSCLC cell lines. These results suggest that Mig-6 is involved in mediating the response to gefitinib in NSCLC cell lines. Additionally we demonstrated that Mig-6 could reverse gefitinib resistance through inhibition of EGFR/ERK pathway in NSCLC cells. Our work uncovered that Mig-6 may be an effective therapeutic target in gefitinib-resistant lung cancer patients.     

FHITlow/pHER2high signature in non-small cell lung cancer is predictive of anti-HER2 molecule efficacy

Despite its efficacy in solid tumours, in particular HER2⁺ breast cancer, HER2-targeted therapy has given rise to disappointing results in non-small cell lung cancer (NSCLC). With the aim of refining the target population for anti-HER2 therapies in NSCLC, we investigated the relationships between HER2 and the tumour suppressor fragile histidine triad (FHIT) in lung tumour cells. First, we observed a negative correlation between FHIT expression and the activated form of HER2 (pHER2) in NSCLC samples and in lung tumour cell lines. Moreover, the silencing or overexpression of FHIT in lung cell lines led to an increase or decrease of HER2 activity, respectively. We also demonstrated that two anti-HER2 drugs, irbinitinib and trastuzumab, restore a more epithelial phenotype and counteract cell invasiveness and growth of FHIT-silenced tumour cell lines. Finally, we showed that the FHIT^low/pHER2^high phenotype predicts sensitivity to an anti-HER2 therapy in primary tumour cells from NSCLC patients. Our results show that FHIT regulates the activity of HER2 in lung tumour cells and that FHIT-inactivated tumour cells are sensitive to HER2 inhibitors. A new subclass of patients with NSCLC may be eligible for an anti-HER2 therapy. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Elevated expression of Fn14 in non-small cell lung cancer correlates with activated EGFR and promotes tumor cell migration and invasion

Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs.