Endobronchial Mucosa Invasion Predicts Survival in Patients with Small Cell Lung Cancer

Background

Current staging system for small cell lung cancer (SCLC) categorizes patients into limited- or extensive-stage disease groups according to anatomical localizations. Even so, a wide-range of survival times has been observed among patients in the same staging system. This study aimed to identify whether endobronchial mucosa invasion is an independent predictor for poor survival in patients with SCLC, and to compare the survival time between patients with and without endobronchial mucosa invasion.

Methods

We studied 432 consecutive patients with SCLC based on histological examination of biopsy specimens or on fine-needle aspiration cytology, and received computed tomography and bone scan for staging. All the enrolled patients were assessed for endobronchial mucosa invasion by bronchoscopic and histological examination. Survival days were compared between patients with or without endobronchial mucosa invasion and the predictors of decreased survival days were investigated.

Results

84% (364/432) of SCLC patients had endobronchial mucosal invasion by cancer cells at initial diagnosis. Endobronchial mucosal involvement (Hazard ratio [HR], 2.01; 95% Confidence Interval [CI], 1.30–3.10), age (HR, 1.04; 95% CI, 1.03–1.06), and extensive stage (HR, 1.39; 95% CI, 1.06–1.84) were independent contributing factors for shorter survival time, while received chemotherapy (HR, 0.32; 95% CI, 0.25–0.42) was an independent contributing factor better outcome. The survival days of SCLC patients with endobronchial involvement were markedly decreased compared with patients without (median 145 vs. 290, p<0.0001). Among SCLC patients of either limited (median 180 vs. 460, p<0.0001) or extensive (median 125 vs. 207, p<0.0001) stages, the median survival duration for patients with endobronchial mucosal invasion was shorter than those with intact endobronchial mucosa, respectively.

Conclusion

Endobronchial mucosal involvement is an independent prognostic factor for SCLC patients and associated with decreased survival days.     

IscU2对非小细胞肺癌增殖、迁移、侵袭能力的影响及其作用机制的研究

该文通过shRNA干扰技术敲低IscU2干扰细胞IscU2的表达,研究了干扰IscU2对非小细胞肺癌(NSCLC)细胞NCI-H520增殖、迁移及侵袭能力的影响。构建了稳定低表达IscU2的非小细胞肺癌细胞系NCI-H520;采用CCK-8和平板克隆实验检测细胞的增殖能力;流式细胞仪检测细胞周期、凋亡、ROS、线粒体膜电位变化情况;Transwell实验检测细胞迁移及侵袭能力;Western blot检测相关蛋白的表达。结果表明,干扰IscU2后,非小细胞肺癌细胞的增殖及克隆形成能力降低;细胞周期停滞在G1/G0期,同时伴随有p-AKT和Cyclin D1蛋白含量的下降;细胞晚期凋亡率明显增加,凋亡蛋白Cleaved-caspase3和Cleaved-PARP表达上调;细胞迁移和侵袭能力降低,上皮标志物E-Cadherin表达上调,间质标志物N-Cadherin和Snail表达下调;细胞ROS积累和线粒体膜电位下降。该研究结果表明,干扰IscU2显著抑制非小细胞肺癌的增殖、迁移、侵袭能力和上皮–间质转化,这为非小细胞肺癌的诊断和治疗提供了新的潜在靶点和视角。     

MicroRNA-2 Suppresses Lewis Lung Cancer Cells Proliferation,Invasion, and Migration in Tumor-Bearing Mice

We sought to find the biological effects of MicroRNA-2 in suppressing Lewis lung cancer cells proliferation, invasion, and migration in tumor-bearing mice. MicroRNA-2 was transfected into Lewis lung cancer cells of tumor-bearing mice by gene transient transfection technique and these Lewis-microRNA-2 cells were taken as MicroRNA transfection group. At the same time, Lewis cells were taken as control group and Lewis-EGFP cells as empty plasmid group. The growth curves of cells in the three groups were drawn by manual counting method, while the invasiveness of cells in the three groups was compared by transmembrane cell invasion assay. The three kinds of cells were seeded into BALB/Nude SPF level nude mice to detect the formation of tumors and the number of metastases by Xenograft experiments. The result showed that the MicroRNA transfection group has the lowest vitality of cells proliferation, fewest cells passed through matrigel matrix protein layer, and lowest cells invasive rate. Mice with Lewis-microRNA-2 cells apparently had a longer time of tumor formation. The average tumor mass and the number of metastases were significantly lower than the other two groups. MicroRNA-2 significantly inhibited Lewis lung cancer cell proliferation, invasion and migration in tumor-bearing mice, which may be associated with the regulation of target genes PLK1 and TGF-β.     

Effects of Cinobufagin on the Proliferation,Migration, and Invasion of H1299 Lung Cancer Cells

Cinobufagin (CB), with its steroidal nucleus structure, is one of the major, biologically active components of Chan Su. Recent studies have shown that CB exerts inhibitory effects against numerous cancer cells. However, the effects of CB regarding the metastasis of non-small cell lung cancer (NSCLC) and the involved mechanisms need to be further studied. The purpose of the present study aimed to report the inhibitory function of CB against proliferation and metastasis of H1299 cells. CB inhibited proliferation of H1299 lung cancer cells with an IC₅₀ value of 0.035±0.008 μM according to the results of MTT assays. Antiproliferative activity was also observed in colony forming cell assays. In addition, 5-ethynyl-2’-deoxyuridine (EdU) retention assays revealed that CB significantly inhibited the rate of DNA synthesis in H1299 cells. Moreover, results of the scratch wound healing assays and transwell migration assays displayed that CB exhibited significant inhibition against migration and invasion of H1299 cells. Furthermore, CB could concentration-dependently reduce the expression of integrin α2, β-catenin, FAK, Src, c-Myc, and STAT3 in H1299 cells. These western blotting results indicated that CB might target integrin α2, β-catenin, FAK and Src to suppress invasion and migration of NSCLC, which was consistent with the network pharmacology analysis results. Collectively, findings of the current study suggest that CB possesses promising activity against NSCLC growth and metastasis.     

沉默胃泌素基因抑制胃癌细胞的增殖、迁移和侵袭

胃癌细胞分泌的胃泌素与胃癌的发生、发展密切相关.为了探讨胃泌素对胃癌细胞增殖、迁移和侵袭的影响,本文构建靶向胃泌素基因的siRNA表达载体, 转染胃癌细胞AGS, 成功获得沉默胃泌素基因的稳转胃癌细胞株AGS/Gas-siRNA. 用MTT实验、软琼脂集落形成实验、细胞伤愈实验、Transwell实验及ELISA检测沉默胃泌素基因后细胞的增殖、迁移、侵袭及转移相关蛋白基质金属蛋白酶-2（MMP-2）和血管内皮生长因子（VEGF）的含量. 结果显示: 与空载体转染的对照细胞比较, 沉默胃泌素基因的细胞, 其增殖率和克隆形成率显著降低,迁移和侵袭到Transwell下室的细胞数分别降低了31.6 %和34 %. 培养上清液中MMP-2和VEGF含量也低于对照细胞. 结果提示,沉默胃泌素基因的胃癌细胞,通过降低MMP 2和VEGF分泌,抑制了细胞的增殖、迁移和侵袭, 这可能是胃泌素促进胃癌侵袭转移的机制之一.     

RNAi干扰Ska2对H1299细胞增殖、迁移及侵袭能力的影响

Ska2（spindle and kinetochore associated complex subunit2）,又称FAM33A（family with sequence similarity33,member A）,是新近发现的一个与细胞周期调控和肿瘤发生发展紧密相关的基瓯且与该团队前期发现的新基NPRR11（proline rich 11）共享一个双向启动子。但是,Ska2在肺癌中的具体作用和分子机制仍不清楚。该研究选用肺癌细胞系H1299,采用RNAi技术构建Ska2基因沉默的稳定细胞株,并进行了细胞表型和潜在分子机制分析。RT-PCR和Western blot结果表明,Ska2在mRNA和蛋白质水平上的表达均被有效抑制。细胞增殖、细胞迁移和侵袭实验结果表明,与对照细胞相比,Ska2基因沉默稳定细胞株的细胞增殖能力、细胞迁移和侵袭能力均显著降低。此外,Ska2基因被沉默后,CCNA1基因的表达显著下调。该研究的结果提示,Ska2与其对侧基因PRR11的功能高度相关,可能与PRR11共同参与肺癌细胞增殖、迁移和侵袭行为的调节。     

过表达胃泌素促进胃癌细胞的增殖、迁移和侵袭

胃癌患者转移淋巴结中胃泌素基因的表达量是原发胃癌组织的42倍,推测胃泌素可能与胃癌转移密切相关. 本文通过构建含胃泌素基因的真核表达载体,成功获得过表达胃泌素的稳转胃癌细胞株AGS和SGC-7901, 并用MTT、细胞伤愈实验、Transwell 小室实验及ELISA检测过表达胃泌素对细胞迁移、侵袭及转移相关蛋白基质金属蛋白酶2（MMP-2）分泌能力的影响. 结果显示,过表达胃泌素稳转细胞的相对增殖率、 迁移入细胞致伤区的相对距离比对照组高,迁移和侵袭到Transwell下室面的细胞, 以及培养液中每mg蛋白质的MMP-2浓度也高于对照组的细胞. 结果提示,胃泌素通过促进胃癌细胞分泌MMP-2来增强细胞的迁移和侵袭能力. 该研究对揭示胃癌转移的分子机制具有重要意义.     

PFTK1 Promotes Gastric Cancer Progression by Regulating Proliferation,Migration and Invasion

PFTK1, also known as PFTAIRE1, CDK14, is a novel member of Cdc2-related serine/threonine protein kinases. Recent studies show that PFTK1 is highly expressed in several malignant tumors such as hepatocellular carcinoma, esophageal cancer, breast cancer, and involved in regulation of cell cycle, tumors proliferation, migration, and invasion that further influence the prognosis of tumors. However, the expression and physiological significance of PFTK1 in gastric cancer remain unclear. In this study, we analyzed the expression and clinical significance of PFTK1 by Western blot in 8 paired fresh gastric cancer tissues, nontumorous gastric mucosal tissues and immunohistochemistry on 161 paraffinembedded slices. High PFTK1 expression was correlated with the tumor grade, lymph node invasion as well as Ki-67. Through Cell Counting Kit (CCK)-8 assay, flow cytometry, colony formation, wound healing and transwell assays, the vitro studies demonstrated that PFTK1 overexpression promoted proliferation, migration and invasion of gastric cancer cells, while PFTK1 knockdown led to the opposite results. Our findings for the first time supported that PFTK1 might play an important role in the regulation of gastric cancer proliferation, migration and would provide a novel promising therapeutic strategy against human gastric cancer.     

骆驼蓬碱通过LOXL1-AS1影响卵巢癌细胞CAOV3增殖、凋亡、迁移侵袭

该研究探讨了骆驼蓬碱对卵巢癌细胞CAOV3增殖、凋亡、迁移侵袭的影响及分子机制.卵巢癌细胞CAOV3分为对照组,骆驼蓬碱低、中、高剂量组,si-NC组,si-LOXL1-AS1组,骆驼蓬碱高剂量+pcDNA组,骆驼蓬碱高剂量+pcDNA-LOXL 1-AS 1组.用细胞计数试剂盒-8(CCK-8)检测细胞存活率;平板克...     

泛素偶联酶2C促进肺癌细胞增殖和迁移与抑制细胞衰老

泛素偶联酶2C与多种肿瘤细胞的增殖密切相关,但其与肺癌发生和发展的关系尚不明确。 本研究以肺癌A549细胞为材料,通过RT-PCR、Western印迹、免疫荧光、SA-β-Gal细胞衰老染色、细胞划痕和Trans-well实验,阐明UBE2C与肺癌细胞的增殖、衰老和迁移能力的关系。结果显示,UBE2C在肺癌细胞中的表达明显高于正常细胞。利用基因修饰技术瞬时过表达或靶向沉默UBE2C后,在肺癌A549细胞中,UBE2C的mRNA和蛋白质水平显著增加3.5倍或减少0.5倍,显著促进或抑制细胞增殖,进而减少或增加细胞的凋亡率。过表达UBE2C后,显著抑制细胞衰老;但沉默UBE2C后,则增加细胞衰老。此外,过表达UBE2C后,下调转移相关基因E-钙黏着蛋白的mRNA和蛋白质表达水平,且上调波形蛋白基因的表达水平,进而促进肺癌细胞的迁移。但靶向敲除UBE2C后,上调E-钙黏着蛋白,同时下调波形蛋白表达水平,进而抑制肺癌细胞的迁移。本研究的开展将明确UBE2C在肺癌中的作用及其机制,为以UBE2C为靶点,提高病人生存期提供了理论基础。     

Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients

Background

Anaplastic Lymphoma Kinase (ALK) positivity represents a novel molecular target in a subset of Non-Small Cell Lung Cancers (NSCLC). We explore Fluorescence in situ Hybridization (FISH) and Immunohistochemistry (IHC) as diagnostic methods for ALK positive patients and to describe its prevalence and outcomes in a population of NSCLC patients.

Methods

NSCLC patients previously screened for Epidermal Growth Factor Receptor (EGFR) at our institution were selected. ALK positive patients were identified by FISH and the value of IHC (D5F3) was explored.

Results

ninety-nine patients were identified. Median age was 61.5 years (range 35–83), all were caucasians, eighty percent were adenocarcinomas, fifty-one percent were male and thirty-eight percent were current smokers. Seven (7.1%) patients were ALK positive by FISH, thirteen (13.1%) were EGFR mutant, and 65 (65.6%) were negative/Wild Type (WT) for both ALK and EGFR. ALK positivity and EGFR mutations were mutually exclusive. ALK positive patients tend to be younger than EGFR mutated or wt patients. ALK positive patients were predominantly never smokers (71.4%) and adenocarcinoma (71.4%). ALK positive and EGFR mutant patients have a better outcome than negative/WT. All patients with ALK FISH negative tumours were negative for ALK IHC. Out of 6 patients positive for ALK FISH with more tissue available, 5 were positive for ALK IHC and 1 negative.

Conclusions

ALK positive patients represent 7.1% of a population of selected NSCLC. ALK positive patients have different clinical features and a better outcome than EGFR WT and ALK negative patients. IHC is a promising method for detecting ALK positive NSCLC patients.     

miR-101 Inhibiting Cell Proliferation,Migration and Invasion in Hepatocellular Carcinoma through Downregulating Girdin

miR-101 is considered to play an important role in hepato-cellular carcinoma (HCC), but the underlying molecular mechanism remains to be elucidated. Here, we aimed to confirm whether Girdin is a target gene of miR-101 and determine the tumor suppressor of miR-101 through Girdin pathway. In our previous studies, we firstly found Girdin protein was overexpressed in HCC tissues, and it closely correlated to tumor size, T stage, TNM stage and Edmondson-Steiner stage of HCC patients. After specific small interfering RNA of Girdin was transfected into HepG2 and Huh7.5.1 cells, the proliferation and invasion ability of tumor cells were significantly inhibited. In this study, we further explored the detailed molecular mechanism of Girdin in HCC. Interestingly, we found that miR-101 significantly low-expressed in HCC tissues compared with that in matched normal tissues while Girdin had a relative higher expression, and miR-101 was inversely correlated with Girdin expression. In addition, after miR-101 transfection, the proliferation, migration and invasion abilities of HepG2 cells were weakened. Furthermore, we confirmed that Girdin is a direct target gene of miR-101. Finally we confirmed Talen-mediated Girdin knockout markedly suppressed cell proliferation, migration and invasion in HCC while down-regulation of miR-101 significantly restored the inhibitory effect. Our findings suggested that miR-101/Girdin axis could be a potential application of HCC treatment.     

MicroRNA-575靶向抑制BLID促进非小细胞肺癌细胞的增殖和侵袭

目的:通过体外实验探讨miR-575对非小细胞肺癌(NSCLC)细胞增殖与侵袭能力的影响及相关机制。方法:采用实时定量PCR法检测不同非小细胞肺癌细胞系中miR-575、BLID的表达;CCK-8法检测转染miR-575模拟物、抑制因子后不同时间A549细胞增殖情况的变化;Transwell法检测A549细胞的侵袭情况;Targetcan法及双荧光素酶检测miR-575对BLID 3'UTR端的靶向作用;Western blot法检测BLID蛋白的表达。结果:A549、SPC-A1、H1299、H1650等人非小细胞肺癌细胞系中miR-575的表达均显著高于永生化的人支气管上皮细胞系16HBE(P0.001)。MiR-575模拟物转染的A549细胞miR-575的表达明显高于对照组(P0.001),同时细胞的增殖和侵袭力增强(P0.05);反之,miR-575抑制因子转染的A549细胞miR-575的表达显著降低,且细胞的增殖和侵袭力明显降低(P0.01)。Targetscan法预测BLID可能是miR-575的下游靶基因,荧光素酶结果显示miR-575不仅能够有效抑制野生型BLID 3'UTR端的荧光素酶反应(P0.01),而且能够降低BLID的蛋白表达量(P0.01)。实时定量PCR结果显示BLID在NSCLC细胞系中均呈现显著的低表达(P0.001),且转染BLID后,NSCLC细胞的增殖和细胞侵袭被明显抑制(P0.05),而当miR-575与BLID共转染时,miR-575能够逆转BLID所抑制的细胞增殖和侵袭(P0.01)。结论:在NSCLC细胞系中,miR-575的表达上调,且能够通过直接作用于下游靶点抑癌基因BLID从而促非小细胞肺癌细胞增殖及侵袭。     

TRIM59 Promotes the Proliferation and Migration of Non-Small Cell Lung Cancer Cells by Upregulating Cell Cycle Related Proteins

TRIM protein family is an evolutionarily conserved gene family implicated in a number of critical processes including inflammation, immunity, antiviral and cancer. In an effort to profile the expression patterns of TRIM superfamily in several non-small cell lung cancer (NSCLC) cell lines, we found that the expression of 10 TRIM genes including TRIM3, TRIM7, TRIM14, TRIM16, TRIM21, TRIM22, TRIM29, TRIM59, TRIM66 and TRIM70 was significantly upregulated in NSCLC cell lines compared with the normal human bronchial epithelial (HBE) cell line, whereas the expression of 7 other TRIM genes including TRIM4, TRIM9, TRIM36, TRIM46, TRIM54, TRIM67 and TRIM76 was significantly down-regulated in NSCLC cell lines compared with that in HBE cells. As TRIM59 has been reported to act as a proto-oncogene that affects both Ras and RB signal pathways in prostate cancer models, we here focused on the role of TRIM59 in the regulation of NSCLC cell proliferation and migration. We reported that TRIM59 protein was significantly increased in various NSCLC cell lines. SiRNA-induced knocking down of TRIM59 significantly inhibited the proliferation and migration of NSCLC cell lines by arresting cell cycle in G2 phase. Moreover, TRIM59 knocking down affected the expression of a number of cell cycle proteins including CDC25C and CDK1. Finally, we knocked down TRIM59 and found that p53 protein expression levels did not upregulate, so we proposed that TRIM59 may promote NSCLC cell growth through other pathways but not the p53 signaling pathway.     

SULF2 Expression Is a Potential Diagnostic and Prognostic Marker in Lung Cancer

Aims

Lung cancer is one of the most deadly cancers; median survival from diagnosis is less than one year in those with advanced disease. Novel lung cancer biomarkers are desperately needed. In this study, we evaluated SULF2 expression by immunohistochemistry and its association with overall survival in a cohort of patients with non-small cell lung cancer (NSCLC). We also looked for the presence of SULF2 protein in plasma to evaluate its potential as an early detection biomarker for NSCLC.

Methods

We identified patients who underwent surgical resection for pulmonary adenocarcinoma or squamous cell carcinoma at our institution. A section from each paraffin-embedded specimen was stained with a SULF2 antibody. A pathologist determined the percentage and intensity of tumor cell staining. Survival analysis was performed using a multivariate Cox proportional hazards model. Using a novel SULF2 ELISA assay, we analyzed plasma levels of SULF2 in a small cohort of healthy donors and patients with early stage NSCLC.

Results

SULF2 staining was present in 82% of the lung cancer samples. Squamous cell carcinomas had a higher mean percentage of staining than adenocarcinomas (100% vs. 60%; p<0.0005). After adjusting for age, sex, race, histologic type, stage, and neoadjuvant therapy, there was a non-significant (31%; p = 0.65) increase in the risk of death for patients with adenocarcinoma with SULF2 staining in tumor cells. In contrast, there was a significant decrease in the risk of death (89%; p = 0.02) for patients with squamous cell carcinoma with SULF2 staining in tumor cells. SULF2 protein was present in plasma of patients with early stage NSCLC, and soluble SULF2 levels increased with age. Finally, plasma SULF2 levels were significantly elevated in early stage NSCLC patients, compared to healthy controls.

Conclusions

Tumor expression of SULF2 may affect prognosis in NSCLC, while blood SULF2 levels may have a significant role in the diagnosis of this fatal disease.     

非小细胞肺癌患者K-ras基因突变与预后的相关性分析

目的:探讨非小细胞肺癌(NSCLC)患者K-ras基因突变与预后的相关性。方法:回顾分析2006年3月~2008年7月江西省肿瘤医院收治的92例NSCLC患者的临床资料,所有患者均采用蝎形探针扩增阻遏突变系统(ARMS)法行K-ras基因突变检测。结果:92例NSCLC患者中,11例K-ras基因突变阳性,阳性率为11.96%,包括Gly12Cys(GGTTGT)突变3例、Gly12Val(GGTGTT)突变2例、Gly12Ala(GGTGCT)突变2例、Gly12Arg(GGTCGT)突变2例、Gly12Cys(GGTTGT)突变与Gly12Val(GGTGTT)突变并存1例、Gly12Cys(GGTTGT)突变与Gly12Ala(GGTGCT)突变并存1例;K-ras基因突变阳性组与阴性组之间的患者性别、年龄、病理类型、吸烟史相比,差异无统计学意义(P0.05);K-ras基因突变阳性组Ⅲa~Ⅳ期患者中位生存期显著低于K-ras基因突变阴性组Ⅲa~Ⅳ期患者,差异有统计学意义(P0.05)。结论:NSCLC患者K-ras基因突变阳性率较低,但它可以对NSCLC患者预后产生负性影响。