人肺腺癌多药抗药细胞系LC-3/CDDP细胞周期及DNA含量变化

应用流式细胞仪测定人肺腺癌多药抗药细胞系LC－3／CDDP之细胞周期、DNA指数，用3H-胸腺嘧啶核苷掺入法测定DNA合成动态，结果显示，LC－3／CDDP细胞系G2＋M峰较亲代细胞明显增高，G期细胞比例降低，S期、G2＋M期比例升高，DNA指数增加，DNA合成速率较亲代细胞显著增快。提示人肺腺癌多药抗药细胞系LC－3／CDDP对DNA损伤的修复功能增强。     

电离辐射克服NSCLC细胞株H1975吉非替尼耐药的体外研究

目的 探讨电离辐射联合吉非替尼对NSCLC耐药株H1975耐药突变、细胞凋亡及相关蛋白表达的影响及其可能机制。方法 实时荧光定量PCR对不同处理组H1975细胞的T790M突变进行相对定量分析;流式细胞仪检测不同处理组H1975细胞的凋亡率;免疫印迹检测不同处理组凋亡相关蛋白的表达水平。结果 2.5 Gy电离辐射组相较于0 Gy对照组,H1975细胞株T790M突变量降为原来的0.67倍,随电离辐射剂量的增高,T790M突变量降低（P＜0.05）;电离辐射联合吉非替尼组的细胞凋亡率为（44.35±8.49）%,相较于单独电离辐射组（21.84±5.62）%或吉非替尼组（17.38±6.78）%明显升高（P＜0.05）;电离辐射联合吉非替尼可诱导H1975细胞中磷酸化表皮生长因子受体（phosphorylated epidermal growth factor receptor, p-EGFR）、磷酸化蛋白激酶B（phosphorylated protein kinase B, p-AKT）蛋白表达水平明显下调。结论 在吉非替尼耐药的NSCLC细胞株H1975中,电离辐射可以克服吉非替尼耐药,与吉非替尼有良好的协同作用。     

Human T-Cell Leukemia Virus-1-positive Cell Line Established from a Patient with Small Cell Lung Cancer

A stable cell line, KHM-3S, was established from a patient with small cell lung cancer (SCLC), who had a high serum level of soluble interleukin 2 receptors (sIL2-R) and was seropositive for human T cell leukemia virus (HTLV)-l. KHM-3S cells were positive for IL2-R (Tac) and NKH-1, but negative for other lymphocytic markers such as OKT 11, OKT 4, OKT 8, T cell receptor (WT 31), B 1, and B 4. Moreover, the KHM-3S cells were negative for leukocyte common antigen and strongly positive for neuron-specific enolase (NSE). Secretion of sIL2-R and NSE by the KHM-3S line was detected by an enzyme-linked immunosorbent assay. Rearrangement of the T cell receptor gene and monoclonal HTLV-1 integration were found by Southern blot analysis of KHM-3S DNA. However, Northern blot analysis showed no T cell receptor mRNA. KHM-3S may be useful for studies on the role of HTLV-1 in carcinogenesis and IL2-R expression in SCLC.     

Selection of Radioresistant Cells by Vitamin A Deficiency in a Small Cell Lung Cancer Cell Line

Radiation sensitivity of a human small cell lung cancer cell line, Lu-134-B cells, cultured in serum-supplemented medium and of cells transferred to and cultured in delipidized serum-supplemented (vitamin A-deficient) medium was studied. The cells cultured in serum-supplemented medium showed the phenotype of classic small cell lung cancer sensitive to radiation, while cells transferred to delipidized serum-supplemented medium showed partial squamous cell differentiation and became resistant to radiation. These results suggest that some small cell lung cancer cells in vitro change their morphology and radiosensitivity depending on the culture conditions. The change in radiosensitivity was reproducible, and was not reversible by culture of the radioresistant cells in delipidized serum-supplemented medium with addition of retinoic acid (vitamin A-sufficient medium) for two months, although squamous cells disappeared. Acquisition of radioresistancy was considered to occur as the result of clonal selective growth in delipidized medium of a minor cell population in the original cell culture, based on a study of chromosome number. It was also found that there was no association of myc -family oncogenes with the changes of radiosensitivity in this cell line.     

免疫检查点抑制剂在小细胞肺癌中的临床研究进展

小细胞肺癌(small cell lung cancer,SCLC)是分化较差的高级别肺神经内分泌肿瘤,尽管仅占所有肺癌的14％左右,但生长迅速、较早出现转移,复发后缺少有效的治疗手段,改善SCLC治疗迫在眉睫.近年来,肿瘤免疫治疗展现了良好的前景,尤其程序性死亡受体1(programmed death1,PD-1)和细胞毒性T淋巴细胞相关抗原4(cytotoxic T-lymphocyte-associated antigen 4,CTLA-4)抑制剂的研究正在改变多种实体瘤的临床实践.SCLC与吸烟密切相关,具有较高的肿瘤突变负荷,是免疫治疗潜在理想的肿瘤类型.本文将总结免疫治疗在SCLC的临床研究进展,探讨SCLC免疫治疗中存在的问题、面临的挑战以及未来的应用前景.     

Mechanisms of Growth Inhibition of Human Lung Cancer Cell Line, PC-9, by Tea Polyphenols

(–)-Epigallocatechin gallate (EGCG), the main constituent of green tea, and green tea extract show growth inhibition of various cancer cell lines, such as lung, mammary, and stomach. We studied how tea polyphenols induce growth inhibition of cancer cells. Since green tea extract contains various tea polyphenols, such as EGCG, (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG), and (–)-epicatechin (EC), the inhibitory potential of each tea polyphenol on the growth of a human lung cancer cell line, PC-9 cells, was first examined. EGC and ECG inhibited the growth of PC-9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell cycle regulation. Flow cytometric analysis revealed that treatment with 50 μM and 100 μM EGCG increased the percentages of cells in the G₂-M phase from 13.8% to 15.6% and 24.1%, respectively. The DNA histogram after treatment with 100 μM EGCG was similar to that after treatment with genistein, suggesting that EGCG induces G₂-M arrest in PC-9 cells. Moreover, we found by microautoradiography that [³H]EGCG was incorporated into the cytosol, as well as the nuclei. These results provide new insights into the mechanisms of action of EGCG and green tea extract as cancer-preventive agents in humans.     

鸡血藤血清联合顺铂对人肺癌PG细胞增殖及周期影响的研究

[目的]探讨鸡血藤血清对人肺癌PG细胞株的增殖抑制及周期阻滞作用.[方法]将肺癌PG细胞分为空白组、鸡血藤组、鸡血藤+顺铂组、顺铂组,四唑盐比色法(MTTr)实验中各组根据血清浓度的不同分为20％、10％、5％、2.5％、4组,观察各组PG细胞的增殖抑制作用;HE染色法、免疫荧光法和流式细胞术实验中各组血清浓度为20％,采用光镜和激光共聚焦显微镜观察各组PG细胞形态,流式细胞仪检测细胞周期.[结果] MTT法中5％、10％、20％鸡血藤组细胞的增殖抑制明显,具有时间和剂量依赖性,但低于顺铂组和鸡血藤+顷铂组,鸡血藤+顺铂组PG细胞的增殖抑制率与顺铂组比较,差异无统计学意义(P＞0.05).光镜和激光共聚焦显微镜下鸡血藤组细胞的凋亡程度明显,但低于顺铂组和鸡血藤+顺铂组.与空白组比较,鸡血藤组PG细胞的G2/M期比例增加(P＜0.05);顺铂组和鸡血藤+顺铂组的S和G2/M期比例增加(P＜0.01),G0/G1期降低(P＜0.01).顺铂组和鸡血藤+顺铂组较鸡血藤组的S期和G2/M期比例增加(P＜0.01),G0/G1期降低(P＜0.01).鸡血藤+顺铂组PG细胞的各期与顺铂组比较,差异无统计学意义(P＞0.05).[结论]鸡血藤血清通过引起细胞周期G2/M期阻滞,从而抑制细胞增殖,这与顺铂阻滞细胞于G2/M和S期不同.     

MiRNA-25在非小细胞肺癌吉非替尼耐药细胞中的表达及其作用机制

目的 探讨miRNA-25在非小细胞肺癌(NSCLC)吉非替尼耐药细胞中的表达及其作用机制.方法 采用RT-PCR法检测miRNA-25在NSCLC吉非替尼耐药细胞PC9/G及亲本细胞PC9中的表达;采用体外转染法将miRNA-25模拟物或抑制物分别转染PC9和PC9/G细胞,采用CCK8实验检测转染前后细胞对吉非替尼敏感性的变化;并用Westem blot检测转染前后细胞中Bim的表达变化.采用双荧光素酶报告基因验证miRNA-25是否作用于Bim基因的3'-UTR区预测靶位.结果 miRNA-25在PC9/G细胞中的表达水平是PC9细胞的(7.15±1.26)倍(P＜0.01).PC9细胞转染miRNA-25模拟物后,吉非替尼对PC9细胞增殖的抑制率较转染前明显下降(P＜0.01),细胞内Bim表达水平较转染前明显下降(P＜0.05).PC9/G细胞在转染miRNA-25抑制物后,吉非替尼对PC9/G细胞增殖的抑制率与转染前比较明显增加(P＜0.01),细胞内Bim表达水平较转染前明显增高(P＜0.05).经双荧光素酶报告基因验证Bim是miRNA-25的靶基因.结论 miRNA-25在NSCLC耐药细胞PC9/G中异常高表达,下调其表达可部分逆转细胞耐药性,miRNA-25可能通过作用于Bim参与NSCLC细胞吉非替尼耐药的发生和发展.     

Role of Tyrosine Phosphorylation in Radiation-Induced Cell Cycle-Arrest of Leukemic B-Cell Precursors at the G2-M Transition Checkpoint

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34^cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34^cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34^cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34^cdc2. We detected no increase of human WEE 1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34^cdc2 amino-terminal region or (c) recombinant human p34^cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34^cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49^WEE1Hu and enzymes which augment the tyrosine kinase activity of p49^WEE1Hu.     

Involvement of FoxM1 in Non-Small Cell Lung Cancer Recurrence

Background: Predictive biomarkers for lung cancer recurrence after curative tumor resection remainunclear. This study set out to assess the role of FoxM1 in the recurrence of non-small cell lung cancer. Methods:Immunohistochemistry for FoxM1 expression was performed on paraffin-embedded tumor tissues from 165NSCLC patients. Association of FoxM1 expression with clinicopathological parameters and disease free survivalwere evaluated. Results: Our results indicated FoxM1 expression to be significantly associated with poorer tissuedifferentiation (P =0.03), higher TNM stage (P <0.01), lymph node metastasis (P <0.01), advanced tumor stage(P <0.01), and poorer disease free survival (P <0.01). Multivariable analysis showed that FoxM1 expressionincreased the hazard of recurrence (hazard ratio= 1.96, 95% CI, 1.04-3.17, P <0.05), indicating that FoxM1is an independent and significant predictor of lung cancer recurrence. Conclusion: Therefore, FoxM1 is anindependent risk factor for recurrence of NSCLC. Elevated FoxM1 expression could be used as an indicator ofpoor disease free survival.     

Effects of Methylseleninic Acid on the Proliferation and Cell Cycle in Human High Metastatic Large Cell Lung Cancer Cell Line L9981 and Its Molecular Mechanism

Background and Objective Lung cancer is the rst killer of human being in the whole world. Recently, although many treatment strategies have been developed, the anti-cancer effects have     

姜黄素逆转电离辐射诱导的非小细胞肺癌A549细胞上皮间质转化

目的 探讨姜黄素对电离辐射诱导的非小细胞肺癌A549细胞上皮间质转化的影响。方法 用电离辐射诱导A549细胞发生上皮间质转化,将其命名为A549R。CCK8法检测细胞增殖;Western blot检测姜黄素对A549R上皮/间质标志物表达的影响;划痕实验及Transwell实验检测姜黄素对A549R细胞迁移能力的影响。结果 A549R细胞经姜黄素处理后,细胞形态由纺锤形转变为椭圆形上皮形态;E-cadherin表达下调,pan-Keratin表达上调,Vimentin、Twist表达显著下调,N-cadherin表达水平无明显变化;划痕愈合面积随姜黄素浓度的升高而显著下降;A549R细胞迁移能力随姜黄素浓度的升高而显著下降。结论 姜黄素通过下调E-cadherin、Vimentin、Twist的表达,并上调Keratin的表达,从而逆转电离辐射诱导A549细胞发生上皮间质转化。     

Rapid induction of p21WAF1 but delayed down-regulation of Cdc25A in the TGF-beta-induced cell cycle arrest of gastric carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that inhibits cellular proliferation in most epithelial cells. cdk4 and several cyclin-dependent kinase (cdk) inhibitors (p15INK4B, p21WAF1/Cip1 and p27Kip1) have been implicated in the TGF-beta-induced cell cycle arrest. More recently, down-regulation of Cdc25A, a cdk activator, was additionally suggested as a mechanism underlying growth inhibition by TGF-beta. The existence of diverse cellular mediators of TGF-beta, however, raises the question of whether their involvement might occur in a redundant manner or coordinately in a certain cell type. Using two TGF-beta-sensitive gastric carcinoma cell lines (SNU-16 and -620), we addressed the contributory roles of several cdk inhibitors, and of cdk4 and Cdc25A, in TGF-beta-induced cell cycle arrest by comparing their temporal expression pattern in response to TGF-beta. Among the cdk inhibitors examined, p21 mRNA was most rapidly (in less than 1 h) and prominently induced by TGF-beta. In contrast, p15 mRNA was more slowly induced than p21 in SNU-620 cells, and not expressed in SNU-16 cells harbouring homozygous deletion of p15. Western blotting results confirmed the rapid increase of p21, while opposite patterns of p27 expression were observed in the two cell lines. The down-regulation of Cdc25A mRNA occurred, but was more delayed than that of p15 or p21. Until G1 arrest was established, changes in the protein levels of both Cdc25A and cdk4 were marginal. Co-immunoprecipitation with anti-cdk4 antibody showed that induced p21 associates with cdk4 and that its kinase activity is reduced by TGF-beta, which kinetically correlates closely with G1 arrest following TGF-beta treatment of both cell lines. These results suggest that in certain human epithelial cells, p21 may play an early role in TGF-beta-induced cell cycle arrest, and its cooperation with other cdk inhibitors is different depending on cell type. Delayed down-regulation of Cdc25A and cdk4 may contribute to cell adaptation to the quiescent state in the two gastric carcinoma cell lines studied.     

microRNA-130a在非小细胞肺癌中的表达及其意义

目的探讨microRNA-130a在非小细胞肺癌中的表达情况及其临床意义。方法应用Real-time PCR方法检测100例非小细胞肺癌患者组织标本中microRNA-130a的表达水平,结合临床病理资料进行统计学分析。结果非小细胞肺癌组织中microRNA-130a的表达水平升高,而且其表达与淋巴结转移和临床分期有显著相关性（P< 0.05）。此外,microRNA-130a的表达与患者吸烟有显著相关性（P< 0.05）。多因素分析发现临床分期、淋巴结转移、microRNA-130a的表达对预后的影响有显著相关性（P< 0.01）。结论microRNA-130a将来可能作为非小细胞肺癌诊断和预后判断的分子标志物。     

Establishment of a Human Small Cell Lung Cancer Cell Line Producing a Large Amount of Anti-diuretic Hormone

A new cancer cell line (Lu-165) producing a large amount of anti-diuretic hormone (ADH, 2.8 μg/g protein) was established from a 50-year-old small cell lung cancer patient presenting with a syndrome of inappropriate anti-diuretic hormone secretion. These cells grew well in serum-supplemented medium and during more than 100 passages they continued producing a large amount of this hormone. This cell line will be a useful tool for studies of the biochemistry and pathology of ADH-producing cancer.     

Characterization and Purification of an Immunosuppressive Factor Produced by a Small Cell Lung Cancer Cell Line

The present study was undertaken to determine whether small cell lung cancer (SCLC) cell lines produce immunosuppressive factors and, if they do, to characterize the factors. The supernatants of SCLG cell lines, H69 and N857, inhibited not only the blastogenic response of human peripheral blood lymphocytes (PBL) to phytohemagglutinin or concanavalin A, but also the cytotoxic activity of lymphokine-activated killer cells. Neither was inhibited by supernatants from non-SCLC cell lines PC9, QG56, and A549. The immunosuppressive activity of H69 supernatant was stable upon heating to 56°C for 60 min, but labile when heated to 70°C for 10 min. The activity was abolished after dialysis at pH 2.0 or pH 11.0, but not at pH 4.5 or pH 9.0. Digestion with trypsin or proteinase eliminated the immunosuppressive activity, whereas treatment with neuraminidase, mixed glycosidase, DNase or RNase had no effect, suggesting that the immunosuppressive activity in H69 supernatant is due to a protein factor. This H69-derived immunosuppressive factor was isolated by ion exchange chromatography using a gradient of 0.04 to 0.08 M NaCl solution. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the factor to have molecular weights of 98 kD and 102 kD, respectively. These results suggest that SCLC cells produce a potent immunosuppressive factor which may account for the immune deficiency in SCLC patients.     

内皮抑素对人肺腺癌A549细胞周期及HIF-1表达的影响

目的研究内皮抑素在体外对人肺腺癌细胞A549的影响及机制。方法流式细胞术（FCM）检测内皮抑素治疗后,肺腺癌细胞A549的细胞周期分布;酶联免疫吸附法（ELISA）检测处理前后细胞中HIF-1的表达情况。结果内皮抑素处理肺腺癌A549细胞,可以使该细胞S期和G₂/M期细胞比例增多,抑制HIF-1的表达,且随处理时间的延长,HIF-1的抑制率不断提高。结论内皮抑素可以使A549细胞阻滞在G2/M期及S期的比例明显增加,从而为其他手段抑制A549细胞的增殖或直接杀伤A549细胞提供了基础;内皮抑素可以抑制肿瘤细胞的HIF-1表达,HIF-1是肿瘤细胞耐受放化疗的因素之一,因此联合内皮抑素治疗有可能提高放、化疗疗效。     

New Advances in the Second‐Line Treatment of Small Cell Lung Cancer

Lung cancer is the leading cause of cancer‐related death in the U.K., with small cell histology accounting for 15%–20% of cases. Small cell lung cancer (SCLC) is initially a chemosensitive disease, but relapse is common, and in this group of patients it remains a rapidly lethal disease with a particularly poor prognosis. The choice of second‐line chemotherapy for patients with relapsed SCLC has been an area of difficulty for oncologists, and until recently there was no randomized evidence for its use over best supportive care (BSC). Topotecan is currently the only drug licensed in Europe and the U.S. for this indication, having been shown in a phase III trial to lead to longer overall survival and better quality of life than with BSC. In this article, we review the current evidence for the use of second‐line cytotoxic therapy and also the emerging role of novel agents and targeted therapies in this setting. In particular, we explore the role of the Bcl‐2 protein family, which are key regulators of mitochondrial apoptosis and are implicated in resistance to anticancer therapies. SCLC overexpresses antiapoptotic members of the Bcl‐2 family in ～80% of cases. Several Bcl‐2 inhibitors, including obatoclax, are currently entering clinical trials in SCLC and are an exciting area of drug development in the relapsed setting.