小细胞肺癌神经内分泌起源标志物的研究进展

[摘要] 小细胞肺癌（small cell lung cancer,SCLC）具有生长分数高、倍增时间短、侵袭性极高的特点,往往在诊断该病的时候就已发生远处转移,若不对其病情进行及时有效的治疗,患者将在2~3 个月内死亡。如果SCLC能够在局限期得到诊断,那么将有20%～25%的患者在接受联合化疗与放疗后,可以获得一个较长生存期。因此,寻找特异性和敏感性高的血液肿瘤标志物,为SCLC早期诊断提供可靠、简便、安全、耐受性好等依据对治疗SCLC极其重要。本文就近年来SCLC神经内分泌起源标志物嗜铬粒蛋白A、神经特异性烯醇化酶、促胃泌素释放肽、脑型肌酸激酶同工酶BB、神经细胞黏附分子、突触素等的研究进展进行综述。     

Biomarkers for small cell lung cancer: neuroendocrine, epithelial and circulating tumour cells

Small cell lung cancer (SCLC) is characterised by an aggressive clinical course with invariable resistance to chemotherapy despite initially high response rates. There has been little improvement in outcome over the past few decades, with no breakthrough yet in targeted therapies. Recent preclinical data and studies of circulating tumour cells (CTCs) highlight distinct cellular heterogeneity within SCLC. Better understanding of how these phenotypes contribute to metastasis and tumour progression might pave the way for development of more successful targeted therapies. Here we review these studies, their implications for future research and for the incorporation of biomarkers reflecting neuroendocrine, epithelial and mesenchymal phenotypes in clinical studies.     

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 SCLC 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 degrees C for 60 min, but labile when heated to 70 degrees 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.     

过表达Notch1对小细胞肺癌细胞的增殖和神经内分泌标志物表达的抑制作用

目的 研究Notch信号通路对小细胞肺癌(SCLC)细胞增殖及神经内分泌标志物表达的调控作用.方法 采用包含Notch1胞内段(NIC)的重组质粒转染NCI-H446细胞,并建立稳定转染株.实验细胞分为3组,即NIC转染组、空质粒转染组和未转染组.应用二苯基溴化四氮唑蓝(MTT)法,连续6 d测定各组细胞的增殖活性.应用细胞爬片、免疫细胞化学染色法以及Western blot技术,对神经元特异性烯醇化酶(NSE)和嗜铬素蛋白A(CgA)的表达量进行检测.结果 MTT法检测结果 显示,NIC转染组细胞的增殖活性明显降低,其连续6 d测得的吸光度(A)值均小于空质粒转染组和未转染组(均P<0.05).免疫细胞化学染色结果 显示,NIC转染组、空质粒转染组和未转染组cgA染色的阳性单位(Pu)值分别为8.81±0.77、38.10±1.55和38.97±0.80;NSE染色的PU值分别为7.21 ±0.59、28.25±1.46和30.57±1.31;NIC转染组CgA和NSE的Pu值均显著小于空质粒转染组和未转染组(均P<0.01).Westem blot检测结果 显示,NIC转染组、空质粒转染组和未转染组CgA条带的灰度值分别为0.54±0.03、0.99±0.05和1.00;NSE条带的灰度值分别为0.43±0.02、1.07±0.09和1.00;NIC转染组CgA和NSE条带的灰度值均显著小于空质粒转染组和未转染组(均P<0.01).结论 Notch1在SCLC中可能是一种抑癌基因,其介导的Notch信号通路的上调能抑制SCLC细胞的增殖和神经内分泌标志物的表达.     

Establishment of a new cell line with neuronal differentiation derived from small cell neuroendocrine carcinoma of the maxillary sinus

OBJECTIVE: It is well known that small cell neuroendocrine carcinoma (SNEC) arising at extrapulmonary sites has a poor prognosis and an interesting biological characterization. To understand biological characterization and elucidation of the origin of the histogenesis of SNEC, we report the establishment of a new SNEC cell line and characteristics of neuroendocrine properties including neuronal differentiation by treatment with dibutyryl cyclic AMP (db-cAMP). METHODS: We established a new cell line (SNEC-MI) derived from SNEC of the maxillary sinus by a modified spill-out method, and verified neuroendocrine properties including neuronal differentiation by immunocytochemical and immunoblotting methods. RESULTS: The established cell line showed spherical or spindle shape in monolayer culture and was positive for neuron-specific enolase (NSE), neuronal cell adhesion protein (N-CAM, CD56) and gastrin-releasing peptide. NSE was also demonstrated in the cultured medium and dense-core neuroendocrine granules were detected ultrastructurally in the cytoplasm. Treatment of cells with db-cAMP markedly induced the development and elongation of neuronal processes, which formed a netlike arrangement. Characterization of these elongated neuronal processes revealed them immunoreacting intensely with high molecular-weight neurofilament, and a time-dependent increase of microtubule-associated protein-2 in cell lysates. CONCLUSIONS: These findings indicated that this cell line possesses the capability to differentiate into neuronal cells, and supported the hypothesis that extrapulmonary SNEC might be derived from a pluripotent stem cell.     

神经内分泌分化与p53对非小细胞肺癌预后的影响

目的探讨神经内分泌(neuroendocrine,NE)分化标志物嗜铬蛋白A(CgA)、突触素(Syn)、神经内分泌颗粒和p53对非小细胞肺癌(non-small cell lung carcinoma,NSCLC)预后的影响。方法62例NSCLC进行含铂类方案新辅助化疗1~2个周期,然后手术治疗,以免疫组化法检测术后标本中NE分化标志物CgA、Syn及p53,以电镜观察标本中神经内分泌颗粒,随访3年,结果采用SPSS11.0统计软件,进行生存率分析。结果p53及NE分化标志物联合p53对非小细胞肺癌患者的无病生存期在统计学上均有显著性差异(P<0.05),NE( )p53(-)患者生存率较高。说明p53和NE分化联合p53与非小细胞肺癌患者的预后有关。结论神经内分泌分化和p53两者联合,用于非小细胞肺癌患者无进展生存期评价具有一定的临床价值。     

Establishment and characterization of a human large cell lung cancer cell line with neuroendocrine differentiation

Characterization of a human lung cancer cell line is reported. This cell line was established from a patient referred to Nemazi Hospital of Shiraz University of Medical Sciences with a diagnosis of poorly differentiated carcinoma. Sterile sample from peritoneal effusion was taken and immediately cultured in RPMI-1640 medium containing 20% FBS, at 37 degrees C with 5% CO2. This cell line has been in continuous culture for more than one year and has been named as Mehr-80. Several features of the cell line were investigated, including growth characteristics, electron microscopic features, cloning efficiency in soft agar, expression of various antigenic markers, chromosomal and DNA analysis. On the basis of morphological and immunohistochemical analysis of Mehr-80, it is possible to conclude that this cell line is characterized by features similar to those reported for large cell carcinoma with neuroendocrine differentiation (LCCND). This cell line will be a valuable in vitro tool for further studies on lung cancers.     

Expression of p64c-myc and neuroendocrine properties define three subclasses of small cell lung cancer

Twelve human small cell lung cancer (SCLC) cell lines and 6 non-SCLC cell lines were analysed with respect to expression of the c-myc, c-myb, and c-raf1 protooncogenes at the protein level. Analysis of p64c-myc protein expression in 12 SCLC cell lines resulted in the observation that it is present at high levels not only in cells with low, but also in those with moderate neuroendocrine differentiation. Neuroendocrine differentiation was based on parameters such as growth rate, colony formation, L-Dopa decarboxylase (DDC) activity, bombesin, and neurotensin described before. Surprisingly, in two cell lines with low neuroendocrine differentiation but without c-myc protein expression (SCLC-86M1 and NCI-H526) p75c-myb expression was observed which may therefore be able to substitute for the p64c-myc protein. Analysis of p74c-raf1 expression did not result in correlation with any growth or differentiation parameter since it was expressed at low levels in 11 out of 12 cases. We conclude that SCLC in vitro can be classified in three rather than two previously defined subclasses. In addition to the classic subclass with slow growth, high neuroendocrine differentiation, and absent or very low p64c-myc expression and the variant subclass with fast growth, absent to very low neuroendocrine differentiation, and high p64c-myc expression, we suggest a third subclass designated as transitional with moderate growth, moderate neuroendocrine differentiation, and high p64c-myc expression. Data on a small number of non-SCLC cell lines tested showed that high levels of p64c-myc correlate with high in vitro growth rates. This indicates that high p64c-myc levels may be associated with high proliferative activity, and lack of differentiation in lung cancer in general. The p74c-raf1 protein was found in all non-SCLC cell lines. Whether this classification of SCLC cell lines is applicable to SCLC in vivo remains to be determined.     

Role of free radicals in an adriamycin-resistant human small cell lung cancer cell line

In two Adriamycin (Adr) resistant sublines (GLC4-Adr1 and GLC4-Adr2) of a human small cell lung carcinoma cell line, GLC4, cross-resistance for radiation was found. GLC4-Adr1 has an acquired Adr resistance factor of 44 after culturing without Adr for 20 days and GLC4-Adr2, the same subline cultured without Adr for 3 months, has a decreased but stable resistance factor of 8. One of the assumed mechanisms of Adr is that the effect is mediated through the formation of free radicals. Therefore free radical scavenging might play a role in these Adr resistant cell lines. Adr, H2O2, and X-ray induced cytotoxicity were evaluated. Glutathione (GSH) levels and activities of associated enzymes were determined as well as Adr, H2O2, and X-ray induced DNA breaks and repair. GSH level was decreased in GLC4-Adr1, but restored to the normal level in GLC4-Adr2. Superoxide dismutase, catalase, glutathione-peroxidase, and glutathione S-transferase were not elevated in the resistant sublines. Adr induced a decreased amount of DNA breaks in GLC4-Adr1 compared to GLC4. For X-ray and H2O2 a comparable amount of DNA damage was found. GLC4-Adr1 was able to repair DNA breaks induced by Adr, X-ray, and H2O2 better than GLC4. In conclusion, no increased enzyme capacity for detoxification of free radicals could be detected in the cytosol of the resistant cells. The resistance against free radicals in the GLC4-Adr1 line may at least in part be a result of increased DNA repair.     

Livin在小细胞肺癌组织中的表达

目的：探讨凋亡抑制蛋白（inhibitor of apoptosis protein,IAP）家族中的新成员Livin在小细胞肺癌（small cell lung cancer,SCLC）组织中的表达及其与临床病理的关系。方法：采用反转录-聚合酶链反应（RT-PCR）技术检测SCLC组织中LivinαmRNA和LivinβmRNA的表达,并用western blot分析Livin蛋白的表达。结果：30例SCLC组织中有19例Livin表达,阳性率为63.33%,其表达与患者年龄、性别、肿瘤的TNM分期、淋巴结转移和肿瘤大小均无关（P〉0.05）。而16例癌旁正常肺组织中Livin低表达,阳性率为12.5%（P〈0.01）。结论：Livin在SCLC组织中异常表达,有望成为小细胞肺癌诊断和基因治疗的新靶点。     

Livin在小细胞肺癌组织中的表达

目的:探讨凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族中的新成员Livin在小细胞肺癌(small cell lung cancer,SCLC)组织中的表达及其与临床病理的关系.方法:采用反转录-聚合酶链反应(RT-PCR)技术检测SCLC组织中LivinαmRNA和LivinβmRNA的表达,并用western blot分析Livin蛋白的表达.结果:30例SCLC组织中有19例Livin表达,阳性率为63.33%,其表达与患者年龄、性别、肿瘤的TNM分期、淋巴结转移和肿瘤大小均无关(P>0.05).而16例癌旁正常肺组织中Livin低表达,阳性率为12.5%(P<0.01).结论:Livin在SCLC组织中异常表达,有望成为小细胞肺癌论断和基因治疗的新靶点.     

CEA、VEGF标志物与小细胞肺癌预后相关性

目的:探讨不同评价标志物与小细胞肺癌预后相关性。方法:64例小细胞肺癌患者以及同期36例行肺叶切除术的正常患者,分为观察组与对照组。免疫组化SP法测定LRP,酶联免疫吸附法(ELISA)对血液中血清癌胚抗原（CEA）、血清血管内皮生长因子(VEGF)进行检测,同时测定观察组在化疗前后LRP阳性率、以及CEA与VEGF的水平变化情况。结果:小细胞肺癌患者与肺部良性病变的VEGF检测值分别为:(290.7±48.52)pg/ml、(123.5±31.48)pg/ml,CEA检测值分别为:(9.78±2.58)ng/L、(4.06±0.84)ng/L,LRP阳性率分别为:53.1%、5.6%,两组比较有显著性差异（P<0.05)。观察组化疗前后的VEGF检测值分别为:(286.7±47.52)pg/ml、(167.5±34.48)pg/ml,CEA检测值分别为:(9.68±2.48)ng/L、(5.16±1.25)ng/L,LRP阳性率分别为:51.6%、15.6%,两组比较有显著性差异（P<0.05)。结论:LRP、VEGF以及CEA等标记物在肺癌组织的表达水平显著高于正常组织,可用于对肺癌疾病的诊断以及对小细胞肺癌预后预测。     

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)-1. 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.     

Small cell lung cancer cell line derived from a primary tumor with a characteristic deletion of 3p

Chemotherapy plus surgery is feasible and potentially effective in selected patients with small cell lung cancer (SCLC) and provides a unique opportunity to study SCLC early in its biological history. The in vitro characteristics of a SCLC cell line derived from a resected lung primary tumor after treatment with 3 courses of chemotherapy is described. The original SCLC cell line UMC-SCLC-1 exhibited features of classic SCLC with typical morphology and growth characteristics, high levels of dopa decarboxylase, bombesin-like peptides, neuron-specific enolase and calcitonin, and the presence of neurosecretory granules and demonstrated the deletion of the short arm of chromosome 3. After multiple passages, UMC-SCLC-1 gradually changed its culture characteristics to a cell line, UMC-SCLC-1A, with morphological features of large cell anaplastic carcinoma, an altered growth pattern, decrease in calcitonin, and increase in radioresistance but retained the other biochemical markers of classic SCLC (bombesin and dopa decarboxylase production). Serial DNA content analyses showed that increased aneuploidy during continuous culture in vitro was associated with the morphological changes. Both UMC-SCLC-1 and UMC-SCLC-1A demonstrated the deletion of chromosome 3p, amplification and abundant expression of N-myc, and increased expression of c-raf. Chemotherapy sensitivities were stable throughout multiple passages and correlated with in vivo response. UMC-SCLC-1A represents a unique SCLC cell line with heterogeneous properties of both classic and morphological variant SCLC cell lines. In addition, the characteristic deletion of 3p, previously described in cultures derived from metastatic lesions and heavily pretreated patients, is seen in a primary lesion early in the natural history of SCLC.     

Establishment and identification of small cell lung cancer cell lines having classic and variant features

Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.