AKR1C1 在非小细胞肺癌的表达及功能分析

目的 探讨醛酮还原酶家族1成员C1（AKR1C1）在非小细胞肺癌（NSCLC）组织和细胞株中的表达及其临床意义。方法 采用免疫组化SP法检测50例NSCLC组织芯片中肿瘤及癌旁组织中AKR1C1的表达。选取NCI-H460细胞分别转染AKR1C1 siRNA（AKR1C1干扰组）和AKR1C阴性对照siRNA（阴性对照组）慢病毒载体,Western blotting验证转染效率, MTT法、低密度细胞集落形成实验、Transwell实验和划痕试验检测干扰AKR1C1表达对细胞增殖、集落形成、侵袭能力的影响,同时另设空白对照组（未转染慢病毒）。结果 NSCLC组织中AKR1C1的高表达率为94.0％（47／50）,高于癌旁组织的6.0％（3／50）,差异有统计学意义（P＜0.05）。AKR1C1干扰组细胞中AKR1C1表达量较阴性对照组下调（P＜0.05）。MTT法显示AKR1C1干扰组与阴性对照组细胞增殖率无显著性差异（P＞0.05）;AKR1C1干扰组、阴性对照组和空白对照组NCI-H460细胞的克隆数分别为69.60±4.03、69.00±1.63和70.33±2.05,组间差异无统计学意义（P＞0.05）;Transwell实验显示,AKR1C1干扰组穿膜细胞数为15.30±2.50,低于阴性对照组的30.00±2.20和空白对照组的31.30±2.40,差异有统计学意义（P＜0.05）。划痕试验显示,AKR1C1干扰组细胞迁移相对距离为0.13±0.05,低于阴性对照组的0.56±0.05和空白对照组的0.60±0.08,差异有统计学意义（P＜0.05）。结论 AKR1C1在NSCLC组织中高表达,其可能与NSCLC转移相关。     

非小细胞肺癌组织中AKR1C3水平及其临床意义

目的 探讨非小细胞肺癌（NSCLC）组织中醛酮还原酶1 C3（AKR1C3）表达及其临床意义。方法 收集2013年1月至2016年3月88例手术切除的NSCLC组织及83例癌旁正常组织,采用实时荧光定量PCR（QPCR）法检测上述组织中的AKR1C3表达水平,分析AKR1C3表达与NSCLC临床病理参数（性别、年龄、TNM分期、肿瘤大小、组织学类型及淋巴结转移）的关系;根据随访资料分析AKR1C3表达与预后的关系,采用受试者工作特征曲线（ROC）评价AKR1C3表达在NSCLC早期诊断中的效能。结果 NSCLC组AKR1C3表达水平为0.187±0.175,低于癌旁正常组织的1.079±0.384,差异有统计学意义（P＜0.05）;AKR1C3诊断NSCLC的曲线下面积为0.982（95％CI：0.966～0.998）。AKR1C3表达与NSCLC性别、年龄、组织学类型及淋巴结转移无关,与肿瘤大小及TNM分期有关。肿瘤大小＞3 cm的AKR1C3表达量为0.095±0.055,低于肿瘤大小≤3 cm的0.340±0.201,而Ⅲ、Ⅳ期的AKR1C3表达量为0.094±0.058,低于Ⅰ、Ⅱ期的0.265±0.202,差异有统计学意义（P＜0.05）。全组NSCLC患者的中位总生存期（OS）为17.60个月。AKR1C3高表达者的中位OS为20.60个月,高于低表达者的11.90个月,差异有统计学意义（P＜0.05）;Ⅰ+Ⅱ期患者的中位OS为21.65个月,高于Ⅲ+Ⅳ期的16.20个月,差异有统计学意义（P＜0.05）;肿瘤大小≤3 cm者的中位OS为18.40个月,高于＞3 cm者的15.70个月,差异有统计学意义（P＜0.05）。结论 AKR1C3在NSCLC组织中表达降低,且与TNM分期、肿瘤大小及预后有关,可能与NSCLC发生发展有关,在NSCLC诊断及病情评估有一定价值。     

非小细胞肺癌耐药分子指标研究进展

化疗在治疗非小细胞肺癌(NSCLC)中占有重要地位，但其联合化疗的有效率也只有30％～40％，并且这个水平已达到了一个新的平台阶段。影响肺癌患者化疗疗效的主要原因是肿瘤细胞对抗癌药的抗药性，其耐药机制非常复杂，与耐药基因：MDRI、MRP、LRP，GST、拓扑异构酶Ⅱ质和量的改变、β-微管蛋白基因突变、FGF等多种因素有关。前瞻性进行分子指标的测定，是指导肺癌患者化疗用药、进行个体化治疗、提高化疗疗效的关键。     

非小细胞肺癌组织免疫微环境的特点及其临床意义

目的：探讨基于非小细胞肺癌（non-small cell lung cancer,NSCLC）患者原发灶组织中程序性死亡受体-配体1（programmed death-ligand 1,PD-L1）表达和间质中CD8+T细胞浸润的免疫微环境分型的特点及其临床意义。方法:回顾性分析2016年1 月到2018 年7 月空军特色医学中心收治的74 例NSCLC患者的石蜡组织标本及临床病理资料,所有患者均有EGFR基因检测结果、未接受放化疗及靶向治疗。采用免疫组化方法检测组织中PD-L1 表达及间质中CD8+T细胞浸润,分别分析PD-L1、CD8+T细胞及基于两者的免疫微环境分型与病理参数和患者生存的关系。结果:NSCLC患者原发灶组织中PD-L1 表达在不同性别、病理类型、吸烟史、EFGR突变组中有明显差异（均P<0.05）,CD8+T细胞浸润在不同TNM分期、淋巴结转移组织各组中有明显差异（均P<0.05）;PD-L1 表达与EGFR突变显著相关（P=0.000）,而CD8+T细胞浸润与EGFR突变不相关（P=0.605）。EGFR野生型患者免疫微环境以CD8+ PD-L1（+ Ⅰ型）为主、突变型以CD8- PD-L1（- Ⅱ型）及CD8+ PD-L1（- Ⅳ型）为主。免疫微环境分型在不同EGFR突变、吸烟史、病理分化程度的各组中分布有明显差异（均P<0.05）,且与EGFR突变显著相关（P<0.05）。随访显示处于无病生存期、复发转移和死亡患者中分别以Ⅰ型、Ⅱ型和Ⅰ型最多。结论:本组NSCLC患者肿瘤免疫微环境分型分布主要以Ⅰ型最多、Ⅲ型最少,其分型与EGFR突变、吸烟史及病理分化有关;EGFR突变患者以CD8+ PD-L1（- Ⅱ）型和CD8+ PD-L1（- Ⅳ型）为主,且与PD-L1低表达相关。     

奈达铂治疗耐药性鼻咽癌和非小细胞肺癌

目的：研究国家二类新药奈达铂单药治疗顺铂(DDP)耐药性晚期鼻咽癌和非小细胞肺癌的临床疗效及安全性。方法：奈达铂单药治疗耐药性鼻咽癌鼻咽癌6例、非小细胞肺癌3例。奈达铂100mg／m^2加入500ml无菌生理盐水中，静脉滴注2小时，每3--4周重复一次。结果：9例入组患者均可评价疗效。鼻咽癌6例中1例CR，2例PR，有效率为50％；非小细胞肺癌3例中有1例PR。总RR为45．5％。主要毒副作用是对血小板及白细胞生成的抑制作用，恶心、呕吐反应较小，对肾脏的损伤、胃肠道性毒性作用及周围末梢神经等毒性较轻，肝肾功能影响不明显。结论：奈过铂单药对DDP耐药的鼻咽癌或非小细胞肺癌仍有较高的疗效，且多数患者耐受良好．     

CFL1 expression levels as a prognostic and drug resistance marker in nonsmall cell lung cancer

BACKGROUND:

Nonsmall cell lung cancer (NSCLC) is the major determinant of overall cancer mortality worldwide. Despite progress in molecular research, current treatments offer limited benefits. Because NSCLC generates early metastasis, and this behavior requires great cell motility, herein the authors assessed the potential value of CFL1 gene (main member of the invasion/metastasis pathway) as a prognostic and predictive NSCLC biomarker.

METHODS:

Metadata analysis of tumor tissue microarray was applied to examine expression of CFL1 in archival lung cancer samples from 111 patients, and its clinicopathologic significance was investigated. The robustness of the finding was validated using another independent data set. Finally, the authors assayed in vitro the role of CFL1 levels in tumor invasiveness and drug resistance using 6 human NSCLC cell lines with different basal degrees of CFL1 gene expression.

RESULTS:

CFL1 levels in biopsies discriminate between good and bad prognosis at early tumor stages (IA, IB, and IIA/B), where high CFL1 levels are correlated with lower overall survival rate (P < .0001). Biomarker performance was further analyzed by immunohistochemistry, hazard ratio (P < .001), and receiver‐operating characteristic curve (area = 0.787; P < .001). High CFL1 mRNA levels and protein content are positively correlated with cellular invasiveness (determined by Matrigel Invasion Chamber System) and resistance (2‐fold increase in drug 50% growth inhibition dose) against a list of 22 alkylating agents. Hierarchical clustering analysis of the CFL1 gene network had the same robustness for stratified NSCLC patients.

CONCLUSIONS:

This study indicates that the CFL1 gene and its functional gene network can be used as prognostic biomarkers for NSCLC and could also guide chemotherapeutic interventions. Cancer 2010. © 2010 American Cancer Society.     

外泌体非编码RNA在非小细胞肺癌获得性耐药中的研究进展

非小细胞肺癌(non-small cell lung cancer,NSCLC)是肺癌中最常见的类型,对化疗及靶向药物的获得性耐药严重影响NSCLC患者的生存期,NSCLC获得性耐药机制复杂,确切机制仍不清楚.肿瘤来源或与肿瘤相关的外泌体是参与调控NSCLC获得性耐药的重要机制,可以通过传递核酸、蛋白质等赋予敏感细胞耐...     

三维适形放疗和同步化疗治疗局部晚期非小细胞肺癌的临床研究

目的 :比较三维适形放疗与普通放疗结合同步化疗治疗局部晚期非小细胞肺癌(NSCLC)的疗效及毒副反应。方法 :64例ⅢA/ⅢB期NSCLC随机分成两组 ,三维适形放疗组采用三维适形放疗技术 ,每次 3～ 6Gy ,1次 /d ,照射 10～ 2 0次 ,肿瘤灶总生物有效量为 80～ 90Gy。普通放疗组放疗量在60～ 66Gy。两组同时给予长春瑞滨 2 5mg/m2 ,放疗开始的d1、d8、d2 9、d3 6、d57、d64、d85、d92 静脉滴入 ;卡铂 3 0 0mg/m2 ,放疗开始的d1、d2 9、d57、d85静脉滴入。结果 :三维适形放疗组有效率 96 88% (3 1/ 3 2 ) ,其中CR为17例 ,PR为 14例。普通放疗组有效率为81 2 5 % ,其中CR为 9例 ,PR为 15例。三维适形放疗组的有效率明显高于普通放疗组 ,P =0 0 19。三维适形放疗组和普通放疗组的 1、2年生存率分别为 84 3 8%、40 63 %和 78 13 %、2 3 5 6% ,中位生存时间分别为19个月、16个月 ,其差异有统计学意义 ,P =0 0 189。适形放疗组III度放射性食管炎、放射性肺炎发生率分别为 6 2 5 % (2 / 3 2 )、3 13 % (1/ 3 2 ) ,低于普通放疗组的 46.9%(15 / 3 2 )和 18.8% (6/ 3 2 ) ,两组间差异有统计学意义 ,P =0 0 0 0 ,P =0 0 0 1。结论 :三维适形放疗和同步化疗是ⅢA/ⅢB期非小细胞肺癌安全有效的治疗手段 ,值得进一步临     

ABCG4在肺鳞癌、腺癌中的表达及临床意义

目的:检测耐药蛋白ABCG4在肺鳞癌、腺癌中的表达,分析其表达率与病理分级和临床分期间的关系,为研究ABCG4在肿瘤表达及耐药相关性提供基础。方法:采用免疫组织化学EnVinsion法、免疫荧光法检测ABCG4在肺鳞癌、腺癌中的表达和激光共聚焦显微镜观察其荧光定位。结果:ABCG4在肺鳞癌、腺癌中高表达,鳞癌总阳性率67.3%、腺癌总阳性率为81.4%,两者差异性显著(P=0.001);中分化、低分化鳞癌阳性率分别为41.7%、92.0%,差异性非常显著(P=0.000);腺癌中分化阳性率76.7%、低分化腺癌92.3%,差异性显著(P=0.033);鳞癌和腺癌的各临床分期的Kruskal-Wallis秩和检验差异性显著,P值分别为0.039、0.048。ABCG4主要定位在胞膜上和胞质中。结论:ABCG4在肺鳞癌、腺癌中高表达,表达的高低与病理分级、临床分期有关,为进一步研究ABCG4在肿瘤中的表达及可能耐药作用提供实验依据。     

肺腺癌药物耐受蛋白ABCG2表达细胞系的功能研究

目的：通过观察A549-ABCG2细胞系生物学特性变化,验证ABCG2蛋白过表达肺癌细胞对各种化疗药物的耐药性及其相关性,为谨慎选择化疗药物及制定临床试验方案提供依据。方法：MTT法检测ABCG2高表达细胞的药物敏感性,检测ABCG2蛋白ATP激酶活性,分秽淇与耐药之间的相关性。结果：A549-ABCG2细胞药物敏感性较A549细胞明显降低,而其ATP激酶活性明显升高。结论：可以通过研究ABCG2高表达细胞系A549-ABCG2来筛选化疗药物为临床治疗服务。     

奥曲肽对紫杉醇抑制非小细胞肺癌细胞的增效作用

目的: 探讨生长抑素类似物奥曲肽（octreotide）对紫杉醇抑制非小细胞肺癌细胞的增效作用及其可能的机制。方法: RTPCR检测非小细胞肺癌细胞株A549和H157 生长抑素受体（somatostatin receptor, SSTR）1～5 mRNA的表达；检测不同时间和浓度梯度的奥曲肽对两株细胞的抑制率，光镜下观测奥曲肽作用后细胞形态学的变化；MTT法检测奥曲肽与紫杉醇对两细胞株增殖的抑制是否具有协同作用；采用流式细胞仪检测奥曲肽、紫杉醇以及两者联合作用时A549细胞的凋亡率;荧光实时定量PCR检测奥曲肽作用后细胞多药耐药相关基因的改变。结果: A549细胞检测到SSTR1～SSTR5 mRNA的表达，而H157细胞只有SSTR1和SSTR4 mRNA的表达。奥曲肽对A549细胞增殖具有抑制作用并具有时间（24、48、72 h）和浓度(1～1 000 nmol/L)依赖性，在光镜下可观测到细胞细胞受损伤的形态学改变；而对H157细胞无抑制作用。对于A549细胞，125、250、500 nmol/L奥曲肽作用48 h可明显增加其对紫杉醇化疗的敏感性（P<0.01）,并能诱导细胞凋亡，但未增加与紫杉醇联合作用后细胞的凋亡率；而对H157细胞，奥曲肽未发现化疗增敏作用。奥曲肽可下调A549细胞多药耐药相关基因MDR1、MRP1的表达(P<0.05, P<0.01)，而对H157细胞多药耐药相关基因的表达无影响。结论: 奥曲肽可提高SSTR受体阳性的非小细胞肺癌细胞对紫杉醇的敏感性，下调多药耐药相关基因的表达是其可能的机制之一。     

Reverse of non‐small cell lung cancer drug resistance induced by cancer‐associated fibroblasts via a paracrine pathway

The tumor microenvironment orchestrates the sustained growth, metastasis and recurrence of cancer. As an indispensable component of the tumor microenvironment, cancer‐associated fibroblasts (CAF) are considered as an essential synthetic machine producing various tumor components, leading to cancer sustained stemness, drug resistance and tumor recurrence. Here, we developed a sustainable primary culture of lung cancer cells fed with lung cancer‐associated fibroblasts, resulting in enrichment and acquisition of drug resistance in cancer cells. Moreover, IGF2/AKT/Sox2/ABCB1 signaling activation in cancer cells was observed in the presence of CAF, which induces upregulation of P‐glycoprotein expression and the drug resistance of non‐small cell lung cancer cells. Our results demonstrated that CAF cells constitute a mechanism for cancer drug resistance. Thus, traditional chemotherapy combined with insulin‐like growth factor 2 (IGF2) signaling inhibitor may present an innovative therapeutic strategy for non‐small cell lung cancer therapy.     

Acquisition of cancer stem cell‐like properties in non‐small cell lung cancer with acquired resistance to afatinib

Afatinib is an irreversible epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR‐TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non‐small cell lung cancer. In this study, we established various afatinib‐resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib‐resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827‐ACR, was established from one of the MET amplified‐cell lines. Several afatinib‐resistant cell lines including HCC827‐ACR displayed epithelial‐to‐mesenchymal transition (EMT) features and epigenetic silencing of miR‐200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib‐resistant cells and found that MET amplification, EMT, and stem cell‐like features are observed in cells with acquired resistance to EGFR‐TKIs. This finding may provide clues to overcoming resistance to EGFR‐TKIs.     

多药耐药相关蛋白基因在非小细胞肺癌表达的临床意义

目的 探索多药耐药相关蛋白（ＭＲＰ）在非小细胞肺癌（ＮＳＣＬＣ）的表达,以及与病理类型、病期及预后的关系。方法 以原位杂交法检测冰冻ＮＳＣＬＣ组织。结果 全组５８例ＮＳＣＬＣ、ＭＲＰＭＲＮＡ总表达率为７４．１％,鳞癌为７２．０％,腺癌为７３．３％。ＭＲＰ的表达与病理类型、ＴＮＭ发期及分化程度无关。４７例接受化疗的ＮＳＣＬＣ中,３５例ＭＲＰ表达者生存率明显低于ＭＲＰ表达者,中位生存期分别为８．７个月     

Akt1 inhibition by RNA interference sensitizes human non-small cell lung cancer cells to cisplatin

Akt/protein kinase B signaling is very important for cancer cell survival and growth when cells are exposed to various apoptotic stimuli. Akt is constitutively activated in NSCLC cells and is a potential target for enhancing the cytotoxicity of chemotherapeutic agents in treatment of NSCLC. In our study, we investigated whether down-regulating Akt1 using RNAi techniques can enhance sensitivity to cisplatin in NSCLC cells. An siRNA targeting Akt1 significantly decreased the protein level of Akt1 and the activity of ERK. Treatment of these cells with 20 muM cisplatin increased apoptotic cell death approximately 2.6-fold compared to cells transfected with a scrambled siRNA. While Akt activity was slightly reduced, ERK activity was greatly increased in cells treated with cisplatin alone. Pretreatment of these cells with the selective MEK inhibitor U0126 effectively reduced the level of cisplatin-induced apoptosis. These results imply that cisplatin-induced MEK/ERK activation appears to mediate apoptotic cell death, but that constitutively activated Akt1 and/or ERK pathway may mediate resistance to cisplatin in NSCLC cells. Taken together, our data demonstrate that down-regulation of Akt1 using RNAi enhances the chemosensitivity of NSCLC cells to cisplatin.     

Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin, a coumermycin antibiotic

Breast cancer resistance protein (BCRP/ABCG2) of an ATP-binding cassette half-transporter confers resistance against mitoxantrone and camptothecin derivatives of topotecan and irinotecan. Novobiocin, a coumermycin antibiotic, is known to enhance anticancer drug sensitivity of cancer cells in vitro and in vivo, the mechanism of which remains undetermined. Here we focused on drug efflux pump and examined whether novobiocin reversed drug resistance in multidrug-resistant cells highly expressing BCRP. To explore the reversal mechanisms, intracellular drug accumulation was measured by flow cytometry, and a topotecan transport study using plasma membrane vesicles was performed. We used PC-6/SN2-5H2 small cell lung cancer and MCF-7/MX breast cancer cells selected with SN-38 of the active irinotecan metabolite and mitoxantrone, respectively, and the BCRP cDNA transfectant MCF-7/clone 8 cells. These cells expressed high levels of BCRP mRNA but not other known transporters. Compared to the parental PC-6 cells, PC-6/SN2-5H2 cells were 141-, 173- and 57.2-fold resistant to topotecan, SN-38 and mitoxantrone, respectively. Novobiocin at 60 microM decreased the degree of the above resistance by approximately 26-fold in PC-6/SN2-5H2 cells, and similarly reversed resistance in MCF-7/MX, MCF-7/clone 8 and un-selected NCI-H460 cells highly expressing BCRP. Furthermore, novobiocin increased the intracellular topotecan accumulation in these cells and inhibited the topotecan transport into the membrane vesicles of PC-6/SN2-5H2 cells. No effects of novobiocin in these assay were observed in the parental PC-6 and MCF-7 cells. The kinetic parameters in the transport study indicated that novobiocin was a inhibitor for BCRP, resulting in competitive inhibition of BCRP-mediated topotecan transport. These findings suggest that novobiocin effectively overcomes BCRP-mediated drug resistance at acceptable concentrations.     

FZDl基因对小细胞肺癌多药耐药作用研究

目的：探讨FZDl在小细胞肺癌多药耐药中的作用。方法：运用QRT—PCR和蛋白质印迹法,分别从基因和蛋白水平检测小细胞肺癌敏感细胞株H69及耐药细胞株H69AR中FZDl的差异表达,同时运用QRT—PCR检测化疗敏感及耐药患者血液标本中FZDl的差异表达;采用siRNA抑制耐药细胞株H69AR中FZDl的表达,通过CCK8检测细胞对各种化疗药物（ADM,DDP,Vp-16）敏感性的变化,流式细胞仪检测细胞凋亡率的变化。结果：无论是基因水平还是蛋白水平,FZDl在H69AR细胞中的表达明显高于H69细胞,差异有统计学意义,P值分别为0．003和〈0．001;FZDl在化疗耐药患者血液标本中的表达较化疗敏感者明显增高,差异有统计学意义,P〈O．001。下调H69AR细胞株中FZDl的表达能够增加细胞对化疗药物的敏感性,差异有统计学意义,P=0．020;H69AR细胞组的凋亡率为（2．037土0．49）％,较H69细胞组（9．63±0．67）％明显降低,差异有统计学意义,P=0．002。下调H69AR细胞中FZDl的表达后,H69AR—Si—FZDl组细胞的凋亡率为（17．093±0．904）％,明显高于空白对照H69AR及阴性对照H69AR—NC组的（2．25±0．967）％,差异有统计学意义,P=0．007。结论：FZDl参与调节小细胞肺癌的多药耐药,下调FZDl基因的表达能够增加细胞对化疗药物的敏感性,FZDl可能通过抑制细胞凋亡而影响小细胞肺癌的多药耐药。