EGFR inhibitors sensitize non-small cell lung cancer cells to TRAIL-induced apoptosis

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be regulated by the epidermal growth factor (EGF) signaling pathway. In this study, recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter (AdTRAIL) was combined with drugs including gefitinib, elotinib, and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer (NSCLC) cell lines to investigate their antitumor activity. In vitro, compared to single reagent, AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460, A549, and SW1573 cell lines. Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT. In vivo, AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice. Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL. These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.     

Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion, and promotion of tumor cell resistance to apoptosis. In our previous studies using non-small cell lung cancer (NSCLC) cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations, we demonstrated that constitutive overexpression of COX-2 leads to stabilization of the inhibitor of apoptosis protein survivin resulting in the elevated apoptosis resistance of COX-2-overexpressing cells. Genetic or pharmacologic suppression of COX-2 activity increased proteasomal degradation of survivin and cellular response to apoptosis induction. Our data show that expression of survivin in non-small cell lung cancer cells can be significantly down-regulated by RNA interference. Whereas COX-2-overexpressing NSCLC cells have significantly higher apoptosis resistance than the parental cells, inhibition of survivin expression by small interfering RNA decreases apoptosis resistance to the level of the parental non-small cell lung cancer. We conclude that COX-2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.     

凋亡抑制因子（IAPs）在非小细胞肺癌患者中的表达及意义

 目的 探讨凋亡抑制基因（IAPs）家族在非小细胞肺癌（NSCLC）患者组织中的表达及其意义。方法 采用半定量反转录聚合酶链反应（RT-PCR）及Western blotting分别从mRNA和蛋白水平对36例NSCLC、36例肺部良性病变进行研究，检测生存蛋白（survivin）（human IAP-1）hIAP-1，（human IAP-2）hIAP-2及（X chromosome-linked IAP）XIAP表达情况。结果 36例NSCLC组织中有32例sur-vivin mRNA表达阳性，而对照组无一例阳性表达；26例癌组织中XIAP mRNA处于高表达，与对照组比较差异有统计学意义（P＜0.05）；hIAP-1 mRNA仅仅在腺癌组织中高表达（P＜0.05），具有组织学类型特异性。Western blotting显示在survivin mRNA阳性组织中均可检测到survivin蛋白的表达；XIAP和hIAP-1 mRNA高表达的组织同样可检测到相应蛋白。相关性分析表明只有hIAP-1的表达与NSCLC组织学类型相关。结论 survivin mRNA高表达可以作为NSCLC辅助诊断的重要指标，hIAP-1可用来协助鉴定NSCLC亚型，survivin和XIAP在肿瘤基因治疗中具有潜在的应用价值和前景。     

Cyclooxygenase inhibitors induce apoptosis in sinonasal cancer cells by increased expression of nonsteroidal anti-inflammatory drug-activated gene

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) has recently been shown to be induced by nonsteroidal anti-inflammatory drugs (NSAIDs) and to have proapoptotic and antitumorigenic activities. Although sulindac sulfide induced apoptosis in sinonasal cancer cells, the relationship between NAG-1 and NSAIDs has not been determined. In this study, we investigated the induction of apoptosis in sinonasal cancer cells treated by various NSAIDs and the role of NAG-1 expression in this induction. The effect of NSAIDs on normal human nasal epithelial (NHNE) cells was also examined to evaluate their safety on normal cells. Finally, the in vivo anti-tumorigenic activity of NSAIDs in mice was investigated. In AMC-HN5 human sinonasal carcinoma cells, indomethacin was the most potent NAG-1 inducer and caused NAG-1 expression in a time- and dose-dependent manner. The induction of NAG-1 expression preceded the induction of apoptosis. Conditioned medium from NAG-1-overexpressing Drosophila cells inhibited proliferation of sinonasal cancer cells and induced apoptosis. In addition, in NAG-1 small interfering RNA-transfected cells, apoptosis induced by indomethacin was suppressed. In contrast, NAG-1 expression and apoptosis were not induced by NSAIDs or conditioned medium in NHNE cells. Furthermore, indomethacin induced a dose-dependent in vivo increase in the expression of NAG-1 mRNA in the mice tumors and the volume of xenograft tumors of AMC-HN5 cells in indomethacin-treated nude mice was reduced compared to that in control mice. In conclusion, indomethacin exerts proapoptotic and antitumorigenic effects in sinonasal cancer cells through the induction of NAG-1 and can be considered a safe and effective chemopreventive agent against sinonasal cancer.     

Gemcitabine (Gemzar) in non-small cell lung cancer

Of the new chemotherapeutic substances of the last decade, gemcitabine (Gemzar, Eli Lilly) is probably the most valuable for the treatment of early and advanced stage non-small cell lung cancer (NSCLC). When used as a single agent in both chemotherapeutically pretreated and chemotherapy-naive patients, gemcitabine shows an objective tumor regression rate of approximately 20%. Gemcitabine's unique mechanism of action and its lack of overlapping toxicity with other cytotoxic agents also define it as an ideal candidate for combination therapy. Early clinical development has included single-agent first- and second-line treatment, doublet combination regimens and incorporation into multimodality treatment strategies for operable and inoperable locally advanced nonmetastatic NSCLC. Gemcitabine/platinum-based combination chemotherapy has become the most attractive treatment standard for NSCLC patients in good clinical condition. The role of gemcitabine in the concurrent or sequential application of chemo- and radiotherapy for inoperable locally advanced NSCLC has also been addressed in several Phase I and II studies. Based on data available, gemcitabine can be safely administered in combination with radiotherapy. This review summarizes results from representative Phase I, II and III studies in order to underline gemcitabine's clinical importance for patients suffering from early and advanced NSCLC.     

Angiogenesis inhibitors in the treatment of non-small cell lung cancer

A therapeutic plateau seems to have been reached with the standard treatment of cytotoxic chemotherapy alone for advanced stage non-small cell lung cancer (NSCLC) and new treatment options are urgently needed. Recent insight into the molecular biology of cancer has identified angiogenesis as one of the key biological processes. The major player in tumor angiogenesis is the vascular endothelial growth factor (VEGF) pathway. VEGF is expressed in the majority of NSCLC and overexpression is associated with a poor prognosis. The VEGF pathway can be inhibited in two main ways: targeting VEGF directly or inhibiting the VEGF receptors. The development of angiogenesis inhibitors has shown great promise in the treatment of NSCLC. Bevacizumab, an anti-VEGF antibody, has been approved for the treatment of advanced NSCLC and other drugs are undergoing phase III investigation. However, a number of unresolved issues remain. In this review, we discuss the main angiogenesis inhibitors in development for the treatment of NSCLC focusing on the VEGF pathway.     

PI3K抑制剂与非小细胞性肺癌

磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路是人体中的重要信号通路,通过逐级磷酸化下游蛋白发挥作用.非小细胞肺癌(non-small cell lung cancer,NSCLC)中存在该信号通路的异常活化.活化的信号通路可以改变NSCLC细胞的增殖、凋亡、侵袭和迁移等生物学特性从而影响其对治疗药物的反应及预后.针对该通路抑制剂的研究很多,部分抑制剂已进入临床研究阶段.PI3K抑制剂包括广谱PI3K抑制剂、亚型特异性的PI3K抑制剂和PI3 K/mTOR双重抑制剂.PI3K抑制剂单药有效率较低,但与化疗药物或其他靶向药物联用取得了不错的临床效果,目前往往将P IK3 CA突变以及抑癌基因PTEN的缺失作为预测疗效的指标,但准确性欠佳,进一步筛选疗效预测指标是目前面临的重要问题.     

加速超分割术前放疗诱导非小细胞肺癌细胞凋亡的研究

目的　探索放射线诱导非小细胞肺癌细胞凋亡及对凋亡相关蛋白Bcl 2、Bax表达的影响。方法　 81例初治的非小细胞肺癌患者 ,分为加速超分割术前放疗组 ( 2 0例 )和单纯手术组 ( 61例 )。术前放疗组采用前后对穿照射 ,2 .5Gy/次 ,2次 /天 ,总量 2 5Gy/10次 /5～ 7天 ,放疗后 2周内手术。用间接免疫荧光法和流式细胞术定量分析细胞凋亡指数 (AI)、细胞周期分布和凋亡相关蛋白Bcl 2和Bax的表达。结果　单纯手术组AI为 4.6%± 2 .3 % ,术前放疗组为 12 .8%± 4.3 % (P <0 .0 0 1)。单纯手术组S期细胞比例 (Sphasefraction ,SPF)为 16.3 %± 4.6% ,术前放疗组为 14 .9%± 4.1% (P >0 .0 5 )。单纯手术组Bcl 2、Bax蛋白免疫荧光指数 (FI)和Bcl 2 /Bax比值分别为 1.3 3± 0 .2 1、1.0 5± 0 .13和 1.2 9± 0 .2 3 ,术前放疗组分别为 1.14±0 .2 6、1.19± 0 .16和 0 .96± 0 .2 3 ,术前放疗组Bcl 2蛋白表达水平下降 (P <0 .0 1) ,Bax蛋白表达水平升高 (P<0 .0 0 1) ,Bcl 2 /Bax比值明显下降 (P <0 .0 0 1)。AI与Bax蛋白表达呈正相关 (P <0 .0 0 1) ,与Bcl 2 /Bax比值呈负相关 (P <0 .0 1)。结论　加速超分割术前放疗使Bcl 2蛋白表达水平下降并诱导Bax蛋白表达水平升高 ,诱发了较高水平的细胞凋亡 ,但是否能提高     

ZD1839 (Iressa) in non-small cell lung cancer

Despite the advent of cisplatin-based combination chemotherapy for advanced non-small cell lung cancer (NSCLC), the prognosis for this patient population remains poor. Novel biologically targeted agents currently in development have the potential for greater efficacy against NSCLC, and possibly less toxicity than is associated with conventional cytotoxic chemotherapy. The epidermal growth factor receptor (EGFR) is recognized as a potentially useful target, and the small molecule, orally active EGFR-tyrosine kinase inhibitor ZD1839 (Iressa) is currently the furthest along in clinical development of the anti-EGFR agents. This review summarizes the currently available clinical data on the use of ZD1839 in the treatment of NSCLC.     

Gefitinib (Iressa) trials in non-small cell lung cancer

Gefitinib (Iressa) is a synthetic anilinoquinazoline capable of inhibiting the epidermal growth factor receptor tyrosine kinase in vitro at nanomolar concentrations. In phase I trials, gefitinib was well tolerated at doses above that required to induce antitumor effects in vitro. Notably, antitumor activity was observed in lung cancer patients. These findings resulted in the initiation of phase II trials employing gefitinib monotherapy in patients with recurrent non-small cell lung cancer (the so-called IDEAL trials). Study participants were randomized to 250 mg or 500 mg of gefitinib per day. Objective response rates between 10 and 20% were achieved with minimal host related toxicity (mainly acne like rash and mild diarrhea). Median survivals ranged between 6 and 8 months. Subsequently, phase III trials (the so-called INTACT trials) combined gefitinib and chemotherapy in chemonaive patients with advanced non-small cell lung cancer. These trials failed to demonstrate a survival advantage with the addition of gefitinib to standard platinum-based chemotherapy regimens. However, overall host related toxicities were not substantially worsened with the addition of gefitinib to chemotherapy. Further studies employing single agent gefitinib as well as regimens employing a different sequencing of chemotherapy and gefitinib are planned in recurrent and previously untreated lung cancer patients.     

miR-223-3p通过调控ECT2诱导非小细胞肺癌细胞凋亡的实验研究

目的：探讨miR-223-3p对非小细胞肺癌细胞增殖与凋亡的影响及其可能的作用机制。方法：选取24例非小细胞肺癌患者的癌及癌旁组织,实时定量PCR检测miR-223-3p的相对表达水平,免疫组化检测分析上皮细胞转化序列2（ECT2）蛋白的表达水平,并对两者进行相关性分析。将人非小细胞肺癌A549细胞分为正常对照组、转染对照组、miR-223-3p过表达组,其中正常对照组细胞未作任何处理,转染对照组细胞转染空质粒载体,miR-223-3p过表达组转染miR-223-3p mimics。MTT实验和平板集落形成实验检测各组细胞的增殖率,流式细胞术检测各组细胞的凋亡率,Western blot实验检测ECT2蛋白的表达水平;采用siRNA转染法沉默A549细胞中的ECT2后,检测细胞增殖及凋亡的变化。结果：miR-223-3p在癌组织中表达显著低于癌旁组织,而癌组织中ECT2蛋白表达高于癌旁组织,二者存在负相关关系（r=-0.666,P < 0.05）;与正常对照组和转染对照组细胞相比,miR-223-3p过表达组细胞的增殖率降低（P < 0.05）,ECT2蛋白表达显著降低,而细胞凋亡率显著升高（P < 0.05）;沉默ECT2对细胞增殖和凋亡的影响与过表达miR-223-3p基本一致。结论：miR-223-3p可能通过负向调控ECT2的表达,进而抑制非小细胞肺癌细胞增殖并诱导其凋亡,其作用机制有待进一步研究。     

Bag3 promotes resistance to apoptosis through Bcl-2 family members in non-small cell lung cancer

In non-small cell lung cancer (NSCLC) certain molecular characteristics, which are related to molecular alterations have been investigated. These are responsible for both the initiation and maintenance of the malignancy in lung cancer. The aim of this study was to evaluate the influence of Bag3 (Bcl-2 associated athanogene 3) in the regulation of apoptosis on NSCLC. Bag3 and Hsp70 expression were examined by immunohistochemistry to confirm their potential roles in the prevalence of NSCLC. We also established human normal bronchial epithelial cells and HOP-62 cell line as the model to analyze cell apoptosis and the expression of Hsp70, Bcl-XL and Bcl-2, which were affected by Bag3. In this study, we found that Bag3 and Hsp70 are highly expressed in few tissues and cell lines of NSCLC. Bag3 inhibits apoptosis in human normal bronchial epithelial cell lines and sustain the survival of NSCLC cells. Bag3, Hsp70, Bcl-XL and Bcl-2 are up-regulated in NSCLC cell lines. At the same time, the silencing of Bag3 results in diminishing protein levels of Bcl-XL and Bcl-2. The results of immunoprecipitation identified that Bag3 could interact with Hsp70, Bcl-XL and Bcl-2 NSCLC cells directly or indirectly. We conclude that NSCLC cells were protected from apoptosis through increasing Bag3 expression and consequently promoted the expression of Bcl-XL and Bcl-2.     

Rapamycin induces p53-independent apoptosis through the mitochondrial pathway in non-small cell lung cancer cells

The mammalian target of rapamycin (mTOR) is a key kinase acting downstream of growth factor receptor PI3K and AKT signaling, leading to processes resulting in increased cell size and proliferation through translation control. Rapamycin, a specific inhibitor of mTOR, results predominately in G1 cell cycle arrest through translation control and occasionally, cell type-dependent apoptosis by an unknown mechanism. In this study, we investigated the effect and mechanism of action of rapamycin on non-small cell lung cancer (NSCLC) cell lines with p53 mutations. Cell proliferation was evaluated by modified MTT assay. The apoptotic effect of rapamycin was measured by caspase-3 activation and flow cytometric analysis of Annexin V binding. The expression of Bcl-2 and the release of cytochrome?c from mitochondria were evaluated by western blotting. We found that rapamycin induced apoptosis in NSCLC cell lines with p53 mutations. Western blot analysis demonstrated that rapamycin downregulates the expression levels of Bcl-2, which leads to increased cytochrome c release from mitochondria and subsequent activation of caspase cascades. These findings suggest that rapamycin induces p53-independent apoptosis through downregulation of Bcl-2 and the mitochondrial pathway in NSCLC cell lines as a novel antitumor mechanism.     

The potential role of mTOR inhibitors in non-small cell lung cancer

The mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a downstream mediator in the phosphatidylinositol 3-kinase/Akt signaling pathway, which plays a critical role in regulating basic cellular functions including cellular growth and proliferation. Currently, the mTOR inhibitor rapamycin and its analogues (CCI-779, RAD001, AP23573), which induce cell-cycle arrest in the G(1) phase, are being evaluated in cancer clinical trials. The mTOR inhibitors appear to be well tolerated, with skin reactions, stomatitis, myelosuppression, and metabolic abnormalities the most common toxicities seen. These adverse events are transient and reversible with interruption of dosing. Several pieces of evidence suggest a certain antitumor activity, including tumor regressions and prolonged stable disease, which has been reported among patients with a variety of malignancies, including non-small cell lung cancer (NSCLC). These promising preliminary clinical data have stimulated further research in this setting. Here, we review the basic structure of the pathway together with current results and future developments of mTOR inhibitors in the treatment of NSCLC patients.     

姜黄素诱导NSCLC细胞凋亡机制探讨

目的 肺癌位居我国居民癌症发病率和死亡率的首位,其中非小细胞癌(non-small cell lung cancer,NSCLC)约占80%,筛选高效低毒的抗癌药物尤为迫切.本研究拟探讨姜黄素对NSCLC细胞的可能作用机制.方法 用不同浓度的姜黄素(0、10、20、30 μmol/L)或活性氧清除剂(CAT和NAC)加姜黄素处理肺癌细胞A549和SPC-A1,采用流式细胞术检测细胞周期、细胞凋亡率、活性氧(reactive oxygen species,ROS)水平和线粒体膜电位的变化,蛋白质印迹法检测自噬相关蛋白LC3、P62和细胞凋亡相关蛋白PARP、Caspase-3和Caspase-9的表达变化.结果 姜黄素抑制非小细胞肺癌细胞株A549和SPC-A1增殖及克隆形成,主要将细胞阻滞在G2/M期,0、10、20和30 μmol/L姜黄素处理的A549细胞G2/M期细胞百分比分别为(12.67±2.52)%、(22.67±2.52)%、(27.00±2.01)%和(42.33±4.04)%,SPC-A1细胞G2/M期细胞百分比分别为(9.33±2.52)%、(18.33±1.53)%、(20.67±2.52)%和(30.67±1.53)%.0、10、20和30 μmol/L姜黄素处理A549细胞凋亡率分别为(4.40±1.02)%、(7.31±1.52)%、(9.32±1.08)%和(13.97±1.98)%,P<0.05;SPC-A1细胞凋亡率分别为(4.38±1.22)%、(5.98±0.75)%、(9.42±1.25)%和(16.13±3.09)%,P<0.05.姜黄素导致ROS水平增高、线粒体膜电位降低和线粒体自噬的发生,并且呈剂量依赖性,而应用ROS清除剂可以减弱以上药物作用.相关信号转导通路蛋白表达与以上细胞生物行为改变表现一致.结论 姜黄素通过ROS途径诱导NSCLC细胞发生线粒体自噬,是一种有潜力的抗癌药物.     

Targeting apoptosis pathways in lung cancer

Lung cancer is a devastating disease with a poor prognosis. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) represent different forms of lung cancer that are associated with distinct genetic causes and display different responses to therapy in the clinic. Whereas SCLC is often sensitive to chemotherapy at start of treatment, NSCLC are less chemo-sensitive. In NSCLC different histological subtypes are distinguished and increasing efforts are made to identify subtypes that respond to specific therapies, such as those harbouring epidermal growth factor receptor (EGFR) mutations that have benefit from treatment with EGFR inhibitors. Targeting of the apoptotic machinery represents another approach that aims to selectively kill cancer cells while sparing normal ones. Here we describe different ways that are currently explored to induce apoptosis in lung cancer cells, specifically pathways controlled by TNF-related apoptosis-inducing ligand (TRAIL), BCL-2 family members and apoptosis inhibitory proteins (IAPs). Preclinical studies are discussed and for some agents results from early clinical studies and future perspectives are considered.     

Angiogenesis inhibitors in the treatment of small cell and non-small cell lung cancer

Angiogenesis is believed to play a critical role in cancer; however, antiangiogenic therapy has not been demonstrated to improve the survival of patients who have lung cancer. In this article, the evidence that supports a role for angiogenesis in the pathogenesis of lung cancer, trials of antiangiogenic agents in lung cancer performed to date, and the lessons learned from these studies are discussed.