Combinatorial action of the HDAC inhibitor trichostatin A and etoposide induces caspase-mediated AIF-dependent apoptotic cell death in non-small cell lung carcinoma cells

Commonly used regimens in cancer therapy rely on the induction of apoptotic cell death, and drug resistance can be attributed, at least in part, to a disabled apoptotic program. Non-small cell lung carcinomas (NSCLC), exhibit an intrinsic resistance to chemotherapy. Here, we show that co-treatment with etoposide (VP16) and the pan-histone deacetylase (HDAC) inhibitor trichostatin A (TSA), but not valproic acid (VPA), induced apoptotic cell death in drug-resistant NSCLC cells. Co-treatment, but not single treatment, with VP16 and TSA induced apoptosis in a caspase-dependent manner accompanied by a crucial decrease in Bcl-xL expression allowing Bax activation and subsequent initiation of the apoptosis inducing factor (AIF)-dependent death pathway. Importantly, AIF proved to be required for the effects of TSA/VP16 as RNA knockdown of AIF resulted in a complete abolishment of TSA/VP16-induced apoptotic cell death in drug-resistant NSCLC cells. Our results thus provide evidence for the requirement of both caspase-dependent and caspase-independent apoptotic pathways in TSA/VP16-mediated death of drug-resistant NSCLC cells, and extend previous suggestions that HDAC inhibitors in combination with conventional chemotherapeutic drugs could be valuable in the treatment of NSCLC cancer and other malignancies in which Bcl-xL is overexpressed.     

Vacuolar H+-ATPase inhibitors overcome Bcl-xL-mediated chemoresistance through restoration of a caspase-independent apoptotic pathway

The anti-apoptotic oncoproteins Bcl-2 and Bcl-xL play crucial roles in tumorigenesis and chemoresistance, and are thus therapeutic cancer targets. We searched for small molecules that disturbed the anti-apoptotic function of Bcl-2 or Bcl-xL, and found vacuolar H⁺-ATPase (V-ATPase) inhibitors, such as bafilomycin A1 (BMA), that showed such activity. Bcl-xL-overexpressing Ms-1 cells displayed resistance to anticancer drugs, but underwent apoptosis following treatment with a combination of V-ATPase inhibitors at doses similar to those that caused inhibitory activities of V-ATPase. We investigated the apoptosis mechanism induced by cotreatment of Bcl-xL-overexpressing Ms-1 cells with BMA as a V-ATPase inhibitor and taxol (TXL) as an anticancer drug. With BMA, TXL triggered mitochondrial membrane potential loss and cytochrome c release, whereas downstream caspase activation was not observed. In contrast, pronounced nuclear translocation of mitochondrial apoptosis-inducing factor and endonuclease G, known as effectors of caspase-independent apoptosis, was observed with BMA and TXL cotreatment. Moreover, depletion of apoptosis-inducing factor and endonuclease G using each siRNA significantly rescued cells from BMA- and TXL-induced apoptosis. Hence, the apoptosis-inducing factor- and endonuclease G-dependent pathway was critical for apoptosis induction by BMA and TXL cotreatment. Our data suggest that V-ATPase inhibitors could not only suppress anti-apoptotic Bcl-2 nor Bcl-xL but could also facilitate the caspase-independent apoptotic pathway. V-ATPase inhibition will be a promising therapeutic approach for Bcl-2- or Bcl-xL-overexpressing malignancies. ( Cancer Sci 2009)     

Tudor staphylococcal nuclease drives chemoresistance of non-small cell lung carcinoma cells by regulating S100A11

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC), the major lung cancer subtype, is characterized by high resistance to chemotherapy. Here we demonstrate that Tudor staphylococcal nuclease (SND1 or TSN) is overexpressed in NSCLC cell lines and tissues, and is important for maintaining NSCLC chemoresistance. Downregulation of TSN by RNAi in NSCLC cells led to strong potentiation of cell death in response to cisplatin. Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level. Downregulation of S100A11 by RNAi resulted in enhanced sensitivity of NSCLC cells to cisplatin, oxaliplatin and 5-fluouracil. AACOCF₃, a phospholipase A₂ (PLA₂) inhibitor, strongly abrogated chemosensitization upon silencing of S100A11 suggesting that PLA₂ inhibition by S100A11 governs the chemoresistance of NSCLC. Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF₃, as well as N-acetyl-L-cysteine, which also mimicked the effect of PLA₂ inhibitor on NSCLC chemosensitization upon S100A11 silencing. Thus, we present the novel TSN-S100A11-PLA₂ axis regulating superoxide-dependent apoptosis, triggered by platinum-based chemotherapeutic agents in NSCLC that may be targeted by innovative cancer therapies.     

Interaction between CD44 and hyaluronate induces chemoresistance in non-small cell lung cancer cell

CD44s is a principle hyaluronate (HA) receptor and has been reported to play an important role in cancer cell invasion and metastasis. The aim of our study is to determine if the interaction between HA and CD44s influences in vitro chemosensitivity of non-small cell lung cancer (NSCLC). NSCLC cell line, H322 cells, transfected with the CD44s gene (H322/CD44s) cultured on HA coated plates were more resistant to cisplatin (CDDP) than that on bovine serum albumin. Multidrug resistance protein2 (MRP2) expression was induced in H322/CD44s cells cultured on HA. MRP2 inhibitor, MK571, not only suppressed MRP2 expression but also reversed CDDP resistance. These results suggest that the interaction between CD44s and HA play a pivotal role in acquired resistance to CDDP in NSCLC and MRP2 could be involved in this potential mechanism.     

Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells

Plumbagin (PLB) has shown anti-cancer activity but the mechanism is unclear. This study has found that PLB has a potent pro-apoptotic and pro-autophagic effect on A549 and H23 cells. PLB arrests cells in G2/M phase, and increases the intracellular level of reactive oxygen species in both cell lines. PLB dose-dependently induces autophagy through inhibition of PI3K/Akt/mTOR pathway as indicated by reduced phosphorylation of Akt and mTOR. Inhibition or induction of autophagy enhances PLB-induced apoptosis. There is crosstalk between PLB-induced apoptosis and autophagy. These findings indicate that PLB initiates both apoptosis and autophagy in NSCLC cells through coordinated pathways.     

Rho GTPases途径在非小细胞肺癌中的表达及其临床意义

目的:探讨Rho GTPases在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及其临床意义.方法:应用RT-PCR和免疫组织化学法检测36例NSCLC组织Rho GTPases信号转导途径中的RhoC、E-钙黏附素、基质金属蛋白酶-2(matrix metalloproteinases-2, MMP-2)和MMP-9的表达.通过绘制Kaplan-Meier生存曲线分析RhoC mRNA的表达与患者预后的关系.结果: RhoC mRNA在NSCLC组织和癌旁正常组织中的表达存在差异(P<0.01),RhoC mRNA的表达与患者的性别、年龄、肿瘤浸润程度、淋巴结转移、肿瘤大小、组织学类型和分化程度无关,而与不同TNM分期有关(P<0.05).RhoC蛋白与MMP-2蛋白的表达呈正相关(r=0.474,P=0.003).RhoC mRNA低表达组患者的生存时间长于高表达组患者,但2者之间差异无统计学意义. 结论:RhoC mRNA的高表达可能与NSCLC的发生和发展有关,也可能与NSCLC的早期及中期浸润和转移有关.     

Oncoprotein HBXIP promotes tumorigenesis through MAPK/ERK pathway activation in non-small cell lung cancer

Objective:The oncoprotein,hepatitis B X-interacting protein(HBXIP),has been reported to play an important role in human malignancies.However,its functions in non-small cell lung cancer(NSCLC)are poorly understood.The goal of the present study was to identify the role of HBXIP in the regulation of NSCLC development.Methods:The level of HBXIP expression in NSCLC tissue was assessed by immunohistochemical and Western blot analyses,and its relationships with clinicopathological features and outcomes were statistically evaluated.The effects of HBXIP on NSCLC cell progression were assessed through cell viability,colony formation,and flow cytometry analyses in vitro.The mechanism by which HBXIP regulated the MAPK pathway was studied by Western blot,immunofluorescence,and immunoprecipitation assays.In addition,in vivo experiments were performed to evaluate the progression of NSCLC and ERK signaling pathway activation after HBXIP knockdown.Results:HBXIP was overexpressed in human NSCLC and was correlated with the invasiveness of NSCLC.The high expression of HBXIP in NSCLC was significantly correlated with gender(P=0.033),N stage(P=0.002),and tumor-node-metastasis stage(P=0.008).In vitro experiments using an NSCLC cell line revealed that HBXIP knockdown resulted in the suppression of cell proliferation and colony formation,which was consistent with the enhanced cell cycle arrest in G1 phase.The results of a mechanistic investigation suggested that binding of HBXIP to MEK1 protein promoted MAPK/ERK signaling pathway activation in NSCLC by preventing the proteasome-mediated degradation of MEK1.In addition,the results obtained using in vivo subcutaneous tumor xenografts confirmed that HBXIP deficiency decreased MEK1 protein levels and NSCLC tumor growth.Conclusions:Taken together,our results showed that the HBXIP-MEK interaction promoted oncogenesis via the MAPK/ERK pathway,which may serve as a novel therapeutic target for cancers in which MAPK/ERK signaling is a dominant feature.     

Defining target volumes for non-small cell lung carcinoma

The definition of target volumes for non-small cell lung carcinoma (NSCLC) remains a controversial topic as tradition-based approaches of the past are being critically re-evaluated in the light of the advent of three-dimensional treatment techniques, by the awareness of the poor local control achieved using current treatment fields and radiation doses, and by the major improvements in noninvasive staging that enable the gross tumor volume to be established with greater accuracy. This article reviews the current knowledge and remaining uncertainties in defining target volumes for NSCLC. The use of these new approaches may allow for improvements in the therapeutic ratio of radiotherapy in NSCLC.     

Combined modality therapy for non-small cell lung carcinoma

Combined modality therapy is frequently used to treat non-small cell lung carcinoma (NSCLC). Surgical resection is the mainstay of treatment for Stages I and II NSCLC. Little evidence supports the use of adjuvant therapy In patients with Stage I NSCLC. Adjuvant radiotherapy to the chest after resection for Stage II and IIIA NSCLC remains controversial but may be appropriate for many patients with resected Stage IIIA disease or inadequately pathologically staged II disease. For Stage IIIA NSCLC, surgery appears suboptimal, though no other single modality appears superior. Recent studies of neoadjuvant therapy for Stage IIIA NSCLC yielded promising results. The standard of care for Stage III unresectable NSCLC and strategies to improve local-regional control in Stage III NSCLC are discussed, as are the importance of toxicity protection with chemoradiation and the potential utility of prophylactic cranial irradiation for preventing brain relapse. Targeted therapies appear promising, and expedited evaluation in Stage III NSCLC is warranted.     

非小细胞肺癌组织ABCC10的表达及其与耐药相关性的研究

目的:检测ABCC10在非小细胞肺癌(NSCLC)组织中的表达,探讨其与组织学类型、分级和TNM分期的关系,检测相应癌组织对化疗药物的敏感性.方法:采用免疫组化EnVinsion法检测ABCC10在75例NSCLC组织中的表达,进行统计学分析.采用MTT法检测相应癌组织对化疗药物的敏感性,分析其与ABCC10表达的相关性.结果:ABCC10主要定位于胞膜上和胞质中.鳞癌、腺癌阳性率分别为77.5%和85.7%,两者差异有统计学意义,P=0.007.鳞癌、腺癌不同病理分级、TNM分期间ABCC10表达差异均有统计学意义(P=0.000、0.003、0.009和0.032).NSCLC组织的耐药性试验结果与其相应组织中ABCC10的表达做相关性检验,多西他赛、紫杉醇、长春瑞滨、长春新碱、顺铂差异有统计学意义(P=0.000),相关系数分别为0.674、0.633、0.610、0.565和0.532.结论:ABCC10在NSCLC组织中高表达,其中腺癌表达高于鳞癌,表达的高低与组织学类型、分级,TNM分期有关,很可能与NSCLC化疗药物耐药有关.     

Defects in apoptotic signal transduction in cisplatin-resistant non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Anticancer drugs exert at least part of their cytotoxic effect by triggering apoptosis. In order to understand the molecular alterations leading to heterogeneous cisplatin sensitivity and apoptosis inducibility in NSCLC cells, we analyzed various apoptotic pathways, including the activation of caspase-8, -9 and -3, the release of cytochrome c from mitochondria and the expression levels of pro- and anti-apoptotic proteins such as Bax, Bad, Bcl-2, Bcl-xL, Fas and p53 using heterogeneously apoptosis-sensitive cells (Ma-10, Ma-31 and Ma-46). Cisplatin treatment induced the activation of caspase-8, -9 and -3 and the release of cytochrome c in apoptosis-sensitive Ma-46. The expression of Bcl-xL was the highest and p53 was not expressed in apoptosis-resistant Ma-31, and Fas was not expressed in Ma-46. These expression levels were not correlated with the apoptosis inducibility of the three cell lines. These results suggest that blockage of the apoptotic signal from mitochondria is responsible for apoptosis resistance in NSCLC cell lines. Our findings also indicate that anti-apoptotic Bcl-xL and pro-apoptotic p53 are necessary but not sufficient for resistance to cisplatin-induced apoptosis in NSCLC cells.     

桥接整合因子1通过AKT-mTOR通路诱导非小细胞肺癌H1975细胞周期阻滞

目的：研究桥接整合因子1（bridging intergrator 1,Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒,并转染H1975细胞（Bin1+组）,另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组）,利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化,Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较,Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）; H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%,均P<0.05\]; Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05）,AKT、mTOR表达变化无统计学差异（P>0.05）;周期蛋白D1、CDK4的表达量均明显下调(P<0.05),Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞,其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。     

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.     

Integrin expression in non-small cell carcinoma of the lung

Summary Alteration of integrin expression in a number of different malignant diseases has been recognized, with a trend of downregulation of collagen-laminin binding integrin expression in epithelial tumor types noted. This study evaluated the expression of a panel of integrin subunits that included subunits that form receptors that bind to collagen and laminin (₂, ₃, _{6 4}) and subunits that form receptors that bind to fibronectin and fibrinogen (₅, _v, ₃, ₆) in 51 specimens of non-small cell carcinoma (NSCCA) of the lung by use of immunohistochemistry. Integrin expression was then correlated with histologic type (squamous vs. adenocarcinoma), absence or presence of hilar or mediastinal nodal metastasis at resection, and cellular differentiation (well or poorly differentiated). In general, downregulation of the collagen-laminin binding subunits was noted in tumor cells of the NSCCA specimens when compared to the progenitor normal bronchial epithelium. No differences were noted in integrin expression between squamous cell and adenocarcinoma or between node-positive or node-negative tumors. However, downregulation of the integrin subunit ₃ was noted to be significantly more common in poorly differentiated tumors (p = 0.02) and several of the other collagen-laminin binding subunits also appeared to be more downregulated in poorly differentiated tumors. No upregulation was seen in the ₅ subunit of the fibronectin receptor or the ₃ subunit of the vitronectin receptor, however, approximately 50% of tumors showed upregulation of the ₆ subunit, the great majority of these being well-differentiated, node-negative tumors. Downregulation of the collagen-laminin integrins may thus be associated with differentiation of NSCCA, but not metastasis, and may serve as an adjunctive prognostic marker of disease. The ₆ subunit appears to be associated with malignant transformation, but may serve as a positive prognostic factor.