Growth Of Human Lung Adenocarcinoma Research Articles

Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells.

Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.

Fungal Immunomodulatory Protein from Nectria haematococca Suppresses Growth of Human Lung Adenocarcinoma by Inhibiting the PI3K/Akt Pathway

Lung cancer is a common disease that is associated with poor prognosis. Fungal immunomodulatory protein from Nectria haematococca (FIP-nha) has potential as a lung cancer therapeutic; as such, illuminating its anti-tumor mechanism is expected to facilitate novel treatment options. Here, we showed that FIP-nha affects lung adenocarcinoma growth ex vivo and in vivo. Comparative quantitative proteomics showed that FIP-nha negatively regulates PI3K/Akt signaling and induces cell cycle arrest, autophagy, and apoptosis. We further demonstrated that FIP-nha suppresses Akt phosphorylation, leading to upregulation of p21 and p27 and downregulation of cyclin B1, cyclin D1, CDK2, and CDK4 expression, ultimately resulting in G1/S and G2/M cell cycle arrest. Meanwhile, FIP-nha-induced PI3K/Akt downregulation promotes A549 apoptosis by increasing the expression ratio of Bax/Bcl-2 and c-PARP and autophagy by decreasing the phosphorylation of mTOR. Thus, we comprehensively revealed the anti-tumor mechanism of FIP-nha, which inhibits tumor growth by modulating PI3K/Akt-regulated cell cycle arrest, autophagy, and apoptosis, and provided the basis for further application of fungal immunomodulatory proteins, especially FIP-nha.

LncRNA LINC00460 promotes tumor growth of human lung adenocarcinoma by targeting miR-302c-5p/FOXA1 axis

Accumulating evidence has shown that long non-coding RNAs (lncRNAs) had malfunctioning roles in the development of human cancers, especially lung adenocarcinoma (LC). In the present study, we aimed to investigate the role and potential mechanism of lncRNA long intergenic non-protein coding RNA 460 (LINC00460) in LC progression using human tissues and cell lines. We observed that LINC00460 was increased in lung adenocarcinoma tissues and cells in comparison to their corresponding controls. Moreover, overexpression of LINC00460 indicated the poor prognosis of lung adenocarcinoma patients. In addition, silencing LINC00460 was able to suppress lung adenocarcinoma cell growth in vitro and in vivo. Rescue assay confirmed that LINC00460 contributed to lung adenocarcinoma progression by regulating miR-302c-5p/FOXA1 signal pathway. In conclusion, LINC00460 promotes LC progression by competitively binding miR-302c-5p and regulating FOXA1 signal pathway. Our findings reveal that LINC00460 may be a potential prognostic biomarker and a candidate target for LC therapy.

Cytoplasmic liver kinase B1 promotes the growth of human lung adenocarcinoma by enhancing autophagy.

Liver kinase B1 (LKB1) as a tumor suppression gene that is associated with various kinds of cancers, including lung cancer. In this study, we found that the effect of LKB1 on tumor growth was dependent on its subcellular expression in A549 and HCC827 cells. Full‐length LKB1 decreased the proliferation and clonogenicity of A549‐LKB1 and HCC827‐LKB1 cells, but increased their apoptosis. Opposite effects were observed in A549‐LKB1s and HCC827‐LKB1S cells that overexpressed truncated LKB1 without the nuclear localization sequence. The truncated cytoplasmic LKB1 enhanced the growth of implanted tumors in vivo. The truncated cytoplasmic LKB1 promoted autophagy, which was independent of AMP‐activated protein kinase and mTOR signaling in A549 and HCC827 cells. Further characterization indicated that higher levels of cytoplasmic LKB1 expression were associated with advanced TNM stage and reduced overall survival (OS) in 190 patients with adenocarcinoma. In contrast, high nuclear expression of LKB1 is associated with early TNM stage and longer OS. The high level of cytoplasmic LKB1 expression was an independent risk factor for poor overall survival in patients with adenocarcinoma. Together, our results revealed that cytoplasmic LKB1 promotes the growth of lung adenocarcinoma and could be a prognostic marker for lung adenocarcinoma.

The upregulation of TMPRSS4, partly ascribed to the downregulation of miR‑125a‑5p, promotes the growth of human lung adenocarcinoma via the NF‑κB signaling pathway.

In this study, with the aid of microarray technology, transmembrane protease serine 4 (TMPRSS4), a novel member of the serine protease family, was found to be upregulated in the majority of lung adenocarcinoma (LUAD) tissues compared to normal lung tissues. Of note, the clinical significance of TMPRSS4 in LUAD has not yet been reported, at least to the best of our knowledge. Through immunohistochemistry assays, we found that TMPRSS4 was overexpressed in LUAD tissues and that the TMPRSS4 expression level was also proportionally associated with the AJCC clinical stage, T stage and pathological grade. Moreover, a high expression of TMPRSS4 was found to be associated with adverse outcomes and was a significant independent factors predicting a poor prognosis. To elucidate the possible mechanisms responsible for the overexpression of TMPRSS4, we examined at microRNAs (miRNAs or miRs), which are small non-coding RNAs commonly dysregulated in human malignancies and are known to promote carcinogenesis by interacting with other types of RNAs. By means of bioinformatics analysis, a miRNA potentially targeting TMPRSS4 mRNA, namely miR-125a-5p, was selected. Dual luciferase reporter gene assays were then performed to verify the interaction. The results of MTT assays and apoptotic assays revealed that miR-125a-5p significantly inhibited cell growth and enhanced apoptosis, and the silencing of TMPRSS4 had similar effects. Furthermore, we observed that either the overexpression of miR-125a-5p or the silencing of TMPRSS4 prevented the activation of the nuclear factor (NF)-κB signaling pathway. On the whole, our findings illustrate that TMPRSS4 may be a candidate oncogene and may thus serve as a prognostic biomarker for LUAD, and its overexpression may be partly ascribed to the downregulation of miR-125a-5p. The dysregulation of miR-125a-5p and TMPRSS4 affect the biological function of LUAD cells via the NF-κB signaling pathway. The miR-125a-5p/TMPRSS4/NF-κB axis may thus provide novel insight into the pathogenic mechanisms of LUAD and may be used in the development of novel treatment strategies for LUAD.

179P The cytoplasmic LKB1 promotes the growth of human lung adenocarcinoma by enhancing autophagy

Background: Liver Kinase B1 (LKB1), as a tumor suppression gene, is associated with various kinds of cancers, including lung cancer. However, it is unclear whether LKB1 can be also located in the nucleus of NSCLC cells and if yes, what its function is during the development and progression of NSCLC. Methods: Full length LKB1 (LKB1L) and LKB1 without nuclear localization signal (LKB1S) were introduced into lung adenocarcinoma cell A549 respectively. MTT assays, colony formation assays, flow cytometry analysis, transmission electron microscopy and western blot were used to evaluate the effect of LKB1L and LKB1S in vitro. The subcutaneous tumor model in nude mice was used to evaluate the effect of LKB1L and LKB1S in vivo. The expression profile of LKB1 was examined in 190 lung adenocarcinoma tissues by immunohistochemistry and was analyzed combined with clinical parameters. Results: In this study, we found that cytoplasmic LKB1 promoted the growth of lung adenocarcinoma by enhancing autophagy and it was independent of the AMPK/mTOR signaling. In 190 lung adenocarcinoma samples, we found that LKB1 was expressed in both the nucleus and cytoplasm of lung adenocarcinoma cells and the levels of nuclear LKB1 were inversely associated the levels of cytoplasmic LKB1 in lung adenocarcinoma tissues. Furthermore, the different subcellular localizations of LKB1 were correlated with opposite clinical parameters in this population. While the cytoplasmic LKB1 expression was associated with poor differentiation and advanced TNM stage, the nuclear LKB1 expression was related to well differentiation and early TNM stage. Moreover, the cytoplasmic LKB1 was associated with poor OS in this population and was an independent risk factor for prognosis. Conclusions: In summary, our data demonstrated that the cytoplasmic LKB1 without nuclear localization signal promoted A549 cell survival in vitro and lung tumor growth in vivo. Furthermore, the cytoplasmic LKB1 was associated with poor prognosis in lung adenocarcinoma cancer. Therefore, our findings may provide new insights into the pathogenesis of lung adenocarcinoma and novel biomarkers for prognosis of lung adenocarcinoma. Legal entity responsible for the study: The First Affiliated Hospital of Xi'an Jiaotong University Funding: Has not received any funding Disclosure: All authors have declared no conflicts of interest.

A cassaine diterpene alkaloid, 3β-acetyl-nor-erythrophlamide, suppresses VEGF-induced angiogenesis and tumor growth via inhibiting eNOS activation.

Angiogenesis is one of the hallmarks of cancer, playing an essential role in tumor growth, invasion, and metastasis. 3β-Acetyl-nor-erythrophlamide (3-ANE), a cassaine diterpene alkaloid compound from Erythrophleum fordii, exerts various pharmacological effects, including antitumor activity. However, the effects of 3-ANE on tumor angiogenesis and its potential molecular mechanism are still unknown. Here, we demonstrated that 3-ANE inhibited the vascular endothelial growth factor (VEGF)-mediated proliferation, migration, invasion, and capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs), without inducing apoptosis. We also found that 3-ANE blocked angiogenesis in vivo, and suppressed tumor angiogenesis and human lung adenocarcinoma growth in the xenograft tumor model. Furthermore, we showed that 3-ANE blocked VEGF-mediated endothelial nitric oxide synthase (eNOS) phosphorylation, vascular permeability and NO production in HUVECs, via disrupting the VEGF-induced association of eNOS and heat-shock protein 90 (HSP90). Our studies therefore provide the first evidence that 3-ANE inhibits tumor angiogenesis by inhibiting the VEGF-mediated eNOS activation and NO production, and 3-ANE could be a potential candidate in angiogenesis-related disease therapy.

Down-regulation of lipocalin 2 suppresses the growth of human lung adenocarcinoma through oxidative stress involving Nrf2/HO-1 signaling.

Lipocalin 2 (LCN2), a multifunctional secretory protein known as neutrophil gelatinase-associated lipocalin (NGAL), is expressed in a variety of cancers. However, little is known about the biological functions of NGAL in the development of lung adenocarcinoma. In the present study, we primarily found that NGAL expression was up-regulated in human lung adenocarcinoma tissues. Additionally, depletion of NGAL expression decreased the ability of cell proliferation and induced cell apoptosis. Furthermore, with the addition of N-acetylcysteine, a scavenger of reactive oxygen species (ROS), it was found that NGAL depletion was sufficient to cause apoptosis of lung adenocarcinoma cells by generating ROS through the inhibition of the nuclear factor E2-related factor 2/heme oxygenase-1 anti-oxidant pathway. Finally, the effect of NGAL down-regulation on the growth of human lung adenocarcinoma was determined in BALB/c nude mice. These findings demonstrate that NGAL may be a potential therapy target for patients with lung adenocarcinoma.

New cyclopentaquinoline derivatives with fluorobenzoic acid induce G1 arrest and apoptosis in human lung adenocarcinoma cells

Non-small cell lung cancer accounts for 80–85% of all lung cancer cases and is the leading cause of cancer death indicating inefficient current treatment. Acridine derivatives interact with DNA and inhibit topoisomerase leading to cell growth arrest or cell death. The aim of this study was to evaluate the effects of new synthesized sixteen 2,3-dihydro-1H-cyclopenta[b]quinoline derivatives (cyclopentaquinoline), a member of acridine-based compounds, on the survival and growth of human lung adenocarcinoma, A549 cells. Anticancer activity of eight new cyclopentaquinoline derivatives with hydrazinonicotinic acid (compounds 1–8) and eight with fluorobenzoic acid (compounds 9–16) were screened using WST-1 assay. Interestingly, cyclopentaquinoline derivatives with fluorobenzoic moiety were found to have a higher anticancer activity than derivatives with hydrazinonicotinic acid. Four out of eight tested compounds with fluorobenzoic acid inhibited 50% cancer cell growth at concentration below 20μM. Moreover, the efficacy of cyclopentaquinoline derivatives containing fluorobenzoic acid correlated with increasing number of carbon atoms in the aliphatic chain. The most effective compounds (6, 15, 16) were selected to determine molecular mechanisms of their anticancer action. The results indicated that inhibition of A549 cell growth by compounds 15 and 16 was associated with a cell cycle arrest at G0/1 phase and with induction of caspase 3-dependent apoptosis. Compound 6 also caused A549 cells death due to apoptosis, however, it had no significant effect on a cell cycle progression. These findings suggest that cyclopentaquinoline derivatives containing fluorobenzoic acid with 8 and 9 carbon atoms in aliphatic chain may be promising candidate for treatment of lung cancer.

Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo

BackgroundThe human coagulation trigger tissue factor (TF) is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi) technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo.MethodsThe specific small interfering RNA (siRNA) designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated.ResultsTF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma.ConclusionsDown-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

Inhibition of human lung adenocarcinoma growth using survivint34a by low-dose systematic administration

Anti-apoptosis plays an important role in tumour formation and development. Survivin is a member of the inhibitor of apoptosis (IAP) family, which is a target for anti-cancer drug exploitation was replaced as development. We investigated the role of the homo dominant-negative mutant Survivin-T34A in suppressing human lung adenocarcinomas (A549). The anti-tumour activity of HSurvivinT34A plasmid was evaluated in the A549 cell line and nude mice bearing A549 subcutaneous tumours. Low-dose systemic administration was continuously used. The HSurvivinT34A plasmid (5 meu g/one) complexed with a cationic liposome (DOTAP/Chol) significantly inhibited tumour growth in our model. We observed microvessel density degradation by CD31 immunohistochemistry and apoptotic cell increase by TUNEL assay, PI staining and flow cytometric analysis in the treated group. The present findings suggest that the HSurvivinT34A plasmid complexed with a cationic liposome may provide an effective approach to inhibit the growth of human lung adenocarcinomas in vitro and in vivo.

IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

BackgroundNon small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas.Aim of the study was to investigate i) IL-12Rβ2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC.Methodology/Principal FindingsStage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rβ2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R+ neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/β2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/β2+ cells inhibited angiogenesis in vitro. Tumors formed by Calu6/β2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines.ConclusionsThis study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial.

Laminin 5 expression protects against anoikis at aerogenous spread and lepidic growth of human lung adenocarcinoma

Adenocarcinoma of the lung is characterized by frequent aerogenous spread (AE) and advancement along the alveolar wall (BAC growth). To elucidate the mechanism of AE metastasis and BAC growth in human lung adenocarcinoma, we established an in vivo orthotopic animal model and an in vitro culture. Investigation of expression levels of integrins, laminins and Type IV collagens, which are the major regulating molecules for cell attachment and anoikis was carried out and a clear correlation between the expression level of laminin 5 (LN5) and the BAC growth was observed using an orthotopic animal model. Introduction of LN5 cDNA to A549 cells increased anoikis resistance in an expression dependent manner. Cells with LN5 overexpression resisted with anoikis after treatment with PI3K-Akt and ERK inhibitors. The amount of phosphorylated focal adhesion kinase (FAK) was also higher in LN5 overexpressing cells. Major tyrosine residues of the EGF receptor at 1068, 1086 and 1173, except at 1148, remained phosphorylated only in the LN5 overexpressing cells even without EGF stimulation, that indicates the ligand independent activation of EGF receptor. BAC growth ratio and AE was confirmed to be significantly correlated with LN5 expression in surgically resected human lung adenocarcinomas by immunohistochemistry. Our results indicate that the activation of the EGF receptor by overexpressing LN5-integrin-FAK signaling pathway may play a crucial role in BAC growth and AE metastasis in human lung adenocarcinoma.

Adoptive immunotherapy using specific cytotoxic T lymphocytes against human lung-cancer-engrafted severe combined immunodeficiency mice.

We studied the ability of human lung-cancer-specific cytotoxic T lymphocytes to suppress the growth of human lung adenocarcinoma (PC-9) engrafted in severe combined immunodeficiency mice. PC-9-specific cytotoxic T lymphocytes were generated by multiple stimulation with irradiated PC-9 cells of regional lymph node lymphocytes from lung cancer patients expressing the same human leukocyte antigen-A locus haplotype as PC-9 following expansion due to the administration of immobilized anti cluster of differentiation 3 mAb and interleukin-2. Cytotoxic T lymphocytes showed specific cytotoxicity against PC-9 cells in vitro. Severe combined immunodeficiency mice with a subcutaneous graft of PC-9 were treated with a PC-9-specific cytotoxic T lymphocyte by i.v. injection and/or with interleukin-2 by s.c. injection. Cytotoxic T lymphocyte treatment suppressed PC-9 graft growth significantly an effect, significantly enhanced when combined with interleukin-2 injection. To evaluate the in vivo specificity of anti-PC-9 cytotoxic T lymphocytes, each mouse was subcutaneously inoculated in the right flank with PC-9, and in the left flank with A549 or Sq-1. Cytotoxic T lymphocytes plus interleukin-2 treatment was found to suppress PC-9 growth selectively, but not A549 or Sq-1 growth. These results provide sufficient rationale for conducting further clinical trials on immunotherapy using cytotoxic T lymphocyte for lung cancer patients.

Toxin gene-mediated growth inhibition of lung adenocarcinoma in an animal model of pleural malignancy.

Transduction of malignant cells with toxin genes provides a novel strategy by which to promote tumor cell destruction. Whereas the capacity of the toxin gene/prodrug combination cytosine deaminase/fluorocytosine to inhibit growth of human metastatic pulmonary adenocarcinoma cell lines in vitro is established, the in vivo efficacy of this binary system has not yet been determined. For the development of toxin gene therapy for the treatment of lung adenocarcinoma metastatic to the pleural space, a reliable, disease-specific model is required. The serosa of the rat small intestine resembles the basal lamina of the pleura and provides the basis for a more convenient model than direct injection of tumor into the pleural space. Adenocarcinoma cells are inoculated into everted denuded rat intestine configured as a sac. Immunocytochemical and histological analyses show rapid cell growth with characteristics that mimic nodular metastatic intrapleural disease. In the context of this model, systemically delivered fluorocytosine significantly inhibits the growth of cytosine deaminase-expressing human lung adenocarcinoma cells. The dosing schedule required 30 days; neither addition of an enzyme inhibitor that increases the half-life of fluorocytosine nor intralumenal drug delivery is effective in shortening (to 15 days) the protocol. We conclude that CD continues to hold promise as a toxin gene for lung adenocarcinoma gene therapy, and that prolonged prodrug administration may be required for maximum efficacy.