Human Pulmonary Adenocarcinoma Cell Line Research Articles

Antiproliferative Effects of Phytoestrogens Formononetin and Resveratrol Against Human Pulmonary Adenocarcinoma Cells A 549

Phytoestrogens are phytoalexins, plant secondary metabolites produced in response to microbial attacks and other forms of external stress stimuli. These compounds are devoid of the steroidal structure of endogenous estrogens but could bind with estrogen receptors with great affinity as they have structural similarity with endogenous/synthetic estrogens. Phytoestrogens are reported to possess several health benefits including cardio protective, antidiabetic, antioxidant and anti osteoporotic properties. Phytoestrogens like genistein, daidzein, and equol have been reported to possess excellent anti proliferative and anticancer properties. Formononetin, is a phytoestrogen, isoflavone widely present in red clover (Trifolium pratense) and resveratrol is a phytoestrogen which is widely present in red grapes (Vitis vinifera). The cardio protective property of red wine has been attributed to the presence of resveratrol. Although formononetin and resveratrol have been investigated extensively for several pharmacological properties, reports on their anti proliferative effects especially against human pulmonary carcinoma cell lines are very less. Hence the current study aims to understand the influence of these two compounds on the growth of human pulmonary adenocarcinoma cell line A 549. MTT assay, cytomorphology analysis by phase contrast microscopy, DNA topology assay and DPPH assay were few assays which were performed to understand the antiproliferative and antioxidant effects of these compounds. Results of the study indicated the potent antiproliferative effects of these compounds against human pulmonary carcinoma cell lines implicating that these compounds could be potential therapeutic molecules with multifaceted roles as anticancer agents, potent antioxidants and anti inflammatory agents.

Fusion with human lung cancer cells elongates the life span of human umbilical endothelial cells and enhances the anti-tumor immunity.

Human umbilical endothelial cells (HUVECs) have been proved as an effective whole-cell vaccine inhibiting tumor angiogenesis. However, HUVECs divide a very limited number of passages before entering replicative senescence, which limits its application for clinical situation. Here, we fused HUVECs with human pulmonary adenocarcinoma cell line A549s and investigated the anti-tumor immunity of the hybrids against mice Lewis lung cancer. HUVECs were fused with A549s using polyethylene glycol and were sorted by flow cytometry. The fusion cells (HUVEC-A549s) were confirmed by testing the expression of telomerase and VE-cadherin, the senescence-associated β-galactosidase activity, and tube formation ability. HUVEC-A549s were then irradiated and injected into the C57BL/6 mice of protective, therapeutic, and metastatic models. The mechanism of the anti-tumor immunity was explored by analyzing mice sera, spleen T lymphocytes, tumor microenvironment, and histological changes. HUVEC-A549s coexpressed tumor and endothelial markers and maintained the vascular function of tube forming at passage 30 without showing signs of senescence. HUVEC-A549s could induce protective and therapeutic anti-tumor activity for LL(2) model and presented stronger activity against metastasis than HUVECs. Both humoral and cellular immunity were participated in the anti-angiogenic activity, as HUVECs-neutralizing IgG and HUVECs-toxic lymphocytes were increased. Angiogenic mediators (VEGF and TGF-β) and tumor microenvironment cells MDSCs and Tregs were also diminished. Our findings might provide a novel strategy for HUVECs-related immunotherapy, and this vaccine requires lower culture condition than primary HUVECs while enhancing the anti-tumor immunity.

ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells.

Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30–40% corresponding to significant depletion (approximately 70–80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.

Effect of lentivirus-induced small interfering RNA targeted against toll-like receptor 3 on the proliferation and immune escape of human pulmonary adenocarcinoma cell line A549

Objective Toll-like receptors (TLR) are very important innate immunity molecules.Recently the relationship between TLR and human tumors had been gradually focused on by more and more researchers.This study was to investigate the effect of TLR3 on the cell proliferation,anti-apoptosis and immune escape in human pulmonary adenocarcinoma cells.Methods A549 cells were transfected with TLR3-RNA interference (RNAi) lentiviral vector,blank vector or non-transfected,then incubated with TLR3 ligand Poly (I∶ C).The expression of B7-H1,prostaglandin E synthase-2 (PGE-2) and phosphorylated p38 were respectively evaluated by flow cytometry,real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.Cells apoptosis was detected by flow cytometry with Annexin V fluoresceine isothiocyanate (FITC)/propidium iodide (PI) double staining.Results Under the same time and concentration stimulation with Poly (I∶ C),the percentage of B7-H1-positive cells,the expression of PGE-2 mRNA and the relative percentage of phosphoryated p38 in TLR3-RNAi lentiviral vector transfected A549 cells were significantly lower than those in the non-transfected and blank vector transfected A549 cells (P ＜ 0.01).The percentage of the apoptotic cells in TLR3-RNAi lentiviral vector transfected A549 cells [(44.40 ± 1.41) ％] was significantly higher than that in the non-transfected [(34.70 ± 0.89) ％] and blank vector transfected [(36.50 ± 1.12) ％] A549 cells (P ＜ 0.01).Conclusion Down-regulation of TLR3 could inhibit the expression of B7-H1,PGE-2 and phosphorylated p38 in A549 cells,and weaken the anti-apoptosis ability of A549 cells. Key words: Lung cancer; Toll-like receptor 3; Apoptosis

ZnO nanoparticles induced cytotoxicity on human pulmonary adenocarcinoma cell line LTEP-a-2

Novel nanoparticles (NPs) such as zinc oxide (ZnO) NPs are widely produced and applied in our daily lives at a rapid pace. Thus, the toxicity of ZnO NPs should be monitored as an important standard for environmental risk assessment. Here we assessed the in vitro cytotoxicity of ZnO NPs on human pulmonary adenocarcinoma cells LTEP-a-2 by tetrazolium salt colorimetric assay of cell proliferation in the presence or absence of ZnO NPs. ZnO NPs-induced morphological changes in LTEP-a-2 cells were examined by light and scanning electron microscopy. The mechanism by which ZnO NPs impose the cytotoxic effect was investigated by a combination of active oxygen test, lactose dehydrogenase-release assay, and apoptosis detection. Results showed that ZnO NPs significantly inhibited the proliferation and induced evident morphological changes (cell shrinkage and chromosome condensation) in LTEP-a-2 cells. Additionally, ZnO NPs increased the level of intracellular reactive oxygen species and induced the formation of apoptotic vesicles as well as the lysis of cell nuclei. Zn2+ ions released from ZnO NPs into aqueous solution are important components that exert cytotoxic effects on LTEP-a-2 cells. This study provides new insights to the cytotoxicity of ZnO NPs against human health.

Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells.

Background:Methyl protodioscin (MPD) is a furostanol bisglycoside with antitumor properties. It has been shown to reduce proliferation, cause cell cycle arrest.Objective:The present study elucidates the mechanism underlying MPD's apoptotic effects, using the A549 human lung cancer cell line.Materials and Methods:The human pulmonary adenocarcinoma cell line A549 was obtained from the Cell Bank of the Animal Experiment Center, North School Region, Sun Yat-Sen University. All of the cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), penicillin (10,000 U/l), and streptomycin (100 mg/l) at 37°C in a 5% CO2 humidified atmosphere. The induction of apoptosis was observed in flow cytometry and fluorescent staining experiments.Results:MPD showed growth inhibitory effects in A549 cells in a dose- and time-dependent manner. The significant G2/M cell cycle arrest and apoptotic effect were also seen in A549 cells treated with MPD. MPD-induced apoptosis was accompanied by a significant reduction of mitochondrial membrane potential, release of mitochondrial cytochrome c to cytosol, activation of caspase-3, downregulation of Bcl-2, p-Bad, and upregulation of Bax.Conclusion:Our results show that the induction of apoptosis by MPD involves multiple molecular pathways and strongly suggest that Bcl-2 family proteins signaling pathways. In addition, mitochondrial membrane potential, mitochondrial cytochrome c and caspase-3 were also closely associated with MPD-induced apoptotic process in human A549 cells.

Screening for transcription factors and their regulatory small molecules involved in regulating the functions of CL1-5 cancer cells under the effects of macrophage-conditioned medium

Many reports have inferred that macrophages can interact with tumor cells in the tumor microenvironment (TME) in a vicious cycle of tumor development; however, the changes in gene expression in tumor cells under the effects of macrophages are still largely unknown. The present study was carried out to illustrate the changes in the gene expression profile in lung cancer cells under the effects of macrophage-conditioned medium. Gene expression profile data were derived from the GEO database GSE9315. The GSM234968 sample was derived from a highly invasive human pulmonary adenocarcinoma cell line, CL1-5, and was treated with conditioned medium (supernatant of a culture solution of human monocyte THP-1). The GSM234967 sample that was not treated with the conditioned medium was used as a control. GO and KEGG enrichment analyses were carried out using DAVID software, and visualization networks were constructed using Cytoscape software. The results showed that 40 differentially expressed genes were annotated. Five differentially expressed transcription factors were identified, EIF2B4, EIF2B5, JUNB, GNG11 and HMGB2, which were all related to 'stress' and 'responses'. The gene cluster of JUNB was mainly enriched in cancer-related pathways, 'Wnt signaling pathway' and 'MAPK signaling pathway'. Finally, 10 small molecules, thioridazine, resveratrol, astemizole, ciclopirox, calmidazolium, etoposide, anisomycin, pyrvinium, azacyclonol and terfenadine, which may act on transcription factors, were identified using the CMap database. In conclusion, we identified transcription factors playing key roles in tumor cells under the effects of macrophages in order to provide new clues for blocking this vicious cycle of tumor development.

Establishment of a human lung cancer cell line with high metastatic potential to multiple organs: Gene expression associated with metastatic potential in human lung cancer

Convenient and reliable multiple organ metastasis model systems might contribute to understanding the mechanism(s) of metastasis of lung cancer, which may lead to overcoming metastasis and improvement in the treatment outcome of lung cancer. We isolated a highly metastatic subline, PC14HM, from the human pulmonary adenocarcinoma cell line, PC14, using an in vivo selection method. The expression of 34,580 genes was compared between PC14HM and parental PC14 by cDNA microarray analysis. Among the differentially expressed genes, expression of four genes in human lung cancer tissues and adjacent normal lung tissues were compared using real-time reverse transcription polymerase chain reaction. Although BALB/c nude mice inoculated with parental PC14 cells had few metastases, almost all mice inoculated with PC14HM cells developed metastases in multiple organs, including the lung, bone and adrenal gland, the same progression seen in human lung cancer. cDNA microarray analysis revealed that 981 genes were differentially (more than 3-fold) expressed between the two cell lines. Functional classification revealed that many of those genes were associated with cell growth, cell communication, development and transcription. Expression of three upregulated genes (HRB-2, HS3ST3A1 and RAB7) was higher in human cancer tissue compared to normal lung tissue, while expression of EDG1, which was downregulated, was lower in the cancer tissue compared to the normal lung. These results suggest that the newly established PC14HM cell line may provide a mouse model of widespread metastasis of lung cancer. This model system may provide insights into the key genetic determinants of widespread metastasis of lung cancer.

STAT3 activation and aberrant ligand-dependent sonic hedgehog signaling in human pulmonary adenocarcinoma

The Sonic hedgehog (SHH) signaling and STAT3 pathways play important roles during carcinogenesis with possible interaction. To determine the association of the activation of SHH signaling pathway and STAT3 pathway in carcinogenesis of human non-small cell lung carcinomas (NSCLC), firstly we examined the expression of SHH signaling molecules including SHH, Gli1(glioma-associated oncogene homolog 1) and HHIP (Hh interacting protein), as well as p-STAT3 (phosphorylation at Tyr705) by immunohistochemistry in 87 cases of NSCLC, 12 atypical adenomatous hyperplasia (AAH) and 20 adjacent normal lung tissues. The expression of SHH, Gli1, HHIP and p-STAT3 was detected respectively in 87/87(100%), 74/87(85.1%), 75/87(86.2%) and 54/87(62.1%) of the NSCLC cases, but not in the adjacent normal lung parenchyma. Ligand-dependent SHH pathway activation and STAT3 signaling activation were observed in most cases of NSCLC, and the high SHH pathway activation rate and STAT3 activation rate were significantly higher in adenocarcinoma compared with squamous cell carcinomas and large cell carcinomas (P<0.05). Both SHH and STAT3 pathway activation level correlated with histological grade in adenocarcinoma, being higher in well-differentiated types (P<0.05). Furthermore, high SHH pathway activation and p-STAT3 expression were also detected in 10/12(83.3%) of AAH cases as well as in most cases of early-stage adenocarcinoma — adenocarcinoma in situ (AIS) and minimally invasive adenocarcinomas (MIA). Correlation analysis showed that p-STAT3 protein expression level was correlated positively with ligand-dependent activation level of SHH signaling in adenocarcinoma (rs=0.585, P=0.000) and AAH (rs=0.996, P=0.000). We speculated that activation of STAT3 could up-regulate SHH gene expression directly or indirectly, and thereby activated SHH signaling resulting in lung tumor cell ontogeny. To explore the interactional mechanism, we then performed serial transient co-transfection assay in human pulmonary adenocarcinoma cell line H441 cells, and the results confirmed STAT3 activation can up-regulate SHH gene expression indirectly. In conclusion, aberrant ligand-dependent SHH signaling activation occurs frequently in NSCLC. The signaling plays a more active role in adenocarcinoma, and is an early event in carcinogenesis of lung adenocarcinoma. The involvement of STAT3 pathway activation might function in inducing the process.

Effects of antidiabetic drug metformin on the migration and invasion abilities of human pulmonary adenocarcinoma A549 cell line in vitro.

There is growing evidence that metformin, a clinically widely used drug in the treatment of type II diabetes, may impede the growth of human tumors. However in a recent study it was found that metformin treatment might result in promotion of the angiogenic phenotype and promote early tumorigenic progression. In order to evaluate the relevance between metformin and tumor metastases, we investigated the effects of metformin on the migration and invasion abilities of human pulmonary adenocarcinoma cell line A549 in vitro and explored the possible underlying mechanisms. A549 cells were treated with 0.5 mmol/L, 2 mmol/L and 8 mmol/L metformin for 72h. The laterad-migration and invasion abilities of the cells in vitro were evaluated by scratch assay and Boyden-Chamber assay, respectively. Expressions of MMP2 and MMP9 mRNA of the cells before and after metformin treatment were measured by Real-Time PCR. The migration rate of A549 cells was increased after metformin treatment at the concentration of 8mmol/L. The invasion ability was also significantly increased from 37.4±4.6 to 59.8±7.2(P<0.05) by 8mmol/L metformin treatment. No significant difference of the migration and invasion abilities was observed between the Group 0.5mmol/L, 2mmol/L and the Control. The expressions of MMP2 and MMP9 mRNA were both up-regulated after metformin treatment, while in the 8mmol/L Group the genes changes were the most significant. Metformin can increase the migration speed and enhance invasion abilities of A549 cells in vitro, which may be attributed to the up-regulation of MMP2 and MMP9.

Enhancing Radiosensitivity of Human Pulmonary Adenocarcinoma Cell Line A549 by CpG ODN1826

Preclinical and early clinical trials indicate that synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) have potent immunostimulatory effects and can enhance the anticancer activity of a variety of cancer treatments. In this study, CpG ODN1826 was used to increase the radiosensitivity of human pulmonary adenocarcinoma cell line A549, and some underlying mechanisms were also detected. With the treatment of CpG ODN on A549 cells, there was an upregulation of CD40, CD80, CD86, and MHC-II on A549 cells in association with Th1 cytokine production. Further, the protein level of toll-like receptor (TLR)-4 was decreased, whereas TLR-9 was enhanced. Flow cytometry results showed that CpG ODN could increase mitochondrial membrane potential and subsequently promote the apoptosis of A549 cells, resulting in increased radiosensitivity. These results suggest a regulatory role of CpG ODN1826 in enhancing the radiosensitivity of A549 cells by activating the adaptive and innate immune responses, thereby providing a new potential strategy to the treatment of malignant tumors.

Empirical study on chemotherapeutic susceptibility of SP cells in human pulmonary adenocarcinoma cell line A549 in Vitro

Objective To explore the chemotherapeutic susceptibility of SP cells in human pulmonary adenocarcinoma cell line A549 and the possible mechanism of multidrug resistance

Study of regulatory effect of insulin on expression of epithelial sodium channel alpha subunit in A549 cells

To observe the effects of insulin on the expression of epithelial sodium channel alpha subunit (ENaC-alpha) in human pulmonary adenocarcinoma cell line (A549 cells) and its impact on apoptosis of the cells. A549 cells were cultured with insulin in a concentration of 30 U/L for 30 minutes or 60 minutes. Then ENaC-alpha, serum and glucocorticoid-inducible kinase-1 (SGK-1) protein and mRNA were determined with Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). Then chelidonii, a blocking agent of protein kinase C (PKC), which could inhibit the effect of SGK-1, was added to the cells culture for 60 minutes, and ENaC-alpha protein was then determined. Cells were cultured in a hypoxia circumstance for 30 minutes, to induce apoptosis, the rate of which was detected with terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), in cells cultured with or without insulin. Insulin could up-regulate the protein and mRNA expression of ENaC-alpha and SGK-1 in A549 cells, and there was a significant difference at 30 minutes, and it gradually increased with prolongation of time. When A549 cells were cultured without insulin, the protein expression of A549 cells was 100%. The protein expression of ENaC-alpha and SGK-1 was 124% and 135% at 30 minutes after being cultured with insulin, and 186% and 176% at 60 minutes, respectively (all P<0.05). Insulin also could inhibit apoptosis which was induced by hypoxia (10.3% vs. 21.6%, P<0.05). Insulin can up-regulate expression of the ENaC-alpha in A549 cells via PKC and SGK-1 pathways, also insulin can inhibit hypoxia induced apoptosis. So insulin can be beneficial in the prognosis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS).

Comparison Study of the Lethal Effects of Nonfocused Ultrasound on Human Pulmonary Adenocarcinoma Cell Line (GLC-82, LLC) and Immortalization Bronchus Epithelial Cell Line (HBE) in Suspension

Objective: To explore the universality and difference in the lethal effects of non-focused ultrasound (NFU) on malignant cells in suspension and provide new evidence for the therapeutic regimen on hydrothorax and ascites from cancer. Patients and Methods: We took the pulmonary adenocarcinoma cell line (GLC-82, LLC) and bronchus epithelial cell line (HBE) as the experimental subject and divided into GLC-82, LLC and HBE 3 cell groups, The NFU dosage used was 30 W/cm2 × 5s. Then we tested and compared the instant and 24-hour survival rates, growth rates, and apoptosis rates after 24 hours under 0 C and 37 C, respectively. Instant and 24-hour survival rates were determined based on the trypan blue exclusive assay, growth rates were detected by MTT after 24 hours, and the percentage changes of apoptosis were analyzed with flow cytometry. Results: After NFU performance, there is no significant difference on instant and 24-hour survival rates, growth rates, and apoptosis rates among these three groups, whereas 24-hour distributional percentage of cell cycle is. Compared with the GLC-82 group and HBE group, the percent of cell number in group 1 increased in LLC group, but the percentage of cell number in group 1 decreased in LLC group. There is significant difference on survival rates, growth rates, and apoptosis rates among the control group, NFU0°C group, and the NFU37 C group. In other words, the survival rates and growth rates of the NFU0 C group and NFU37 C group are lower than that of the control group, and the NFU37°C group is lower than the NFU0 C group. However, the apoptosis rate of the NFU0°C group and NFU37 C group is higher than that of the control group, and the NFU37 C group is higher than the NFU0 C group slightly. However, there is no significant difference on 24-hour distributional percentage of cell cycle among these three treatments. Conclusion: NFU has a lethal effect on GLC-82, LLC and HBE in suspensions, which shows a general significance; the effectiveness, which is better on 37 C, is temperature related. The lethal effect of NFU on cancer cells in suspensions is not only transient but also persistent.

Aression of TLR9 in human pulmonary adenocarcinoma cell line A549

Objective Being considered as a bridge between the innate immunity and acquired immunity, Toll-like receptors (TRLs) are very important innate immunity moleculars. Recent researchs on the innate immunity have focused on the relationship between TLRs and human tumor. This paper investgated the expression and significance of TLR9 in human pulmonary adenocarcinoma cell (A549 cell) and human bronchial epithelial cell (HBE cell).

Novel Ras pathway inhibitor induces apoptosis and growth inhibition of K-ras-mutated cancer cells in vitro and in vivo

MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind.

Fas Ligand Released by Activated Monocytes Causes Apoptosis of Lung Epithelial Cells in Human Acute Lung Injury Model in Vitro

Alveolar epithelial cell death plays a crucial role in the progression of acute lung injury. We have demonstrated up-regulation of Fas expression on alveolar epithelial cells, and soluble Fas ligand secretion from inflammatory cells upon acute lung injury. Here we show that the lipopolysaccharide-stimulated human monocyte cell line THP-1 releases Fas ligand, and that conditioned medium from lipopolysaccharide-stimulated THP-1 cells induces apoptosis of the human pulmonary adenocarcinoma cell line A549. Activation of caspase-3 and -8 is associated with the apoptosis. Gene targeting on Fas in A549 cells by specific small interfering RNA impairs apoptosis induced by conditioned medium from activated THP-1, while that on Fas ligand in THP-1 cells impairs the apoptosis-inducing activity of the conditioned medium produced by lipopolysaccharide-stimulated cells. These results suggest that Fas ligand released by monocytes causes alveolar epithelial cell death through a Fas-dependent apoptotic mechanism in the development of acute lung injury.

Overexpressed Raf‐1 and phosphorylated cyclic adenosine 3′‐5′‐monophosphatate response element‐binding protein are early markers for lung adenocarcinoma

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer and has a high mortality. The tobacco carcinogen nicotine-derived nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) stimulates the proliferation of human PAC cells and small airway epithelial cells through beta-1 adrenorecptor-mediated transactivation of the epidermal growth factor receptor (EGFR). Using the NNK hamster PAC model and human PAC tissue arrays with matched and unmatched normal lung tissues, the authors tested the hypothesis that Raf-1, an effector of the EGFR, and P-CREB, an effector of the beta-adrenoreceptor, are overexpressed in a significant subset of human PACs and are early markers of PAC development. Western blots from respiratory epithelial cells and microadenomas harvested by laser-capture microdissection from hamster lungs accompanied by immunostains were used to monitor the expression levels of Raf-1 and P-CREB after 5 weeks, 10 weeks, and 20 weeks of NNK treatment. Expression levels of these markers in human PAC tissue arrays were assessed by immunostains. Reverse-phase proteomics, Western blot analysis, and immunoprecipitation in immortalized human small-airway epithelial cells and in a human PAC cell line in the presence and absence of dominant-negative Raf were used to determine Raf dependence of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation in response to NNK or isoproterenol. The data showed a time-dependent increase in the expression of Raf-1 and P-CREB after NNK treatment in small-airway epithelial cells and microadenomas of hamsters. The majority of human lung adenocarcinomas simultaneously overexpressed Raf-1 and P-CREB. Dominant-negative Raf completely abrogated ERK1/2 activation by NNK and isoproterenol. The current results indicated that RAF-1 and P-CREB may contribute to the development of a significant subset of human lung adenocarcinomas and may offer promising targets for early detection and treatment.

Inhibition of expression of vascular endothelial growth factor and its receptors in pulmonary adenocarcinoma cell by TNP-470 in combination with gemcitabine

Angiogenesis is required for solid tumor growth and facilitates tumor progression and metastasis. The inhibition effects of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), an angiogenesis inhibitor, and gemcitabine, a chemotherapeutic agent, on expression of growth factors were investigated using human pulmonary adenocarcinoma cell line, A549. The A549 cells were divided into four groups: control group, 10(-6) mg/ml gemcitabine treated group, 10(-4) mg/ml TNP-470 treated group and gemcitabine+TNP-470 treated group. The mRNA and protein expression of vascular endothelial growth factor (VEGF) and its receptors, FMS-like tyrosine kinase-1 (FLT-1) and kinase insert domain-containing receptor (KDR), in different groups were measured. The growth of A549 cell cultured with gemcitabine or TNP-470 was inhibited in an almost dose-dependent manner. Although gemcitabine (10(-6) mg/ml) alone and TNP-470 (10(-4) mg/ml) alone had no effect on the mRNA and protein expression of VEGF and its receptors (FLT-1, KDR) in A549 cells compared to the control (P>0.05), 10(-6) mg/ml gemcitabine in combination with 10(-4) mg/ml TNP-470 had significant effect (P<0.01). Moreover, combination of the two drugs significantly inhibited the mRNA expression of VEGF, FLT-1 and KDR compared to either drug alone (P<0.05). This study suggests that combined treatment with TNP-470 plus gemcitabine may augment the antiangiogenic and antineoplastic effects in lung cancer cells in vitro.

Pulmonary fibroblasts stimulate the proliferation of cell lines from human lung adenocarcinomas

Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.