Editorial Expression of Concern: n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2α and telomerase activity.

Bone is a common site for metastasis in lung adenocarcinoma, but the mechanism behind lung adenocarcinoma bone metastasis is still unclear. And currently, there is a lack of easily traceable and stable lung adenocarcinoma bone metastasis cell models, which limits the research on the mechanism of lung adenocarcinoma bone metastasis. The establishment of human lung adenocarcinoma cell line that are highly metastatic to bone, labeled with green fluorescent proteins (GFP) and fireflies luciferase (LUC), along with transcriptomic characterization, would be beneficial for research on lung adenocarcinoma bone metastasis and provide new experimental methods. The human lung adenocarcinoma cell line A549-GFP-LUC was injected into nude mice via the left ventricle to construct a bone metastasis model, and was domesticated in vivo for three consecutive times to obtain the human high bone metastasis lung adenocarcinoma cell line A549-GFP-LUC-BM3; cell counting kit-8 (CCK-8), colony formation assay, scratch wound assays, Transwell assay and Western blot were used to compare the proliferation and invasion abilities of A549-GFP-LUC-BM3 with the parental cells. A549-GFP-LUC-BM3 cells and parental cells were further analyzed by transcriptomic sequencing. Human high-bone metastatic lung adenocarcinoma cells A549-GFP-LUC-BM3 was successfully established. Compared to parental cells, this cells exhibited a significantly higher incidence of bone metastasis and enhanced in vitro proliferation, migration, and invasion abilities. Transcriptomic sequencing results revealed that the A549-GFP-LUC-BM3 cell line had 2954 differentially expressed genes compared to the parental cells, with 1021 genes up-regulated and 1933 genes down-regulated. Gene Ontology (GO) functional enrichment analysis indicated that the differentially expressed genes were primarily localized in cellular components such as the cell periphery. The molecular functions identified as significantly enriched included signaling receptor activity, calcium ion binding, and extracellular matrix structural constituent. Additionally, the biological processes found to be enriched were cell adhesion and biological adhesion. The enrichment analysis conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the differentially expressed genes were primarily involved in the metabolism of xenobiotics by cytochrome P450, retinol metabolism, drug metabolism-cytochrome P450, cell adhesion molecules, steroid hormone biosynthesis, and the nuclear factor kappa B (NF-κB) signaling pathway. The highly bone-metastatic human lung adenocarcinoma cell line with GFP and luciferase double labeling was successfully established. The biological behavior and transcriptome sequencing of the cell line suggest that it has a high bone-metastatic potential.

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1(N17)) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.

This study was aimed at identifying the molecular mechanisms by which ceramide inhibits telomerase activity in the A549 human lung adenocarcinoma cell line. C(6)-ceramide (20 microm) caused a significant reduction of telomerase activity at 24 h as detected using the telomeric repeat amplification protocol, and this inhibition correlated with decreased telomerase reverse transcriptase (hTERT) protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses showed that C(6)-ceramide significantly decreased hTERT mRNA in a time-dependent manner. Electrophoretic mobility shift and supershift assays demonstrated that the binding activity of c-Myc transcription factor to the E-box sequence on the hTERT promoter was inhibited in response to C(6)-ceramide at 24 h. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box deleted sequences cloned upstream of a luciferase reporter gene. Further analysis using RT-PCR and Western blotting showed that c-Myc protein but not its mRNA levels were decreased in response to C(6)-ceramide at 24 h. The effects of ceramide on the c-Myc protein were shown to be due to a reduction in half-life via increased ubiquitination. Similar results were obtained by increased endogenous ceramide levels in response to nontoxic concentrations of daunorubicin, resulting in the inhibition of telomerase and c-Myc activities. Furthermore, the elevation of endogenous ceramide by overexpression of bacterial sphingomyelinase after transient transfections also induced the inhibition of telomerase activity with concomitant decreased hTERT and c-Myc protein levels. Taken together, these results show for the first time that both exogenous and endogenous ceramides mediate the modulation of telomerase activity via decreased hTERT promoter activity caused by rapid proteolysis of the ubiquitin-conjugated c-Myc transcription factor.

Objective To study the killing effects of corilagin on human lung adenocarcinoma cell line A549.Methods Cell culture,methyl thiazol tetrazolium (MTT) assay,Western blotting and flow cytometry were used to detect the cell cycle,apoptosis,Notch signaling pathway proteins of human lung adenocarcinoma cell line A549 treated with corilagin.Results 35 μmol/L corilagin could inhibit the proliferation of human lung adenocarcinoma cell line A549.20 μmol/L corilagin increased early apoptosis from 2.0％ to 5.4％,and late apoptosis from 3.1％ to 12.8％.40 μmol/L corilagin increased early apoptosis to 7.0％ and late apoptosis to 13.7％.Corilagin could inhibit the expression of Notch1,NICD and mammalian target of rapamycin (mTOR) protein in A549 cells.Conclusion Corilagin could inhibit the proliferation of human lung adenocarcinoma cell line A549 and induce apoptosis possibly through the Notch signaling pathway. Key words: Corilagin; Lung adenocarcinoma; Notch signaling pathway

The aim of the present study was to assess the expression of microRNA-146a (miR-146a) in human lung adenocarcinoma cells, its effect on cellular behaviors, and the underlying molecular mechanisms. Reverse transcription-quantitative PCR (RT-qPCR) was used to measure miR-146a expression in the human normal lung epithelial cell line, BEAS-2B, and human lung adenocarcinoma cell lines, A549, PC-9 and H1299, to determine whether miR-146a acts as an oncogene or anti-oncogene. miR-146a mimics were transfected into target cells to observe the proliferation, apoptosis, invasion and migration of human lung adenocarcinoma cells. The target genes of miR-146a were predicted using bioinformatics analysis, and binding sites were validated by dual-luciferase reporter assay. Target gene expression at the mRNA and protein levels was measured by RT-qPCR and western blot analysis, respectively. The expression levels of miR-146a in human lung adenocarcinoma cell lines were lower than its expression in BEAS-2B (P<0.01). A549 cell line is a EGFR wild-type lung adenocarcinoma cell line, which is also the most widely studied in NSCLC, and therefore this was chosen as the target cell line for further investigation. Overexpression of miR-146a in A549 cells can inhibit cell proliferation (P<0.05), promote apoptosis (P<0.05), and reduce the cells' migratory ability (P<0.01). Bioinformatics prediction indicated that interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6) are the target genes of miR-146a. Dual-luciferase reporter assay showed that miR-146a could specifically bind to 3′-untranslated regions of IRAK1 and TRAF6. The protein and mRNA levels of IRAK1 and TRAF6 were significantly downregulated after miR-146a overexpression in A549 cells (P<0.01). The results of this study demonstrated that the expression of miR-146a in human lung adenocarcinoma cells was significantly lower than in normal lung epithelial cells, indicating that miR-146a acts as an anti-oncogene. miR-146a suppresses the proliferation and migration of human lung adenocarcinoma cells by downregulating the expression of IRAK1 and TRAF6.

Aurora kinase A (AURKA) is an oncogenic serine/threonine kinase, it plays important roles in tumorigenesis and chemoresistance. In this study, we investigated the expression of AURKA in lung adenocarcinoma tissues, the role of small interference RNA targeting AURKA on growth, cell cycle, and apoptosis of lung adenocarcinoma cell lines invitro. The AURKA is highly expressed in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines. Lentivirus-mediated short hairpin RNA (shRNA) was used to knock down AURKA expression in human lung adenocarcinoma cell lines H1299 and A549. The results indicated that depletion of AURKA could inhibit cell growth, cause cell cycle arrest and apoptosis. The potential mechanisms of AURKA inhibition induced cell cycle arrest and apoptosis are associated with downregulated RAF-1, CCND2, CCND3, CDK4, PAK4, EGFR and upregulated WEE1 expression. Furthermore, AURKA knockdown cooperated with vincristine (VCR) to repress A549 cell proliferation. Therefore, AURKA plays important roles in the proliferation of human lung adenocarcinoma cells, which suggests that AURKA could be a promising tool for lung adenocarcinoma therapy.

ROS mediated apoptosis and cell cycle arrest in human lung adenocarcinoma cell line by silver nanoparticles synthesized using Swietenia macrophylla seed extract

To observe the effects of photodynamic therapy (PDT) on human lung adenocarcinoma cell line A549 in vitro. Human lung adenocarcinoma cell line A549 was used to investigate the killing role of PDT with hematoporphyrin derivative (HPD) as photosensitizer and semiconductor successive laser as light source. The cultured cell was treated with different concentrations of HPD and different do-sages of laser, then MTT colorometric assay was applied to measure the OD492. 5 mg/L of HPD was confirmed no effect. 10 mg/L of HPD could produce a marked therapeutical effect under the condition of same laser dosage. When the concentration of HPD was increased, the effect was not risen obviously. 10 J/cm² of laser could lead OD492 to decrease to platform under the condition of same HPD concentration. So the killing effect was saturated when the PDT parameters were selected as 10 mg/L of HPD and 10 J/cm² of laser. Under the same laser capacity density of 10 J/cm², A549 cells were exposured to three different duty-time fabrications which were 214 mw×10 min, 428 mw×5 min and 714 mw×3 min.The outcomes showed that OD492 had no statistical difference. PDT has a significant killing effect on human lung adenocarcinoma cell line A549. 10 mg/L of HPD and 10 J/cm² of laser were the best parameters of this experiment. The PDT effect is not influenced by different duty-time fabrications under the best laser capacity density.

To determine the effect of exogenous GM3 on proliferation, apoptosis and VEGF expression in human lung adenocarcinoma cell line A549 cells. A549 cells were treated with GM3 at different concentrations for 48 hours. MTT assay was used to detect the cell proliferation and flow cytometry was applied to analyze cell apoptosis. RT-PCR was used to detect the expression level of VEGF mRNA and confocal laser scanning microscopy was applied to observe the localization and fluorescence intensity of VEGF. Comparing with the control, being treated with higher than 10 µmol/L GM3 significantly inhibited A549 cell proliferation (P < 0.05), and the suppressive effect could be enhanced following increasing doses. The IC(50) was 412 µmol/L. Comparing with the control, being treated with higher than 40 µmol/L GM3 significantly promoted the apoptotic rate of A549 cells (P < 0.05). Comparing with the control, being treated with higher than 40 µmol/L GM3 significantly decreased the VEGF mRNA level of A549 cells (P < 0.05), and the fluorescence intensity of VEGF distinctly weakened. Exogenous ganglioside GM3 can inhibit the proliferation, promote apoptosis, and down-regulate the VEGF expression level in A549 cells. This may be considered as two mechanisms of GM3 for its anti-tumor effect by modulating cell apoptosis and angiogenesis.

p90 ribosomal S6 kinase (p90RSK) constitutes a family of serine/threonine kinases that have been shown to be involved in cell proliferation of various malignancies via direct or indirect effects on the cell-cycle machinery. We investigated the role of p90RSK in lung adenocarcinomas and whether the inhibition of p90RSK diminishes cancer progression. Moreover, we investigated the involvement of glycogen synthase kinase-3β (GSK-3β) and osteopontin (OPN) in the p90RSK-induced lung adenocarcinoma progression. p90RSK, OPN, and GSK-3β protein expressions were examined in the A549 human lung adenocarcinoma cell line in the presence and absence of BI-D1870 (BID), a p90RSK inhibitor. Gene expression of anti-apoptotic and pro-apoptotic markers namely Bcl2 and Bax, respectively, were studied by reverse transcription polymerase chain reaction. In addition, the A549 lung adenocarcinoma cell line was characterized for cell proliferation using the MTT assay and cell migration using the scratch migration assay. Our study revealed that total RSK1 protein expression is over expressed in the A549 human lung adenocarcinoma cell line, an effect which is significantly reduced upon pretreatment with BID (69.32±12.41% of control; P<0.05). The inhibition of p90RSK also showed a significant suppression of cell proliferation (54.3±6.73% of control; P<0.01) and cell migration (187.90±16.10% of control; P<0.01). Treatment of the A549 cells with BID regressed the expression of Bcl2 mRNA (56.92±6.07% of control; P<0.01). BID also regressed protein expression of OPN (79.57±5.32% of control; P<0.05) and phospho-GSK-3β (73.04±8.95% of control; P<0.05). The p90RSK has an essential role in promoting tumor growth and proliferation in non-small cell lung cancer (NSCLC). BID may serve as an alternative cancer treatment in NSCLC.

To investigate the role of hTERT gene expression and AP-2alpha in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells. Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining. The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BP-induced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2alpha. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT. The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.

Mitochondria play important roles in the cellular response to various types of stress, including that triggered by ionizing radiation. We have previously reported that the mitochondrial ribosomal protein death-associated protein 3 (DAP3) regulates the radioresistance of human lung adenocarcinoma (LUAD) cell lines A549 and H1299. However, the underlying mechanism of this regulation remains to be elucidated. To this end, we have herein investigated the role of DAP3 in the cell cycle regulation after irradiation. Notably, the DAP3 knockdown attenuated the radiation-induced increase of the G2/M cell population. Furthermore, western blotting analysis has revealed that the DAP3 knockdown decreased the expression of proteins related to the G2/M arrest, such as those of the phosphorylated cdc2 (Tyr15) and the phosphorylated checkpoint kinase 1 (Ser296), in irradiated A549 cells and H1299 cells. Moreover, by using a chk1 inhibitor, we were able to demonstrate that chk1 is involved in the radiation-induced G2/M arrest in both A549 and H1299 cells. Notably, the chk1 inhibitor was able to enhance the radiosensitivity of H1299 cells, while both chk1 inhibitor-abolished G2 arrest and inhibition of chk2-mediated events such as downregulation of radiation-induced p21 expression were required for enhancing radiosensitivity of A549 cells. Collectively, our findings reveal a novel role of DAP3 to regulate G2/M arrest through pchk1 in irradiated LUAD cells and suggest that chk1-mediated G2/M arrest regulates the radioresistance of H1299 cells, whereas both the chk1-mediated G2/M arrest and the chk2-mediated events contribute to the radioresistance of A549 cells.

p53 gene is the most commonly mutated gene in lung cancer. p53 mutation results in insensitivity of cells when exposed to chemotherapy. It has been reported that adenovirus-mediated wild-type p53 gene transfection into lung cancer cells can enhance the cytotoxic effect of anti-cancer drugs. The aim of this study is to evaluate the effects of domestic recombinant adenovirus-p53 (Ad-p53, Gendicine) on growth and chemosensitivity of human lung adenocarcinoma cell lines. Human lung adenocarcinoma cell lines GLC-82 (including mutant p53) and A549 (including wild-type p53) were treated with Ad-p53, cisplatin (DDP) or Ad-p53+DDP respectively. p53 expression was detected by Western blot. The cell growth inhibition was assessed by MTT, and cell cycle and apoptosis were detected by flow cytometry. High-level p53 expression was detected in Ad-p53 infected GLC-82 and A549 cells by Western blot. There was a dose-dependent and time-dependent inhibition of cell proliferation by Ad-p53. After combined treatment with Ad-p53 (100MOI) and DDP (0.5mg/L) for 72h, the growth inhibition rate of A549 cells was 43.13%±0.72%, which was significantly higher than that in Ad-p53 group ( 23.44%±0.54%, P < 0.001) and DDP group (14.17%±1.39%, P < 0.001); and the growth inhibition rate of GLC-82 cells was 63.73%±0.92%, which was significantly higher than that in Ad-p53 group ( 41.51%±0.59%, P < 0.001) and DDP group (56.11%±1.12%, P < 0.001). Combined administration of Ad-p53 and DDP remarkably arrested A549 and GLC-82 cells in G0-G1, and cells in S phase significantly decreased. Meanwhile the apoptotic rate of A549 cells was 28.99%±1.07% in Ad-p53+DDP group, which was significantly higher than that in Ad-p53 group (15.35%±1.31%, P < 0.001) and DDP group (1.74%±0.77%, P < 0.001). The apoptotic rate of GLC-82 cells was 62.98%±2.43% in Ad-p53+DDP group, which was significantly higher than that in Ad-p53 group (20.88%±0.71%, P < 0.001) and DDP group (6.91%±1.52%, P < 0.001). Ad-p53 (Gendicine) can inhibit the growth of human lung adenocarcinoma cell lines irrespective of the status of endogenous p53 gene. Its combination with DDP may significantly enhance the chemosensitivity of human lung adenocarcinoma cells to DDP.

The vasoactive intestinal peptide receptor-1(VIPR1) has prominent growth effects on a number of common neoplasms. However, there were contradictions in the effect cross different cancers. We aimed to explore the effect of VIPR1 overexpression on a human lung adenocarcinoma cell line H1299. GEO dataset was used to screen differentially expressed genes in lung adenocarcinoma tissues. The expression of VIPR1 mRNA was determined in the cancer Genome Atlas (TCGA). Immunohistochemical analysis was performed to determine VIPR1 protein expression in lung adenocarcinoma and corresponding adjacent tissues (n = 22). Fluorescence real-time quantitative PCR detected the expression of VIPR1 in human normal lung epithelial cell line BEAS-2B and lung adenocarcinoma cell line H1299. Overexpression strategies were employed to assess functions of VIPR1 expression on several malignant phenotypes in H1299. The expression of VIPR1 was lower in lung adenocarcinoma tissues than that in adjacent tissues. Compared with the normal lung epithelial cells BEAS-2B, VIPR1 was down-regulated in lung cancer cells H1299 (P < 0.05). After the overexpression of VIPR1, we found that VIPR1 significantly inhibited growth, migration, and invasion of H1299 cells (P < 0.05). Our findings point out the tumor suppressor roles of VIPR1 in human LUAD pathogenesis.

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor 3H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.