Migration Ability Of A549 Cells Research Articles

Endoplasmic reticulum stress mediates nickel chloride-induced epithelial‑mesenchymal transition and migration of human lung cancer A549 cells through Smad2/3 and p38 MAPK activation

BackgroundThe endoplasmic reticulum (ER) is a cellular membrane-bound organelle whereby proteins are synthesized, folded and glycosylated. Due to intrinsic (e.g., genetic) and extrinsic (e.g., environmental stressors) perturbations, ER proteostasis can be deregulated within cells which triggers unfolded protein response (UPR) as an adaptive stress response that may impact the migration and invasion properties of cancer cells. However, the mechanisms underlying the nickel compounds on lung cancer cell migration and invasion remain uncertain. ObjectiveWe aimed to study whether Nickel chloride (NiCl2) induces ER stress in lung cancer cells, and whether ER stress is involved in modulating epithelial-mesenchymal transition (EMT) and migration by Smads and MAPKs pathways activation following NiCl2 treatment. MethodsA549 cells were treated with NiCl2 to determine the cell viability using MTT assay. The wound healing assay was used to evaluate cell migration ability. ER ultrastructure was observed by transmission electron microscopy. Western blotting assay was performed to evaluate the protein levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 for ER stress and UPR, E-cadherin and Vimentin for EMT, p-Smad2/3, p-ERK, p-JNK, and p-P38 for activation of Smads and MAPKs signaling pathways. ResultsThe expression levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 were significantly increased following treatment with NiCl2 in time- and dose-effect relationship. The ER stress inhibitor 4-PBA downregulated the expression levels of the above five proteins, and reversed the decrease in E-cadherin protein level and the increase in vimentin protein expression and cell migration abilities caused by NiCl2. Furthermore, 4-PBA significantly reduced nickel chloride-induced Smad2/3 and p38 MAPK pathway activation, while not affected ERK and JNK MAPK pathways. ConclusionNiCl2 triggers ER stress and UPR in A549 cells. Moreover, 4-PBA alleviates NiCl2-induced EMT and migration ability of A549 cells possibly through the Smad2/3 and p38 MAPK pathways activation, rather than ERK and JNK MAPK pathways.

Inhibitory role of puerarin on the A549 lung cancer cell line.

Although more and more drugs had been proved to be effective in controlling tumor cells, lung cancer was still the leading cause of cancer-related deaths all over the world. This study aimed to investigate the effect and mechanism of puerarin on the invasion and metastasis of A549 lung cancer cell line. A medium containing puerarin was prepared according to the gradient concentration, and 10, 20, and 40 µmol/L were selected as the experimental group (low, medium, and high concentration groups, respectively) according to the cytotoxicity experiment. Meanwhile, 0 µmol/L was used as the control group. Following administration, metastasis-related indexes were detected by the cell scratch test, cell migration test, gene difference detection, and western blotting. 24 hours after administration, the cell scratch and Transwell showed that the migration ability of A549 cells decreased with the increasing puerarin concentration. The polymerase chain reaction (PCR) and western blotting results demonstrated that the expression of the cell invasion and metastasis-related factor, matrix metallopeptidase 9 (MMP9), was negatively correlated with drug concentration. Further investigation demonstrated that the phosphorylation of extracellular signal-regulated kinase (ERK) was also inhibited. Puerarin can inhibit the expression of invasion and metastasis-related factors by inhibiting the phosphorylation of ERK.

Role and mechanism of benzo[a]pyrene in the transformation of chronic obstructive pulmonary disease into lung adenocarcinoma.

This experiment is explores the genes that play a key role, their expression changes and the biological processes in the transformation of chronic obstructive pulmonary disease (COPD) into lung adenocarcinoma (LAC). Meanwhile, identify the effects of Benzo[a]pyrene (BaP) in the conversion of COPD into LAC. 1. Differential expression genes of COPD and LAC were screened and analyzed by high-throughput microarray data between the two diseases and their respective control groups. 2. The screened genes were used for routine bioinformatics analysis such as functional analysis, expression verification, protein interaction analysis and functional enrichment. 3. Cigarette smoke extract (CSE) combined with lipopolysaccharide (LPS) was used to establish an in vitro COPD model. 4. MTT assay was used to detect the influence of B(a)P in effect on A549 cell proliferation. CCK-8, Transwell invasion test and scratch test were used to detect the cell proliferation, invasion and migration ability, while qPCR and Western Blot tests were used to observe the cell proliferation, apoptosis and changes in related indicators such as EMT. 5. Experimental method of separately adding agonists (tBHQ) and inhibitors (DIC) of NQO1 was used to confirm the effect of NQO1 on A549 cell proliferation, apoptosis, migration and invasion. 6. To further clarify whether BaP exerted effect on cell proliferation, apoptosis, migration and invasion through NQO1, we knocked down NQO1 gene and then infecting cells with BaP. 1. We screened genes of COPD and LAC using datasets from GSE151052, GSE118370, and GSE140797. After screening, the genes upregulated in COPD and downregulated in LAC were RTKN2, SLC6A4, and HBB, the gene downregulated in COPD and upregulated in LAC was NQO1, the genes downregulated in both COPD and LAC were FPR1, LYVE1 and PKHD1L1. 2. The main signaling pathways in which the target genes were enriched are cell cycle, EMT, PI3K/AKT, and apoptosis. In the data included GEPIA, PKHD1L1, FPR1, LYVE1, RTKN2, HBB, and SLC6A4 were significantly downregulated and NQO1 was upregulated in LAC relative to controls. In addition, there were 46 interaction proteins in the target genes, and the functions they enriched included hydrogen peroxide catabolism, etc. 3. When A549 cell was stimulated with 100ng/mL LPS+ 10% CSE, the COX-2 expression indicated that COPD model in vitro was successfully established. 4. The optimal dose and action time were screened which were 1μM and 24h. Compared to the control group, COPD and BaP group increased cell proliferation and invasion capabilities. On the basis of COPD, adding BaP could further increase the proliferation and migration capabilities. Interestingly, the levels of NQO1 decreased in COPD models, while increased by BaP. 5. tBHQ can increase the proliferation and migration capacity of A549 cells, which is inhibited by the addition of DIC. 6. The enhanced proliferation, migration and invasion of A549 cells by BaP were attenuated after knockdown of NQO1. Our study reveals that PKHD1L1, FPR1, LYVE1, RTKN2, HBB, SLC6A4 and NQO1 may play an important role in the conversion of COPD to LAC. High NQO1 expression may increase the proliferation and migration ability of A549 cells, and BaP may promote the EMT state by increasing the expression of NQO1, thereby making the COPD model in vitro expose the tumor characteristics.

Enhanced Inhibitory Effect of DC-CIK Cells on Lung Adenocarcinoma via Anti-Tim-3 Antibody and Antiprogrammed Cell Death-1 Antibody and Possible Mechanism.

Objective To investigate the effect and mechanism of blocking the signaling pathways of the T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and programmed death protein 1 (PD-1) in dendritic cell-cytokine induced killer (DC-CIK) cells on human lung adenocarcinoma A549 cells. Methods Peripheral blood mononuclear cells (PBMCs) were isolated and induced into mature DC-CIK cells by cytokines in vitro. After blocking the Tim-3 and PD-1 signaling transduction pathways with anti-Tim-3 and anti-PD-1 antibodies, DC-CIK cells were coincubated with A549 cells. The killing effect of DC-CIK cells against A549 cells was measured by a CCK-8 assay. The impact of DC-CIK cells on the invasion and migration ability of A549 cells was detected by the Transwell test. The apoptosis rate of DC-CIK cells and the ratio of CD4+, CD8+, and DC-CIK cell subsets were determined by flow cytometry. The cell proliferation of DC-CIK was detected by the CCK-8 assay. Results The antibodies of anti-Tim-3 antibody and anti-PD-1 could block Tim-3+ and PD-1+ DC-CIK cells and could significantly increase the killing effect of DC-CIK cells on A549 cells. The number of A549 cells under the microporous membrane of the Transwell chamber was reduced considerably in invasion and migration tests. Anti-Tim-3 and anti-PD-1 antibodies significantly reduced apoptosis of DC-CIK cells. No significant differences were observed in the ratios of CD4+ and CD8+ DC-CIK cell subsets or the proliferation capacity of DC-CIK cells in each group. Conclusion Blocking the Tim-3 and PD-1 signaling pathways of DC-CIK cells with antibodies can enhance the killing ability of DC-CIK cells in A549 cells and significantly suppress the invasion and migration ability of A549 cells. The potential mechanism may be related to reduced apoptosis of DC-CIK cells.

Nanog suppression enhanced the chemosensitivity of human non-small-cell lung cancer cells to Cisplatin and inhibited cell migration

Lung cancer is the leading cause of cancer-associated death in the world. As one of the leading transcription factors in controlling stemness features, Nanog was shown to promote cancer progression, metastasis, and drug resistance. Considering that, this research was conducted to evaluate the effect of Nanog suppression using specific siRNA on the chemosensitivity of lung cancer cells to Cisplatin through inhibition of cell proliferation, migration, and stemness as well as apoptosis induction. Then, A549 lung cancer cells were transfected with Nanog siRNA and treated with Cisplatin individually or combined. Subsequently, to investigate cell proliferation and apoptosis induction, MTT assay and Annexin V/PI staining were performed, respectively. Also, colony formation assay was carried out to evaluate cell stemness features, and migration ability of A549 cells was followed using a wound-healing assay. Gene expression was quantified via qRT-PCR. The obtained results illustrated that siRNA-mediated Nanog suppression remarkably increased the chemosensitivity of A549 cells to Cisplatin through apoptosis induction. Consistently, Nanog suppression combined with Cisplatin led to upregulation of Caspase-3 apoptotic gene and Bax/Bcl-2 ratio. Besides, Nanog knockdown, individually or combined with Cisplatin, prevented colony formation ability of A549 cells by downregulating Sox2 and CD44 genes. It was also indicated that the combination therapy remarkably downregulated MMP9 expression and subsequently suppressed A549 cell migration. A significant reduction was also observed in c-Myc and PD-L1 gene expression levels. In conclusion, the findings of the current study demonstrated that silencing Nanog combined with Cisplatin could be a potent treatment approach for lung cancer patients.

Antimicrobial peptide 17BIPHE2 inhibits the proliferation of lung cancer cells in vitro and in vivo by regulating the ERK signaling pathway.

In 2018, there were 18.1 million new cancer cases and 9.6 million cancer-related deaths worldwide, among which the incidence rate of lung cancer (11.6%) and fatality rate (18.4%) both ranked first. The antimicrobial peptide LL-37 is an important component of the natural immune system and possesses several biological properties, including antibacterial, antiviral and anticancer effects. The antimicrobial peptide 17BIPHE2, the shortest synthetic peptide derivative of LL-37, exhibits biological activities similar to those of LL-37. The objective of the present study was to investigate the mechanism of action of exogenous 17BIPHE2 against lung cancer cells. The human lung adenocarcinoma cell line A549 was treated with 17BIPHE2. Changes in cell proliferation, migration, invasion, mitochondrial membrane potential (ΔΨm), and the levels of reactive oxygen species (ROS), Ca2+ and apoptosis-related proteins, including BAX, BCL-2 and ERK, were detected using flow cytometry, transmission electron microscopy and western blotting. The results showed that 17BIPHE2 significantly increased the apoptosis rate of A549 cells and elevated BAX expression, ERK phosphorylation, and ROS and Ca2+ levels, but decreased the expression of BCL-2, ERK and Ki67. In addition, the peptide reduced ΔΨm and the cell migration ability of A549 cells and inhibited tumor growth. ERK inhibition significantly attenuated the anticancer effect of 17BIPHE2. The present observations suggested that 17BIPHE2 can effectively inhibit cancer cells by regulating the ERK signaling pathway.

Antiproliferative effect and autophagy induction of curcumin derivative ZYX02-Na on the human lung cancer cells A549.

At present, a large number of curcumin derivatives had been produced and identified aiming to replace the curcumin in view of its low bioavailability and stability. Here, a novel curcumin derivative ZYX02-Na was first used to reduce the cell viability of human non-small cell lung cells A549, which was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry and Western blot analysis showed that ZYX02-Na could lead to cell cycle arrest in G0/G1 phase, which demonstrated that ZYX02-Na inhibited the proliferation of A549 cells. Furthermore, the AMPK/mTOR/4E-BP1 signaling pathway was activated in ZYX02-Na-treated A549 cells. Besides, wounding healing and transwell experiments showed that ZYX02-Na could also inhibited the migration ability of A549 cells. Moreover, we also found that ZYX02-Na could induce autophagy of A549 cells by acridine orange staining, GFP-LC3 subcellular localization observation and Western blotting analysis, respectively. In short, our current studies indicated that ZYX02-Na possessed the antiproliferation effect and autophagy induction on A549 cells, while in vivo anticancer study of ZYX02-Na needs to be done in future.

Facile synthetic nano-curcumin encapsulated Bio-fabricated nanoparticles induces ROS-mediated apoptosis and migration blocking of human lung cancer cells

Lung cancer is one of the major life-threatening cancers, with a higher mortality rate and morbidity. Curcumin acts as a potential anticancer agent but it shows less aqueous solubility so its clinical application is limited. In this present study, we developed curcumin-surface decorated Zinc Oxide nanoparticles ZnO@Cur to enhance its aqueous formulation and improve the anti-cancer activity of curcumin. The synthesized ZnO@Cur confirmed by various spectroscopies (UV–vis and fluorescence) and electroscopic techniques (Powder XRD, SEM, TEM, and DLS). ZnO@Cur dramatically suppressed the proliferation of A549 and HEL-299 cells and showed low toxicity in MTT assays. Compared with free ZnO and curcumin, ZnO@Cur significantly inhibited the migration ability of A549 cells. Moreover, the induction of apoptosis in A549 cells by ZnO@Cur was resulted by AO-EB staining. ZnO@Cur activated to promote intracellular ROS overproduction and induced apoptosis. ZnO@Cur shows excellent biocompatibility compared to free ZnO and Curcumin, the present study explained that ZnO@Cur as a safe and hopeful strategy for chemotherapeutics of lung cancer therapy and deserve for further clinical evaluations.

Sesamin induces A549 cell mitophagy and mitochondrial apoptosis via a reactive oxygen species-mediated reduction in mitochondrial membrane potential.

Sesamin, a lipid-soluble lignin originally isolated from sesame seeds, which induces cancer cell apoptosis and autophagy. In the present study, has been reported that sesamin induces apoptosis via several pathways in human lung cancer cells. However, whether mitophagy is involved in sesamin induced lung cancer cell apotosis remains unclear. This study, the anticancer activity of sesamin in lung cancer was studied by reactive oxygen species (ROS) and mitophagy. A549 cells were treated with sesamin, and cell viability, migration ability, and cell cycle were assessed using the CCK8 assay, scratch-wound test, and flow cytometry, respectively. ROS levels, mitochondrial membrane potential, and apoptosis were examined by flow cytometric detection of DCFH-DA fluorescence and by using JC-1 and TUNEL assays. The results indicated that sesamin treatment inhibited the cell viability and migration ability of A549 cells and induced G0/G1 phase arrest. Furthermore, sesamin induced an increase in ROS levels, a reduction in mitochondrial membrane potential, and apoptosis accompanied by an increase in cleaved caspase-3 and cleaved caspase-9. Additionally, sesamin triggered mitophagy and increased the expression of PINK1 and translocation of Parkin from the cytoplasm to the mitochondria. However, the antioxidant N-acetyl-L-cysteine clearly reduced the oxidative stress and mitophagy induced by sesamin. Furthermore, we found that cyclosporine A (an inhibitor of mitophagy) decreased the inhibitory effect of sesamin on A549 cell viability. Collectively, our data indicate that sesamin exerts lethal effects on lung cancer cells through the induction of ROS-mediated mitophagy and mitochondrial apoptosis.

IL-6 stimulates lncRNA ZEB2-AS1 to aggravate the progression of non-small cell lung cancer through activating STAT1.

To illustrate the role of interleukin 6 (IL-6) in the progression of non-small cell lung cancer (NSCLC) via activating STAT1. The level of IL-6 mRNA in 48 paired NSCLC tissues and matched normal ones was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Kaplan-Meier curves were depicted for assessing the overall survival of NSCLC patients with high or low level of IL-6 mRNA. Subsequently, the ZEB2-AS1 level in A549 cells treated with different doses of IL-6 for different time points was determined. After A549 cells were treated with different doses of IL-6, wound closure assays were performed to assess the migration of cells. Protein levels of pSTAT1 and STAT1 in IL-6-treated A549 cells were detected by Western blot. The regulatory effect of STAT1 on IL-6-induced migration of A549 cells was also evaluated. The interaction between ZEB2-AS1 and STAT1 was explored through Chromatin Immunoprecipitation (ChIP) assay. Finally, the role of ZEB2-AS1/STAT1 axis in regulating NSCLC cells was investigated through rescue experiments. Our results indicated that IL-6 was upregulated in NSCLC tissues and cancer cell lines. NSCLC patients with T3-T4 or accompanied with lymphatic metastasis had a higher IL-6 abundance than those with T1-T2 or without metastatic foci. The worse prognosis was identified in NSCLC patients with high expression of IL-6 compared to those with low expression. ZEB2-AS1 showed dose-dependent and time-dependent increase in IL-6-treated A549 cells. IL-6 treatment gradually enhanced the migration ability of A549 cells in a dose-dependent manner. In IL-6-treated A549 cells, protein level of pSTAT1 was remarkably upregulated, and knockdown of STAT1 significantly reversed the promotive effect of IL-6 on migration ability of A549 cells. The results of ChIP assay verified the interaction between ZEB2-AS1 and STAT1. In addition, ZEB2-AS1 could reverse the regulatory effect of STAT1 on the migration ability of A549 cells. IL-6 was upregulated in NSCLC and accelerated the progression of NSCLC via activating STAT1/ ZEB2-AS1.

CaSR Induces Osteoclast Differentiation and Promotes Bone Metastasis in Lung Adenocarcinoma.

Objective: Explore the mechanism of CaSR's involvement in bone metastasis in lung adenocarcinoma.Methods: Immunohistochemistry (IHC) was used to detect the expression of calcium-sensing receptor (CaSR) in 120 cases of lung adenocarcinoma with bone metastasis. Stably transfected cell lines with CaSR overexpression and knockdown based on A549 cells were constructed. The expression of CaSR was verified by western blot and qPCR. The proliferation and migration abilities of A549 cells were tested using cholecystokinin-8 (CCK-8) and Transwell assays, respectively. Western blotting was used to detect the expression of matrix metalloproteinases MMP2, MMP9, CaSR, and NF-κB. The supernatant from each cell culture group was collected as a conditional co-culture solution to study the induction of osteoclast precursor cells and osteoblasts. Western blot and qPCR were used to validate the expression of bone matrix degradation-related enzymes cathepsin K and hormone calcitonin receptor (CTR) and osteoblast-induced osteoclast maturation and differentiation enzyme receptor activator of nuclear factor-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and PTHrP. Immunofluorescent staining was used to detect F-actin ring formation and osteocalcin expression. Western blot results for NF-κB expression identified a regulatory relationship between NF-κB and CaSR.Results: CaSR expression in lung cancer tissues was significantly higher than that in adjacent and normal lung tissues. The expression of CaSR in lung cancer tissues with bone metastasis was higher than that in non-metastatic lung cancer tissues. The proliferation and migration ability of A549 cells increased significantly with overexpressed CaSR. The co-culture solution directly induced osteoclast precursor cells and the expression of bone matrix degradation-related enzymes significantly increased. Osteoblasts were significantly inhibited and osteoblast-induced osteoclast maturation and differentiation enzymes were significantly downregulated. It was found that the expression of NF-κB and PTHrP increased when CaSR was overexpressed. Osteoclast differentiation factor expression was also significantly increased, which directly induces osteoclast differentiation and maturation. These results were reversed when CaSR was knocked down.Conclusions: CaSR can positively regulate NF-κB and PTHrP expression in A549 cells with a high metastatic potential, thereby promoting osteoclast differentiation and maturation, and facilitating the occurrence and development of bone metastasis in lung adenocarcinoma.

Exogenous hydrogen sulfide donor NaHS alleviates nickel-induced epithelial-mesenchymal transition and the migration of A549cells by regulating TGF-β1/Smad2/Smad3 signaling.

Nickel compounds are known to be common environmental and occupational carcinogens which also promote the migration of lung cancer cells. However, the molecular mechanism yet remains to be clarified. Hydrogen sulfide (H2S) is involved in cancer biological processes. However, the exact effect and functionality of H2S on nickel, towards the promotion of the migration ability of lung cancer cells, remains to be unknown. In this study, we have found that the nickel chloride (NiCl2) treatment significantly downregulates the protein levels of endogenous H2S enzyme cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-Mercaptopyruvate sulfurtransferase (3-MST). A correlation between NiCl2-induced epithelial-mesenchymal transition (EMT) and the migration ability of lung cancer A549cells has been observed. Exogenous H2S donor, sodium hydrogen sulfide (NaHS) (100μmol/L), can reverse NiCl2-induced EMT as well as the migration ability of A549cells. NiCl2 treatment is able to upregulate the protein level of transforming growth factor-β1 (TGF-β1), p-Smad2, p-Smad3, p-JNK, p-ERK and p-P38 in a time-dependent fashion, indicating that both TGF-β1/Smad2/Smad3 and mitogen-activated protein kinase (MAPK) signaling cascades (a non-Smad pathway) may play essential roles in NiCl2-dependent EMT as well as cell migration of human lung cancer cells. Furthermore, exogenous NaHS alleviates the NiCl2-induced EMT and the migration ability of A549cells only by regulating TGF-β1/Smad2/Smad3, rather than the MAPK, signaling pathway. These results indicate that the exogenous administration of NaHS might be a potential therapeutic strategy against nickel-induced lung cancer progression.

Vitamin D3 inhibits invasion and migration of lung cancer A549 cells via nuclear factor-κB/GPX1 pathway

Objective To observe the effect of Vitamin D3 on the invasion and migration of lung cancer A549 cells and to explore its mechanism. Methods A549 cells were treated with Vitamin D3 at a concentration of 60 nmol/L for 5 d, and 10 μmol/L BAY11-7082 for Ah cells for 1h. A549 cells were transfected with small interfering RNA (siRNA) targeting GPX1, and A549 was detected by Transwell method. The ability of cells to invade and migrate was detected by Western blotting to detect the expression levels of nuclear factor-κB (NF-κB) and GPX1 proteins. Results After treatment with Vitamin D3, the migration and invasion ability of A549 cells decreased significantly [P(Blank)<0.01; p(AA)<0.05], and the expression of NF-κB and GPX1 decreased. After treatment with NF-κB inhibitor BAY11-7082, the migration and invasion ability of A549 cells decreased significantly (P<0.01), and the expression of NF-κB and GPX1 was observed to decrease. After the expression of GPX1 was decreased by RNA interference, the migration and invasion ability of A549 cells decreased significantly [P(Blannk)<0.01, P(Scramble)<0.05], but there was no significant change in NF-κB. Conclusion Vitamin D3 can inhibit the invasion and migration of lung cancer A549 cells through NF-κB/GPX1 pathway. Key words: RNA interference; Glutathione peroxidase-1; Vitamin D3; Non-small cell lung cancer

SKA3 Up-regulation Promotes Lung Adenocarcinoma Growth and is a Predictor of Poor Prognosis.

ObjectiveThe objective of this research is to investigate the expression and function of SKA3 in lung adenocarcinoma.MethodsThe mRNA expression level of SKA3 in lung adenocarcinoma and its association with clinic-pathological factors were analyzed using data obtained from the TCGA database. Small interfering RNA (siRNA) for SKA3 (si-SKA3) was used to down-regulate SKA3 in A549 cells. pcDNA3.1- SKA3 was used to overexpress SKA3 in A549 cells. The proliferation ability of A549 cells was determined via MTT assay and colony formation assay. A wound healing assay was performed to examine the migration ability of A549 cells. The protein expression of p-MEK, MEK, p-ERK and ERK were determined by western blot.ResultsWe found that SKA3 is up-regulated in lung adenocarcinoma compared to the normal lung tissues. Kaplan-Meier analysis showed that high SKA3 expression is markedly associated with poor prognosis in lung adenocarcinoma patients. SKA3 expression is significantly correlated with age, gender, pathologic-stage, pathologic-N and pathologic-M. Moreover, depleting SKA3 obviously inhibited A549 cell proliferation and migration in vitro, while overexpression of SKA3 notably increased A549 cell proliferation and migration. Western blot analysis showed that the protein expression ratio of p-MEK/MEK and p-ERK/ERK decreased noticeably after depleting SKA3.ConclusionSKA3 expression was enhanced and associated with poor prognosis in lung adenocarcinoma patients, and it might play a facilitating role in cell growth and motility by regulating the MAPK signaling pathway.

MiR-448 promotes progression of non-small-cell lung cancer via targeting SIRT1.

Deregulation of microRNAs (miRs) has been demonstrated to be involved in both the initiation and the development of non-small-cell lung cancer (NSCLC). miR-448 has been identified as a tumor suppressor in several cancer types. The aim of the present study was to explore the role of miR-448 in NSCLC. Tumor tissues and paired normal tissues were obtained from patients with NSCLC. The viability and migration of A549 cells were determined by the Cell Counting kit-8 and wound-healing assays, respectively. Gene and protein levels were detected by reverse transcription-quantitative polymerase chain reaction analysis and western blotting, respectively. The interaction between the 3' untranslated region of sirtuin1 (SIRT1) and miR-448 was predicted by TargetScan and verified by dual luciferase reporter assay. miR-448 levels were revealed to be decreased whereas SIRT1 levels were increased in NSCLC tissues compared with normal tissues. Pearson's correlation analysis demonstrated that there was a negative correlation between miR-448 and SIRT1 mRNA levels. Overexpression of miR-448 led to reduced growth and migration ability of A549 cells. In addition, overexpression of miR-448 decreased SIRT1 mRNA and protein levels, thereby inhibiting epithelial-mesenchymal transition (EMT) and affecting EMT-associated molecules, including vimentin and E-cadherin. Dual luciferase reporter assay confirmed that SIRT1 was a direct target of miR-448. Notably, activation of SIRT1 by resveratrol treatment partially reversed the cell growth inhibition induced by miR-448 mimics. These findings suggested that the progression of NSCLC may be controlled by miR-448, which appears to hold promise as a therapeutic target for patients with NSCLC.

Cytochalasin H isolated from mangrove‑derived endophytic fungus induces apoptosis and inhibits migration in lung cancer cells.

CytochalasinH(CyH) has been shown to exhibit promising anticancer activities against various types of cancers; however, the underlying mechanisms remain unknown. In a previous study, we isolated CyH from the mangrove‑derived endophytic fungus Phomopsis sp. in Zhanjiang, China. In the present study, we further explored the effect of CyH on apoptosis and migration in the human lung adenocarcinoma cell line A549. Cell Counting kit‑8(CCK‑8) assay was used to observe the effects of CyH on the growth of A549 cells. The cell cycle and apoptosis were determined using flow cytometry. The effect of CyH on cell migration was observed by scratch wound healing and chamber migration assays. Western blotting was used to detect the expression of apoptosis‑and metastasis‑associated proteins. Our results showed that CyH exhibited cytotoxicity to A549 cells. The treatment of CyH arrested A549 cells at the G2/M phase. Furthermore, sub‑G1 peaks and fragmented DNA ladders were observed, and the mitochondrial transmembrane potential was also decreased in CyH‑treated A549 cells. CyH significantly increased Bax, P53, and cleaved caspase‑3 (17kDa) protein expression and decreased Bcl‑xL, Bcl‑2, and full‑length caspase‑3 (35kDa) protein expression, resulting in an increased ratio of the pro‑apoptosis/anti‑apoptosis proteins Bax/Bcl‑2. Additionally, CyH treatment inhibited the migration ability of A549 cells in a dose‑dependent manner. Taken together, our results suggest that CyH may be a potential chemopreventive drug for the treatment of lung cancer.

Matrine reduces the proliferation of A549cells via the p53/p21/PCNA/eIF4E signaling pathway.

The aim of the present study was to investigate how matrine affects the proliferation of A549 human lung adenocarcinoma cells via the p53/p21/proliferating cell nuclear antigen (PCNA)/eukaryotic translation initiation factor 4E (eIF4E) signaling pathway. The effect of different concentrations of matrine on the proliferation of A549 cells was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The migration of A549 cells following exposure to varied concentrations of matrine was detected using a Transwell cell migration assay. The effect of 240 mg/l matrine on the apoptotic rate of A549 cells was determined using flow cytometry. The change in the mRNA and protein expression levels of p53, p21, PCNA and eIF4E following exposure to matrine were detected using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The increase of matrine from 60–240 mg/l led to reduced cell migration and inhibition of A549 cell proliferation. The apoptotic rate of A549 cells when treated with 240 mg/l matrine was significantly different when compared with the untreated control. The mRNA expression levels of p53 and p21 in the group treated with 240 mg/l matrine were significantly higher compared with the control group. The mRNA expression levels of PCNA and eIF4E were significantly lower in the 240 mg/l matrine-treated group compared with the control. The protein expression levels of p53 and p21 were significantly higher in the 240 mg/l matrine group compared with the control group. Treatment with 240 mg/l matrine reduced the protein expression levels of PCNA and eIF4E. Matrine also reduced the migration ability of A549 cells and inhibited their proliferation, which may be associated with the overexpression of p53 and p21, and the reduction of PCNA and eIF4E expression levels.

Synthesis of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one derivatives as potential anticancer and apoptosis inducing agents

A series of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one derivatives were synthesized and characterized by 1H and 13C NMR, ESI-MS and HRMS. All the synthesized compounds were evaluated for their anticancer activity against HT-29, PC-3, A549 and U87MG human tumor cell lines. Most of the synthesized compounds displayed potent growth inhibition selectively of A549 cancer cells and did not show significant toxicity to the non-cancerous HaCaT cells. Some of the representative compounds, particularly, 16, 22 and 28 exhibited potent growth inhibition with IC50 values in the range of 1.25–3.98 μM, which are comparable or even better than the standard chemotherapeutic drug 5-fluorouracil. Preliminary mechanistic studies revealed that these compounds could effectively inhibit the migration ability of A549 cells. Flow-cytometry analysis revealed that the compounds treatment led to G2/M cell cycle arrest. Moreover, the compounds induced apoptosis in A549 cells through depolarization of mitochondrial membrane potential (DΨm) and increased reactive oxygen species (ROS) production, suggesting their potential to act as promising lead compounds for the development of cancer chemotherapeutics.

Influence of propofol on proliferation and apoptosis of human lung cancer A549 cells

Objective To investigate the influence of propofol on lung cancer cell proliferation and apoptosis in vitro. Methods MTT assay was used to detect the inhibition effect of propofol on the growth of A549. Flow cytometry was applied to detect the impact of propofol on apoptosis of A549 cells. Western blotting was used to determine the effect of propofol on aquaporin 1(AQP1) protein expression in A549 cells, and the migration ability of A549 cells was investigated by Transwell chamber. Results MTT assay and flow cytometry showed that the rate of inhibition and apoptosis in the experiment group after propofol acting on A549 were(52.8±5.2)%,(61.2±5.9)%, (69.4±6.1)%;(42.8±4.1)%,(59.8±11.2)%,(71.4±13.4)%, respectively. The expression of AQP1 protein was 0.87±0.12、0.54±0.07、0.31±0.04;and the migration ability of A549 cells was 1.35±8.98、51.78±7.58、40.35±5.12. Conclusion Propofol can significantly inhibit the proliferation, apoptosis and migration ability of human lung cancer A549 cells, which may be associated with the down- regulation of AQP1 protein. Key words: Propofol; Lung cancer; Apoptosis; Aquaporin 1

A Microarray-based Approach Identifies ADP Ribosylation Factor-like Protein 2 as a Target of microRNA-16

microRNAs (miRNAs) are generally thought to negatively regulate the expression of their target genes by mRNA degradation or by translation repression. Here we show an efficient way to identify miRNA target genes by screening alterations in global mRNA levels following changes in miRNA levels. In this study, we used mRNA microarrays to measure global mRNA expression in three cell lines with increased or decreased levels of miR-16 and performed bioinformatics analysis based on multiple target prediction algorithms. For further investigation among the predicted miR-16 target genes, we selected genes that show an expression pattern opposite to that of miR-16. One of the candidate target genes that may interact with miR-16, ADP-ribosylation factor-like protein 2 (ARL2), was further investigated. First, ARL2 was deduced to be an ideal miR-16 target by computational predictions. Second, ARL2 mRNA and protein levels were significantly abolished by treatment with miR-16 precursors, whereas a miR-16 inhibitor increased ARL2 mRNA and protein levels. Third, a luciferase reporter assay confirmed that miR-16 directly recognizes the 3'-untranslated region (3'-UTR) of ARL2. Finally, we showed that miR-16 could regulate proliferation and induce a significant G0/G1 cell cycle arrest, which was due at least in part, to the down-regulation of ARL2. In summary, the present study suggests that integrating global mRNA profiling and bioinformatics tools may provide the basis for further investigation of the potential targets of a given miRNA. These results also illustrate a novel function of miR-16 targeting ARL2 in modulating proliferation and cell cycle progression.