Migration Of A549 Lung Cancer Cells Research Articles

Pharmacological Properties of White Mulberry (Morus alba L.) Leaves: Suppressing Migratory and Invasive Activities Against A549 Lung Cancer Cells.

Morus alba known as a white mulberry is a medicinal plant that has been used in food ingredients and traditional medicine. M. alba leaves contain various bioactive phenolic compounds, in particular chlorogenic acid (CGA), which is a major bioactive ingredient. Their anticancer potency of M. alba leaf extracts derived from Soxhlet extraction was evaluated based on cytotoxicity and antimigratory and antiinvasive properties. The dichloromethane extract exhibited the highest nitric oxide radical scavenging activity with a half-maximal inhibitory concentration (IC50) value of 780μg/mL, promising cytotoxicity against HuCCA-1, MCF-7, and A-549 cells with IC50 values of 59.18, 62.20, and 103.25μg/mL, respectively. CGA selectively inhibited the growth of MCF-7 cells with an IC50 value of 26.75μg/mL and showed potent radical scavenging activity against DPPH radicals (IC50 = 18.85μg/mL). An ethanolic extract derived from the gradient Soxhlet extraction suppressed A549 lung cancer cell migration and invasion more effectively than CGA with no migratory inhibition effect on noncancerous HaCaT cells. Furthermore, the ethanolic extract and CGA accelerated HaCaT wound closure at 20µg/mL, which was the same as allantoin. Bioactive ingredients including triterpenes, steroids, phenolics, and flavonoids were mainly detected in all extracts. The highest content of CGA (52.23g/100g dry weight) was found in the ethanolic extract derived from the gradient Soxhlet extraction. These findings show the potency of the dichloromethane extract as a cytotoxic agent against various cancer types and the ethanolic extract as an antimetastatic agent by their antimigratory and antiinvasive activities.

The p38 MAPK/snail signaling axis participates in cadmium-induced lung cancer cell migration and invasiveness.

To determine that p38 MAPK activation contributes to the migration and invasion of lung cancer cells caused by cadmium (Cd). A549 lung cancer cell migration and invasion were assessed using a transwell plate system, and the role of p38 was determined by knocking down p38 activity with two different inhibitors of p38. The activity of p38 was measured by western blot analysis using phospho-specific p38 antibodies and normalized to blots using antibodies directed to total p38 proteins. Snail transcripts were measured using qRT-PCR. The inhibition of p38 blocked Cd-induced migration and invasion, which correlated with an increased activation of p38 as a function of dose and time. Furthermore, Cd-induced activation of p38 MAPK controlled the increase of snail mRNA expression. The p38 MAPK/snail signaling axis was involved in Cd-induced lung cancer cell migration and invasion.

Phenylethanoid Glycoside-Enriched Extract Prepared from Clerodendrum chinense Leaf Inhibits A549 Lung Cancer Cell Migration and Apoptosis Induction through Enhancing ROS Production

This study aims to investigate the antioxidant and anti-cancer activities of Clerodendrum chinense leaf ethanolic extract. The phenylethanoid glycoside-enriched extract, namely verbascoside and isoverbascoside, was determined in the ethanolic C. chinense leaf extract using the validated HPLC method. The ethanolic extract showed DPPH and ABTS free radical scavenging activities with the IC50 values of 334.2 ± 45.48 μg/mL and 1012.77 ± 61.86 µg/mL, respectively, and a FRAP value of 88.73 ± 4.59 to 2480.81 ± 0.00 µM. C. chinense leaf extract exhibited anti-proliferative activity against A549 lung cancer cells in a dose- and time-dependent manner, with the IC50 value of 340.63 ± 89.43, 210.60 ± 81.74, and 107.08 ± 28.90 µg/mL after treatment for 24, 48, and 72 h, respectively. The IC50 values of verbascoside, isoverbascoside, and hispidulin were 248.40 ± 15.82, 393.10 ± 15.27, and 3.86 ± 0.87 µg/mL, respectively, indicating that the anti-proliferative effects of the C. chinense leaf extract mainly resulted from hispidulin and verbascoside. The selectivity index (SI) of C. chinense leaf extract against A549 lung cancer cells vs. normal keratinocytes were 2.4 and 2.8 after incubation for 24 and 48 h, respectively, suggesting the cytotoxic selectivity of the extract toward the cancer cell line. Additionally, the C. chinense leaf extract at 250 µg/mL induced late apoptotic cells up to 21.67% with enhancing reactive oxygen species (ROS) induction. Furthermore, the lung cancer cell colony formation was significantly inhibited after being treated with C. chinense leaf extract in a dose-dependent manner. The C. chinense leaf extract at 250 µg/mL has also shown to significantly inhibit cancer cell migration compared with the untreated group. The obtained results provide evidence of the anti-lung cancer potentials of the C. chinense leaf ethanolic extract.

Activation of GRP78 ATPase suppresses A549 lung cancer cell migration by promoting ITGB4 degradation

ABSTRACT Hypochlorous acid (HOCl) is an essential signal molecule in cancer cells. Activated GRP78 ATPase by a HOCl probe named ZBM-H inhibits lung cancer cell growth. However, the role and underlying mechanism of GRP78 ATPase in lung cancer cell migration have not been established. Here, we reported that activation of GRP78 ATPase by ZBM-H suppressed A549 cell migration and inhibited EMT process. Notably, ZBM-H time-dependently decreased the protein level of integrin β4 (ITGB4) in A549 cells. Combinatorial treatment of 3BDO (an autophagy inhibitor) and ZBM-H partially rescued the protein level of ITGB4. Consistently, 3BDO partially reversed ZBM-H-inhibited cell migration. Furthermore, ZBM-H promoted the interaction between ANXA7 and Hsc70, which participated in the regulation of selective autophagy and degradation of ITGB4.

Inhibition of A549 Lung Cancer Cell Migration and Invasion by Ent-Caprolactin C via the Suppression of Transforming Growth Factor-β-Induced Epithelial-Mesenchymal Transition.

The epithelial–mesenchymal transition (EMT) of cancer cells is a crucial process in cancer cell metastasis. An Aquimarina sp. MC085 extract was found to inhibit A549 human lung cancer cell invasion, and caprolactin C (1), a new natural product, α-amino-ε-caprolactam linked to 3-methyl butanoic acid, was purified through bioactivity-guided isolation of the extract. Furthermore, its enantiomeric compound, ent-caprolactin C (2), was synthesized. Both 1 and 2 inhibited the invasion and γ-irradiation-induced migration of A549 cells. In transforming growth factor-β (TGF-β)-treated A549 cells, 2 inhibited the phosphorylation of Smad2/3 and suppressed the EMT cell marker proteins (N-cadherin, β-catenin, and vimentin), as well as the related messenger ribonucleic acid expression (N-cadherin, matrix metalloproteinase-9, Snail, and vimentin), while compound 1 did not suppress Smad2/3 phosphorylation and the expression of EMT cell markers. Therefore, compound 2 could be a potential candidate for antimetastatic agent development, because it suppresses TGF-β-induced EMT.

Sec3 knockdown inhibits TGF-β induced epithelial-mesenchymal transition through the down-regulation of Akt phosphorylation in A549 cells

The exocyst, an evolutionarily conserved octomeric protein complex, has been demonstrated as an essential component for vesicle tethering during cell exocytosis, and participates in various physiological processes in the cell. Although subunits of the exocyst complex have been reported to be involved in the regulation of TGF-β induced cancer cell migration and epithelial-mesenchymal transition (EMT), the potential function of Sec3 in these regulated processes remains unclear. Here, we show that Sec3 knockdown abolishes TGF-β stimulated A549 lung cancer cell migration in vitro and causes defects in the regulated EMT process. In addition, we find that depletion of Sec3 significantly inhibits TGF-β stimulated Akt phosphorylation in A549 cells, whereas the increase of Smad2 phosphorylation is unaffected. Furthermore, replenishment of an RNAi-resistant form of Sec3 is shown to restore the defects of TGF-β induced cell migration, EMT and Akt signaling activation. In summary, our study provides evidence that Sec3 is involved in TGF-β induced cell migration and EMT processes, presumably through the regulation of PI3K/Akt signaling activation in A549 cancer cells.

MicroRNA-520e inhibited the migration and invasion of lung cancer A549 cells by regulating Zinc finger protein 367

Objective To investigate the underlying molecular mechanisms of microRNA-520e (miR-520e) in the migration and invasion of lung cancer cell line A549. Methods The reverse transcription-polymerase chain reaction (RT-PCR) was adopted to detect the expression differences of miR-520e in lung cancer cell line A549 and normal lung epithelial cell line BEAS-2B. Transfected miR-520e mimics to obtain miR-520e overexpressing cell lines. Using migration assay and Transwell assay to detect the effect of miR-520e on the migration and invasion of A549 cells. Bioinformatics was employed to predict the miR-520e-targeted Zinc finger protein 367 (ZNF367) gene. Dual luciferase assay was adopted to analyze the mechanism of miR-520e regulating ZNF367. Western blotting was used to detect the changes of ZNF367 in lung cancer cells. The effect of ZNF367 on the migration and invasion of A549 cells was studied by small interfering RNA (siRNA). Results The expression of miR-520e was 0.342 ± 0.214 in A549 cells compared with that of BEAS-2B cells (1.000 ± 0.135, P=0.000). After transfection with miR-520e mimic, compared with untransfected group [ normal control (NC) group: 1.000 ± 0.273] the expression of miR-520e in mimic group was 2.617±0.124 (P=0.000). After 24 hours of incubation, compared with NC group [(100.000 ± 12.347)%], the cell migration rate in mimic group was [(43.727 ± 16.746)%] and in scramble group was [(91.722 ± 7.631)%], overexpression of miR-520e inhibits lung cancer cell migration (P=0.000). Invasion experiments show that, the number of invasive cells in the mimic group [(36.458±21.301)%]was lower compared with the miR-520e scramble group [(93.783±9.125)%] and the NC group [(100.000±15.114)%, P=0.000]. Luciferase reporter gene results showed that transfection of miR-520e mimics inhibited luciferase activity by 0.374±0.241 (P=0.000). The expression of ZNF367 was down-regulated by miR-520e mimics (0.426 ± 0.153, P=0.000; 0.368±0.127, P=0.000) by RT-PCR and Western blotting. Further experiments showed that miR-520e targets ZNF367 gene to regulate the migration and invasion of lung cancer cells. Conclusion miR-520e inhibits the migration and invasion of lung cancer A549 cells by regulating ZNF367 Key words: MicroRNA-520e; Zinc finger protein 367; Lung cancer; Migration; Invasion

Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBPECP, Depends on a Single Tryptophan.

A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBPECP solution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBPECP bound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We developed a molecular model showing that the two effects synergistically promote the penetration. The loss of either effect would abolish the penetration. GBPECP has been proven to enter cells through macropinocytosis. The GBPECP treatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be modulated by GBPECP internalization. The intracellular penetration of GBPECP leading to inhibition of epithelial cell migration and invasion depends on the presence of the tryptophan residue in its sequence compared with similar derivative peptides. Therefore, GBPECP shows substantial potential as a novel delivery therapeutic through rapid and effective internalization and interference with cell mobility.

Marsdenia tenacissima extract suppresses A549 cell migration through regulation of CCR5-CCL5 axis, Rho C, and phosphorylated FAK

Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.

Effect of Inhibiting NGAL Gene Expression on A549 Lung Cancer Cell Migration and Invasion

背景与目的检测NGAL在肺癌组织的表达，观察小干扰RNA（siRNA）沉默NGAL基因表达对肺癌细胞增殖及侵袭能力的影响。方法应用免疫组织化学检测肺癌组织中NGAL表达。通过RNAi技术干扰NGAL后，运用定量PCR、Western blot技术，观察干扰NGAL基因效率。干扰NGAL后，MTT检测细胞增殖、Transwell实验检测细胞迁移侵袭能力，细胞划痕愈合能力，免疫荧光及Western blot方法检测上皮细胞-间质细胞转化（epithelial-mesenchymal transition, EMT）相关的蛋白E-cadherin及Vimentin的表达情况。结果肺癌中NGAL阳性率高于癌旁组织（P < 0.01）。NGAL-siRNA转染组，癌细胞中NGAL基因及蛋白水平表达都受到明显的抑制。NGAL-siRNA转染组细胞增殖能力，迁移及侵袭能力相对于对照组都降低，差异具有统计学意义（P < 0.05）。干扰NGAL，E-cadherin的表达升高，Vimentin的表达水平降低。干扰NGAL，MMP-2和MMP-9蛋白表达水平降低。结论NGAL在肺癌组织高表达，且在肺癌细胞的增殖、侵袭和迁移及EMT过程中起着重要作用。NGAL可能是肺癌浸润和转移的一个重要参考指标，可为肺癌治疗提供潜在的靶点。

Suppression of A549 lung cancer cell migration by precursor let-7g microRNA

Let-7g miRNAs, short non-coding RNAs approximately 21 nucleotides long, repress protein translation by binding to the 3'UTR of target mRNAs. Aberrant expression of let-7g is associated with the poor prognosis of lung cancer patients. Compared to normal lung cells, let-7g expression is absent in non-small cell lung cancer (NSCLC) cells. Furthermore, K-Ras and HMGA2 are well known as targets of let-7g. In this study, we evaluated the potential role of precursor (pre)-let-7g in lung cancer cell metastasis, focusing on the two targets of let-7g, HMGA2 and K-Ras. We found that pre-let-7g inhibited the migration of A549 lung cancer cells through HMGA2-mediated E2F1 down-regulation. Thus, our results suggest that pre-let-7g could be used as a suitable target for the suppression of lung cancer cell migration.

Lung cancer cell migration is regulated via repressing growth factor PTN/RPTP β/ζ signaling by menin

Menin encoded by the multiple endocrine neoplasia type 1 (MEN1) gene is associated with chromatin and the nuclear matrix and exerts multiple biological functions including regulation of cell proliferation and adhesion. Men1 mutations increase the likelihood of lung cancer development in mice. Menin expression is reduced in certain human non-small cell lung cancer cells, and reduction of menin is closely correlated with increased lung cancer metastasis to lymph nodes. However, it is poorly understood whether menin affects migration of lung cancer cells. In this study, we show that menin-regulated A549 lung cancer cell migration, which was mediated by growth factor pleiotrophin (PTN) and its cell surface receptor, protein tyrosine phosphatase beta/zeta (RPTP β/ζ). Ectopic menin expression significantly repressed PTN transcription, but indirectly inhibited RPTP β/ζ expression through repressing PTN expression. Further studies revealed that menin-regulated cell migration through PTN/RPTP β/ζ, in conjunction with integrin αvβ3, focal adhesion kinase, phosphatidylinositol 3-kinase and phosphorylated extracellular signal regulated kinase 1/2. These findings provide mechanistic insights into the molecular basis for menin/PTN-mediated regulation of A549 lung cancer cell migration.

Highlights of This Issue

Available clinical data establish a protective effect of COX-2 inhibition on human cancer progression, but the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer therapeutics. Kim and colleagues report that the COX-2 effector PGE2, which activates four cognate receptors, signals selectively via EP4 to enhance A549 lung cancer cell migration. They further find that this mode of signaling requires the presence of βArrestin1 and the tyrosine kinase c-Src. Hence, this study provides preclinical-based rationale for the selective targeting of EP4 to inhibit PGE2-induced lung cancer cell migration.Cancer progression is governed by two parallel yet interdependent mechanisms: intrinsic, lineage-specific transcriptional programs and external microenvironmental cues. In this article, specific contributions of adhesive interactions with fibronectin to the transcriptional program of malignant B cells were assessed. It was found that highly adherent B cells express higher levels of genes associated with progression and poor prognosis of multiple myeloma. In contrast, B cells that display poor adhesive properties and lower tumorigenic potential express genes associated with higher levels of B-cell differentiation. These results further indicate that adhesive interactions can modulate the gene expression program of malignant B cells, affecting genes associated with tumorigenesis.MicroRNAs (miRNA) negatively regulate gene expression through binding to a target mRNA. The miRNA profile of prostate carcinoma was compared to normal tissue by deep sequencing. Szczyrba and colleagues found a deregulation by at least 1.5-fold for only 33 miRNAs confirmed for selected miRNAs by Northern blotting and/or qRT-PCR. These data will help to define novel therapeutic targets, either proteins or perhaps the miRNAs themselves. For example, the mRNA of the MYO6 gene was determined to be a target for both miR-145 and miR-143, which are both downregulated by about 75% in prostate carcinoma compared to noncancerous prostate tissue. This is in line with higher levels of myosin IV protein in the tumour cells.The TGFβ regulates many cancer phenotypes, including the cell cycle, migration, and epithelial-to-mesenchymal transition (EMT). Mullenders and coworkers used a small interfering RNA library targeting some 700 kinases and kinase-related genes to find novel modulators of the TGFβ response. They identified several genes that were previously not linked to the TGFβ signaling, including the IRAK2 kinase. Loss of this kinase resulted in a reduction of nuclear accumulation and phosphorylation of SMAD2. In addition, suppression of IRAK2 expression led to a partial override of a TGFβ-induced cell cycle arrest. These data show that IRAK2 is a genuine component of TGFβ signaling.

Prostaglandin E2 Promotes Lung Cancer Cell Migration via EP4-βArrestin1-c-Src Signalsome

Many human cancers express elevated levels of cyclooxygenase-2 (COX-2), an enzyme responsible for the biosynthesis of prostaglandins. Available clinical data establish the protective effect of COX-2 inhibition on human cancer progression. However, despite these encouraging outcomes, the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer drugs. Hence, a better understanding of the molecular signals downstream of COX-2 is needed for the elucidation of drug targets that may improve cancer therapy. Here, we show that the COX-2 product prostaglandin E(2) (PGE(2)) acts on cognate receptor EP4 to promote the migration of A549 lung cancer cells. Treatment with PGE(2) enhances tyrosine kinase c-Src activation, and blockade of c-Src activity represses the PGE(2)-mediated lung cancer cell migration. PGE(2) affects target cells by activating four receptors named EP1 to EP4. Use of EP subtype-selective ligand agonists suggested that EP4 mediates prostaglandin-induced A549 lung cancer cell migration, and this conclusion was confirmed using a short hairpin RNA approach to specifically knock down EP4 expression. Proximal EP4 effectors include heterotrimeric Gs and betaArrestin proteins. Knockdown of betaArrestin1 expression with shRNA significantly impaired the PGE(2)-induced c-Src activation and cell migration. Together, these results support the idea that increased expression of the COX-2 product PGE(2) in the lung tumor microenvironment may initiate a mitogenic signaling cascade composed of EP4, betaArrestin1, and c-Src which mediates cancer cell migration. Selective targeting of EP4 with a ligand antagonist may provide an efficient approach to better manage patients with advanced lung cancer.