Invasion Ability Of A549 Cells Research Articles

The expression and the role of PIWI like RNA-mediated gene silencing 3 (PIWIL3) in lung cell line.

BackgroundTo determine the expression and role of PIWI-like RNA-mediated gene silencing 3 (PIWIL3) in human lung cancer.MethodsImmunohistochemistry was performed to measure the expression of PIWIL3 in 30 of pairs lung cancer and corresponding paracancer tissues. Quantitative PCR (qPCR) was conducted to analyze the expression of PIWIL3 in four breast cancer cell lines. siRNA was used to silence PIWIL3 in the lung cancer cells (A549). Methylthiazoletetrazolium (MTT) proliferation assay and colony formation assay were conducted to detect the growth of A549 cells. Fluorescence-activated cell sorting (FACS) was performed to measure the apoptosis of A549 cells. Transwell migration assay and wound scratch healing assay were used to analyze the migration and invasion ability of A549 cells. SPSS 20.0 was used to analyze the data.ResultsThe expression of PIWIL3 protein was higher in tumor tissues than that in paracancer tissues. In addition, PIWIL3 mRNA was highly expressed in all four lung cancer cell lines. Furthermore, RPS15A knockdown significantly suppressed cell proliferation (P<0.01), induced apoptosis (P<0.01) and inhibited metastasis (P<0.01) of A549 cells.ConclusionsPIWIL3 was highly expressed in lung cancer tissues and could promote the progression of lung cancer.

MiR-296-3p may affect the proliferation and migration of non-small cell lung cancer cells via regulating RABL3.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. The pathogenesis of NSCLC has not yet been fully understood, and the therapeutic efficacy of current anti-NSCLC medication remains unsatisfactory. Previous studies indicated that miR-296-3p was down-regulated in NSCLC, suggesting that miR-296-3p may participate in the pathogenesis of NSCLC; however, the specific mechanisms still need to be further explored. The aim of this work is to investigate the roles of miR-296-3p in NSCLC and the related mechanism. Thirty NSCLC tissue and paired adjacent tissue were collected, and Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was performed to examine the expression of miR-296-3p in cancer tissue and the adjacent tissue. Next, A549 cells were cultured and transfected with miR-296-3p mimics, and cell migration and invasion were determined using scratch wound-healing and transwell assays. Moreover, Western blot assay was performed to determine the effect of miR-296-3p on the expression of Rab-like 3 (RABL3), Matrix metallopeptidase (MMP)-2, Janus kinase (JAK) and Signal transducer and activator of transcription 3 (STAT3); next, Dual-Luciferase reporter assay has been conducted to prove the direct targeting relationship between miR-296-3p and RABL3. Finally, the cells of different treatments were subcutaneously implanted into nude mice to investigate the effect of miR-296-3p mimics in the xenograft mice tumor models. Our data indicated that miR-296-3p was significantly down-regulated and RABL3 was markedly up-regulated in NSCLC tissue compared with the adjacent tissue. Moreover, transient over-expression of miR-296-3p in A549 cells induced a significant decrease in the proliferation and invasion ability of A549 cells, as well as decreased expression of RABL3, MMP-2, JAK and STAT3. Furthermore, the Dual-Luciferase reporter assay confirmed that RABL3 is a direct target of miR-296-3p. Finally, the results of animal studies indicated that miR-296-3p can regulate the tumorigenesis of A549 cells in vivo. Our findings proved that miR-296-3p may play a role as a tumor suppressor in NSCLC both in vitro and in vivo, and we first reported that miR-296-3p can regulate the migration and invasion of A549 cells via targeting RABL3. Our data suggested that miR-296-3p may serve as a potential therapeutic target for treating NSCLC.

Alisol B 23‑acetate inhibits the viability and induces apoptosis of non‑small cell lung cancer cells via PI3K/AKT/mTOR signal pathway.

The aim of the present study was to investigate the effects of alisol B 23-acetate (AB23A) on inhibiting the viability and inducing apoptosis of human non-small cell lung cancer (NSCLC) cells and the anticancer mechanisms of AB23A in vitro. The viability of A549 cells following treatment with different doses of AB23A was examined using a Cell Counting Kit-8 assay. Subsequently, apoptosis and the cell cycle were detected using flow cytometric analysis. The effect of AB23A on migration and invasion of A549 cells was detected by wound healing and Transwell assays. Western blotting was performed to determine the relative expression of Bax/Bcl-2, phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR). AB23A markedly inhibited the viability enhanced apoptosis of A549 cells and arrested the cell cycle in G1 phase. Additionally, AB23A upregulated the ratio of Bax/Bcl-2 in the A549 cells in a concentration-dependent manner. The results of wound healing and Transwell assays indicated that AB23A also suppresses the migration and invasion ability of A549 cells. Furthermore, AB23A reduced the protein levels of phosphorylated AKT, PI3K and mTOR. In conclusion, AB23A exerted anti-cancer activity via inhibiting cells viability, migration and invasion, and promoting apoptosis. Therefore, AB23A is a potential antitumor drug for the treatment of NSCLC.

Icotinib inhibits proliferation and epithelial-mesenchymal transition of non-small cell lung cancer A549 cells.

Objective: To investigate the inhibitory effect of Icotinib on proliferation and epithelial-mesenchymal transition of A549 cells. Methods: A549 cells were treated with Icotinib of different concentrations. The morphology of the cells was observed by inverted phase contrast microscope. The effect of different concentrations of Icotinib on the proliferation inhibition rate of A549 cells was detected by MTT assay. Flow cytometry was used to detect effect of Icotinib on the apoptosis rate of A549 cells. ELISA assay was used to detect the expression of EMT-related proteins, E-cadherin, N-cadherin, Vimentin and fibronectin, in A549 cells cultured supernatant. Transwell assay was used to examine the effects of different concentrations of Icotinib on the migration and invasion abilities of A549 cells. Results: Morphological changes were observed after A549 cells exposed to different concentrations of Icotinib for 48 hours. The inhibition rate of proliferation for each treatment group increased from MTT assay results. Flow cytometry results showed that cell apoptosis rate was significantly increased. Expression of E-cadherin was up-regulated and expression of N-cadherin, Vimentin and fibronectin were down-regulated from ELISA results. A549 cell migration and invasion abilities were significantly suppressed by Icotinib at different concentrations. Conclusion: Icotinib inhibits A549 cell proliferation, migration and invasion in both concentration and time-dependent manners, in turn it promotes A549 cell apoptosis in the same way. Icotinib also inhibits epithelial-mesenchymal transition by regulating EMT-related proteins expression in A549 cells.

CTEN induces epithelial-mesenchymal transition (EMT) and metastasis in non small cell lung cancer cells.

To explore the effects and mechanism of CTEN (COOH-terminus tensin-like molecule) on EMT, cell migration and invasion of Human lung adenocarcinoma cells. The pCMV-vector, pCMV-CTEN, Control-shRNA, and CTEN-shRNA were transfected into A549 and NCI-H1299 cells by Lipofectamine 2000. Transforming growth factor-β1(TGF-β1)and epithelial-mesenchymal transition (EMT) -related biomarkers were detected by eliseand western blot. The migration and invasion ability of A549 cells and NCI-H1299 were examined by scratch-wound assay and transwell assay respectively. We found compare with control group, the expression of TGF-β and mesenchymal markers in CTEN overexpression group were increased, and the epithelial marker was decreased, which induced the EMT process. Meanwhile, scratch-woundassay showed that the migration efficiency of A549 and NCI-H1299 cells in CTEN overexpression group were higher than that in control group.Transwell assay demonstrated that the number of cells that migrated and invaded through the membrane were obviously more than those in control group.Furthermore, Knockdown of CTEN partially reversed transforming growth factor-β1(TGF-β1)-induced changes in EMT markers. In conclusion, CTEN activated the expression of TGF-β1, thereby prompting EMT in lung adenocareinma cancer cells.

MicroRNA-21 promotes the proliferation, migration and invasion of non-small cell lung cancer A549 cells by regulating autophagy activity via AMPK/ULK1 signaling pathway.

The present study investigated the expression of microRNA (miR)-21 in non-small cell lung cancer (NSCLC) tissues, its biological functions and mechanism of autophagy regulation. A total of 46 patients with NSCLC were enrolled in the present study. To measure the expression of miR-21, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed. NSCLC A549 cells were transfected with miR-negative control (NC), miR-21 mimics or inhibitor. The CCK-8 assay was used to investigate the proliferation of A549 cells. To study migration and invasion abilities of A549 cells, The Transwell assay was performed. In addition, to determine the expression levels of ULK1, LC3B, AMPKα, p-AMPKα and p62 proteins, western blotting was conducted and laser confocal microscopy was performed to observe the formation of autophagosomes in A549 cells. To explore whether miR-21 regulates the biological functions of A549 cells via autophagy, an autophagy inhibitor, 3-MA, or agonist, rapamycin, were used in a rescue assay. Results indicated that miR-21 expression in NSCLC tissues was enhanced, and closely correlated with the occurrence and development of NSCLC. In vitro experiments showed that miR-21 mimics promoted the proliferation, migration and invasion of A549 cells, while miR-21 inhibitor inhibited these biological functions. Western blotting indicated that miR-21 upregulated autophagy marker LC3BII protein, but downregulated p62 protein. Laser confocal microscopy showed that miR-21 activated autophagy of A549. Rescue experiments indicated that autophagy reversed the effect of miR-21 on the proliferation, migration and invasion of A549 cells. Western blotting data suggested that autophagy-related AMPK/ULK1 signaling pathway was activated by miR-21, and interference or overexpression of ULK1 reversed the biological functions of miR-21. The present study demonstrated that miR-21 expression in NSCLC tissues was upregulated and positively correlated with lymphatic metastasis and clinical staging. In addition, miR-21 regulated autophagy activity of NSCLC A549 cells via AMPK/ULK1 signaling pathway, and promoted the proliferation, migration and invasion of NSCLC A549 cells.

Effect of microRNA- 193a on the proliferation and migration ability of human lung carcinoma A549 cells by promyosin

Objective To investigate the effect and the related mechanism of microRNA (miRNA, miR)- 193a on the proliferation and migration ability of human lung carcinoma A549 cells by promyosin (TPMF). Methods Western blotting was used to select the cell lines with the lowest expression of TPMF in lung cancer cell lines. The TPMF gene was silenced through TPMF Silencing Lentivirus (LV3- TPMF). green fluorescent protein (GFP) fluorescence detector and Western bolting were used to detect the efficiency of gene silence. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the correlation between miR- 193a and TPMF. Effect of TPMF on the invasion ability of A549 cells was examined by Transwell matrigel invasion assays. Effect of TPMF on the A549 cells proliferation ability was examined by cell clone assays. The correlation between miR- 193a and TPMF was analyzed by Western blotting and RT- PCR. The Effect of TPMF Silencing on Proliferation of Tumor in Nude Mice Survival Rate was detected. Results LV3- TPMF lentivirus could effectively inhibit the expression of TPMF [(88.29±8.62)% vs. (17.82±4.91)%, P=0.010]. Silencing the expression of TPMF could inhibit the invasion ability of human lung carcinoma A549 cells [(351.23±6.92)% vs. (83.26±3.57)%, P=0.023]. Silence of TPMF could inhibit the proliferation of human lung carcinoma A549 cells [(103.82±5.12)% vs. (22.34±1.91)%, P=0.030]. Silence of TPMF could extend the survival rates of nude mice. Conclusion MiR- 193a can inhibit the invasion and proliferation of lung carcinoma A549 cells by regulating the expression of TPMF. Key words: MicroRNA-193a; Lung carcinoma; Transwell; Promyosin; Survival rate

Hypoxic microenvironment promotes proliferation and invasion of non-small cell lung cancer A549 Cells through Wnt/β-catenin signaling pathway

Objective: To study the effect of Wnt/β-catenin signaling pathway on hypoxia induced proliferation and invasion in lung cancer cell line A549. Methods: Cultured lung cancer A549 cells were treated with hypoxia for 24 h. The proliferation rates were detected by CCK8; the mRNA and protein expression levels of HIF-1α and β-catenin were detected by RT-PCR and Western blot; the effect of hypoxia on invasion ability of A549 cells were detected with Transwell method. Furthermore, β-catenin siRNA was used to investigate the role of β-catenin on hypoxia induced proliferation and invasion. Results: The results demonstrated that hypoxia could promote A549 cells proliferation and invasion. The mRNA and protein level of HIF-1α and β-catenin was upregulated by hypoxia treatment, whereas β- catenin siRNA could block these processes. Conclusion: Hypoxia promotes osteosarcoma cell line A549 proliferation and invasion by activating Wnt/β-catenin signaling pathway.

JAK2 variations and functions in lung adenocarcinoma.

Lung cancer ranks as the first most common cancer and the first leading cause of cancer-related death in China and worldwide. Due to the difficulty in early diagnosis and the onset of cancer metastasis, the 5-year survival rate of lung cancer remains low. JAK2 has emerged as pivotal participant in biological processes, often dysregulated in a range of cancers. Recently our study found that JAK2 might play an important role in lung cancer pathogenesis. While our understanding of JAK2 in the onset and progression of lung cancer is still in its infancy, there is no doubt that understanding the variations and functions of JAK2 will certainly secure strong biomarkers and improve treatment options for lung cancer patients. The expression level of JAK2 mRNA was assayed using RT-PCR. JAK2 mutations and amplification were detected using next-generation sequencing (NGS). The shRNA and overexpression plasmids of JAK2 were conducted. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide) assay, Trans-well migration and Matrigel invasion assay were conducted to study the proliferation, migration and invasion abilities of lung adenocarcinoma cells independently. We found that JAK2 mRNA was up-regulated in lung adenocarcinoma tissues when compared with their adjacent non-tumor tissues, and was associated with lymph node metastasis ( p < 0.05). JAK2 V617F and N30S mutations and JAK2 amplification were detected by NGS in plasmid samples in patients with lung adenocarcinoma. Downregulation of JAK2 inhibited the proliferation, migration and invasion abilities of lung adenocarcinoma A549 cells. Moreover, overexpression of JAK2 induced the proliferation, migration and invasion abilities of A549 cells. Thus, the up-regulation, mutation and amplification of JAK2 detected in lung adenocarcinoma may participate in lung cancer progression by regulating cancer cells' proliferation, migration and invasion.

Ovol2 gene inhibits the Epithelial-to-Mesenchymal Transition in lung adenocarcinoma by transcriptionally repressing Twist1

BackgroundAssociated with recent achievements in therapy for advanced lung adenocarcinoma, there will still be an unmet medical need for effective treatment of stage IIIb/IV, and the prognosis of lung cancer is not optimistic till now. ObjectiveIn order to obtain some essential evidences for a potential targeted therapy in lung adenocarcinoma, the effects of Ovol2 gene on Epithelial-to-Mesenchymal Transition (EMT) was observed and the probable mechanisms were analyzed. MethodsOvol2 expression was previously evaluated by immunochemistry in lung adenocarcinoma tissue, and Ovol2 was overexpressed by lentivirus infection in A549 cells. Subsequently, the migration and invasion ability of A549 cells was tested by Transwell and Wound healing experiments. The mRNA level of genes correlated to EMT was detected by Real-time PCR, and the expression of reasonable makers was probed by Western Blot. Finally, rescue experiment, Luciferase assay, and chromatin immunoprecipitation assay were performed to explore the probable mechanisms. ResultsAfter treated with Ovol2 overexpression, the expression level of E-cadherin was increased, while the expression level of Vimentin and Twist1 was declined not only in the mRNA level but also in the protein level. Moreover, we found that Ovol2 represses transcription of Twist1 by binding to its promoter directly. Wound healing and Transwell assays indicate that the migration and invasion ability were downregulated by Ovol2 in A549 cells. ConclusionOvol2 can suppress migration and invasion ability of A549 cells, and prevent EMT by inhibition of Twist1 transcription directly.

Tanshinone IIA combined with adriamycin inhibited malignant biological behaviors of NSCLC A549 cell line in a synergistic way.

BackgroundThe study was designed to develop a platform to verify whether the extract of herbs combined with chemotherapy drugs play a synergistic role in anti-tumor effects, and to provide experimental evidence and theoretical reference for finding new effective sensitizers.MethodsInhibition of tanshinone IIA and adriamycin on the proliferation of A549, PC9 and HLF cells were assessed by CCK8 assays. The combination index (CI) was calculated with the Chou-Talalay method, based on the median-effect principle. Migration and invasion ability of A549 cells were determined by wound healing assay and transwell assay. Flow cytometry was used to detect the cell apoptosis and the distribution of cell cycles. TUNEL staining was used to detect the apoptotic cells. Immunofluorescence staining was used to detect the expression of Cleaved Caspase-3. Western blotting was used to detect the proteins expression of relative apoptotic signal pathways. CDOCKER module in DS 2.5 was used to detect the binding modes of the drugs and the proteins.ResultsBoth tanshinone IIA and adriamycin could inhibit the growth of A549, PC9, and HLF cells in a dose- and time-dependent manner, while the proliferative inhibition effect of tanshinone IIA on cells was much weaker than that of adriamycin. Different from the cancer cells, HLF cells displayed a stronger sensitivity to adriamycin, and a weaker sensitivity to tanshinone IIA. When tanshinone IIA combined with adriamycin at a ratio of 20:1, they exhibited a synergistic anti-proliferation effect on A549 and PC9 cells, but not in HLF cells. Tanshinone IIA combined with adriamycin could synergistically inhibit migration, induce apoptosis and arrest cell cycle at the S and G2 phases in A549 cells. Both groups of the single drug treatment and the drug combination up-regulated the expressions of Cleaved Caspase-3 and Bax, but down-regulated the expressions of VEGF, VEGFR2, p-PI3K, p-Akt, Bcl-2, and Caspase-3 protein. Compared with the single drug treatment groups, the drug combination groups were more statistically significant. The molecular docking algorithms indicated that tanshinone IIA could be docked into the active sites of all the tested proteins with H-bond and aromatic interactions, compared with that of adriamycin.ConclusionsTanshinone IIA can be developed as a novel agent in the postoperative adjuvant therapy combined with other anti-tumor agents, and improve the sensibility of chemotherapeutics for non-small cell lung cancer with fewer side effects. In addition, this experiment can not only provide a reference for the development of more effective anti-tumor medicine ingredients, but also build a platform for evaluating the anti-tumor effects of Chinese herbal medicines in combination with chemotherapy drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2921-x) contains supplementary material, which is available to authorized users.

Oncogenic role of epithelial cell transforming sequence 2 in lung adenocarcinoma cells.

Lung adenocarcinoma, which is the most common non-small cell lung cancer, is the leading cause of death from cancer worldwide. Epithelial cell transforming sequence 2 (ECT2) is frequently upregulated and acts as an oncogene in various human cancers. In addition, ECT2 was reported to be upregulated in early stage lung adenocarcinoma. However, the detailed role of ECT2 in mediating the malignant phenotypes of lung adenocarcinoma cells has not previously been elucidated. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to examine ECT2 mRNA and protein expression levels, respectively. MTT, wound healing and Transwell assays were conducted to determine cell proliferation, migration and invasion abilities, respectively. In the present study, ECT2 was significantly upregulated in lung adenocarcinoma cell lines (H650, EKVX, HCC4006, HCC827, HCC2935, Hop62 and A549), as compared with a normal lung epithelial cell line (BEAS-2B). Moreover, knockdown of ECT2, induced by transfection with ECT2 siRNA, significantly inhibited the proliferation of lung adenocarcinoma A549 cells, whereas overexpression of ECT2 enhanced A549 cell proliferation. Furthermore, knockdown of ECT2 expression suppressed the migration and invasion of A549 cells, whereas overexpression of ECT2 enhanced the migration and invasion abilities of A549 cells. Notably, inhibition of ECT2 also suppressed the expression levels of N-cadherin and vimentin, whereas it enhanced the expression level of E-cadherin, indicating that ECT2 is associated with the epithelial-mesenchymal transition in A549 cells. On the contrary, overexpression of ECT2 enhanced the expression levels of N-cadherin and vimentin, whereas it reduced the expression level of E-cadherin in A549 cells. In conclusion, the results of the present study suggest that ECT2 has an oncogenic role in lung adenocarcinoma cells. Therefore, ECT2 may be a potential novel target for the treatment of lung adenocarcinoma.

Abstract 1678: DRAM1 regulates EMT of lung cancer A549 cells via SQSTM1/p62 in vitro

Abstract Epithelial-to-mesenchymal transition (EMT) has been linked to the regulation of non-small cell lung cancer (NSCLC) progression, migration, invasion, metastasis, and chemoresistance as well. Recent studies showed that autophagy was a novel target to mediate EMT of cancer cells through regulating the migration and invasion in cancer. The DNA Damage-Regulated Autophagy Modulator 1 (DRAM1) is an evolutionarily conserved transmembrane protein and localizes predominantly to lysosomes, while also colocalizing with the autophagosome marker LC3. Previous investigation discovered that DRAM1 played an important role in regulating autophagy, inducing apoptosis of NSCLC cells. However, the function and mechanism of DRAM1 mediated autophagy in regulating EMT in NSCLC remains poorly understood. In this study, transforming growth factor-β (TGF-β) and starvation were employed to induce autophagy for investigating EMT in NSCLC A549 cells. The results showed that after exposure to TGF-β 24h, the protein levels of DRAM1, autophagy marker SQSTM1/p62, LC3I/II and EMT marker vimentin significantly increased while another EMT marker E-cadherin protein decreased obviously compared with the control by western blot. Also the similar results were observed after starvation 48h in this study. To further illustrate the role of DRAM1 in regulation of autophagy and EMT, wildtype DRAM1 overexpression plasmid and DRAM1 siRNA was transfected into A549 cells respectively to check the autophagy markers and EMT markers expression, meanwhile wound healing test and cell invasion transwell chamber test were assayed after the gene manipulation. These experiments results demonstrated that DRAM1 overexpression dramatically increased the SQSTM1/p62, LC3I/II and vimentin protein levels and inhibited the expression of E-cadherin, promoting the migration and invasion abilities of A549 cells. Conversely, down-regulation of DRAM1 restrained SQSTM1/p62, LC3 I/II and vimentin protein levels and elevated the expression of E-cadherin, leading to the inhibition of migration and invasion abilities of A549 cells. Interestingly, our further study showed that after p62 siRNA transfected into A549 cells E-cadherin upregulated while vimentin reduced compared to the control group. Consistently, knockdown of p62 suppressed the migration and invasion abilities of A549 cells and down regulated the expression of twist, which is an EMT related transcription factor. These data suggested that DRAM1 played an important role in regulating the EMT of lung cancer A549 cells through SQSTM1/p62 modulating, which might be a novel potential target for lung cancer treatment. Citation Format: Binfeng He, Jiajing Cai, Guangcheng Huang, Fan Chang, Lei Xu, Dongsheng Wang, Yibin Deng, Xiaolan Guo. DRAM1 regulates EMT of lung cancer A549 cells via SQSTM1/p62 in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1678.

Expression and pathological mechanism of MMP-9 and HIF-2α in CD133(+) lung cancer stem cells

To investigate the expression and pathological mechanism of matrix metalloproteinase (MMP)-9 and hypoxia-inducible factor (HIF)-2α in CD133⁺ lung cancer stem cells. Sixty-two cases of lung cancer paraffin embedding tissues were collected from the First Affiliated Hospital of Soochow University between January 2009 and December 2009. Immunohistochemistry (IHC) was used for detection of CD133 expression in lung cancer tissues and the clinical significance was analyzed. Real-time polymerase chain reaction (PCR) was used for the investigation of expression of tumor metastasis associated genes, including MMP-1, MMP-2, MMP-9, HIF-1α, HIF-1β, HIF-2α and tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, TIMP-4. Scrambled siRNA or CD133 siRNA were used to transfect the lung cancer cell line A549, then Control-si-A549 cells and CD133-si-A549 cells were generated respectively. PCR was used to analysis CD133, MMP-9 and HIF-2α genes expression and transwell invasion assay was used to study the invasion ability of A549 cells in two groups. 51.6% of lung cancer tissues expressed CD133 (P<0.05); the expression level of CD133 was related to tumor metastasis and patients' survival rate (P<0.05). The gene expression of HIF-2α and MMP-9 were increased in CD133⁺ lung cancer cells compared with CD133⁻ cancer cells (1.58 ± 0.39 vs 1.10 ± 0.31, 1.67 ± 0.38 vs 1.05 ± 0.21, all P<0.05), whereas no difference was found in gene expression of MMP-1, MMP-2, HIF-1α, HIF-1β and TIMP-1, TIMP-2, TIMP-3, TIMP-4 (all P>0.05). Compared with the Control-si-A549 cell, the expression of CD133, HIF-2α and MMP-9 (0.24 ± 0.10 vs 0.85 ± 0.23, 0.19 ± 0.09 vs 0.54 ± 0.18, 0.31 ± 0.17 vs 1.12 ± 0.31, all P<0.05) in CD133-si-A549 cell were remarkably decreased. The number of CD133-si-A549 cells migrated to below room was significantly smaller than that of Control-si-A549 cells (207 ± 25 vs 82 ± 10, P<0.05). The CD133⁺ lung cancer stem cell is correlated to the tumor metastasis and patients' survival. CD133⁺ tumor stem cell can promote the tumor invasion and metastasis via the up-regulation of HIF-2α and MMP-9 expression.

Paris Saponin VII Inhibits the Migration and Invasion in Human A549 Lung Cancer Cells.

Metastasis is the main cause of death in lung cancer. Targeting the process of metastasis is a strategy to lung cancer treatment. Trillium tschonoskii Maxim., a traditional Chinese medicine, has been used for treatment of many diseases, including cancer. This study aims to determine the anti-metastatic effect of paris saponin VII (PS VII) which was extracted from T. tschonoskii Maxim. by using human lung cancer cell line A549 cells. Our results showed that PS VII could significantly suppress the viability as well as cell migration and invasion abilities of A549 cells in a concentration-dependent manner. PS VII reduced the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 by elevating the expression of TIMP1/2. These data indicated that PS VII could reduce the metastatic capability of A549 cells, probably through up-regulating the expression of TIMP1/2. These findings demonstrated a new therapeutic potential for PS VII in anti-metastatic therapy of lung cancer. Copyright © 2015 John Wiley & Sons, Ltd.

CCL19/CCR7 upregulates heparanase via specificity protein-1 (Sp1) to promote invasion of cell in lung cancer

CCL19/chemokine receptor 7 (CCR7) has been found to be associated with tumor growth, angiogenesis, invasion, and lymph node metastasis. Our previous study demonstrated that CCR7 overexpressed in non-small cell lung cancer (NSCLC) and had close relationship with tumor invasion and lymph node metastasis. However, the molecular mechanism of CCR7 promoting invasion of human NSCLC cells is still unclear. In this study, we demonstrated that human lung adenocarcinoma A549 cells treated with recombinant human CCL19 could obviously upregulate the expression of Sp1 and heparanase at both the mRNA and protein levels. After blockage of CCR7, Sp1 and heparanase expressions were inhibited. Following inhibition of Sp1, heparanase expression was downregulated. The analysis showed the promoter region of heparanase gene containing a number of potential sp1 binding sites (5'-GGGGC-3'). Chromatin immunoprecipitation analysis demonstrated that Sp1 could bind to the heparanase promoter. Cell invasion assays showed that the invasion ability of A549 cells was increased with CCL19 incubation compared to the control cells. These results suggested that CCL19/CCR7 may upregulate the expression of heparanase via Sp1 and contribute to the invasion of A549 cells.

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.

Resveratrol inhibits EGF-induced invasion of human lung adenocarcinoma A549 cells

背景与目的侵袭转移是人肺腺癌死亡的主要原因，表皮生长因子（epidermal growth factor, EGF）可通过活化ERK和PI3K-Akt信号通路，促进人肺腺癌A549细胞侵袭。本实验旨在研究白藜芦醇抑制EGF诱导体外A549细胞侵袭的作用，并初步探讨其机制。方法采用MTT法确定白藜芦醇对A549细胞无明显毒性的浓度范围，并以该浓度范围内的白藜芦醇作用EGF诱导的A549细胞，Transwell小室法检测其抑制细胞侵袭作用，明胶酶谱法分析细胞的MMP-2活性变化，Western blot检测EGF信号通路相关蛋白表达。结果30 μM内的白藜芦醇无明显的细胞毒性，20 μM处理的受EGF诱导的A549细胞侵袭能力明显降低，MMP-2分泌减少，胞内p-ERK1/2和PI3K（0.5 h-6 h）含量下降。结论20 μM白藜芦醇可有效抑制A549细胞侵袭，其机制可能是通过抑制ERK通路和PI3K-Akt通路中相关蛋白激活，最终降低MMP-2的分泌。