Salvianolic acid A reverses cisplatin resistance in lung cancer A549 cells by targeting c-met and attenuating Akt/mTOR pathway.

Drug resistance is one of the leading causes of chemotherapy failure in non-small cell lung cancer (NSCLC) treatment. The purpose of this study was to investigate the role of c-met in human lung cancer cisplatin resistance cell line (A549/DDP) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. In this study, we found that A549/DDP cells exert up-regulation of c-met by activating the Akt/mTOR signaling pathway. We also show that SAA could increase the chemotherapeutic efficacy of cisplatin, suggesting a synergistic effect of SAA and cisplatin. Moreover, we revealed that SAA enhanced sensitivity to cisplatin in A549/DDP cells mainly through suppression of the c-met/AKT/mTOR signaling pathway. Knockdown of c-met revealed similar effects as that of SAA in A549/DDP cells. In addition, SAA effectively prevented multidrug resistance associated protein1 (MDR1) up-regulation in A549/DDP cells. Taken together, our results indicated that SAA suppressed c-met expression and enhanced the sensitivity of lung adenocarcinoma A549 cells to cisplatin through AKT/mTOR signaling pathway.

Integrated microRNA and gene expression profiling reveals the crucial miRNAs in curcumin anti-lung cancer cell invasion.

BACKGROUND: Curcumin (diferuloylmethane) has chemopreventive and therapeutic properties against many types of tumors, both in vitro and in vivo. Previous reports have shown that curcumin exhibits anti-invasive activities, but the mechanisms remain largely unclear. METHODS: In this study, both microRNA (miRNA) and messenger RNA (mRNA) expression profiles were used to characterize the anti-metastasis mechanisms of curcumin in human non-small cell lung cancer A549 cell line. RESULTS: Microarray analysis revealed that 36 miRNAs were differentially expressed between the curcumin-treated and control groups. miR-330-5p exhibited maximum upregulation, while miR-25-5p exhibited maximum downregulation in the curcumin treatment group. mRNA expression profiles and functional analysis indicated that 226 differentially expressed mRNAs belonged to different functional categories. Significant pathway analysis showed that mitogen-activated protein kinase, transforming growth factor-ß, and Wnt signaling pathways were significantly downregulated. At the same time, axon guidance, glioma, and ErbB tyrosine kinase receptor signaling pathways were significantly upregulated. We constructed a miRNA gene network that contributed to the curcumin inhibition of metastasis in lung cancer cells. let-7a-3p, miR-1262, miR-499a-5p, miR-1276, miR-331-5p, and miR-330-5p were identified as key microRNA regulators in the network. Finally, using miR-330-5p as an example, we confirmed the role of miR-330-5p in mediating the anti-migration effect of curcumin, suggesting the importance of miRNAs in the regulation of curcumin biological activity. CONCLUSION: Our findings provide new insights into the anti-metastasis mechanism of curcumin in lung cancer.

Exploration of inhibitory mechanisms of curcumin in lung cancer metastasis using a miRNA- transcription factor-target gene network.

The present study was aimed to unravel the inhibitory mechanisms of curcumin for lung cancer metastasis via constructing a miRNA-transcription factor (TF)-target gene network. Differentially expressed miRNAs between human high-metastatic non-small cell lung cancer 95D cells treated with and without curcumin were identified using a TaqMan human miRNA array followed by real-time PCR, out of which, the top 6 miRNAs (miR-302b-3p, miR-335-5p, miR-338-3p, miR-34c-5p, miR-29c-3p and miR-34a-35p) with more verified target genes and TFs than other miRNAs as confirmed by a literature review were selected for further analysis. The miRecords database was utilized to predict the target genes of these 6 miRNAs, TFs of which were identified based on the TRANSFAC database. The findings of the above procedure were used to construct a miRNA-TF-target gene network, among which miR-34a-5p, miR-34c-5p and miR-302b-3p seemed to regulate CCND1, WNT1 and MYC to be involved in Wnt signaling pathway through the LEF1 transcription factor. Therefore, we suggest miR-34a-5p/miR-34c-5p/miR-302b-3p -LEF1-CCND1/WNT1/MYC axis may be a crucial mechanism in inhibition of lung cancer metastasis by curcumin.

miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting MET.

MicroRNAs (miRNAs) play a critical role in drug resistance and epithelial-mesenchymal transition (EMT). The aims of this study were to explore the potential role of miR-206 in governing cisplatin resistance and EMT in lung cancer cells. We found that both lung adenocarcinoma A549 cisplatin-resistant cells (A549/DDP) and H1299 cisplatin-resistant cells (H1299/DDP) acquired mesenchymal features and were along with low expression of miR-206 and high migration and invasion abilities. Ectopic expression of miR-206 mimics inhibited cisplatin resistance, reversed the EMT phenotype, decreased the migration and invasion in these DDP-resistant cells. In contrast, miR-206 inhibitors increased cisplatin resistance, EMT, cell migration and invasion in non-DDP-resistant cells. Furthermore, we found that MET is the direct target of miR-206 in lung cancer cells. Knockdown of MET exhibited an EMT and DDP resistant inhibitory effect on DDP-resistant cells. Conversely, overexpression of MET in non-DDP- resistant cells produced a promoting effect on cell EMT and DDP resistance. In lung adenocarcinoma tissues, we demonstrated that low expression of miR-206 were also correlated with increased cisplatin resistance and MET expression. In addition, we revealed that miR-206 overexpression reduced cisplatin resistance and EMT in DDP-resistant cells, partly due to inactivation of MET/PI3K/AKT/mTOR signaling pathway, and subsequent downregulation of MDR1, ZEB1 and Snail expression. Finally, we found that miR-206 could also sensitize A549/DDP cells to cisplatin in mice model. Taken together, our study implied that activation of miR-206 or inactivation of its target gene pathway could serve as a novel approach to reverse cisplatin resistance in lung adenocarcinomas cells.

MiR-206 inhibits HGF-induced epithelial-mesenchymal transition and angiogenesis in non-small cell lung cancer via c-Met /PI3k/Akt/mTOR pathway.

MiR-206 is low expression in lung cancers and associated with cancer metastasis. However, the roles of miR-206 in epithelial-mesenchymal transition (EMT) and angiogenesis in lung cancer remain unknown. In this study, we find that hepatocyte growth factor (HGF) induces EMT, invasion and migration in A549 and 95D lung cancer cells, and these processes could be markedly inhibited by miR-206 overexpression. Moreover, we demonstrate that miR-206 directly targets c-Met and inhibits its downstream PI3k/Akt/mTOR signaling pathway. In contrast, miR-206 inhibitors promote the expression of c-Met and activate the PI3k/Akt/mTOR signaling, and this effect could be attenuated by the PI3K inhibitor. Moreover, c-Met overexpression assay further confirms the significant inhibitory effect of miR-206 on HGF-induced EMT, cell migration and invasion. Notably, we also find that miR-206 effectively inhibits HGF-induced tube formation and migration of human umbilical vein endothelial cells (HUVECs), and the mechanism is also related to inhibition of PI3k/Akt/mTOR signaling. Finally, we reveal the inhibitory effect of miR-206 on EMT and angiogenesis in xenograft tumor mice model. Taken together, miR-206 inhibits HGF-induced EMT and angiogenesis in lung cancer by suppressing c-Met/PI3k/Akt/mTOR signaling. Therefore, miR-206 might be a potential target for the therapeutic strategy against EMT and angiogenesis of lung cancer.

Identification of carcinogenic potential-associated molecular mechanisms in CD133(+) A549 cells based on microRNA profiles.

This study aimed to identify carcinogenic potential-related molecular mechanisms in cancer stem cells (CSCs) in lung cancer. CD133(+) and CD133(-) subpopulations were sorted from A549 cells using magnetic-activated cell sorting. The abilities to form sphere and clone, proliferate, migrate, and invade were compared between CD133(+) and CD133(-) cells, as well as drug sensitivity. Thereafter, microRNA (miRNA) profiles were performed to identify differentially expressed miRNAs between CD133(+) and CD133(-) subpopulation. Following, bioinformatic methods were used to predict target genes for differentially expressed miRNAs and perform enrichment analysis. Furthermore, the mammalian target of rapamycin (mTOR) signaling pathways and CSC property-associated signaling pathways were explored and visualized in regulatory network among competitive endogenous RNA (ceRNA), miRNA, and target gene. CD133(+) subpopulation showed greater oncogenic potential than CD133(-) subpopulation. In all, 14 differentially expressed miRNAs were obtained and enriched in 119 pathways, including five upregulated (hsa-miR-23b-3p, -23a-3p, -15b-5p, -24-3p, and -4734) and nine downregulated (hsa-miR-1246, -30b-5p, -5096, -6510-5p, has-miR-7110-5p, -7641, -3197, -7108-5p, and -6791-5p). For mTOR signaling pathway, eight differential miRNAs (hsa-miR-23b-3p, -23a-3p, -15b-5p, -24-3p, -4734, -1246, -7641, and -3197) and 39 target genes (e.g., AKT1, AKT2, PIK3CB, PIK3CG, PIK3R1, PIK3CA, and PIK3CD) were involved, as well as some ceRNAs. Besides, for CSC property-related signaling pathways, six miRNAs (hsa-miR-1246, -15b-5p, -30b-5p, -3197, -4734, and -7110-5p) were dramatically enriched in Hedgehog, Notch, and Wnt signaling pathways via regulating 108 target genes (e.g., DVL1, DVL3, WNT3A, and WNT5A). The mTOR and CSC property-associated signaling pathways may be important oncogenic molecular mechanisms in CD133(+) A549 cells.

Comprehensive gene and microRNA expression profiling reveals miR-206 inhibits MET in lung cancer metastasis.

MiRNAs associated with the metastasis of lung cancer remain largely unexplored. In this study, gene and miRNA expression profiling were performed to analyze the global expression of mRNAs and miRNAs in human high- and low-metastatic lung cancer cell strains. By developing an integrated bioinformatics analysis, six miRNAs (miR-424-3p, miR-450b-5p, miR-335-5p, miR-34a-5p, miR-302b-3p and miR-206) showed higher target gene degrees in the miRNA-gene network and might be potential metastasis-related miRNAs. Using the qRT-PCR method, the six miRNAs were further confirmed to show a significant expression difference between human lung cancer and normal tissue samples. Since miR-206 showed lower expression both in lung cancer tissues and cell lines, it was used as an example for further functional verification. The wound healing assay and transwell invasion assay showed that miR-206 mimics significantly inhibited the cell migration and invasion of the high-metastatic lung cancer 95D cell strain. One of its predicted targets in our miRNA-gene network, MET, was also obviously decreased at the protein level when miR-206 was overexpressed. Instead, miR-206 inhibitors increased MET protein expression, cell migration and invasion of the low-metastatic lung cancer 95C cell strain. Meanwhile, the luciferase assay showed that MET was a direct target of miR-206. Furthermore, MET gene silence showed a similar anti-migration and anti-invasion effect with miR-206 mimics in 95D cells and could partially attenuate the migration- and invasion-promoting effect of miR-206 inhibitors in 95C cells, suggesting that miR-206 targets MET in lung cancer metastasis. Finally, we also demonstrated that miR-206 can significantly inhibit lung cancer proliferation and metastasis in mouse models. In conclusion, our study provided a miRNA-gene regulatory network in lung cancer metastasis and further demonstrated the roles of miR-206 and MET in this process, which enhances the understanding of the regulatory mechanism in lung cancer metastasis.

Curcumin inhibits proliferation-migration of NSCLC by steering crosstalk between a Wnt signaling pathway and an adherens junction via EGR-1.

A microarray analysis of differential genes by curcumin treatment was performed and the crucial pathways associated with non-small cell lung cancer (NSCLC) were investigated. Total RNAs from 0, 10 or 20 µM curcumin treated NSCLC 95D cells were used to prepare microarray chips. The differentially expressed genes (DEGs) were identified using the RankProducts package and their function was predicted by DAVID and gene set enrichment analysis. The pathway crosstalk was analyzed by mapping the gene expression profiles into protein-protein interaction databases. Validation of the microarray results was performed by cell viability, cell migration and western blot analyses. A total of 486 (10 µM) and 264 (20 µM) DEGs were screened between the 95D cells in the presence and absence of curcumin. Function enrichment analysis indicated the DEGs were mainly involved in the steroid biosynthetic process and regulation of autophagy. Pathway crosstalk analysis suggested there was a significant interaction between NSCLC and adherens junctions (or Wnt signaling pathways, which are important for cancer cell proliferation and invasion) in both 10 µM and 20 µM curcumin treated 95D cells. Furthermore, early growth response (EGR-1) was demonstrated to regulate the crosstalk between adherens junctions and Wnt signaling pathways, indicating that EGR-1 may also regulate cell proliferation and migration. This hypothesis was validated by in vitro experiments: EGR-1 was decreased after curcumin treatment. Curcumin exhibited a significant anti-proliferation and anti-migration activity in NSCLC 95D cells, possibly by steering the crosstalk between the Wnt signaling pathway and adherens junction via EGR-1.

Curcumin inhibits lung cancer cell migration and invasion through Rac1-dependent signaling pathway.

Curcumin, a natural and crystalline compound isolated from the plant Curcuma longa with low toxicity in normal cells, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about antimetastasis effects and mechanism of curcumin in lung cancer. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis. In this study, we examined the influence of curcumin on in vitro invasiveness of human lung cancer cells and the expressions of Rac1. The results indicate that curcumin at 10 µM slightly reduced the proliferation of 801D lung cancer cells but showed an obvious inhibitory effect on epidermal growth factor or transforming growth factor ß1-induced lung cancer cell migration and invasion. Meanwhile, we demonstrated that the suppression of invasiveness correlated with inhibition of Rac1/PAK1 signaling pathways and matrix metalloproteinase (MMP) 2 and 9 protein expression by combining curcumin treatment with the methods of Rac1 gene silence and overexpression in lung cancer cells. Laser confocal microscope also showed that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. At last, through xenograft experiments, we confirmed the connection between Rac1 and the growth and metastasis inhibitory effect of curcumin in vivo. In summary, these data demonstrated that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through inhibition of Rac1/PAK1 signaling pathway and MMP-2 and MMP-9 expression, which provided a novel insight into the molecular mechanism of curcumin against lung cancer.

Functional and pathway enrichment analysis for integrated regulatory network of high- and low-metastatic lung cancer.

Metastasis is a common feature of lung cancer, involving relationships between genes, proteins and miRNAs. However, lack of early detection and limited options for targeted therapies are weaknesses that cantribute to the dismal statistics observed in lung cancer metastasis. In this paper, gene expression profiling analysis for genes differentially expressed between high- (95D) and low-metastatic lung cancer cell lines (95C) was performed using gene annotation, pathway analysis, literature mining, and the integrated regulatory network as well as motif analysis of miRNA-DEG and TF-DEG. In addition, the expression of EGR-1 (early growth reponse-1) in surgically resected lung squamous carcinomas, adenocarcinomas and normal lung tissue was detected by immunohistochemistry to reveal the relationships between EGR-1 and lung cancer metastasis. A total of 570 different expressed genes (DEGs) were screened, the vast majority of up-regulated DEGs were connected to cell adhesion and focal adhesion. EGR-1 was observed in the center node of the regulatory network, which seems to play a role in the process of cancer metastasis, and further immunohistochemistry detection confirmed this reasoning. Besides EGR-1, several significant module-related DEGs were enriched in the pathway within cancer and focal adhesion according to KEGG pathway enrichment analysis of network modules. The construction of an integrated regulatory network and the functional prediction of EGR-1 provided us with the cytological basis of lung cancer metastasis research and an understanding of the mechanism of metastasis in lung cancer. EGR-1 should be considered as a potential target gene in therapeutic agent for lung cancer metastasis.

Silence of ezrin modifies migration and actin cytoskeleton rearrangements and enhances chemosensitivity of lung cancer cells in vitro.

Ezrin, primarily acts as a linker between the plasma membrane and the cytoskeleton, is involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion, motility, and modulation of signaling pathways. Although ezrin is now recognized as a key component in tumor metastasis, its roles and the underlying mechanisms remain unclear. In the present study, we chose highly metastatic human lung carcinoma 95D cells, which highly express the ezrin proteins, as a model to examine the functional roles of ezrin in tumor suppression. An ezrin-silenced 95D cell line was established using lentivirus-mediated short hairpin RNA method. CCK-8 assay and soft agar assay analysis showed that downregulation of ezrin significantly suppressed the tumorigenicity and proliferation of 95D cells in vitro. cell migration and invasion studies showed that ezrin-specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. In parallel, it also induced rearrangements of the actin cytoskeleton. Flow cytometry assay showed that changes in the ezrin protein level significantly affected the cell cycle distribution and eventual apoptosis. Furthermore, further studies showed that ezrin regulated the expression level of E-cadherin and CD44, which are key molecules involved in cell growth, migration, and invasion. Meanwhile, the suppression of ezrin expression also sensitized cells to antitumor drugs. Altogether, our results demonstrated that ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will benefit the development of therapeutic strategy for lung cancer.

[Expression of ezrin in human non-small cell lung cancer and its relationship with metastasis and prognosis].

OBJECTIVE: To explore the expression of ezrin protein in human non-small cell lung cancer (NSCLC) tissues and lung cancer cell lines, and the association between the expression of ezrin protein and the expression of E-cadherin and CD44V6 proteins. METHODS: The expression of ezrin protein and mRNA in lung cancer cell lines was detected by RT-PCR and Western blotting. Ezrin, E-cadherin and CD44V6 were detected by immunohistochemical SP staining in tumor tissues from 150 lung cancer cases and in adjacent normal lung tissues from 30 patients. Furthermore, the expression of ezrin in 30 freshly-taken NSCLC tissues was also detected by Western blot. RESULTS: The expression of ezrin protein and mRNA was up-regulated in highly metastatic human lung cancer. The positive rate of ezrin, E-cadherin and CD44V6 expression in the lung cancer was 61.3%, 54.0% and 58.7%, respectively. The up-regulation of ezrin expression was significantly correlated with lymph node metastasis, but not correlated with age, sex, tumor size, histological type, clinical TNM system and pathological grade. Western blot analysis showed that the level of ezrin in the NSCLC tissues was significantly higher than that in the normal tissues (t = 5.013, P < 0.01). Survival analysis showed that the 5-year survival rate of patients with negative ezrin expression was 29.3%, significantly higher than that of patients with positive ezrin expression (15.2%, χ(2) = 4.128, P = 0.042). Multivariate Cox regression analysis showed that ezrin expression (RR = 3.012, P = 0.047) and lymph node metastasis (RR = 4.827, P = 0.035) were significantly independent prognostic factors for patients with lung cancer. Furthermore, a negative correlation was observed between the expressions of ezrin and E-cadherin in lung cancer, and a positive correlation between the expressions of ezrin and CD44V6 in lung cancer. CONCLUSIONS: Ezrin, E-cadherin and CD44V6 play an important role in the regulation of growth and meastasis of lung cancer. Combined detection of ezrin, E-cadherin and CD44V6 expression is helpful in evaluating the metastasis and prognosis of non-small cell lung cancer.

Expression analysis of Cdc42 in lung cancer and modulation of its expression by curcumin in lung cancer cell lines.

Cdc42, a Rho GTPase family member, is involved in cell transformation, proliferation, survival, invasion and metastasis of human cancer cells. Overexpression of Cdc42 has been reported in several types of human cancer. However, the underlying mechanisms are not well understood. The present study showed that Cdc42 was overexpressed in 80 of 110 primary lung cancer patients, and overexpression of Cdc42 was significantly associated with high TNM stage and lymph node metastasis. Moreover, RNAi-mediated suppression of Cdc42 expression reduced actin filopodia formation, migration and invasion potential of a highly metastatic lung cancer cell line, 801D. In parallel, 801D cells were treated with curcumin and the effect on the expression of the Cdc42 gene at the transcriptional and translational levels was analyzed by RT-PCR and Western blotting. Curcumin inhibited cell migration, invasion and downregulated Cdc42 gene and Cdc42-related target gene expression in 801D cells. It also induced rearrangements of the actin cytoskeleton. These effects mimicked those of Cdc42 knockdown. Furthermore, xenograft experiments confirmed the suppression of tumor growth and invasion in vivo, which was due to the effect of curcumin and the inhibition of Cdc42 by curcumin. Our results showing the downregulation of Cdc42 expression by curcumin in lung cancer cells taken together with the clinical data suggest a potential therapeutic role for curcumin in inducing Cdc42-mediated inhibition of invasion of lung cancer cells.

Lysosomal membrane permeabilization is involved in curcumin-induced apoptosis of A549 lung carcinoma cells.

We previously reported that curcumin inhibited lung cancer A549 cells growth and promoted cell apoptosis in vitro. In this study, we further examined the apoptosis-related parameters, including lysosomal damage and cathepsin activation, in A549 cells exposed to curcumin. We found that curcumin caused lysosomal membrane permeabilization (LMP) and cytosolic relocation of cathepsin B (cath B) and cathepsin D (cath D). However, only Z-FA-fmk (a cath B inhibitor) but not pepstatin A (a cath D inhibitor) inhibited curcumin-induced cell apoptosis, mitochondrial membrane potential loss, and cytochrome c release. The antioxidant N-acetylcysteine and glutathione attenuated LMP, suggesting that lysosomal destabilization was dependent on the elevation of reactive oxygen species and which precedes mitochondrial dysfunction. These findings indicated a novel pathway for curcumin regulation of ROS-lysosomal-mitochondrial pathway and provided the key mechanism of regulation of LMP in cell apoptosis, which may be exploited for cancer treatment.

Silencing of Rac1 modifies lung cancer cell migration, invasion and actin cytoskeleton rearrangements and enhances chemosensitivity to antitumor drugs.

Rac1, an intracellular signal transducer, regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. Overexpression of Rac1 has been reported in several human cancers. However, the underlying mechanisms are not well understood. In the present study, we evaluated the possibility of Rac1 as an appropriate molecular target for cancer gene therapy. The expression of Rac1 in 150 primary non-small cell lung cancer tissues (NSCLC) and 30 normal paraneoplastic lung tissues was determined by immunohistochemical staining, and the correlation of Rac1 overexpression with clinicopathological factors was evaluated. Overexpression of Rac1 was detected in 94 of 150 lung cancer specimens, the incidence rate being higher than that in normal lung tissue specimens. In addition, overexpression of Rac1 was also associated with poor differentiation, high TNM stage, and lymph node metastasis in NSCLC patients. Moreover, RNAi-mediated suppression of Rac1 expression reduced lamellipodia formation, migration and invasion potential of a lung cancer cell carcinoma cell line, 801D. Down-regulation of Rac1 expression also reduced the expression of Pak1. NSC23766, an inhibitor of Rac1 activity, could also inhibit lung cancer cell migration, invasion and induce rearrangements of the actin cytoskeleton. Furthermore, the suppression of Rac1 expression also sensitized cells to antitumor drugs. These results indicate that the overexpression of Rac1 is tightly associated with an aggressive phenotype of lung cancer cells. Therefore, we proposed that Rac1 could be a potential molecular target of gene therapy by RNAi-targeting in lung cancer cells.