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.

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.

Improved Biocompatibility and Angiogenesis of the Bone Titanium Scaffold through ERK1/2 Signaling Mediated by an Attached Strontium Element.

The promotion of early osseointegration is crucial for the success of biomedical titanium implants. Physical and chemical modifications to the material surface can significantly compensate for the lack of biocompatibility and early osseointegration of the implant. In this study, we implanted strontium onto titanium plates and analyzed the effect of strontium-doped materials on angiogenesis and biocompatibility in the human bone structure. Our findings demonstrated that strontium-loaded titanium sheet materials effectively promote human umbilical vein endothelial cell (HUVEC) biocompatibility and vascular differentiation ability, as evidenced by proliferation-apoptosis assays, RT-qPCR for vascular neogenesis markers, ELISA for vascular endothelial growth factor (VEGF) levels, and nitric oxide (NO) analysis. Mechanism studies based on RNAseq and Western blotting analysis revealed that strontium can promote titanium material biocompatibility with HUVEC cells and vascular neovascularization ability by activating the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Meanwhile, blocking the ERK1/2 signaling pathway could reverse the promotional effect of vascular formation. Overall, we have successfully fabricated a multifunctional biocompatible bone implant with better histocompatibility and angiogenesis compared to uncoated implants.

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.

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.

[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.

One-step laparoscopic pancreatic necrosectomy verse surgical step-up approach for infected pancreatic necrosis: a case-control study.

BACKGROUND: The surgical step-up approach often requires multiple debridements and might not be suitable for infected pancreatic necrosis (IPN) patients with various abscesses or no safe route for percutaneous catheter drainage (PCD). This case-control study aimed to investigate the safety and effectiveness of one-step laparoscopic pancreatic necrosectomy (LPN) in treating IPN. METHODS: This case-control study included IPN patients undergoing one-step LPN or surgical step-up in our center from January 2015 to December 2020. The short-term and long-term complications after surgery, length of hospital stay, and postoperative ICU stays in both groups were analyzed. Univariate and multivariate logistic regression analyses were performed to explore the risk factors of major complications or death. RESULTS: A total of 53 IPN patients underwent one-step LPN and 37 IPN patients underwent surgical step-up approach in this study. There was no significant difference in the incidence of death, major complications, new-onset diabetes, or new-onset pancreatic exocrine insufficiency between the two groups. However, the length of hospital stay in the one-step LPN group was significantly shorter than that in the surgical step-up group. Univariate regression analysis showed that the surgical approach (one-step/step-up) was not the risk factor for major complications or death. Multivariate logistic regression analysis indicated that computed tomography (CT) severity index, American Society of Anesthesiologists (ASA) class IV, and white blood cell (WBC) were the significant risk factors for major complications or death. CONCLUSION: One-step LPN is as safe and effective as the surgical step-up approach for treating IPN patients, and reduces total hospital stay.

[Correlation of the expression of III ß-tubulin and MDR1 protein with biological features of non-small cell lung cancer].

OBJECTIVE: To explore the expression of III ß-tubulin and MDR1 protein in patients with non-small cell lung cancer (NSCLC), and to clarify its clinical significance. METHODS: Paraffin embedded tissues from 158 primary non-small cell lung cancers and para-cancerous lung tissues were investigated for the expression of III ß-tubulin and MDR1 protein by immunohistochemistry, as well as in freshly-taken NSCLC tissues by Western blot. The relationship between the expression of III ß-tubulin and MDR1 and the biological features of lung cancer was analyzed. RESULTS: The positive rate of III ß-tubulin and MDR1 protein expression in lung cancer tissues was 65.19% and 51.27%, respectively. Western blot analysis showed that the level of of III ß-tubulin and MDR1 protein in NSCLC tissues was remarkably higher than that in normal tissues (P < 0.01). The expression of III ß-tubulin in stage III-IV cases was significantly higher than that in stage I-II cases (P < 0.05), while the expression of MDR1 protein showed no significant difference (P > 0.05). The positive rate of III ß-tubulin expression in well-moderate pathological grades was lower than that in poor ones. The positive rate of MDR1 expression in adenocarcinoma was higher than that in squamous cell carcinoma and large cell undifferentiated cancers (P < 0.01). The positive rate of expression of MDR1 protein and III ß-tubulin was not correlated with sex, age, tumor size and lymph node metastasis (P > 0.05). CONCLUSION: The expression of III ß-tubulin and MDR1 may play an important role in the development and progression of human non-small cell lung cancer, and could be looked as an important index for judging the prognosis of lung cancer.

[Expression of alpha-tubulin and MDR1 and their correlation with the biological features of non-small cell lung carcinoma].

OBJECTIVE: To detect the expression of alpha-tubulin and MDR1 in human non-small cell lung carcinoma (NSCLC), and to clarify their clinical significance. METHODS: Paraffin embedded tissues from 158 primary non-small lung carcinomas and 30 paracancerous lung tissues were examined for expression of alpha-tubulin and MDR1 by immunohistochemistry (SP method). 30 freshly taken NSCLC tissues were examined by Western blot analysis. The relationship between alpha-tubulin and MDR1 expression and the biological features of lung carcinoma was analyzed. RESULTS: The positive rate of alpha-tubulin and MDR1 expressions in the lung carcinomas was 65.2% and 51.3%, respectively. There was no expression of either of them in 30 paracancerous lung tissues. Western blot analysis showed that the level of alpha-tubulin and MDR1 expressions in NSCLC tissues were 0.49 +/- 0.06 and 0.56 +/- 0.04, respectively, significantly higher than that in paracancerous tissues (0.07 +/- 0.01) (t = 3.693 and t = 6.769, P < 0.01). The positive rate of alpha-tubulin expression was gradually increased with tumor progression, significantly higher in III-IV stage cancers and in poorly differentiated carcinomas (both P < 0.01). There was a distinct statistically significant difference between stage I, stage II and III, and stage IV. The positive rate of alpha-tubulin in well-moderately differentiated carcinomas was lower than that in poorly differentiated ones. There was no significant correlation with age, sex, tumor size, histological type, clinical TNM system and lymph node metastasis. The positive rate of MDR1 was not correlated with sex, age, tumor size, pathological grading, clinical TNM stages and lymph node metastasis. But the positive rate of MDR1 in adenocarcinoma was significantly higher than that in squamous carcinoma and undifferentiated large cell carcinomas (P < 0.01). alpha-tubulin and MDR1 expression had no impact on the outcome of chemotherapy (chi(2) = 0.69 and 1.30, P > 0.05, respectively). Univariate analysis showed that the 5-year survival rate of patients with negative alpha-tubulin and MDR1 expression was 30.7% and 28.5%, respectively, significantly higher than that of patients with positive alpha-tubulin and MDR1 expression (13.5% and 11.8%, respectively) (chi(2) = 20.69 and 15.52, P < 0.01, respectively), and multivariate Cox regression analysis showed that alpha-tubulin (RR = 3.287, P = 0.006) and clinical TNM stage (RR = 1.954, P = 0.025) were significantly independent predictive factor for patients with lung cancer, MDR1 and other factors could not be used as an independent predicitive factors. However, there was no significant correlation between the expression of alpha-tubulin and MDR1 in lung carcinoma(r = 0.093, P > 0.05). CONCLUSION: The expression of alpha-tubulin and MDR1 may play an important role in the development and progression of human non-small cell lung carcinoma. Combined detection could be considered as an important index for predicting prognosis of lung carcinoma.

LncRNA-MTA2TR functions as a promoter in pancreatic cancer via driving deacetylation-dependent accumulation of HIF-1α.

Rationale: Hypoxia has been proved to contribute to aggressive phenotype of cancers, while functional and regulatory mechanism of long noncoding RNA (lncRNA) in the contribution of hypoxia on pancreatic cancer (PC) tumorigenesis is incompletely understood. The aim of this study was to uncover the regulatory and functional roles for hypoxia-induced lncRNA-MTA2TR (MTA2 transcriptional regulator RNA, AF083120.1) in the regulation of PC tumorigenesis. Methods: A lncRNA microarray confirmed MTA2TR expression in tissues of PC patients. The effects of MTA2TR on proliferation and metastasis of PC cells and xenograft models were determined, and the key mechanisms by which MTA2TR promotes PC were further dissected. Furthermore, the expression and regulation of MTA2TR under hypoxic conditions in PC cells were assessed. We also assessed the correlation between MTA2TR expression and PC patient clinical outcomes. Results: We found that metastasis associated protein 2 (MTA2) transcriptional regulator lncRNA (MTA2TR) was overexpressed in PC patient tissues relative to paired noncancerous tissues. Furthermore, we found that depletion of MTA2TR significantly inhibited PC cell proliferation and invasion both in vitro and in vivo. We further demonstrated that MTA2TR transcriptionally upregulates MTA2 expression by recruiting activating transcription factor 3 (ATF3) to the promoter area of MTA2. Consequentially, MTA2 can stabilize the HIF-1α protein via deacetylation, which further activates HIF-1α transcriptional activity. Interestingly, our results revealed that MTA2TR is transcriptionally regulated by HIF-1α under hypoxic conditions. Our clinical samples further indicated that the overexpression of MTA2TR was correlated with MTA2 upregulation, as well as with reduced overall survival (OS) in PC patients. Conclusions: These results suggest that feedback between MTA2TR and HIF-1α may play a key role in regulating PC tumorigenesis, thus potentially highlighting novel avenues PC treatment.

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.

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.

[Expression of epithelial-cadherin, CD44v6 and connexin43 in hepatocellular carcinoma].

OBJECTIVE: To study the expression of epithelial-cadherin (E-cad), CD44v6 and Cx43 in hepatocellular carcinoma (HCC) and its relationship with sex and age of patients, as well as tumor histopathologic grades. METHODS: Double immunofluorescent staining and laser scanning confocal microscopy was used to study the expression of E-cad, CD44v6 and Cx43 in 30 cases of normal liver tissue, 25 cases of benign hepatic lesions and 38 cases of HCC. In the HCC group, correlation of antigen expression with sex and age of patients and tumor histopathologic grades was studied by T-test. RESULTS: Significant decrease in expression of E-cad and Cx43 was noted in HCC group, as compared to normal liver tissue and benign hepatic lesion (P<0.05). On the other hand, CD44v6 expression was higher in HCC group than in the other two groups (P<0.05). In HCC group, the expression of E-cad and Cx43 did not correlate with sex, age and histopathologic grades (P>0.05). However, CD44v6 expression positively correlated with higher tumor histopathologic grades (P<0.05) but not sex and age of patients (P>0.05). In HCC group, the expression of E-cad positively correlated with that of Cx43, while the expression of CD44v6 negatively correlated with that of E-cad and Cx43. CONCLUSIONS: Tumor immunophenotype alters during development and progression of HCC. Low expression of E-cad and Cx43 and high expression of CD44v6 may be related to aggressive clinical behavior of HCC, moreover, high expression of CD44v6 correlated with high tumor grades. Detection of E-cad, CD44v6 and Cx43 expression may thus be useful in predicting prognosis of HCC.

Curcumin inhibited HGF-induced EMT and angiogenesis through regulating c-Met dependent PI3K/Akt/mTOR signaling pathways in lung cancer.

The epithelial-mesenchymal transition (EMT) and angiogenesis have emerged as two pivotal events in cancer progression. Curcumin has been extensively studied in preclinical models and clinical trials of cancer prevention due to its favorable toxicity profile. However, the possible involvement of curcumin in the EMT and angiogenesis in lung cancer remains unclear. This study found that curcumin inhibited hepatocyte growth factor (HGF)-induced migration and EMT-related morphological changes in A549 and PC-9 cells. Moreover, pretreatment with curcumin blocked HGF-induced c-Met phosphorylation and downstream activation of Akt, mTOR, and S6. These effects mimicked that of c-Met inhibitor SU11274 or PI3 kinase inhibitor LY294002 or mTOR inhibitor rapamycin treatment. c-Met gene overexpression analysis further demonstrated that curcumin suppressed lung cancer cell EMT by inhibiting c-Met/Akt/mTOR signaling pathways. In human umbilical vein endothelial cells (HUVECs), we found that curcumin also significantly inhibited PI3K/Akt/mTOR signaling and induced apoptosis and reduced migration and tube formation of HGF-treated HUVEC. Finally, in the experimental mouse model, we showed that curcumin inhibited HGF-stimulated tumor growth and induced an increase in E-cadherin expression and a decrease in vimentin, CD34, and vascular endothelial growth factor (VEGF) expression. Collectively, these findings indicated that curcumin could inhibit HGF-promoted EMT and angiogenesis by targeting c-Met and blocking PI3K/Akt/mTOR pathways.

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.

[Construction of PTEN eukaryotic expression plasmid and its effects on breast carcinoma cell line MDA468].

OBJECTIVE: To investigate the effects of exogenous wild PTEN gene stably transfection on growth of breast cancer cells in vitro. METHODS: At first, a recombinant eukaryotic expression plasmid pcDNA3.1-PTEN was constructed. Human breast cancer cell line MDA468 was transfected with pcDNA3.1-PTEN or mock transfected plasmid pcDNA3.1(-) with lipofectamine. RT-PCR, immunohistochemical staining and Western blot were used to determine target gene expression. Cell viability was tested by MTT assay. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. RESULTS: The PTEN stably transfected cells demonstrated the integration of the exogenous target gene and corresponding mRNA and protein over-expression. There was a significant decline in cell viability of pcDNA3.1-PTEN transfected MDA468 cells in comparison with the mock-transfected ones (P < 0.01). The PTEN-trasfected MDA468 cells also showed an increase in the rate of apoptosis, compared with parental and mock-trasfected cells (P < 0.001). CONCLUSION: Stable expression of exogenous PTEN can suppress the malignant phenotypes of the human breast cancer cell line MDA468.

[Exogenous PTEN gene induces apoptosis in breast cancer cells].

OBJECTIVE: To investigate the effects of exogenous PTEN gene on apoptosis of breast cancer cells. METHODS: Human breast cancer cells of the line MDA468 were cultured. Recombinant plasmid pcDNA3.1-PTEN was constructed and transfected into the breast cancer cells with the lipofectAMINE 2000 transfection technique. Parental MDA468 cells and parental MDA468 cells transfected with blank vector pcDNA3.1(-) were used as control groups. RT-PCR was used to detect the expression of the PTEN mRNA and Western blotting was used to determine the expression of PTEN protein. Epithelial growth factor (EGF) was added into the cultures of MDA468 cells transfected with pcDNA3.1-PTEN or blank vector. Then Western blotting was used to detect the expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by EGF. The aapoptosis of the MDA468 cells was determined by flow cytometry with the double-staining method using FITC-conjugated annexin V and PI. RESULTS: Expressions of PTEN mRNA and protein were shown in the MDA468 cells transfected with pcDNA3.1-PTEN by RT-PCR and Western blotting. Both the expression of p-AKT and that of p-FAK were down-regulated in the MDA468 cells transfected with pcDNA3.1-PTEN in comparison with those in the control cells. The apoptotic rate of the MDA468 cells transfected with PCDNA3.1-PTEN was 21.68%, significantly higher than those of the blank control cells (1.17%) and pcDNA3.1(-)-transfected cells (3.55%, both P < 0.01). CONCLUSION: Exogenous PTEN suppress the growth of human breast cancer cell and induces apoptosis by phosphatase activity, which provides a new clue to gene therapy for breast cancer.

[Study on mechanism of tea polyphenols in inducing human lung cancer cell apoptosis in vitro].

OBJECTIVE: To investigate the apoptosis inducing effect of tea polyphenols (TPP) on human lung cancer cell (LCC) and its associative mechanism. METHODS: The apoptosis inducing effect of TPP on LCC in vitro, and its influence on expression of the related gene were determined by MTT assay, laser scanning confocal microscopy and flow cytometry. RESULTS: TPP in different concentration (50,100,200 and 400 microg/ml) had dose-dependent inhibitory effect on LCC, the inhibitory rate was 28.69+/-1.27% ,46. 19+/-1.79% ,64.61+/-1.29%, 75.90+/-1.96%, respectively. The inhibited LCC were blocked in (G0/G1 phase, and could not transferred to S and G2/ M phase of cell cycle. Meanwhile, TPP could induce apoptosis of LCC, the apoptotic rate being 4.76+/-0.11 %, 5.78+/-0.38 %, 10.06+/-0.67 %, 24.44+/-0.44 %, respectively. Morphologic changes of cells were seen in laser scanning confocal microscopy observation. Compared to the control group, intracellular Ca2+ concentration, Annexin V expression, phospatase and tensin homologe deleted on chromosome ten (PTEN) protein and expression gradually increased, while Cyclin D1 protein expression gradually decreased in the TPP treated groups along with the increasing of TPS concentration. CONCLUSION: TTP can induce LCC apoptosis, the mechanism is related to the change of intracellular Ca2+ concentration, PTEN protein and Cyclin D1 protein expression.