Effects of p21-activated kinase 6 on invasion and migration of human non-small-cell lung cancer A549 cells

AIM：To investigate the effects of p21-activated kinase 6 (PAK6) on the invasive and migratory abilities of human non-small-cell lung cancer A549 cells. METHODS：The expression of PAK6 mRNA in A549 cells, human bronchial epithelial (HBE) cells, non-small-cell lung cancer tissues and paired adjacent non-tumor tissues was measured by real-time PCR. After A549 cells were transfected with siRNA-PAK6 (siPAK6) or negative control (NC) for 48 h, the expression of PAK6 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively. The invasion and migration of A549 cells were detected by Matrigel invasion assay and Transwell migration assay. The cytoskeletal changes were observed with FITC-phalloidin staining under confocal microscope. RESULTS：The level of PAK6 mRNA in A549 cells was higher than that in HBE cells (3.50±1.16 vs 1.12±0.42, P<0.05). The level of PAK6 mRNA in non-small-cell lung cancer tissues was higher than that in paired adjacent non-tumor tissues (5.13±1.33 vs 1.08±0.37, P<0.05). The expression of PAK6 protein decreased by 72% in A549 cells transfected with siPAK6 (P<0.05), and the level of PAK6 mRNA significantly decreased in A549 cells transfected with siPAK6 (3.72±0.75 vs 0.69±0.21, P<0.05). Matrigel invasion assay and Transwell migration assay demonstrated that knockdown of PAK6 markedly attenuated the invasion and migration of A549 cells (P<0.05). The cytoskeletal actin remodeling and reduction of stress fibers in A549 cells transfected with siPAK6 were observed under confocal microscope. CONCLUSION：PAK6 may affect the invasive and migratory abilities of non-small-cell lung cancer cells by cytoskeletal actin remodeling.     

Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

Effect of BMP9 over-expression on migration and invasion of human lung squamous-cell carcinoma NCI-H520 cells

AIM: To investigate the effect of bone morphogenetic proteins 9(BMP9) on the migration and invasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism. METHODS: The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial (HBE) cells was detected by RT-PCR and Western blot. The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expression of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot. The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays. The mRNA and protein levels of the migration-related factor matrix metalloproteinase 2(MMP2) were detected by RT-PCR and Western blot. The level of phosphorylated Smad1/5(p-Smad1/5) was detected by Western blot. Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9. The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays. RESULTS: The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells. After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly decreased, and the mRNA and protein levels of MMP2 were decreased. Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability. CONCLUSION: Over-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells. The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.     

Effect of PAK4-LIMK1-cofilin signaling on non-small-cell lung cancer migration and invasion

AIM: To explore the mechanism of p21-activated kinase 4 (PAK4) on non-small-cell lung cancer (NSCLC) migration and invasion.METHODS: After A549 and NCI-H520 cell lines were transfected with PAK4-siRNA or negative control, the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The invasion and migration of A549 cells and NCI-H520 cells were measured by Matrigel invasion assay and Transwell migration assay. LIMK1, cofilin, and their respective phosphorylation were examined by Western blot. The interaction of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay. The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A549 cells and NCI-H520 cells. The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot. A549 cells and NCI-H520 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion. RESULTS: After A549 cells and NCI-H520 cells were transfected with PAK4-siRNA for 48 h, the expression of PAK4 at mRNA and protein levels, and the numbers of invasion and migration cells in PAK4-siRNA group were lower than those in control group. Compared with control group, the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group, whereas the total expression levels of LIMK1 and cofilin did not change. The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-H520 cells. LIMK1 phosphorylation in the presence of PAK4 (K350M) was significantly lower than that in the presence of PAK4 (WT) or PAK4 (S445N) in the protein kinase assay. The PAK4 upregulation was positively correlated with the level of p-LIMK1 (P<0.05). After A549 cells and NCI-H520 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid, the migration and invasion cell numbers in co-transfection group were higher than those in PAK4-siRNA transfection group. CONCLUSION: PAK4 promotes the invasive and migratory abilities of NSCLC, which is mediated by LIMK1 phosphorylation.     

Effects of tangeretin on growth and invasion of human non-small-cell lung cancer cells

AIM: To study the influences of tangeretin (TGN) on the growth and invasion of non-small-cell lung cancer (NSCLC) cells, and to explore the molecular mechanisms. METHODS: The A549 cells were treated with different concentrations of TGN in vitro. The relative cell activity was determined by MTT assay. The apoptotic rate was analyzed by flow cytometry with Annexin V-FITC/PI staining. The number of the invasive cells was measured by Transwell assay. The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR, and the protein levels of Ki67, Cyt C, caspase-3, cleaved caspase-3, MMP-2, MMP-9, Akt, p-Akt and p-PI3K were determined by Western blotting analysis. RESULTS: TGN inhibited the proliferation of A549 cells in a dose-dependent manner (P<0.05) along with the low expression level of proliferation biomarker Ki67. TGN up-regulated the protein levels of Cyt C, caspase-3 and cleaved caspase-3 (P<0.01) and promoted the apoptosis of A549 cells in a dose-dependent manner. Moreover, TGN down-regulated the invasion-related molecules MMP-2 and MMP-9 at the mRNA and protein levels, and the number of invasive cells reduced with the increase in the concentration of TGN. The protein levels of p-Akt and p-PI3K in the A549 cells was reduced (P<0.05), and no difference of the cell viability in the cells treated with different concentrations of TGN was observed after blocking PI3K/Akt signaling pathway using LY294002. CONCLUSION: TGN inhibits the growth and invasion of A549 cells and promotes the cell apoptosis by potentially inhibiting PI3K/Akt signaling pathway activation. Therefore, this study will provide a new target for the prevention and control of NSCLC.     

Effect of PDK1 on biological characteristics of lung cancer A549 cells

AIM: To investigate the effects of 3-phosphoinositide-dependent protein-1 (PDK1) on the biological characteristics of non-small-cell lung cancer cell line A549 and the underlying mechanisms.METHODS: The expression levels of PDK1 in lung normal epithelial cell line BEAS-2B and different lung cancer cell lines H460, SPCA1 and A549 were determined by Western blot and real-time PCR. Small interfering RNA was used to down-regulated PDK1 expression in the A549 cells, and then cell viability and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The expression of cell cycle-and apoptosis-related molecules at protein level and the activation of Akt/FoxO1 pathway were measured by Western blot. Insulin-like growth factor-1 (IGF-1, one of the most potent Akt activators) was used to evaluate the interaction between PDK1 and Akt/FoxO1 pathway.RESULTS: Compared with lung normal epithelial cell line BEAS-2B, PDK1 expression in the lung cancer cell lines was obviously increased (P<0.05). Knockdown of PDK1 suppressed cell viability and cell cycle, but promoted the apoptosis of the A549 cells. The results of Western blot showed that the protein levels of cyclin D1, CDK4, p-Rb, Bcl-2, p-Akt and cytoplasmic p-FoxO1 were significantly decreased after knockdown of PDK1, with increases in the protein levels of P27, cleaved caspase-3 and nuclear FoxO1. Pre-incubation with IGF-1 partly reversed the effect of PDK1 knockdown on Akt/FoxO1 pathway and increased the viability of A549 cells.CONCLUSION: In human non-small-cell lung cancer A549 cells, knockdown of PDK1 suppresses cell viability and promotes cell apoptosis by regulating the expression of cell cycle-and apoptosis-related molecules via Akt/FoxO1 pathway, suggesting that PDK1 may be a potential target for diagnosis and theatment of lung cancer.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Mechanism of microRNA-138-5p inhibiting proliferation,migration and invasion of lung cancer cells

AIM: To investigate the mechanism of microRNA-138-5p (miR-138-5p) inhibiting the proliferation, migration and invasion abilities of lung cancer cells.METHODS: The lung cancer A549 and H460 cells were transfected with miR-NC (control group) or miR-138-5p (experimental group). The bioinformatic analysis was performed to predict the target genes of miR-138-5p.The expression levels of miR-138-5p, forkhead box protein C1 (FOXC1) mRNA and vimentin mRNA were detected by RT-qPCR. The protein expression of FOXC1, vimentin, E-cadherin, N-cadherin and β-catenin was determined by Western blot. MTS method and colony formation assay were used to detect cell viability and proliferation ability. Wound healing assay and Transwell assay were used to detect cell migration and invasion ability.RESULTS: Over-expression of miR-138-5p significantly reduced the expression of FOXC1 and vimentin at mRNA and protein levels (P<0.05). The expression of E-cadherin and β-catenin were up-regulated and the expression of N-cadherin was down-regulated. The proliferation, migration and invasion abilities of the lung cancer cells were inhibited by the over-expression of miR-138-5p.CONCLUSION: miR-138-5p inhibits the proliferation, migration and invasion abilities of lung cancer cells by targeting FOXC1 and vimentin. It may be a potential target for lung cancer gene therapy.     

CASC2/miR-18a/BTG3 signaling inhibits cell migration and invasion of non-small cell lung cancer

AIM: To explore the function and molecular mechanism of long non-coding RNA CASC2 in non-small-cell lung cancer (NSCLC) cell migration and invasion. METHODS: RT-qPCR and Western blot were used to detect the expression of CASC2, microRNA-18a (miR-18a) and BTG3 in human bronchial epithelial cell line 16-HBE, and NSCLC cell lines A549 and H1299. The interaction between CASC2 and miR-18a or miR-18a and BTG3 was predicted by bioinformatics software and verified by double-luciferase reporter assays. Transwell assays were performed to detect the migration and invasion abilities of the NSCLC cells. RT-qPCR and Western blot were used to determine the regulatory effects of CASC2 on miR-18a and BTG3 expression. RESULTS: Compared with 16-HBE cells, the expression of CASC2 and BTG3 was significantly down-regulated in the NASCL cells, while miR-18a was significantly over-expressed (P<0.05). CASC2 acted as a molecular sponge for miR-18a, and BTG3 was verified to be a target gene of miR-18a. Up-regulation of CASC2 inhibited the migration and invasion abilities of NSCLC cells, while exogenous restoration of miR-18a stimulated cell migration and invasion abilities. In addition, exogenously over-expressed miR-181a reversed the promoting effect of CASC2 on BTG3 protein expression. CONCLUSION: CASC2 promotes BTG3 expression by negatively regulating miR-18a, and then inhibits the migration and invasion abilities of NSCLC cells.     

Down-regulation of lncRNA PCAT1 suppresses oral squamous cell carcinoma cell proliferation,growth, invasion,migration and epithelial-mesenchymal transition

AIM: To investigate the effects of the long non-coding RNA (lncRNA) PCAT1 on the oral squamous cell carcinoma (OSCC) cell proliferation, growth, invasion and migration, and to explore the underlying mechanisms. METHODS: The PCAT1 siRNA was transfected by Lipofectmine 2000, and RT-qPCR and Western blot were performed to determine the mRNA and protein expression of relevant genes, respectively. CCK-8 assay and colony formation assay were used to measure OSCC cell proliferation and growth, respectively. The cell invasion and migration assays were used to measure the invasive and migratory abilities of the OSCC cells, respectively. RESULTS: PCAT1 was significantly up-regulated in OSCC tissues and cells compared with normal adjacent tissues and normal human oral keratinocyte cells, respectively (P<0.05). PCAT1 siRNA transfection suppressed the expression of PCAT1 in Tca8113 and TSCCa cells (P<0.05). Knockdown of PCAT1 in Tca8133 cells and TSCCa cells significantly suppressed the cell proliferation, invasion and migration abilities (P<0.05). In addition, knockdown of PCAT1 in Tca8133 cells and TSCCa cells also suppressed the mRNA and protein levels of ZEB-1, N-cadherin and vimentin, and increased the mRNA and protein expression of E-cadherin (P<0.05). CONCLUSION: Knockdown of PCAT1 suppresses cell proliferation and migration abilities, and the effect of PCAT1 on OSCC cells may be associated with epithelial-mesenchymal transition.     

miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

High mobility group box 1 protein promotes proliferation,migration and invasion of colon cancer cells

AIM: To investigate the expression of high mobility group box 1 protein (HMGB1) in colon can-cer cells, and to determine its regulatory roles in colon cancer cell proliferation, migration and invasion. METHODS: qPCR and Western blot were used to quantify the mRNA and protein expression levels of HMGB1 in human colon cancer SW620 cells and normal colonic epithelial FHC cells. HMGB1 shRNA was transfected into the SW620 cells to establish the stable HMGB1-downregulating colon cancer cells (shHMGB1 group), and negative control (shNC) group and blank control (blank) group were also set up. The proliferation, migration and invasion of the cells were determined by CCK-8 assay, colony formation experiment and Transwell chamber assays. Western blot was used to determine the protein levels of p-ERK, ERK, c-Myc, matrix metalloproteinase (MMP)-2/9, E-cadherin, N-cadherin, Bcl-2 and Bax. RESULTS: Both of the mRNA and protein levels of HMGB1 in colon cancer cells were higher than those in the normal colonic epithelial cells (P<0.05). HMGB1 gene was successfully knocked down in SW620 cells. Compared with blank group and shNC group, the proliferation, migration and invasion abilities of the cells in shHMGB1 group were significantly inhibited (P<0.05). The protein levels of MMP-2, MMP-9, N-cadherin, c-Myc, Bcl-2 and p-ERK were reduced notably, while the expression of Bax protein was increased (P<0.05) in shHMGB1 group compared with shNC group and blank group.CONCLUSION: HMGB1 effectively promotes the proliferation, migration and invasion of colon cancer cells through ERK/c-Myc signaling pathway.     

Effect of HC-1119 on biological behaviors of triple-negative breast cancer BT549 cells

AIM: To explore the effect of new artificially synthesized androgen receptor (AR) antagonist HC-1119 on the biological function of triple-negative breast cancer (TNBC) BT549 cells and the molecular mechanism. METHODS: The AR expression was assessed in different human breast cancer cell lines MDA-MB-231, T47D, MCF-7, SKBR3 and BT549 by Western blot. The TNBC BT549 cells with AR positive expression were treated with HC-1119. The cell viability was measured by CCK-8 assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. The migration and invasion abilities were detected by Transwell assay in vitro. The protein expression of E-cadherin, vimentin and P21 was determined by Western blot. RESULTS: AR was positively expressed in BT549 cells. HC-1119 inhibited the cell viability in a time-and dose-dependent manner (P<0.05), increased the percentage of apoptotic cells and the percentage of S-phase cells significantly, repressed the migration and invasion abilities (P<0.05), and decreased P21 expression at protein level (P<0.01). No influence on the expression of E-cadherin and vimentin in the BT549 cells was observed. CONCLUSION: AR antagonist HC-1119 decreases the viability, migration ability and invasion ability, enhances the apoptosis, and arrests the cell cycle distribution of TNBC BT549 cells. HC-1119 represses the viability of BT549 cells by down-regulating P21 expression, while the process of epithelial-mesenchymal transition is not involved in the inhibition of cell migration.     

Roles of maspin in biological behaviors of A549 cells

AIM: To investigate the roles of maspin in the biological behaviors of non-small-cell lung cancer A549 cells.METHODS: Full-length coding region of maspin was amplified by PCR from human normal breast tissue and cloned into MSCV vector. Virus supernatants was produced by Phoenix A packaging system, and target cell line was infected with the virus supernatants. The transfected cells were screened with puromycin. The cell line with maspin over-expression was identified by real-time PCR and Western blot. Cell growth, migration and invasion were investigated by xCelligence system.RESULTS: Maspin over-expression vector was successfully constructed. The cell line with maspin over-expression was established. No difference of the growth between A549-control cells and A549-maspin cells was observed. The migration and invasion were quite different between A549-control cells and A549-maspin cells. The expression level of integrin β1 in A549-maspin cells was decreased compared with A549-control cells.CONCLUSION: Maspin has no effect on the growth of A549 cells. Maspin suppresses the migration and invasion of A549 cells, in which integrin β1 is involved.     

Effects of TRPC1 on TGF-β1-induced migration of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.     

Salinomycin promotes the apoptosis-inducing effect of gefitinib on human lung adenocarcinoma cell line A549

AIM: To investigate the effect of salinomycin alone or in combination with gefitinib (an inhibitor of epidermal growth factor receptor tyrosine kinase) on the growth and apoptosis of human non-small-cell lung cancer cell line A549. METHODS: The inhibitory effect of salinomycin on the growth of A549 cells was tested by MTT assay. The cell apoptosis and the level of mitochondrial membrane potential were determined by flow cytometry. The activity of caspase-3, -8, and -9 was measured by the method of colorimetry. The protein levels of cytochrome C, Bcl- 2, p-EGFR, p-Akt and p-ERK were detected by Western blotting. RESULTS: Salinomycin or gefitinib alone inhibited the growth of A549 cells in a dose-dependent manner. Salinomycin or gefitinib also induced apoptosis of the cells. Salinomycin combined with gefitinib produced stronger inhibitory effect on the cell proliferation, and a significant increase in cell apoptosis was also observed. Compared with control group, salinomycin alone significantly reduced mitochondrial membrane potential, transitorily increased the levels of intracellular reactive oxygen species (ROS), cytoplasmic cytochrome C and Ca2+, and increased the activity of caspase-3, -8 and -9 in A549 cells. Gefitinib alone inhibited the protein expression of p-EGFR, p-Akt and p-ERK, but no obvious effect on the release of cytochrome C and the activity of caspase-3, -8 and -9 was found. The combination of salinomycin and gefitinib significantly reduced the protein levels of Bcl-2, p-EGFR, p-Akt and p-ERK, but the protein levels of EGFR, Akt and ERK were not obviously changed. CONCLUSION: The synergy of salinomycin and gefitinib is observed. Salinomycin inhibits the growth and induces apoptosis of human lung carcinoma A549 cells through Bcl-2 pathway and mitochondrial apoptosis pathway. Salinomycin also increases the sensitivity of A549 cells to gefitinib.     

Effects of BCL6B on proliferation and migration of human colorectal carcinoma LoVo cells and its potential mechanism

AIM: To detect the endogenous expression of B-cell leukemia/lymphoma 6 member B (BCL6B) in FHC and LoVo cells, and to investigate the effects of BCL6B on proliferation and migration of LoVo cells for further exploring the underlying mechanism. METHODS: The endogenous expression of BCL6B in the FHC and LoVo cells was detected by RT-PCR and Western blot. The methods of MTT assay, colony formation assay, wound healing assay and Transwell chamber experiment were employed to examine the biological functions of BCL6B in the LoVo cells. The mRNA and protein levels of BCL6B, cyclin D1 and matrix metalloproteinase-9 (MMP-9) were determined by RT-PCR and Western blot, respectively. The level of phosphorylated protein kinase B (p-AKT) was detected by Western blot. RESULTS: BCL6B expression was notably repressed in the LoVo cells as compared with the FHC cells, which were significantly increased by transfection with pcDNA3.1-BCL6B. The abilities of proliferation and migration of the LoVo cells at 72 h were inhibited by 28.33%(P<0.01) and 36.11%(P<0.05) in BCL6B group. The mRNA levels of cyclin D1 and MMP-9 in the cells of BCL6B group were decreased by 39.90%(P<0.01) and 77.36% (P<0.05), and the protein levels of cyclin D1, MMP-9 and p-AKT were reduced by 44.00%(P<0.05), 47.06%(P<0.01) and 32.88% (P<0.05), respectively. CONCLUSION: BCL6B inhibits proliferation and migration of the LoVo cells, and the PI3K/AKT signaling pathway is involved in this process.