miR-125a-5p reverses cisplatin resistance of non-small-cell lung cancer A549/DDP cells by targeting LIMK1

AIM:To explore the effect of microRNA-125a-5p (miR-125a-5p) on cisplatin (DDP) resistance of non-small-cell lung cancer A549/DDP cells and its related mechanisms. METHODS:The expression levels of miR-125a-5p and LIM kinase 1 (LIMK1) in non-small-cell lung cancer tissues, A549 cells and A549/DDP cells were detected by RT-qPCR. The A549/DDP cell viability, apoptotic rate and expression of drug resistance-related proteins after over-expression or knockdown of miR-125a-5p and/or LIMK1 expression were detected by MTT assay, flow cytometry and Western blot, respectively. The targeting relationship between miR-125a-5p and LIMK1 was verified by TargetScan online prediction and dual-luciferase reporter system. The cell viability, apoptotic rate and expression of drug resistance-related proteins after co-expression of miR-125a-5p and LIMK1 were also determined. RESULTS:The expression level of miR-125a-5p was down-regulated and LIMK1 expression was up-regulated in non-small-cell lung cancer tissues and cell lines (P<0.05). The results of dual-luciferase assay indicated that miR-125a-5p negatively regulated the expression of LIMK1. The expression of drug resistance-related proteins and the viability of A549/DDP cells were inhibited after over-expression of miR-125a-5p or knockdown of LIMK1, while the apoptosis was enhanced. Over-expression of LIMK1 attenuated the inhibitory effect of miR-125a-5p on A549/DDP cell viability and drug resistance-related protein expression (P<0.05). CONCLUSION:miR-125a-5p reverses the resistance of A549/DDP cells to DDP by inhibiting the expression of LIMK1 and drug resistance-related proteins.     

Expression of miRNA-181a in human lung adenocarcinoma cells and its effect on cell function

AIM: To study the expression of microRNA (miRNA)-181a in different human lung adenocarcinoma cell lines, and to investigate the effect of miRNA-181a on cell function and its mechanism in human lung adenocarcinoma drug resistant cell A549/DDP. METHODS: Real-time PCR was used to detect the expression of miRNA-181a in BEAS-2B cells, A549 cells and A549/DDP cells. The A549/DDP cells were transfected with pGenesil-miRNA-181a eukaryotic expression plasmid. At the same time, the untransfection group and negative transfection group were also set up. The expression of miRNA-181a, cell viability, cell growth inhibition and apoptosis rate during cis-diamminedichloroplatinum (DDP) treatment, cell cycle, cell invasion, the protein expression of miRNA-181a target genes bcl-2 and p53 in the A549/DDP cells were determined by real-time fluorescence quantitative PCR, MTT assay, flow cytometry, Transwell method and Western blot, respectivly. RESULTS: The expression of miRNA-181a in A549 cells and A549/DDP cells was significantly lower than that in BEAS-2B cells, and the lowest expression level was observed in A549/DDP cells (P<0.05). The expression of miRNA-181a in A549/DDP cells was significantly increased after transfection with pGenesil-miRNA-181a (P<0.05). The cell viability, cell cycle and invasion ability of the A549/DDP cells were inhibited after miRNA-181a transfection (P<0.05). The cell growth inhibition rate and apoptotic rate of the A549/DDP cells were increased (P<0.05). The expression of Bcl-2 was reduced, but the expression of P53 was increased after transfection with miRNA-181a in A549/DDP cells (P<0.05). CONCLUSION: miRNA-181a may be correlated with the development of human lung adenocarcinoma. miRNA-181a can serve as a new target for treatment of lung cancer.     

Effect of miR-24 on chemotherapy sensitivity in lung cancer A549 cells

AIM: To study the effect of microRNA (miR)-24 on chemotherapy sensitivity and its possible mechanisms in human lung adenocarcinoma A549 cells. METHODS: The expression of miR-24 in the A549 cells and A549/DDP cells was determined by real-time PCR. Transfection of miR-24 inhibitor was used to down-regulate the miR-24 level in the A549/DDP cells. The viability and apoptosis rate were measured by CCK-8 assay and flow cytometry, respectively. The protein levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cytochrome C (Cyt C), phosphorylated extracellular signal regulated kinase (p-ERK) and P53 were detected by Western blot. Luciferase reporter assay was used to predict and identify the target genes of miR-24. RESULTS: The expression of miR-24 was significantly higher in the A549/DDP cells than that in the A549 cells (P<0.05). miR-24 inhibitor induced cell apoptosis and increased the sensitivity of the A549/DDP cells to cisplatin. Furthermore, miR-24 inhibitor down-regulated the ratio of Bcl-2/Bax, while up-regulated the protein levels of P53, p-ERK, cleaved caspase-9, cleaved caspase-3 and Cyt C. Incubation with U0126, a specific ERK inhibitor, partly reversed the viability of miR-24 inhibitor transfected A549/DDP cells. Bioinformatics analysis demonstrated that p53 was a potential target gene of miR-24. Co-teansfection of miR-24 inhibitor and P53 siRNA in A549/DDP cells partially reversed the effect of miR-24 inhibitor on cell viabiltiy. CONCLUSION: Down-regulation of miR-24 increases the sensitivity of A549/DDP cells to cisplatin. The mechanism may be related to directly targeting p53 gene and over-activation of ERK/P53 signaling pathway, thus promoting apoptosis via mitochondrial apoptosis pathway.     

Salinomycin inhibited proliferation and induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP

AIM: To investigate the effect of salinomycin on the proliferation and apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. METHODS: The inhibitory effect of salinomycin on the growth of A549/DDP cells was tested by MTT method in vitro . The apoptosis and mitochondrial membrane potential (ΔΨ_m) of A549/DDP cells were assayed by flow cytometry. The activity of caspase-3, 8 and 9 was determined by the method of colorimetry. The levels of cytochrome C, Bcl- 2, Bax, β-catenin, and phosphorylated low-density lipoprotein receptor-related protein 6(p-LRP6) were measured by Western blotting. RESULTS: Salinomycin inhibited the growth of A549/DDP cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L decreased ΔΨ_m level, and increased reactive oxygen species (ROS), cytochrome C and cytosolic Ca²⁺ release in the cells. Salinomycin also increased the acti-vity of caspase-3, 8, and 9 in the cells, reduced the ratio of Bcl-2/Bax, and decreased the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin depresses the cell growth by inhibiting Wnt signaling, and induces the apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP via mitochondria-dependent and Bcl-2/Bax pathways.     

Hydrogen inhibits C/EBP homologous protein-mediated macrophage apoptosis by activating autophagy

AIM: To investigate the protective effect of hydrogen (H₂) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H₂-saturated medium was added to murine RAW264.7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine (3-MA) and 3 μmol/L rapamycin (Rap) for 1 h, and then treated with ox-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI staining, respectively. The activity of lactate dehydrogenase (LDH) in medium was detected. The protein levels of beclin-1 (a molecular marker of autophagy) and C/EBP homologous protein (CHOP, a key signaling component of endoplasmic reticulum stress-associaed apoptosis pathway) were determined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope. RESULTS: Hydrogen attenuated the reduction of cell viability, LDH leakage, apoptosis and CHOP upregulation induced by ox-LDL. Hydrogen promoted ox-LDL-induced autophagy in macrophages as assessed by beclin-1 upregulation, and LC3 granulation, and this promotion effect of hydrogen was inhibited by 3-MA (an autophagy inhibitor) and further enhanced by Rap (an autophagy inducer). Moreover, the inhibitory effect of hydrogen on ox-LDL-induced macrophage apoptosis, reduction of cell viability and CHOP upregulation were also blocked by 3-MA and enhanced by Rap. Similar results were obtained in human THP-1-derived macrophages, as assessed by the inhibition of ox-LDL-induced apoptosis and CHOP upregulation, and the promotion of beclin-1 expression by hydrogen. CONCLUSION: Hydrogen may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression, and the upstream mechanism may partially involved in the activation of autophagy.     

Autophagy protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting C/EBP homologous protein expression

AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: The RAW264.7 macrophages were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid (PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of lactate dehydrogenase (LDH) in the medium and caspase-3 in the cells were determined by detection kits. The protein levels of beclin-1 (a molecular marker of autophagy), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein (CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis) were examined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope.RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability, and dramatic elevation in LDH leakage, cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autophagy inducer). ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap. Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL. Moreover, PBA (endoplasmic reticulum stress inhibitor) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granulation of LC3. CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages, and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression.     

Reversing effect of naringin on cisplatin resistance in human lung cancer A549/DDP cells

AIM: To investigate the effect of naringin (NRG) on cisplatin (DDP) resistance in human lung cancer A549/DDP cells and its possible mechanism. METHODS: A549/DDP cells were cultured in vitro and treated with NRG and/or DDP at different concentrations for 24 h, and then the cell viability were measured by CCK-8 assay. The combination index (CI) of NRG and DDP were analyzed by Chou-Talalay method. The apoptosis rate was analyzed by flow cytometry. Western blot was performed to detect the protein levels of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), p-Akt, CXC chemokine receptor 4 (CXCR4), cleaved caspase-3, Bcl-2 and Bax.RESULTS: The protein levels of P-gp, MRP1, p-Akt and CXCR4 in the A549/DDP cells were higher than those in the A549 cells (P<0.05). The cell viability was remarkably reduced in a dose-dependent manner when A549/DDP cells were exposed to NRG and/or DDP (P<0.05), and the IC₅₀ values of NRG and DDP were 36.92 μmol/L and 129.77 μmol/L, respectively. When the inhibition rate exceeded 15%, NRG in combination with DDP produced a synergistic effect (CI<1). Combination treatment with NRG and DDP significantly induced apoptosis (P<0.05), up-regulated the protein levels of cleaved caspase-3 and Bax, and down-regulated the protein level of Bcl-2 (P<0.05). Meanwhile, NRG remarkably down-regulated the protein levels of P-gp, MRP1, p-Akt and CXCR4 in a dose-dependent manner (P<0.05). CONCLUSION: NRG may enhance the sensibility of A549/DDP cells to DDP most likely via up-regulating the protein level of Bax and down-regulating the protein levels of Bcl-2, P-gp, MRP1, p-Akt and CXCR4.     

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.     

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.     

Anticancer function of Shp2 in lung adenocarcinoma A549 cells and rela-ted molecular mechanisms

AIM: To explore the anticancer function of Shp2 in lung adenocarcinoma A549 cells and the related molecular mechanisms. METHODS: The viability and proliferation of A549 cells treated with Shp2 specific inhibitor Phps-1 or cisplatin (DDP) were measured by CCK-8 assay and EdU assay. Annexin V-FITC/PI double staining was applied to detect apoptotic rate of A549 cells with different interventions. The protein levels of caspase-3-17p, Bcl-2, Bax, p-STAT3/STAT3 and p-ERK/ERK were determined by Western blot. RESULTS: Compared with control group, Phps-1 at the concentration of 20 μmol/L significantly increased the viability of A549 cells after 24 h of treatment (P<0.05). Meanwhile, the proliferation rate of A549 cells in Phps-1 20 μmol/L group was significant increased compared with control group (P<0.05). The apoptotic rate of A549 cells in DDP treatment group decreased from 13.01%±2.62% to 3.67%±0.93% after adding Phps-1 (P<0.05). Phps-1 down-regulated the protein levels of caspase-3-17p, Bax and p-ERK, but up-regulated p-STAT3.CONCLUSION: Shp2 is a tumor suppressor in A549 cells, which may be associated with the activation of STAT3 signal pathway.     

Effects of Buzhong Yiqi decoction-medicated serum on drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin

AIM:To investigate the effects of Buzhong Yiqi decoction-medicated serum on the drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin. METHODS:Medicated serum containing Buzhong Yiqi decoction was prepared. The optimal dose of the medicated serum was selected by MTT assay. The A549 cells and A549/DDP cells were treated with the optimal medicated serum and cisplatin at different concentrations. The IC 50, resistance index and reversal index were determined. The cells were divided into control serum group, optimal medicated serum group, LY294002 group, LY294002 + optimal medicated serum group (combination group) and negative group. The expression of PI3K and Akt at mRNA and protein levels was detected by the methods of immunocytochemistry, Western blotting and real-time PCR. RESULTS:Treatment with 10% middle dose of medicated serum for 48 h was the optimal dose and time for medicated serum. The sensitivity of A549/DDP cells to cisplatin was obviously enhanced when the cells were exposed to the optimal medicated serum with the reversal index of 2.46. The expression of PI3K and Akt at mRNA and protein levels in optimal medicated serum group and combination group was significantly decreased. CONCLUSION:Buzhong Yiqi decoction decreases the resistance index by reducing the expression of PI3K in A549/DDP cells, thus increasing the sensitivity of A549/DDP cells to cisplatin.     

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.     

Ursolic acid inhibits migration and invasion of human lung cancer A549 cells by targeting miRNA-133a

AIM: To investigate the effects of ursolic acid (UA) on the migration and invasion of human lung cancer cell line A549, and to explore its mechanism. METHODS: The cell viability was detected by MTT assay. The expression of miRNA-133a was detected in the A549 cells treated with UA by real-time PCR. The miRNA-133a mimics and inhibitor were transfected into the A549 cells, and the transfection efficiency was analyzed by real-time PCR. The cell migratory and invasive abilities were determined by wound healing and Transwell methods, respectively. RESULTS: The viability of the human lung cancer A549 cells was significantly inhibited by UA in a dose-dependent manner (P<0.05). IC₅₀ of UA (24 h) for lung cancer A549 cells was 31.04 μmol/L. UA treatment significantly inhibited the migratory and invasive abilities of A549 cells in a concentration-dependent manner, accompanied by significantly elevation of miRNA-133a expression. The mimics and inhibitor of miRNA-133a significantly upregulated and downregulated the expression of miRNA-133a in the transfected A549 cells, respectively. In addition, the viability of the A549 cells was decreased extremely after tansfected with the miRNA-133a mimics (P<0.01), so did the results of the cell migration and invasion test. The A549 cells tansfected with the miRNA-133a inhibitor showed an opposite changes of the cell viability, migration and invasion. CONCLUSION: UA inhibited the viability, migration and invasion of lung cancer A549 cells by elevating the expression of miRNA-133a.     

Tripchlorolide activates p-ERK and induces autophagy in lung cancer A549 cells

AIM: To investigate the effects of tripchlorolide (TP) on proliferation and autophagy of human lung cancer A549 cells, and explore its mechanism. METHODS: MTT assay was performed to analyze the effect of TP on the viability of human lung cancer A549 cells. The A549 cells were treated with TP, and their autophagy was observed under the fluorescence microscope through acridine orange staining. Green fluorescence spots were observed by fluorescence microscopy through GFP-LC3 plasmid transfection experiment. The levels of LC3 and p-ERK in the A549 cells after TP treatment were determined by Western blot. RESULTS: The viability of human lung cancer A549 cells was significantly inhibited by TP in a dose-time dependent manner (P<0.05). The number of the intracellular acidic follicles dyed with bright red fluorescence was significantly increased after TP treatment in A549 cells. The number of green dot-like congregate autophagosomes in cell cytoplasm was significantly increased after TP treatment in the A549 cells transfected with GFP-LC3 plasmid, while the normal treatment only induced a few cells with autophagosome formation. At the same time, we did not observe the dot-like congregate autophagosomes after TP treatment in the A549 cells transfected with GFP-control plasmid. Compared with control group, the expression of LC3-Ⅱ protein was up-regulated in A549 cells after TP treatment (P<0.01). Furthermore, treatment with TP in A549 cells for 48 h also led to a significant upregulation of phosphorylated form of ERK (P<0.01). In contrast, no significant change in the levels of total ERK protein was observed. Compared with 100 nmol/L TP group, TP+3-MA group down-regulated the protein levels of LC3-Ⅱ (P<0.01) and p-ERK (P<0.01) in the A549 cells. CONCLUSION: TP significantly inhibits the growth of A549 lung cancer cells and induces the autophagy, which may be correlated with upregulation of p-ERK protein.     

Honokiol induces autophagy by inhibiting mTOR signaling pathway in lung cancer A549 cells

AIM:To investigate whether honokiol induces the autophagy of human lung cancer A549 cells and to explore its mechanism. METHODS:The A549 cells were cultured in vitro, and treated with honokiol at different concentrations (0, 10, 20, 40, 60 and 80 μmol/L) for 48 h. MTT assay was performed to analyze the effect of honokiol on the viability of the A549 cells. The formation of autophagosome was observed by staining with acridine orange under fluorescence microscope. The protein levels of autophagy-associated protein LC3, mTOR and p-mTOR in the A549 cells treated with honokiol, or combined with autophagy inhibitor 3-methyladenine (3-MA) were determined by Western blot. RESULTS:Honokiol significantly inhibited the viability of A549 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophageic vacuoles with bright red fluorescence was significantly increased after honokiol treatment. The protein level of LC3-Ⅱ/LC3-I in the A549 cells was significantly enhanced after honokiol (40 μmol/L) treatment, and the ratio of LC3-Ⅱ/LC3-I was significantly decreased by treatment with 3-MA (P<0.05). Furthermore, treatment with honokiol (40 μmol/L) in the A549 cells for 48 h also resulted in significant down-regulation of phosphorylated form of mTOR (P<0.01), while the total protein level was not changed. CONCLUSION:Honokiol significantly inhibits the growth of lung cancer A549 cells and induces the autophagy, which may be correlated with inhibition of mTOR signaling pathway.     

Effect of hTERT antisense oligodeoxynucleotide on Cis-diamminedichicloroplatinum-induced apoptosis in Jurkat cells

AIM:To explore the effect of hTERT antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis induced by chemotherapeutic drugs in Jurkat cell lines. METHODS:Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation, DNA gel electrophoresis and flow cytometry analysis. RESULTS:The survival rates of Jurkat cells cultured with daunorubicin, vincristin, and etoposide, respectively were similar with that cultured with those chemotherapic drugs plus hTERT ASODN. The survival rates of Jurkat cells cultured with cis-diamminedichicloroplatinum(DDP) added 24 hours later were higher than that cultured with hTERT ASODN and DDP added 24 hours later. The survival rates of Jurkat cells cultured with DDP were similar with that cultured with hTERT SODN and DDP. In morphological observation of apoptotic cells using Giemsa staining, cells displayed classic apoptotic changes treated with DDP or DDP combined with hTERT ASODN or SODN at 48 hours. Agarose gel electrophoresis of genomic DNA from Jurkat cells treated with ASODN and DDP combination for 48 hours showed typical DNA "ladder". Neither the DNA from Jurkat cells treated with SODN plus DDP nor the DNA from the cells treated with DDP alone showed ladder pattern. Apoptosis rates of Jurkat cells treated with DDP for 48 hours after 24 hours of exposure to ASODN significantly increased. There was significant difference in the percentage of apoptotic Jurkat cells between hTERT ASODN plus DDP and SODN plus DDP or DDP alone, respectively. CONCLUSION:The hTERT ASODN complementary to the translation initiation region of hTERT mRNA enhanced DDP-induced apoptosis in Jurkat cells.     

Hypoxia-induced caveolin-1 up-regulation is involved in migration and invasion of lung adenocarcinoma A549 cells

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride (CoCl2) on caveolin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcinoma A549 cells. METHODS:The concentrations of Cav-1 and hypoxia-inducible factor (HIF)-1α in pleural effusion of the patients with lung cancer (MPE) or tuberculous pleurisy (TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1α inhibitor YC-1. The concentrations of Cav-1 and HIF-1α in the cell supernatants were measured by ELISA. The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respectively. RESULTS:The levels of Cav-1 and HIF-1α in MPE were significantly higher than those in TBPE. There was a highly positive correlation between Cav-1 and HIF-1α levels in the pleural effusion. CoCl2 induced the generation of Cav-1 and HIF-1α in A549 cells in a concentration- and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration- or time-dependent inhibition was observed. HIF-1α inhibitor YC-1 concentration-dependently inhibited the generation of HIF-1α and Cav-1 induced by CoCl2 in A549 cells. CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1. CONCLUSION: The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells. A possible role for HIF-1α is indicated in Cav-1 generation.     

Cepharanthine promotes A549 cell apoptosis by regulating miR-150 and miR-182

AIM: To investigate the effect of cepharanthine on the growth of human lung carcinoma A549 cells and the underlying mechanism. METHODS: After A549 cells were treated with cepharanthine, the growth inhibitory rate was detected by MTT assay. The cell morphological changes were observed under light microscope. The apoptosis of the A549 cells was analyzed by flow cytometry. The expression of microRNA (miR)-150, miR-182, p53 mRNA and FOXO1 mRNA were detected by real-time PCR. The downstream target genes were predicted by software, and the expression of p53 and FOXO1 was determined by Western blot. RESULTS: After cepharanthine treatment, the growth of A549 cells was inhibited, the apoptosis rate was significantly increased, and the expression levels of miR-150 and miR-182 were significantly decreased. With cepharanthine treatment at 10 μmol/L, the expression levels of p53 and FOXO1 were elevated; however, with cepharanthine at 30 μmol/L, the expression levels of p53 and FOXO1 were decreased. After transfection with miR-150, the expression of p53 was significantly decreased, while the expression of FOXO1 was significantly decreased after transfection with miR-182. CONCLUSION: Cepharanthine inhibits the growth of A549 cells and promotes the apoptosis of A549 cells by inhibiting the expression of miR-150 and miR-182. miR-150 and miR-182 may down-regulate the expression of p53 and FOXO1, respectively.     

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.     

Activation of TLR9 affected chemotherapeutic sensitivity of doctaxel in human non-small cell lung cancer in vitro

AIM: To probe whether CpG oligodeoxyribonucleotides 7909 (CpG ODN7909) combined with Toll like receptor (TLR)9 affected the chemotherapeutic sensitivity of doctaxel (DOC) in human lung cancer A549 and H520 cell lines.METHODS: Sequences of TLR9 siRNAs were designed. A549 and H520 cells were transfected with TLR9 siRNA by lipofectamine. The expression of TLR9 was detected by Western blot. The cell activity was measured by CCK-8 assay. The experiments were divided into blank control group, control siRNA group and TLR9 siRNA interference group. The cell cycle distribution and cell apoptosis were analyzed by flow cytometry. The expression of P38 and Bax was determined by Western blot. The cells in each group were exposed to CpG ODN7909 and/or DOC.RESULTS: In A549 cells and H520 cells, CpG ODN7909 alone had no obvious effect on the cell activity, G₂/M phase arrest and apoptosis, but increased the protein expression of P38 and Bax (P<0.01). In addition, there was no significant changes of the above indexes in CpG ODN7909 treated-TLR9 siRNA group was observed. DOC alone significantly inhibited the cell activity, higher the G₂/M phase fractions, apoptotic rates and Bax expression (P<0.01), but didn't affect the expression of P38 in all 3 groups. Compared with the cells treated with DOC alone, the cells treated with CpG ODN7909 combined with DOC exhibited lower cell activity, higher G₂/M phase fractions, apoptosis rates and more Bax expression (P<0.01), showed no significant change of P38 expression. In addition, there was no significant change of the above indexes in CpG ODN7909 combined with DOC treated-TLR9 siRNA group was observed.CONCLUSION: CpG ODN7909 may enhance the chemotherapeutic sensitivity of DOC in human lung cancer cells by combining with TLR9. The mechanism might be related to enhancing the inhibitory effect and apoptosis of DOC on the cell activity in vitro, arresting the cells at G₂/M phase of the cells.