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.     

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.     

Caspase-3 gene silencing attenuates apoptosis in alveolar epithelial cells after Streptococcus pneumoniae infection

AIM: To investigate the influence of Streptococcus pneumoniae on the apoptosis of alveolar epithelial cell and the modulation of caspases-3 on apoptosis. METHODS: A study was undertaken to determine whether Streptococcus pneumoniae infection induced the apoptosis of A549 and facilitated the expression of pro-inflammative cytokine. After inhibited by siRNA, the expression of caspase-3 and apoptosis grade in A549 were determined. RESULTS: Streptococcus pneumoniae contributed to alveolar epithelial cell apoptosis in a time-dependent manner. Streptococcus pneumoniae facilitated the expression of caspase-3 and IL-6 in A549, while depressed IL-10 concentration in culture medium. SiRNA directly decreased the expression of caspase-3 to rescue A549 from apoptosis but not affected IL-6 and IL-10 concentration. CONCLUSION: caspase-3 gene silencing rescues epithelial cells from apoptosis initiated by Streptococcus pneumoniae infection.     

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.     

Effects of salinomycin combined with L-asparaginase on proliferation and apoptosis of human acute T-cell leukemia Jurkat cells

AIM:To investigate the effects of salinomycin alone or in combination with L-asparaginase on the growth and apoptosis of human acute T-cell leukemia Jurkat cells, and the possible mechanism. METHODS:The growth of Jurkat cells was tested by Cell Counting Kit-8 in vitro. The levels of cytochrome C, Bcl-2, caspase-3, caspase-8 and caspase-9 were measured by Western blotting. Flow cytometry was used to assay cell apoptosis. RESULTS:Salinomycin or L-asparaginase alone inhibited the growth of Jurkat cells in a dose-dependent manner. The IC50 value of L-asparaginase was 8.12 IU/L, while that of salinomycin was 0.75 μmol/L. Salinomycin combined with L-asparaginase induced more significant inhibition of cell proliferation (P<0.05). Western blotting showed that the expression of Bcl-2 protein in combination group was significantly reduced, and the expression of caspase-3, caspase-8, caspase-9 and cytochrome C was significantly increased (P<0.05). Flow cytometry showed that the apoptotic rates of Jurkat cells incubated with salinomycin (0.5 μmol/L), L-asparaginase (2.5 IU/L) and both drugs for 48 h were (7.11±0.23)%, (25.43±0.47)% and (39.12±1.97)%, respectively, and significantly higher than that in control group ［(6.67±0.13)%, P<0.05］.CONCLUSION: Salinomycin synergizes with L-asparaginase-induced cytotoxicity in vitro, and the combined treatment with salinomycin and L-asparaginase induces the apoptosis of Jurkat cells.     

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.     

H IF-1αm ediates effect of interm ittent hypoxia on biological behavior of A 549 cells in vitro

ATM: To investigate whether hypoxia-inducible factor 1α (HIF-1α) mediates the effect of intermittent hypoxia on A549 cell viability, apoptosis and invasive ability METHODS: A549 cells were transfected with HIF-1α-siRNA and cultured under intermittent hypoxia. The expression of HIF-1α and its downstream genes, such as Bcl-2, Bax, P53, P21 and VEGF at mRNA and protein levels was determined by real-time PCR and Western blot. The viability of the A549 cells was measured by MTT assay. The apoptosis and cell cycle distribution of the A549 cells were examined by flow cytometry. The invasive ability of the A549 cells was detected by transwell test. RESULTS: The expression levels of HIF-1α, Bcl-2 and VEGF in non-HIF-1α-siRNA transfected A549 cells cultured in intermittent hypoxia environment[blank controlgroup(IH C),empty vector control group (IH E) and negative control group (IH N)] were higher than those in the A549 cells in normoxia group (RA), but the expression levels of Bax and P21 were lower than those in RA group (P<0.05). The siRNA-mediated HIF-1α gene silencing[intermittent hypoxia silenced group (IHS)] resulted in obvious down-regulation of HIF-1α, Bcl-2 and VEGF, and significant increase in the protein expression of P21 and Bax(P<0.05). The expression level of P53 in intermittent hypoxia groups was significantly higher than that in RA group, and no significant difference of P53 expression in different intermittent hypoxia groups was observed. Compared with normoxia, intermittent hypoxia resulted in significantly enhanced cell viability, decreased apoptosis, and enhanced invasive ability of non-HIF-1α-siRNA transfected A549 cells (P<0.05). The siRNA-mediated HIF-1α gene silencing resulted in significant cell viability inhibition, elevated apoptotic rate and decreased invasive ability under hypoxic condition (P<0.05).CONCLUSION: Intermittent hypoxia promotes the viability and invasion of A549 cells by HIF-1α-mediated downstream gene expression. HIF-1α gene silencing inhibits A549 cell growth and invasion under intermittent hypoxia by inhibition of HIF-1α signal pathways in vitro.     

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.     

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.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Stable reversal of multidrug resistance in A549/DDP cells by shRNA targeting MRP1 gene

AIM: To reverse multidrug resistance (MDR) of A549/DDP cells with short hairpin RNA (shRNA) expression vectors. METHODS: Two multidrug resistance-associated protein 1( MRP1 ) gene-specific shRNA expression plasmids pSilencer 2.1-U6 neo-MRP1 were constructed and introduced into A549/DDP cells. MRP1 mRNA was assayed by real-time fluorescent quantitative PCR. The MRP1 function was determined by rhodamine 123(Rho123) retention and the protein expression of MRP1 was detected by immunofluorescent staining. The viability of A549/DDP cells was evaluated by MTT method. RESULTS: MRP1 shRNA expression plasmids were successfully constructed. The expression of MRP1 at mRNA and protein levels was significantly decreased after sh-MRP1-2.1-1 and sh-MRP1-2.1-2 were transfected into A549/DDP cells. The intracellular accumulation of Rho123 significantly increased from(16.93±0.58)% to (89.02±0.59)% and (82.56±1.37)%. IC₅₀ of cisplatin were decreased from (101.45±0.64) μmol/L to (38.06±0.05) μmol/L and (53.72±0.36) μmol/L. IC₅₀ of 5-fluorouracil were decreased from (263.20±2.00) μmol/L to (98.82±1.16) μmol/L and (141.81±0.49) μmol/L. CONCLUSION: The shRNA expression plasmid pSilencer 2.1-U6 neo-MRP1 can stably and permanently inhibit MRP1 gene. The sensitivity of A549/DDP cells to drug is reversed.     

miR-486 targeted regulation of PTEN on LPS-induced apoptosis of alveolar epithelial cell A549

AIM: To investigate the effect of microRNA-486 (miR-486) on lipopolysaccharide (LPS)-induced apoptosis of alveolar epithelial cell A549. METHODS: A549 cells were treated with LPS, and the expression of miR-486 was detected by RT-qPCR. miR-486 mimics were transfected into LPS-induced A549 cells, and RT-qPCR was used to detect the up-regulation effect. The apoptotic rate was analyzed by flow cytometry and the protein levels of cleaved caspase-3 (C-caspase-3) and C-caspase-9 were determined by Western blot. The target gene prediction software was used to predict the target gene PTEN of miR-486. Luciferase reporter vector was used to identify the target relationship. pcDNA 3.1-PTEN and miR-486 mimics were co-transfected into A549 cells to detect the effect of PTEN up-regulation on apoptosis of miR-486 mimics transfected A549 cells stimulated with LPS. RESULTS: After LPS treatment, the expression of miR-486 in A549 cells was significantly decreased (P<0.05). Transfection of miR-486 mimics significantly up-regulated the expression of miR-486 in A549 cells stimulated with LPS (P<0.05). The apoptotic rate of A549 cells and the protein levels of C-caspase-3 and C-caspase-9 were significantly increased after LPS treatment (P<0.05). Up-regulation of miR-486 significantly down-regulated LPS-induced apoptosis of A549 cells (P<0.05). The expression of PTEN was negatively regulated by miR-486. Transfection of pcDNA 3.1-PTEN significantly increased the expression of PTEN, promoted the apoptosis and increased the protein levels of C-caspase-3 and C-caspase-9 in A549 cells stimulated with LPS after co-transfection with miR-486 mimics(P<0.05). CONCLUSION: miR-486 inhibits PTEN expression and reduces LPS-induced apoptosis of A549 cells.     

Rapamycin enhances chemosensitivity of endometrial cancer cells to adria-mycin

AIM: To explore the therapeutic effect of adriamycin combined with rapamycin on endometrial cancer cells. METHODS: Two endometrial carcinoma cell lines with different PTEN gene states were chosen: HEC-1A (wild type) and Ishikawa (mutant type). Before adriamycin administration, the cells were pretreated with low concentration of rapamycin for 24 h. The cell viability and 50% inhibitory concentration (IC₅₀) of adriamycin at 24 h were determined by MTT assay. Multiple drug effect/combination index (CI) was used to evaluate the interaction between adriamycin and rapamycin. Apoptotic rate was measured by flow cytometry. The effects of the drugs on phosphorylation of PI3K/Akt and apoptosis protein caspase-3 were detected by Western blotting. RESULTS: Both adriamycin and rapamycin showed obvious growth inhibitory effects on the 2 endometrial cancer cell lines in a time- and dose-dependent manner. After pretreated with rapamycin, IC₅₀ of adriamycin decreased sharply. In Ishikawa cells, it decreased from (21.3±3.8) μmol/L to(11.9±1.2) μmol/L,P<0.05. In HEC-1A cells, it decreased from (14.3±2.8) μmol/L to (8.2±0.9) μmol/L,P<0.05. Combination index value of the 2 drugs was more than 1.15 in the 2 endometrial cancer cell lines, indicating synergistic effects. The combination therapy of adriamycin with rapamycin increased apoptotic rates in the 2 cell lines, and induced the down-regulation of phosphorylated Akt and over-expression of caspase-3 as compared with single drug treatment (P<0.05). CONCLUSION: Adriamycin combined with rapamycin significantly enhances the chemosensitivity of endometrial cancer cells and reduces drug resistance, which will become a new trend for treating endometrial cancer.     

Effect of trastuzumab (herceptin) on the apoptosis induced by ZD1839 (iressa) in lung cancer A549 cells

AIM: ZD1839 and trastuzumab are reported to improve the therapeutic efficacy of treatment for non-smallcell lung cancer (NSCLC) and breast cancer, respectively, although the effectiveness of either drug alone is not satisfactory. NSCLC cells often express both EGFR and HER2. We therefore investigated whether a combination of ZD1839 and trastuzumab had an additive or synergistic antitumor effect. METHODS: MTT was used to measure the inhibitory effects of ZD1839 (iressa) and trastuzumab (herceptin) on the growth of A549 cells. The cell apoptosis was studied by DAPI staining, and Annexin V/PI double labeling. RESULTS: The inhibitory action of cell growth was seen in A549 cells dealing with ZD1839 and trastuzumab. They inhibited the growth of the human lung cancer cell line A549 in a concentration and time dependent manners. Compared with either ZD1839 or trastuzumab alone, combination with curcumin respectively increased the growth inhibition rate and increased apoptosis of A549 cells (P<0.05) significantly, suggesting the synergistic actions of the two drugs. CONCLUSION: The results suggest that combination treatment with ZD1839 and trastuzumab might have improved therapeutic efficacy against NSCLC cells expressing both EGFR and HER2.     

Effect of 3,5-hydroxy-6,7,3’,4’-tetramethoxyflavone isolated from Laggera pterodonta on apoptosis of Hep-2 cells in vitro

AIM: To investigate the effect of 3,5-hydroxy-6,7,3’,4’- tetramethoxyflavone (HTMF) isolated from Laggera pterodonta on Hep-2 cell apoptosis and the underlying mechanism. METHODS: The MTT assay was used to observe the cytotoxicity of HTMF to the normal cells and the inhibitory effect of HTMF on the proliferation of tumor cells. The apoptosis was determined by flow cytometry. Western blotting was used to detect the expression of caspase-3 and caspase-9. RESULTS: HTMF significantly inhibited the growth of Hep-2 cells in dose and time dependent manners. HTMF exhibited weak cytotoxicity to the two normal cell lines Vero and EVC304, while showed low effect of anti-proliferation on HepG2 cells and A549 cells. The increase in apoptosis of Hep-2 cells by HTMF was observed with dose and time dependent manners. Western blotting showed that HTMF time dependently increased the expression of caspase-3 and caspase-9 in Hep-2 cells. CONCLUSION: HTMF has high inhibitory effect on the proliferation of Hep-2 cells by induction of apoptosis in the tumor cells through caspase-9 and caspase-3 activation. However, the cytotoxicity of HTMF to the normal cells is low.     

Erianin induces apoptosis of human lung cancer A549 cells via ROS/p38 MAPK pathway

AIM:To investigate the effect of erianin on the viability and apoptosis of human lung cancer A549 cells and its possible mechanism. METHODS:A549 cells and BEAS-2B cells were cultured in vitro and treated with erianin at 10, 20, 40, 80 and 160 nmol/L for 48 h. The cell viability was measured by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. The activity of superoxide dismutase (SOD) was detected by WST-8 method, and the content of malondialdehyde (MDA) was detected by barbituric acid method. The protein levels of nuclear factor E2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), p38 MAPK, p-p38 MAPK, caspase-3 and cleaved caspase-3 were determined by Western blot.RESULTS:Erianin remarkably reduced the viability of A549 cells in a dose-dependent manner (P<0.05) with IC₅₀ at 52.64 nmol/L. Erianin also induced apoptosis (P<0.05), increased ROS level and MDA content (P<0.05), diminished SOD activity (P<0.05), and down-regulated the protein levels of Nrf2, NQO1 and HO-1 (P<0.05), in a dose-dependent manner. Meanwhile, erianin up-regulated the levels of p-p38 MAPK and cleaved caspase-3 (P<0.05), and these effects were inhibited by N-acetyl-L-cysteine and SB203580 (P<0.05).CONCLUSION:Erianin may induce apoptosis of human lung cancer A549 cells most likely via inhibiting SOD activity and down-regulating the protein levels of Nrf2, NQO1 and HO-1, thus resulting in an increase in ROS and activation of p38 MAPK.     

Regulatory effects of miR-195 on biological behaviors of lung cancer A549 cells

AIM: To investigate the regulatory effects of microRNA (miR)-195 on the biological behaviors, such as viability, apoptosis and migration, of lung cancer A549 cells, and to explore the related mechanisms. METHODS: After miR-195 mimics were transfected into the A549 cells, the cell viability, cell cycle distribution and apoptosis were measured by CCK-8 assay and flow cytometry. Transwell assay was used to detect cell migration ability. Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb/Rb were determined by Western blot. Dual-luciferase reporter assay was used to screen and identify the possible target genes of miR-195. RESULTS: Over-expression of miR-195 in the A549 cells inhibited the cell viability and induced cell cycle arrest, accompanied with the decrease in the cell migration ability and the increase in the apoptotic rate (P<0.05). Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased (P<0.05). Dual-luciferase reporter assay demonstrated that MYB was a potential target gene of miR-195. Over-expression of MYB in the A549 cells partially reversed the effects of miR-195 on the cell viability, apoptosis and migration. CONCLUSION: miR-195 inhibits lung cancer A549 cell growth and migration, and promotes cell apoptosis by targeting MYB gene.     

Antisense nucleic acid of K-ras inhibits growth of lung adenocarcinoma cancer cell A549

AIM: To explore the mechanism of inhibiting lung adenocarcinoma cancer mediated by antisense nucleic acid of K-ras.METHODS: The expression of K-ras was detected in A549 cells and 6 lung adenocarinoma samples. The antisense expression vector of K-ras was successfully constucted (named antisense- K-ras-pcDNA3.1). After transfection, the growth curve and Apoptosis were determined by MTT assay and Annexin V staining, respectively. Cyclin A, cyclin D1, cyclin E, CDK2, CDK4, P53, Rb and caspase-3 were detected by Western blotting. RESULTS: K-ras was highly expressed in 4 samples of lung adenocarcinoma and A549 cells. In A549 cells transfected with antisense nucleic acid of K-ras, the cell growth was significantly inhibited and the apoptosis was visible.The expression levels of cyclin A, cyclin D1, cyclin E, CDK2 and CDK4 were significantly decreased, and the expression levels of P53, Rb and caspase-3 were significantly increased. CONCLUSION: The growth inhibition in A549 cells mediated by antisense nucleic acid of K-ras is related to the decreases in the expression of cyclin A, cyclin D1, cyclin E, CDK2 and CKD4 , and the increases in the expression of P53, Rb and caspase-3.     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.