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.     

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.     

miR-134 regulates cisplatin resistance of human lung adenocarcinoma cells

AIM: To investigate the roles of microRNA-134 (miR-134) in the cisplatin resistance of lung adenocarcinoma cells. METHODS: miRNA microarray was applied to compare the miRNA expression profile between A549/CDDP and A549 cells. Real-time PCR was used to confirm the expression of miR-134. miR-134 mimics and inhibitors were transfected into A549/CDDP and A549 cells, respectively. MTT assay was used to detect the sensitivity of lung cancer cells to cisplatin. Western blot was applied to test whether miR-134 regulated forkhead box protein M1 (FOXM1) and multidrug-associated protein 1 (MRP1) expression. RESULTS: Based on the data of miRNA microarray, 13 miRNAs were found to be differentially expressed in A549/CDDP cells compared with A549 cells, among which miR-134 was the most significantly down-regulated one. Compared with control group, A549/CDDP cells transfected with miR-134 mimics showed greatly enhanced sensitivity to cisplatin as indicated by IC₅₀ values (P<0.01). In contrast, suppression of the miR-134 level in the A549 cells resulted in a decreased sensitivity to cisplatin (P<0.01). FOXM1 siRNA down-regulated the protein levels of FOXM1. A549/CDDP cells transfected with si-FOXM1 showed enhanced sensitivity to cisplatin (P<0.01). In addition, the result of Western blot showed that miR-134 repressed MRP1 protein expression. CONCLUSION: miR-134 effectively increases the sensitivity of lung adenocarcinoma cells to cisplatin, and this effect of miR-134 may be partly due to its regulation of FOXM1 and MRP1 expression.     

A 3D cell culture system to study epithelial-mesenchymal transition and drug resistance of lung cancer

AIM To explore the molecular mechanism of transforming growth factor-β1 （TGF-β1）-induced epithelial-mesenchymal transition （EMT） and cisplatin resistance in three-dimensionally cultured lung cancer cells. METHODS Under three-dimensional culture condition, the morphological changes and protein expression changes of human non-small-cell lung cancer 95D cells were observed by inversed fluorescence microscopy, scanning electron microscopy, laser scanning confocal microscopy and Western blot before or after TGF-β1 stimulation. The cisplatin sensitivity was determined by MTT assay. RESULTS Under the three-dimensional culture condition, the structure of 95D cell spheroids after TGF-β1 stimulation collapsed, the cells were dispersed and migrating, and the spheroids merged with each other. The results of laser confocal microscopy showed that E-cadherin protein expression in the 95D cells did not changed after TGF-β1 stimulation, and the protein expression of N-cadherin and vimentin was significantly up-regulated. The results of Western blot showed that the expression of E-cadherin was down-regulated after TGF-β1 stimulation, and the protein levels of N-cadherin, vimentin, phosphorylated AKT and phosphorylated mTOR were up-regulated. LY294002 and rapamycin reversed TGF-β1-induced expression of the above proteins. The results of MTT assay showed that TGF-β1 reduced the sensitivity of three-dimensionally cultured 95D cells to cisplatin, while LY294002 and rapamycin reversed the cisplatin resistance of the 95D cells stimulated by TGF-β1. CONCLUSION TGF-β1 induces the EMT and cisplatin resistance of three-dimensionally cultured lung cancer cells through the activation of PI3K/AKT/mTOR signaling pathway.     

Effect of IL-8 on mitochondrial fusion and fission gene expression in human lung adenocarcinoma cell line A549 during cell migration

AIM: To investigate the changes of migration of lung adenocarcinoma cells promoted by IL-8 and the inner and outer mitochondrial membrane dynamic changes during this process.METHODS: Human lung adenocarcinoma cell line A549 was divided into control group and IL-8 group. Cell migration was analyzed by scratch detection and Transwell assay. The secretion of endogenous IL-8 was detected by ELISA. The protein levels of mitochondrial cytochrome C (Cyt C) and mitochondrial outer membrane protein Tom20 was detected by Western blot. The mRNA expression of mitochondrial fusion genes Mfn1, Mfn2 and OPA1 and fission genes Fis1, Drp1 and MTP18 was detected by RT-PCR. The morphological changes of mitochondria were observed by MitoTracker Red CMXRos dye staining and confocal microscopy.RESULTS: The migratory rate of A549 cells and endogenous secretion of IL-8 in A549 cells were higher than those in SPC-A-1 cells. The migratory rate of A549 cells was improved by IL-8 in a time-dependent manner. Compared with control group, the Tom20 protein expression was increased (P<0.05), and the Cyt C protein expression was decreased (P<0.05). The expression of mitochondrial outer membrane fusion genes Mfn1 and Mfn2 was increased (P<0.05), and the expression of mitochondrial inner membrane fusion gene OPA1 was decreased (P<0.05). The expression of fission genes Drp1 and MTP18 were decreased (P<0.05), while the expression of Fis1 was no change (P>0.05). Under confocal microscope, the punctate aggregates in the mitochondria of the A549 cells treated with IL-8 were observed.CONCLUSION: The migratory rate of A549 cells is increased by IL-8, which is related to the changes of mitochondrial fusion genes and the fission genes.     

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.     

Effect of di-indolyl thiozoline on proliferation of lung cancer A549 cells

AIM: To investigate the effect of di-indolyl thiozoline (DIIT) on the proliferation of human lung cancer A549 cells. METHODS: The effects of DIIT on the proliferation of human lung cancer A549 cell line were determined by CCK-8 assay and EdU assay. The effects of DIIT on the expression of cyclin-dependent kinase 4 (CDK4), cyclin D1, and the phosphorylation of Akt and mTOR were determined by Western blot. RESULTS: After the A549 cells were treated with DIIT at 12.5, 25, 50 and 100 mg/L, the cell viability detected by CCK-8 assay was decreased by 12%, 27% (P<0.01), 33% (P<0.01) and 52% (P<0.01), respectively, compared with DMSO control group. The EdU positive cell number determined by EdU assay was decreased by 10%, 21% (P<0.05), 26% (P<0.05) and 34% (P<0.01), respectively, compared with DMSO control group. Compared with DMSO control group, DIIT inhibited the phosphorylation of Akt and mTOR and the expression of cyclin CDK4 and cyclin D1 (P<0.05). CONCLUSION: Di-indolyl thiozoline inhibits the proliferation of A549 cells, which may be related to the decreases in phosphorylation levels of Akt and mTOR and the inhibition of cell cycle-related protein expression.     

Effects of propofol on lung injury and PI3K/Akt pathway in rats after liver ischemia and reperfusion

AIM：To study the effect of propofol on phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in the model of rat lung injury after hepatic ischemia and reperfusion (IR). METHODS：Sixty-six SD rats were randomly divided into 4 groups：sham operation group (n=6), IR group (n=24), propofol group (n=24) and propofol+wortmannin group (n=12). The rats in IR group and propofol group were further divided into 4 subgroups according to the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The rats in propofol+wortmannin group were also divided into 2 subgroups according to the time points of 3 h and 6 h after reperfusion. The rats in sham group were only dissected porta without ligation. The ligation of hepatic pedicle in the rats in IR group was performed to induce liver ischemia for 30 min and then reperfusion was conduced. The rats in propofol group were given slow injection of propofol (20 mg/kg) from the caudal vein 10 min before ischemia, and then propofol was continuously pumped at dose of 20 mg·kg-1·h-1 until rats' death. Other procedures were the same as the rats in IR group. The rats in propofol+wortmannin group were injected with wortmannin, a blocker of PI3K, at dose of 15 μg/kg before ischemia, and also given the injection of propofol as the rats in propofol group. The lung tissues of the rats were collected at the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The protein levels of total Akt (t-Akt), phosphorylated Akt (p-Akt) and Bcl-2 in the lung tissues were detected by Western blotting. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS：Compared with sham group, the protein levels of p-Akt and Bcl-2, and the cell apoptotic rate in the lung tissues in IR group, propofol group and propofol+wortmannin group increased. Compared with IR group, the protein levels of p-Akt and Bcl-2 increased, and cell apoptotic rate of the lung tissues decreased in propofol group. Compared with propofol group, the protein levels of p-Akt and Bcl-2 decreased, and cell apoptotic rate in the lung tissues increased in propofol+wortmannin group. CONCLUSION：Propofol reduces the lung injury in rats induced by liver ischemia and reperfusion, and its mechanism may be involved in PI3K/Akt signaling pathway.     

Muscarinic receptor 3 agonist promotes epithelial-meschymal transition in human lung cancer A549 cells by activating PI3K/AKT signaling pathway

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transition (EMT) of the lung cancer A549 cells stimulated with muscarinic receptor 3 (M3R) agonist carbachol. METHODS: The lung cancer cells A549 were treated with 400 μmol/L carbachol. The morphological changes of the cells were observed under inverted phase contrast microscope. The migration and invasion abilites were measured by Wound healing and Transwell assays. qPCR was used to detect the mRNA level of vimentin and E-cadherin. The protein levels of p-AKT, vimentin and E-cadherin were determined by Western blot. RESULTS: After treatment with carbachol, the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle-like cells. The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A549 cells were enhanced. Carbachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level (P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated (P<0.05). These changes was inhibited by M3R antagonist 4-DAMP. CONCLUSION: Carbachol promotes EMT in the human lung cancer A549 cells by activating PI3K/AKT signaling pathway.     

S100A6 promotes proliferation and migration of human osteosarcoma cell line 143B through PI3K/Akt signaling pathway

AIM: To investigate the effect of PI3K/Akt signaling pathway on S100A6-induced proliferation and migration of human osteosarcoma cell line 143B. METHODS: Recombinant human S100A6 protein (rhS100A6) was prepared. The 143B cells were treated with rhS100A6 in the presence or absence of PI3K inhibitor (LY294002 or wortmannin) exposure. The final concentrations of rhS100A6, LY294002 and wortmannin were 30 mg/L, 10 μmol/L and 0.5 μmol/L, respectively. The expression levels of total Akt (t-Akt) and phosphorylated Akt (p-Akt) in the 143B cells were analyzed by Western blotting. The cell proliferation and migration were determined by MTT and Transwell assays. RESULTS: rhS100A6 protein was successfully prepared, and significantly increased the proliferation and migration of 143B cells (P<005). rhS100A6 up-regulated the phosphorylation of Akt in 143B cells (P<005). Compared with rhS100A6 group, the level of p-Akt in 143B cells and the proliferation and migration of the cells were decreased in combined treatment group of rhS100A6 with LY294002 or wortmannin (P<005), where the proliferation rate at different time points dropped from 10.3% to 69.7% (P<005), and the migration rate dropped from 34.9% to 47.7% (P<005). CONCLUSION: To some extent, S100A6 promotes proliferation and migration of human ostersarcoma cell line 143B through PI3K/Akt signaling pathway.     

Effects of marrow stromal cell-mediated microenvironment on human lung adenocarcinoma A549 cells

AIM: To investigate the effects of marrow stromal cell line HS-5 on human lung adenocarcinoma A549 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the viability and migration ability of A549 cells were detected by MTT assay and wound-healing assay. After treatment with HS-5-CM, the expression of CX3C chemokine receptor 1 (CX3CR1) at mRNA level in the A549 cells was examined by qPCR. The protein levels of p-ERK and ERK in the A549 cells treated with MAPK/ERK pathway inhibitor U0126 were observed by Western blot, the migration ability of the A549 cells was measured by wound-healing assay, and the protein expression of CX3CR1 was determined by Western blot. RESULTS: HS-5-CM promoted the viability and migration ability of the A549 cells (P<0.01). The expression of CX3CR1 at mRNA level in the A549 cells was increased after treatment with HS-5-CM. MAPK/ERK inhibitor U0126 inhibited the activation of MAPK/ERK signaling pathway (P<0.01), and reduced the migration ability (P<0.01) and the expression of CX3CR1 (P<0.05) in the A549 cells. CONCLUSION: HS-5-CM significantly promotes the A549 cell viability and migration ability. Activation of MAPK/ERK signaling pathway and the expression of CX3CR1 may play a important role in this process.     

Effect of rapamycin on proliferation,invasion, adhesion,apoptosis and autophagy of human lung adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum

AIM: To study the effect of rapamycin (Rap) on the proliferation, invasion, adhesion, apoptosis and autophagy of human adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum (DDP). METHODS: Human adenocarcinoma A549 and resistant A549/DDP cell lines were cultured. The inhibitory effects of Rap alone or combined with DDP on A549 and resistant A549/DDP cells were detected by MTT assay. The in vitroinvasion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by Transwell methods. The in vitroadhesion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by adhesion experiments. The apoptosis of A549 and resistant A549/DDP cells induced by Rap alone or combined with DDP was analyzed by flow cytometry. The cell autophagy marker proteins beclin-1 and LC3 in A549 and resistant A549/DDP cells treated with Rap alone or combined with DDP were detected by Western blotting.RESULTS: Compared with Rap or DDP alone group, the combination of Rap and DDP significantly inhibited the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells in vitro, and promoted the cell apoptosis and autophagy marker proteins beclin-1 and LC3 expression (all P＜0.05).CONCLUSION: Rap enhances the effect of DDP through promoting the cell autophagy, thereby inhibiting the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells and inducing the cell apoptosis with a synergistic effect.     

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.     

Study of establishment of K562/HHT cell line and reversal of multidrug resistance by antiprogestin drug mifepristone

AIM: To study the mechanism of multidrug resistance (MDR) of leukemia cells induced by homoharringtonine (HHT) and the reversal effect of mifepristone on MDR.METHODS: Human leukemia cell line K562 was induced into MDR cell line by intermittent administration of high dose of HHT.MTT assay was used to detect the sensitivity of these MDR cells to all sorts of chemotherapeutic agents with or without mifepristone.The cytotoxicity of mifepristone was also observed.RT-PCR was used to detect the expression of MDR1 gene and glucosylceramide synthase (GCS) gene.Flow cytometry was used to detect the expression of P-glucoprotein and the accumulative value of intracellular daunorubicin (DNR) in these MDR cells with or without mifepristone.Immunohistochemistry was used to detect the expression of Bcl-2,Bax and caspase-3 in these MDR cells with or without mifepristone.RESULTS: MDR cell line K562/HHT was acquired after induced by HHT for 2 months.This MDR cell line possessed the ability of 462.6 fold resistance to HHT and cross-resistance to adriamycin,vincristine and etoposide.The expression of MDR1 gene,GCS gene,P-glucoprotein and Bcl-2/Bax ratio in K562/HHT cells were significantly higher than those in K562 cells (P<0.05).The caspase-3 expression and the accumulative value of intracellular DNR in K562/HHT cells were significantly lower than those in K562 cells (P<0.05).10 μmol/L mifepristone reversed the resistance of K562/HHT cells to HHT,adriamycin,vincristine and etoposide at different levels.The Bcl-2/Bax ratio,caspase-3 expression and accumulative value of intracellular DNR in K562/HHT cells treated with RU486 were significantly different compared with K562/HHT cells without RU486 treatment (P<0.05).CONCLUSIONS: Leukemia cell line K562 can be induced into MDR cell line K562/HHT by HHT.P-glucoprotein,GCS,Bcl-2/Bax ratio and caspase-3 may play an important role in K562/HHT cells.Mifepristone can reverse MDR in K562/HHT cells by decreasing the accumulative value of intracellular drug and regulating the expression of Bcl-2,Bax and caspase-3.     

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.     

Effect of gonadotropin-releasing hormone analogue on human breast cancer cell line in vitro

AIM: To investigate the effects of gonadotropin-releasing hormone (GnRH) analogue on the growth of breast cancer cell lines MCF-7 and MDA-MB-231 in vitro and to explore the related mechanisms with PI3K/Akt or ERK/MAPK pathways. METHODS: The proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231 treatment with triptorelin was detected by MTT assay and the distribution of the cell cycle was determined by flow cytometry. The phosphorylation of the ERK1/2 and Akt was evaluated by Western blotting. RESULTS: Triptorelin inhibited the proliferation of MCF-7 cells at concentration of 10^-5 mol/L after treated for 192 h or at concentration of 10^-4 mol/L after treated for 168 h and 192 h. Triptorelin inhibited the proliferation of MDA-MB-231 cells at concentration of 10^-4 mol/L after treated for 192 h (P<0.05).Treatment with triptorelin for 192 h at concentration of 10^-4 mol/L had no statistical significance effect on phosphorylation of ERK1/2 and Akt(P>0.05).CONCLUSION: Inhibitory effect of GnRH analogue triptorelin on human breast cancer cells is not just the connection with the down-regulation of pituitary hormone, but also a direct inhibitory effect. The role may not be involved in the activation of ERK/MAPK and PI3K/Akt signaling pathways.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Effects of BCL-3 gene silencing on the proliferation and drug sensitivity of human colorectal cancer cell line RKO with high expression of BCL-3

AIM：To construct lentiviral vectors for RNA interference (RNAi) of BCL-3 gene, and to detect the changes of biological behaviors and drug sensitivity of colorectal cancer cells after BCL-3 gene silencing. METHODS：The expression of BCL-3 in five human colorectal cancer cell lines was detected by RT-PCR and Western blotting. Lentiviral vectors for RNAi of BCL-3 gene were constructed and transfected into the human colorectal cancer cell line with high expression of BCL-3, and then the silencing effect was detected by Western blotting. After BCL-3 gene silencing, the change of cell proliferation was detected by MTT assay and soft agar colony formation assay, and the change of drug sensitivity was detected by MTT assay. RESULTS：BCL-3 was highly expressed in human colorectal cancer cell line RKO. Lentiviral vectors for RNAi of BCL-3 gene were successfully constructed, and Western blotting showed that BCL-3-shRNA2 could efficiently inhibit the expression of BCL-3 protein in RKO cells. After BCL-3 gene silencing, the proliferation ability and colony formation rate of RKO cells were decreased, and the median inhibitory concentration of oxaliplatin for RKO cells also decreased significantly. CONCLUSION: Inhibition of BCL-3 gene expression decreases the proliferation ability of human colorectal cell line RKO with high expression of BCL-3, and enhances the sensitivity of RKO cells to oxaliplatin.     

Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro

AIM: To study the reverse effects of saikoside (SS) on the multidrug resistance (MDR) of human leukemic cell line K562/ADM and to investigate the related mechanism. METHODS: K562 cells and K562/ADM cells in the culture were treated with SS at the concentrations of 1~100 mg/L. The inhibitory rate of the cell proliferation was measured by MTT assay. Non-cytotoxic dose of SS was determined. K562/ADM cells were treated with SS at non-cytotoxic doses of 1.25, 2.5 and 5.0 mg/L with different concentrations of adriamycin (ADM,0.05~100 mg/L). The 50% inhibitory concentration (IC₅₀) and the reversal index in all groups were determined. The cell morphology was observed after treated with SS+ADM. The effects of SS on ADM accumulation in K562/ADM cells, the cell cycle profile and apoptosis were examined by flow cytometry. RESULTS: The inhibitory rates were significantly increased in a dose-dependent manner when the cells were treated with different doses of SS (1~100 mg/L). The available reversal concentration of SS was 5.0 mg/L and the reversal index was 21.5 folds for K562/ADM cells. After treated with SS+ADM, the number of tumor cells was decreased and apoptotic cells were increased in a dose-response relationship. ADM accumulation in K562/ADM cells treated with SS was significantly higher than that in control cells (P<0.05). SS may significantly enhanced the apoptosis of K562/ADM cells treated with ADM (P<0.05). K562/ADM cells treated with SS were blocked in the stage of G₀/G₁. CONCLUSION: SS has effect on proliferation inhibition and MDR reversal in K562/ADM cell line. The reversal mechanisms of SS may be due to increasing the accumulation of chemo therapeutics in the cell, inducing the cell apoptosis and arresting the cells in G₀/G₁ phase.