Inhibition of FoxM1 sensitizes leukemia K562 cells to homoharringtonine

AIM: To study whether inhibition of forkhead box protein M1(FoxM1) sensitizes leukemia K562 cells to homoharringtonine (HHT). METHODS: K562 cells were incubated with HHT at different concentrations (0μmol/L, 0.015μmol/L, 0.030μmol/L and 0.045μmol/L) for different time (0 h, 24 h, 48 h and 72 h). The mRNA and protein levels of FoxM1 were detected by real-time PCR and Western blot. FoxM1 siRNA was transfected into K562 cells with 0.015μmol/L HHT after 6 h. After 72 h incubation, the cell proliferation was detected by cell counting and soft agar assay, and the proportion of apoptotic K562 cells was determined by flow cytometry. The expression of c-Myc and Sp1 were detected by real-time PCR and Western blot. RESULTS: FoxM1 expression was reduced time-dependently and dose-dependently, suggesting that HHT mediated the downregulation of FoxM1 in K562 cells. In K562 cells, treatment with FoxM1 siRNA and HHT inhibited the cell proliferation and promoted the apoptosis significantly. Therefore, inhibition of FoxM1 sensitized leukemia K562 cells to HHT. The expression of c-Myc and Sp1 was positively regulated by FoxM1. CONCLUSION: HHT inhibits Forkhead box protein M1 expression in K562 cells. Inhibition of FoxM1 sensitizes K562 cells to HHT.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin

AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner.     

Glucosylceramide synthase upregulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway in leukemia multidrug-resis-tant cell line

AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway, thus enhancing drug resistance of K562/A02 human leukemia multidrug resistant cell line. METHODS: siRNA targeting GCS was transfected into K562/A02 cells. Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting. After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting, respectively. The viability of the cells was evaluated by CCK-8 assay. RESULTS: The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K562/A02 cells by GCS siRNA transfection compared with negative control group. Inactivation of MEK/ERK signaling due to U0126 treatment decreased Bcl-2 mRNA and protein levels in a concentration-dependent manner, and sensitized K562/A02 cells to adriamycin. CONCLUSION: GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway, thus regulating multidrug resistance of human leukemia K562/A02 cells.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

Expression and function of circular RNA_0000231 in non-small-cell lung cancer

AIM: To investigate the expression and function of circular RNA_0000231 (circ_0000231) in non-small-cell lung cancer (NSCLC). METHODS: RT-qPCR was used to detect the expression of circ_0000231 in the NSCLC tissues and cell lines. circ_0000231 small interfering RNA (si-circ_0000231) or negative control siRNA of circ_0000231 (NC) was transfected into the NSCLC cells. The proliferation and apoptosis of the NSCLC cells were detected by CCK-8 assay, colony formation assay and flow cytometry, respectively. The expression of cyclin D1 (CCND1) and anti-apoptotic protein Bcl-2 were determined by RT-qPCR and Western blot. RESULTS: The expression of circ_0000231 in the NSCLC tissues and cell lines was significantly up-regulated compared with precancerous tissues and lung epithelial cells BEAS-2B (P<0.05). After transfection of NSCLC cells with si-circ_0000231, the cell viability, colony formation numbers were significantly decreased, and the apoptotic rate in si-circ_0000231 group was significantly increased as compared with NC group (P<0.01). In addition, the results of RT-qPCR and Western blot showed that transfection of si-circ_0000231 inhibited the expression of CCND1 and Bcl-2 (P<0.01). CONCLUSION: The expression of circ_0000231 is significantly increased in the NSCLC tissues and cells. Knock-down of circ_0000231 expression significantly inhibits the proliferation of NSCLC cells.     

Mitochondrial ceramidase overexpression up-regulates Bcl-2 prot ein level in K562 cells probably through its metabolic product sphingosine-1-ph osphate

AIM:To investigate the role of mitochondri al ceramidase in mitochondrial functions,especially in the regulation of apopto sis.METHODS:pCDNA3.1/His-MtCDase plasmid,containing mitochondrial ceramidase cDNA sequence,was transfected into K562 cells by liposome,and G418 was used to screen the positive clones.A stable transfected K562 cell line was established and defined as ‘K562TC’.The differences between K562 an d K562TC cells in serum withdrawal resistance and Bcl-2 protein expression were evaluated by annexin V/PI test,flow cytometry and Western blotting,respectivel y.RESULTS:K562TC cells with elevated Bcl-2 protein expression lev el identified by FCM or Western blotting showed stronger resistance to apoptosis induced by serum withdrawal than their parental cells.Inhibition of mitochondr ial ceramidase expression in K562TC cells by its specific antisense oligodeoxynu cleotide was correlated with a decrease in Bcl-2 protein level.N,N'-dimethylsp hingosine (DMS),a sphingosine kinase inhibitor,depleted intracellular sphingos ine-1-phosphate (SPP) production,also abrogated Bcl-2 protein expression in K56 2TC cells,while exogenous sphingosine-1-phosphate up-regulated Bcl-2 protein le vel in K562 cells.CONCLUSION:Mitochondrial ceramidase overexpression in K562 cell s leads to markedly elevated level of Bcl-2 protein a nd results in more resistance to serum withdrawal.This effect is initiated no t by sphingosine,the direct metabolite of mitochondrial ceramidase,but via sph ingosine-1-phosphate,its phosphorylated form,indicating that mitochondrial cer amidase,through its sphingoid metabolite sphingosine-1-phosphate,up-regulates Bcl-2 protein expression in K562 cells.     

Up-regulation of mRNA expressions of fas,bcl-2, bim,bax, caspase-3, caspase-9, and bcl2l12 in K562 treated with bortezomib

AIM: To detect the treatment of K562 leukemia cells with bortezomib altering the expression of genes fas, bcl-2, bcl2l12, bim, bax, caspase-9 and caspase-3.METHODS: MTT assay was used to detect the inhibition of proliferation. Apoptosis was detected by Annexin-V staining and mitochondrial transmembrane potential (Δψm). RT-PCR was used to analyze the mRNA expressions of fas, bcl-2, bcl2l12, bim, bax, caspase-3 and caspase-9.RESULTS: Bortezomib caused a time- and dose-dependent inhibition of cell proliferation and IC50 of 24 h and 48 h were 161.41 nmol/L and 96.33 nmol/L, respectively. At the concentration of 104 nmol/L, bortezomib induced apoptosis in a time-dependent manner, including increasing annexin-V positivity and decreasing the Δψm. RT-PCR showed that bortezomib up-regulated the mRNA expression of fas, bcl2l12, caspase-9 and caspase-3, but mRNA expressions of bcl-2, bim and bax did not changed obviously.CONCLUSION: Bortezomib inhibits the proliferation of K562 and induces apoptosis, in which fas, bcl2l12, caspase-9 or caspase-3 gene is one of the main genes taking part in.     

Effect of CIP2A over-expression on proliferation and apoptosis of gastric epithelial cells

AIM: To investigate the effects of cancerous inhibitor of protein phosphatase 2A (CIP2A) over-expression on the proliferation and apoptosis of gastric epithelial cells.METHODS: The mRNA expression of CIP2A and cyclin D1 in the tissues of normal gastric mucosa and gastric polyps was detected by RT-qPCR. The GES-1 cells were divided into control group, Ad-emp group and Ad-CIP2A group. The cell proliferation ability was detected by MTT assay and BrdU assay, and the cell apoptosis was analyzed by flow cytometry. The expression of apoptosis-related molecules was determined by Western blot and RT-qPCR. The levels of inflammatory cytokines were measured by ELISA after GES-1 cells were infected with Ad-emp and Ad-CIP2A. Furthermore, the protein levels of p-Rb, E2F1 and cyclin D1 in the GES-1 cells was determined by Western blot after transfected with CIP2A siRNA.RESULTS: The expression of CIP2A and cyclin D1 in adenomatous gastric polyps tissues was significantly higher than that in normal gastric mucosa tissues, and no significant change of that between hyperplastic gastric polyps tissues and normal gastric mucosa was observed. After transfected with CIP2A, the proliferation ability of GES-1 cells was increased, the cell apoptosis was inhibited, the concentrations of IL-1β and IL-10 was up-regulated and the protein levels of p-Rb, E2F1 and cyclin D1 were increased, while the protein levels of p-Rb, E2F1 and cyclin D1 were significantly decreased after transfected with CIP2A siRNA.CONCLUSION: CIP2A promotes the proliferation and inhibits the apoptosis of GES-1 cells by activating Rb/E2F1.     

Combination of sorafenib and daunorubicin has antileukemic activity on K562 cells

AIM: To investigate the synergetic inhibitory effect of sorafenib and daunorubicin (DNR) on K562 and U937 cells. METHODS: The inhibitory rate of sorafenib or daunorubicin alone, and the combined inhibitory rate of sorafenib and IC10 daunorubicin were measured by MTT assay. Apoptotic rate of single drug or combination was assessed by flow cytometry (Annexin Ⅴ/PI staining) and Hoechst 33258 staining assay. p-ERK1/2 level was detected by Western blotting after the cells were treated with sorafenib, daunorubicin and U0126 or combinations. Synergistic or antagonistic effect of proliferation and apoptosis on K562 and U937 was estimated according to the Jins Method. RESULTS: Combination of sorafenib and DNR showed synergistic growth inhibition (q>1.15, P<0.01) and synergistic promotion of apoptosis (q>1.15, P<0.05) in K562 and U937 cells. The level of p-ERK1/2 in K562 cells was obviously higher than that in U937 cells (P<0.01). p-ERK1/2 expression was completely inhibited in sorafenib or U0126 treated K562 cells for 24 h. Combination of U0126 with DNR inhibited the proliferation of K562 cells synergistically. CONCLUSION: Combination of sorafenib with DNR showed synergistic cell growth inhibition and promotion of apoptosis in K562 and U937 cells. U937 cells were more sensitive to DNR than K562 cells while K562 cells were more sensitive to sorafenib. Sorafenib enhances the anti-leukemic activity of DNR in K562 and U937 cells via down-regulation of p-ERK1/2 expression.     

Inhibition of c-Myc by 10058-F4 overcomes imatinib resistance in chronic myeloid leukemia cells

AIM：To investigate the effect of c-Myc inhibitor 10058-F4 on human chronic myeloid leukemia (CML) K562 cells and imatinib-resistant K562/G cells. METHODS：The protein expression of c-Myc was detected by Western blotting. Cell proliferation was evaluated by MTT assay and colony formation assay. PI staining was used to determine the cell cycle distribution. Annexin V-PI staining was applied for apoptosis detection. RESULTS：Imatinib-resistant K562/G cells displayed lower sensitivity to imatinib than K562 cells with high expression of c-Myc. Treatment with specific c-Myc inhibitor 10058-F4 inhibited the cell proliferation in a dose- and time-dependent manner, and K562/G displayed more sensitivity to 10058-F4 than K562 cells. 10058-F4 also induced cell cycle arrest in G0/G1 phase and induced apoptotic cell death in the 2 cells. Importantly, 10058-F4 suppressed the colony formation ability in K562 and K562/G cells. CONCLUSION：c-Myc is a novel target to overcome imatinib-induced drug resistance, and c-Myc inhibitor provides a new approach in CML therapy.     

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 inhibits proliferation and induces apoptosis of Gleevec-resistant chronic myeloid leukemic cell line K562/Glv

AIM: To study the effect of salinomycin on inhibiting proliferation and inducing apoptosis of Gleevec-resistant chronic myeloid leukemia cell line K562/Glv. METHODS: The inhibitory effect of salinomycin on the growth of K562/Glv cells was detected by CCK-8 assay in vitro. Flow cytometry was used to observe apoptosis, mitochondria membrane potential (ΔΨ_m), reactive oxygen species (ROS) and the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in K562/Glv cells. The activity of caspase-3, -8 and -9 was measured 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 determined by Western blotting. RESULTS: Salinomycin inhibited the growth of K562/Glv cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L inhibited the growth of the cells with the inhibitory rate of (36.70±2.31)%. The cell apoptotic rate was (19.66±2.23)%. Salinomycin at concentration of 0.2 μmol/L decreased the level of ΔΨ_m, and increased the levels of ROS, cytochrome C and[Ca²⁺]_i in the cells. Salinomycin also increased the activity of caspase-3, -8 and -9 in the cells, reduced the ratio of Bcl-2/Bax, and attenuated the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin induces the apoptosis of Gleevec-resistant myeloid leukemia cell line K562/Glv via Bcl-2/Bax and mitochondria-dependent pathways, and inhibits the cell growth through Wnt signal pathway.     

Effect of miRNAs on apoptotic leukemia cells induced by demethylcantharidin

AIM: To explore the role of miRNA expression in the process of antitumor drug demethylcantharidin (DMC)-induced leukemia cell apoptosis. METHODS: K562 cells were treated with DMC to induce apoptosis. Microarray assessment was performed to detect the changes of miRNAs. The expression of miRNAs was further detected by RT-PCR and real-time PCR. The miRNA-relative genes were analyzed by gene information softwares,and their expression was determined by ELISA analysis. RESULTS: The results of microarray showed that more than 290 miRNAs were differentially expressed after DMC treatment. The expression of miR-16, miR-34 and miR-125 increased at more than 1.5 folds in DMC treatment group determined by RT-PCR and real-time PCR analysis, while both miR-106 and miR-150 were down-expressed over 60％. Using microRNA TargetScan and miRanda analysis software, we found that the expression of oncogenes ( bcl-2, E2F1, E2F3 ) and tumor suppressor genes ( RB1, p53 ) may be regulated by the above miRNAs. The expression of RB1 and P53 proteins significantly increased, while Bcl-2, E2F1 and E2F3 proteins were obviously down-regulated after DMC treatment.CONCLUSION: DMC induces K562 cell apoptosis by regulating the expression of miRNAs and their relative genes.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbonate on the proliferation and apoptosis in K562 cells

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB on the proliferation and apoptosis of K562 cells and to explore the anti-tumor mechanism of PDTC.METHODS: Trans AMTM NF-κB p65 kit was used to detect the activity of p65 in K562 cells treated by PDTC. The effect of PDTC on the proliferation of K562 cells was measured by WST-1 method. DNA damage was detected by single cell gel electrophoresis (comet assay). The procaspase-3 and activated protein level of caspase-3 were detected by Western blotting.RESULTS: The activity of p65 in K562 cells was inhibited after treated by PDTC (P<0.01). Simultaneously the cell proliferation was significantly inhibited in a dose-and time-dependent manner (P<0.01). The degree of DNA damage in K562 cells treated with PDTC at concentrations of 25 μmol/L, 50 μmol/L or 100 μmol/L was more severe than that in control. The rates of comet cells in the PDTC-treated groups (43.50％, 84.00％, 95.63％) were significantly higher than those in control (9.75％, P<0.01), and it was also dose-dependent. The expression of procaspase-3 and activated caspase-3 protein were detected in the cytoplasm of the K562 cells treated by PDTC by Western blotting.CONCLUSION: NF-κB plays an important role in regulating cell proliferation and apoptosis in K562 cells. PDTC inhibits NF-κB activity and elevates the expression of caspase-3, which is related to increase in cell apoptosis.     

TPX2 induces apoptosis of rectal cancer HR-8348 cells through p38 MAPK signaling pathway

AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells.METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA (siRNA) served as TPX2 siRNA group. The non-transfected cells were used as control group. The cells transfected with siRNA negative control (siRNA-NC) were used as siRNA-NC group. The TPX2 siRNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 siRNA+SB203580 group. The expression of TPX2 at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK, p-p38 MAPK, cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot.RESULTS: After transfection, the expression of TPX2 at mRNA and protein levels was decreased in TPX2 siRNA-transfected cells (P<0.05). Transfection with siRNA-NC had no effect on TPX2 mRNA and protein levels in the cells. After knockdown of TPX2 expression, the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased, while the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK were increased significantly reduced (P<0.05). Compared with TPX2 siRNA group, the apopto-tic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 siRNA+SB203580 group were significantly decreased, while the viability was significantly increased (P<0.05).CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.     

Cyclooxygenase-2 inhibitors enhance the antileukemia activity of STI571

AIM：To investigate whether celecoxib,a cyclooxygenase-2 (COX-2) inhibitor,potentiates the anti-leukemia activity of STI571 in K562 cells.METHODS：K562 cells were treated with STI571,celecoxib or combination of both at different concentrations in suspension culture.Cell proliferation was documented by MTT assay,and cell apoptosis was determined by flow cytometry and morphology.Meanwhile,RT-PCR was applied to analyze the probable mechanism underlying the effects of the drugs.RESULTS：The combination of STI571 and celecoxib dramatically suppressed the proliferation of K562 cells,in which 0.25 μmol/L STI571 and 40.0 μmol/L celecoxib enhanced the inhibiting rate to 76.1%±1.6%.Furthermore,the combining administration of drugs significantly promoted the apoptosis induced by STI571，which showed characteristic changes of morphologic features and increase in sub-G1 cells.By using RT-PCR technique,the expression of COX-2 had no decline by single administration of celecoxib or STI571.However,a progressive down-regulation was caused by coadministration of two drugs.In contrast with COX-2,the expression of VEGF had no changes at any time.CONCLUSION：The administration of celecoxib alone only inhibits the proliferation of K562 cells.Combination treatment with STI571 and celecoxib promotes the apoptosis induced by STI571.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.