Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Antisense drug targeting with VEGF mRNA designed by computer aid and inhibitory effects on K562 cell line in vitro

AIM:The effective antisense sequences targeted VEGF mRNA with computer software would be screened and designed, and effect of them on growth K562 cells and protein expression of VEGF were studied with experiments.METHODS:Seven antisense sequences were selected and synthesized, which consisted of 18-20 deoxynucleotide acid and were modified with phosphorothioate, according to principle of low free energy of overall △G₃₇ Overall. Cell growth was assayed by trypan blue dye exclusion assay and level of VEGF protien in the media was determined by ELISA.RESULTS:Six of seven sequences were capable of inhibing growth of K562 cells and downregulating the VEGF protein expression significantly, compared with Scrambed control group. It was found that there was a close correlation between low level of overall △G₃₇ and antisense effectiveness (r=0.887,P<0.01).CONCLUSION:VEGF mRNA antisense oliogdeoxynucleotides, which were designed by computer software of RNAstructure, were able to inhibit growth of K562 and its protein expression. The VEGF mRNA may be new target attached by drugs. At same time, the computer aided design is useful methods to obtain the effective antisense.     

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.     

Inhibitory effect of propolis on proliferation of K562 cells in vitro

AIM: To study the effect of propolis on the proliferation of K562 cells.METHODS: K562 cells were cultured in vitro. Cell proliferation was measured by MTT method. The apoptotic rate was determined by flow cytometry. RT-PCR was applied to detect mRNA expression of Nup98. The protein level of Nup98 was determined by Western blotting. RESULTS: The inhibitory rates of proliferation induced by propolis at the concentrations of 2 mg/L, 20 mg/L and 200 mg/L were obviously higher than that in control cells in a time-and dose-dependent manner. The apoptotic rate was increased in a dose-dependent manner. High concentration of propolis down-regulated the expression of Nup98 at mRNA and protein levels. CONCLUSION: Propolis inhibits the proliferation and induces apoptosis in K562 cells. The mechanism may be related with down-regulation of Nup98.     

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.     

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.     

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.     

The effect of antisense bcr-abl oligonucleotides on the growth of K562 cells

AIM:To study the effect of bcr- abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chrenic myelogeneous leukemia (CML) gene therapy.METHODS:Cells were exposed to oligomeis, observed by inverted microscope.Cells inhibitory rate were determined by 0.4 trypan blue exclusion . CFU-K562 were cultured in 0.8 % methylcellulose . P210 was measured by flow cytomety RESULTS: K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than Spznol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h . There was signifi-cant inhibition of cell proliferation in a rang‘cells number from 1×10⁴/mL to 5×10⁴/mL after treatment with 10unol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA.CONCLUSION: bcr-abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.     

Inhibitory effect of different target-side antisense oligonucleotides on bFGF expression in SWO-38 cells

AIM:Three different antisense oligonucleotides complementary to basic fibroblast growth factor (bFGF) mRNA were compared in inhibitory effect on gene targeted expression.METHODS:After transfecting bFGF antisense oligonucleotides (asODN) into SWO-38 cells by lipofectin, the proliferation of cells was identified by MTT method, apoptosis was examined by flow cytometric cell cycle analysis and the expression levers of bFGF were detected by Western-blotting.RESULTS:There were 49%, 33%, 51% inhibition of cell growth and 35%, 27%, 18% cell apoptosis after asODN1, asODN2 and asODN3 treatment.In addition, the decrease in bFGF protein was 63%, 42%, 11%, respectively.CONCLUSION:The data suggeste that asODN1 is a potent target to bFGF mRNA, which inhibits cell growth and induces apoptosis in SWO-38 cells.     

Wild-type PTEN gene enhances the inhibitory effect of artesunate on K562 cell growth

AIM: To investigate the effect of wild-type PTEN transfection on the sensitivity of human leukemia K562 cells to artesunate (ART) and its molecular mechanism. METHODS: The adenovirus containing wild-type PTEN (Ad-WT-PTEN) or empty vectors (Ad) were transfected into K562 cells[with multiplicity of infection (MOI)=200]. The untransfected cells served as normal control. The effect of wild-type PTEN on the inhibition of K562 cell growth by ART was observed. The sensitizing ratio of PTEN combined with ART based on IC₅₀ was calculated. The viability of K562 cells was detected by MTT assay. The apoptosis was analyzed by flow cytometry. The mRNA level of PTEN was assessed by real-time PCR. The protein expression of PTEN, p-Akt and Akt was detected by Western blot. The activity of caspase-3/7 was measured by caspase activity kits. RESULTS: The sensitivity of K562 cells to ART was significantly increased by 2.25 folds after transfected with PTEN based on the IC₅₀. The cell viability in Ad-WT-PTEN+ART group was significantly lower than that in Ad+ART group after transfection for 3 d (P<0.01). The apoptotic rate in Ad-WT-PTEN+ART group was significantly higher than that in Ad+ART group (P<0.01). The expression of PTEN at mRNA and protein levels in the K562 cells after transfection with PTEN was significantly increased, and the protein level of p-Akt and caspase-3/7 activity were down-regulated, particularly in PTEN combined with ART group. CONCLUSION: The wild-type PTEN gene enhances the sensitivity of the K562 cells to ART by down-regulating the level of p-Akt and up-regulating the caspase-3/7 activity.     

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.     

Induction of apoptosis by c-myc antisense oligonucleotide in osteosarcoma cell MG-63

AIM: To study the induction of apoptosis by c-myc antisense oligonucleotide in osteosarcoma cell (MG-63).METHODS: The designed c-myc antisense oligonucleotide fragment was transfected into human osteosarcoma MG-63 cells. The cell growth and apoptosis were measured by the methods of MTT, FCM, HE staining and transmission electron microscopy.RESULTS: The results showed that the proliferation of human osteosarcoma MG-63 cells was inhibited and apoptotic rate was 37.92% when treated with c-myc antisense oligonucleotide at the does of 10.0 μmol/L for 48 h. c-myc antisense oligonucleotide (10.0 μmol/L) also inhibited the expression of c-myc protein.CONCLUSION: c-myc antisense oligonucleotide is able to induce apoptosis in human osteosarcoma MG-63 cells.     

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.     

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.     

Effect of decitabine on resistance of K562/A02 cells to adriamycin

AIM: To investigate the effect of decitabine (DAC) on the resistance of human chronic myeloid leukemia cell line K562/A02 to adriamycin (ADR), and to explore the possible mechanism. METHODS: The K562/A02 cell line and its parental cell line K562 were treated with different concentrations of ADR or DAC alone, or in combination. The cytotoxic effects of these 2 agents were determined by CCK-8 assay. The degree of DNA methylation was evaluated by Sequenom MassARRAY system and colorimetric method. The cell cycle distribution and the apoptotic rate were determined by flow cytometry. RESULTS: K562/A02 cells were more significantly resistant to ADR than K562 cells.The half maximal inhibitory concentration of ADR for 24 h of the K562/A02 cells was about 50 times higher than that of the K562 cells. To DAC, in the concentration range of 0.5~8 μmol/L, K562/A02 cells were more sensitive than K562 cells. As compared with the same concentrations (4.31 μmol/L and 17.24 μmol/L) of ADR alone, the combination with 1 μmol/L DAC significantly improved the sensitivity of K562/A02 cells to ADR. Both DAC and ADR affected the cell cycle progression and apoptotic rate of K562/A02 cells. DAC (1 μmol/L) treatment mainly showed S phase arrest and increased early apoptotic rate for 24 h, and G₂/M phase arrest and increased late apoptosis and necrosis for 48 h in a time-related manner. ADR treatment showed G₂/M phase arrest and increased late apoptosis and necrosis in a concentration-dependent manner. In combination with 1 μmol/L DAC, the effect of ADR on the cell cycle distribution was further enhanced, showing more obvious G₂/M phase arrest, but no significant difference of the apoptotic rate was observed. The degree of methylation in the genome had no significant difference between the 2 cells, and it before and after DAC treatment had no significant change. CONCLUSION: DAC enhances the sensitivity of K562/A02 cells to ADR, showing drug resistance-reversing potential. The mechanism may be related to regulating cell cycle progression and promoting apoptosis and necrosis of K562/A02 cells.     

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.     

Inhibition of VEGF expression and SMMC 7721 cell growth by VEGFsiRNA

AIM: To determine the inhibition of VEGF small interfering RNA (siRNA) on expression of VEGF protein and SMMC 7721 cell growth. METHODS: Nine VEGFsiRNA sequences with nineteen nucleotides were designed under the assistance of computer. The inhibitory effect of VEGFsiRNA was analyzed by CCK8. The protein level of VEGF in the media was determined by ELISA. The change of cell cycle was detected by flow cytometry. RESULTS: All the VEGFsiRNA were capable of inhibiting the proliferation of SMMC 7721 cells significantly compared with control and lipofectamine control, while the inhibitory effects of VEGFsiRNA2, VEGFsiRNA4, VEGFsiRNA6, VEGFsiRNA7, VEGFsiRNA8 and VEGFsiRNA9 were better than that of antisense oligodeoxynucleotide. All the VEGFsiRNA reduced the expression of VEGF protein. The effect of VEGFsiRNA7 and VEGFsiRNA2 were the best with the inhibitory rates of 52.65% and 50.43%， respectively. VEGFsiRNAs induced the S arrest in SMMC 7721 cells. The signs of apoptosis in SMMC 7721 cells induced by VEGFsiRNA were not observed. CONCLUSION: VEGFsiRNA sequences were designed and synthesized successfully. VEGFsiRNA effectively inhibited the proliferation of SMMC 7721 cells and reduced the level of VEGF protein.     

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.     

Preparation of recombinant human soluble TRAIL and its inducing effect on apoptosis of tumor cells by TRAIL combined with bortezomib

AIM: To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to verify the biological activity of TRAIL. METHODS: The prokaryotic expression plasmid pET-28a (+)-TRAIL_114-281 was constructed. Human soluble TRAIL was obtained through optimized inducing protein expression and purification conditions. The biological activity of TRAIL was verified by CCK-8 assay. The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib (Velcade, PS-341) on the tumor cell lines H460(TRAIL-sensitive) and K562(TRAIL-resistance) for 24 h was determined. The apoptotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining. The activities of caspase-8, -9 and -3 in the cells were detected by colorimetric method. The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot. The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry. RESULTS: The recombinant human soluble TRAIL protein with stable bioactivity was successfully acquired, which induced apoptosis in H460 cells and K562 cells. After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL (P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration. Apoptotic rate in combination group was obviously higher than that in single group (P<0.05). In the process of apoptosis, the activities of caspase-8, -9 and -3 in H460 cells and K562 cells were both increased. The expression of Bcl-2 and cFLIP in treatment groups (especially the combination group) was decreased compared with control group. No significant change of the Bax expression level was observed. The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regulated after treated with bortezomib (P<0.05). CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.