Sodium valproate induces cycle arrest,apoptosis and elevates p21WAF1 mRNA expression in chronic myeloid leukemia cell line K562

AIM:To investigate the effects of sodium valproate (VPA) on the cell cycle and apoptosis of chronic myeloid leukemia cell line K562, and to explore the possible mechanisms. METHODS:K562 cells were treated with VPA. Cell cycle and apoptosis were analyzed by flow cytometry. The expression of p21WAF1 mRNA was detected by RT-PCR. RESULTS:After treatment with VPA, cell cycle was arrested obviously at G0/G1 phase ［(82.30±9.41)% vs (40.13±2.12)%, P<0.05］. The apoptotic rate was significantly higher in the cells treated with VPA than that in untreated cells ［(11.47%±0.25%) vs (4.77%±0.40%), P<0.05］. The level of p21WAF1 mRNA was increased ［(1.65±0.91) vs (0.25±0.04), P<0.05］. CONCLUSION:VPA induces elevated expression of p21WAF1 mRNA in K562 cells, resulting in G0/G1 phase arrest and apoptosis in vitro.     

FAK gene silencing enhances chemotherapeutic drug sensitivity in leukemic cells XU Lü-hong1,

AIM: To investigate the effect of focal adhesion kinase (FAK) gene silencing on chemotherapeutic drug sensitivity in leukemic cells. METHODS: Lentiviral-FAK-shRNA was transfected into BCR/ABL-BaF3 leukemic cells. The protein expression of FAK was detected by Western blotting. BCR/ABL-BaF3 leukemic cells were treated with different concentrations of imatinib in vitro, and the apoptosis was determined by labeling with Annexin V. A murine model of leukemia was established and the mice were treated with FAK shRNA and imatinib. Survival time and distribution of leukemic cells in bone marrow and spleen of the mice were monitored. RESULTS: FAK shRNA was successfully constructed and effectively inhibited FAK gene expression. With 5 μmol/L imatinib treatment, the percentages of apoptotic cells in vector control group and FAK shRNA group were (9.76±1.97)% and (21.90±3.20)%, respectively, and significant difference between these 2 groups (P<0.05) was observed. With 50 μmol/L imatinib treatment, the percentages of apoptotic cells in vector control group and FAK shRNA group were (56.10±6.00)% and (82.10±5.70)%, respectively,also with significant difference between these 2 groups (P<0.05). Compared with vector control group, the mice in FAK gene silencing group displayed significantly prolonged survival time. Moreover, 60 days after injection of leukemic cells, the percentages of leukemic cells in bone marrow and spleen of the mice were significantly decreased in FAK gene silencing group as compared with those in vector control group. CONCLUSION: FAK gene silencing promotes the efficacy of chemotherapeutic drug in leukemic cells, indicating that FAK gene silencing might be considered as a new therapeutic strategy for leukemia.     

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.     

Apoptosis induced by berbamine in K562 cells correlates with the expression levels of bcr/abl gene and P210

AIM: To explore the effect of berbamine(BER) on apoptosis in K562 cells and its possible molecular mechanisms. METHODS: The apoptosis rate was measured by flow cytometry while electron microscopy and DNA electrophoresis were used to evaluate the characteristic changes of apoptosis, RT-PCR and Western blot were used to examine the expression levels of apoptosis related gene bcr/abl and BCR/ABL protein. RESULTS: By FCM, the apoptosis rate of K562 cells treated with 8.0 mg/L BER for 24 h and 72 h increased from (29.20±3.82)% to (61.77±4.35)% (P＜0.01); The typical apoptosis morphologic changes and the DNA ladder were more clearly observed. After treated with BER 0 to 16.0 mg/L for 24 h, the expression levels of bcr/abl mRNA and P210 (semiquantity value) decreased quickly from 1.19±0.02 to 0.73±0.02 (P＜0.01) and from 1.04±0.02 to 0.63±0.01 (P＜0.01), respectively. CONCLUSION: BER induces apoptosis of K562 cells in a time-and-concentration-dependent manner, the decline of bcr/abl mRNA and P210 may play an important role in the apoptotic effect of BER in K562 cells. BER could be used as a new clinical trials for bcr-abl⁺ diseases such as CML.     

Inhibitory effect of cytotoxin 1 from Naja atra Cantor venom on K562 cells

AIM: To investigate the inhibitory effect of cytotoxin 1 (CTX1) from Naja atra Cantor venom on human chronic myeloid leukemia cell line K562.METHODS: The MTS and cell counting methods were used to detect cell relative viability and cell numbers of K562 cells treated with CTX1 at different concentrations. In the living culture system, the dead and dying cells stained by PI were observed by inverted fluorescence microscope. After treated with CTX1, the apoptotic cells were detected by flow cytometry with annexinV-FITC and PI double staining.RESULTS: The relative viabilities of K562 cells were (90.50±3.07)%, (58.33±3.08)% and (27.43±1.99)% when the cells were treated with CTX1 for 24 h at the concentrations of 2, 5 and 10 mg/L,respectively. The median inhibitory concentration of CTX1 on K562 cells was 5.77 mg/L after 24-hour treatment. Comparment with control group, the percentages of K562 cells by cell counting were (85.01±3.54)%, (56.65±3.59)% and (43.24±4.15)% after treatment with 8 mg/L of CTX1 for 6 h, 12 h, 24 h,respectively. As treatment concentration of CTX1 was elevated and treatment time was prolonged, the cells stained by PI were remarkably observed under inverted fluorescence microscope. After treatment with CTX1 at 8 mg/L for 6 h, 12 h and 24 h, the incidences of cell necrosis were (0.73±0.06)%, (13.90±0.46)% and (23.33±0.86)%, respectively, and the incidences of late apoptosis were (16.27±0.21)%, (26.90±1.23)% and (18.77±0.81)%, respectively.CONCLUSION: CTX1 possesses obvious inhibitory effect on K562 cells and it mainly causes the late phase of apoptosis and necrosis.     

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.     

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.     

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.     

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.     

Role of miRNA-24 in regulation of endothelial nitric oxide synthase expression and vascular endothelial cell proliferation

AIM：To investigate whether miRNA-24 is involved in the regulation of endothelial nitric oxide synthase (eNOS) expression and vascular endothelial cell proliferation. METHODS：A plasmid that highly expressed miRNA-24 was constructed, and was transfected into the human umbilical vein endothelial cells (HUVECs) by liposome. The cell proliferation was detected by MTT assay. The expression of eNOS and Sp1 at mRNA and protein levels was exa-mined by real-time PCR, immunohistochemistry and Western blotting.RESULTS：Compared with control group, the proliferation of endothelial cells in miRNA-24 group was significantly decreased by 41.97 % (0.47±0.04 vs 0.81±0.03, P<0.01), and the expression of eNOS at mRNA and protein levels was decreased by 44.8% (0.48±0.01 vs 0.87±0.03, P<0.05) and 71.92% (0.16±0.06 vs 0.57±0.08, P<0.05), respectively. Meanwhile, the mRNA and protein levels of Sp1 were significantly decreased by 53.00% (0.45±0.02 vs 0.93±0.01, P<0.05) and by 62.31% (0.13±0.07 vs 0.31±0.09, P<0.05), respectively. In miRNA-24 inhibitor group, the above indexes were decreased compared with control group, but significantly increased compared with miRNA-24 group. CONCLUSION：miRNA-24 significantly inhibits the proliferation of HUVECs and the eNOS expression. Sp1 possibly acts as one of the important factors in the regulation of eNOS expression by miRNA-24.     

Targeted inhibition of microRNA-21 with antisense oligonucleotide and their effects on human leukemic K562 cells

AIM: To explore the inhibitory effect of anti-miRNA-21 oligonucleotide (AMO-miRNA-21) on human leukemic K562 cells. METHODS: K562 cells were transfected with AMO-miRNA-21, which was complementary to the miRNA-21 in a sequence-specific manner. Viability of K562 cells was measured by MTT assay and the optimal concentration for transfection was determined. The inhibitory effect of AMO on the K562 cell growth was examined by trypan blue dye exclusion assay at 24 h, 48 h and 72 h after transfection. Giemsas staining was used to detect morphologic changes of the transfected cells. The cell apoptosis and cell cycle progression were assayed by flow cytometry. Expression of microRNA-21 in the cells was measured by real-time PCR. RESULTS: The growth of cells treated with AMO-miRNA-21 was obviously inhibited compared with that in control groups (P<0.05). Very low cytotoxic and high inhibitory effects of AMO-miRNA-21 were found at concentration of 0.6 μmol/L. The inhibitory effect lasted for 72 h. Apoptotic cells were increased in AMO group and typical morphologic changes were conformed by Giemsas staining. One visible hypodiploid peak was detected in the histogram. However, the cell cycle progression was not inhibited evidently. The expression of microRNA-21 in the transfected cells was down-regulated significantly. CONCLUSION: Targeted inhibition of microRNA-21 with antisense oligonucleotide effectively suppresses leukemic K562 cells growth by inducing apoptosis. miRNA-21 might be a potential target for leukemia therapy.     

Effects of GOLPH3 gene silencing mediated by lentivirus on proliferation,invasion and metastasis of human gastric cancer cell line SGC-7901

AIM：To investigate the effect of Golgi phosphoprotein 3 (GOLPH3) gene silencing on the proliferation, invasion and metastasis of human gastric cancer SGC-7901 cell in vitro. METHODS：SGC-7901 cells were transfected with lentiviral vector carrying GOLPH3 shRNA to construct a stable GOLPH3-silencing cell line LV-GOLPH3-RNAi. The expression of GOLPH3 at mRNA and protein levelss were detected by real-time PCR and Western blotting, respectively. The cell proliferation was analyzed by MTT assay. Transwell migration and invasion experiments were performed to measure the migration and invasion abilities, respectively. RESULTS：The stable GOLPH3-silencing cell line was successfully established. The expression of GOLPH3 at mRNA and protein levels was reduced significantly (P<0.05), leading to the inhibition of cell proliferation in LV-GOLPH3-RNAi group compared with scrambled group and blank control group, as well as the capacities of migration (56.7±1.5 vs 186.0±3.4 and 183.3±4.2, P<0.05) and invasion (33.5±3.0 vs 85.0±3.9 and 83.1±4.4, P<0.05). CONCLUSION：GOLPH3 silencing suppresses the capacities of proliferation, migration and invasion of human gastric cancer SGC-7901 cells, suggesting that GOLPH3 may be a potential tumor marker and independent prognostic factor.     

Insulin-like growth factor I protects neonatal rat cardiomyocytes from apoptosis through improvement of mitochondrial function

AIM：To investigate whether mitochondrial mechanism is involved in the anti-apoptotic effect of insulin-like growth factor I (IGF-I) on cardiomyocytes. METHODS：Primary neonatal rat cardiomyocytes (NRCMs) were cultured and treated with 200 μmol/L hydrogen peroxide (H2O2) to induce apoptosis. Kruppel-like factor 9 (KLF9)-specific siRNA was transfected into the cells by Lipofectamine 2000. The mitochondrial function was measured by JC-1 mitochondrial membrane potential (MMP) assay. The mitochondrial morphology was observed by transmission electron microscopy. Myocardial cell apoptosis was detected by Annexin V-FITC/PI dual staining, caspase-3 activity assay, DNA-ladder analysis and Hoechst 33258 staining. RESULTS：The apoptosis of NRCMs was induced by H2O2, with MMP decreased by (24.0±1.6)% compared with control group. The fall rates of MMP in IGF-I group and KLF9 siRNA group were (18.3±1.2)% and (15.2±1.2)%, respectively (both P<0.01 vs H2O2 group), and improved mitochondrial morphology, decreased caspase-3 activity, attenuated DNA fragmentation and reduced apoptotic bodies were also observed in these two groups. The apoptotic rates of NRCMs in IGF-I group and KLF9 siRNA group were (22.4±4.2)% and (32.5±3.5)%, respectively, both lower than that in H2O2 group ［(42.5±1.8)%, P<0.01］. The anti-apoptotic effect of KLF9 silencing on NRCMs was consistent with that of IGF-I treatment. CONCLUSION：IGF-I protects NRCMs from apoptosis through down-regulating KLF9 expression and improving mitochondrial function.     

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.     

FAK shRNA inhibits growth of leukemic cell line BCR/ABL-BaF3

AIM: To investigate the effect of focal adhesion kinase (FAK) shRNA on the growth of leukemic cells.METHODS: Lentiviral-FAK-shRNA was transfected into BCR/ABL-BaF3 cells, while empty vector was transfected into the same cells for control. The proteins of FAK and other molecules were detected by Western blotting. Cell growth was observed by culturing the leukemic cells in RPMI-1640 medium in vitro, and colony formation was observed by culturing the leukemic cells in methylcellulose medium. To establish a murine model of leukemia, BCR/ABL-BaF3 cells were injected into BALB/c mice through tail vein. Survival time of the leukemic mice was monitored, and the distribution of the leukemic cells in spleen of the mice was also detected. RESULTS: FAK shRNA inhibited the protein expression of FAK, reduced STAT5 phosphorylation and induced caspases-3 activation in BCR/ABL-BaF3 cells. FAK shRNA inhibited the cell growth in vitro. Colony formation experiment showed that the number of colony in vector control group and FAK shRNA group was 215.60±13.01 and 125.00±9.06, respectively, and the difference was statistically significant (P<0.01). The mice in vector control group died between day 21 and day 27, while the mice in FAK shRNA group died between day 52 and day 60, and the difference was statistically significant (P<0.05). Moreover, 25 days after injection of leukemic cells, the percentage of leukemic cells in spleen of the leukemic mice in vector control group and FAK shRNA group was (82.40±6.13)% and (14.50±3.70)%, respectively (P<0.01). CONCLUSION: FAK shRNA inhibits the growth of leukemic cells in vitro and in vivo, indicating that FAK gene silencing might be a new therapeutic strategy for leukemia.     

Effects of gemcitabine on inhibiting proliferation and inducing apoptosis of CD34+CD38- myeloid leukemia stem cells

AIM：To investigate the effects of gemcitabine (GEM), a novel analog of deoxycytidine and nucleoside reductase inhibitor similar to cytarabine (Ara-C) in structure, on the proliferation and apoptosis of myeloid leukemic stem cells (LSCs), CD34+CD38-KG1a cells. METHODS：The expression of CD34 and CD38 on the surface of acute myeloid leukemia KG1a cells was detected by flow cytometry. The effects of GEM at various concentrations for 24 h and sustained medication for 14 d and 21 d on the proliferation and colony-forming ability of KG1a cells were analyzed by soft agar colony-forming experiment. The changes of the cell cycle of KG1a cells treated with various concentrations of GEM were tested by flow cytometry. The apoptosis of KG1a cells was determined by flow cytometry with the staining of Annexin V-FITC and propidium iodide (PI). RESULTS：The percentage of CD34+CD38- cells in acute myeloid leukemia KG1a cells was (98.02±0.72)%.Treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell cycle distribution of the KG1a cells, whereas KG1a cells treated with 0.5 mg/L GEM for 24 h were arrested at G0/G1 phase. After treatment with 0.1 mg/L and 0.5 mg/L GEM for 24 h, the colony numbers at 14 d and 21 d were lower than that in saline control group. No difference of the colony numbers between the cells treated with normal saline and 0.05 mg/L GEM for 14 d and 21 d was observed. After sustained medication with 0.05 mg/L, 0.1 mg/L and 0.5 mg/L GEM and Ara-C for 14 d and 21 d, the colony numbers decreased as compared to saline control group. Treatment with 0.5 mg/L GEM for 24 h increased the apoptotic rate of KG1a cells compared with saline control group, while treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell apoptosis. CONCLUSION：GEM inhibits the proliferation and colony-forming ability, arrests the cell cycle at G0/G1 phase and induces apoptosis of CD34+CD38- acute myeloid leukemia cells.     

Fibrinolytic activity and its mechanisms in various leukemic cell lines

AIM:To study the relation between expression of uPAR and annexinⅡ and fibrinolytic activity in various leukemic cell lines. METHODS:The plasma activity was measured under the reaction between cells of NB4, SHI-1, K562, Jurkat, Raji and plaminogen by chromogenic assay. The protein expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by flow cytometry method. The mRNA expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by RT-PCR. RESULTS:The plasma activity in SHI-1 cells and NB4 cells were higher obviously than that in Raji, K562 and Jurkat cells. The protein expression ratio of uPAR and annexinⅡ in NB4 cells were (13.15±1.61)% and (95.97±1.19)%， respectively, they were (99.00±0.26)%, (90.35±2.15)% respectively in SHI-1 cells, and they were lower in K562, Jurkat, Raji cells. The expression of annexinⅡ mRNA in NB4 cells was higher than that in SHI-1 cells, and they were undectectable in K562 and Jurkat cells. The expression of uPAR mRNA in NB4 and SHI-1 cells were higher than that in Jurkat and K562 cells. The expression of uPAR mRNA in Raji cells was undectectable. CONCLUSION:The primary hyperfibrinolysis in leucocythemia cells was observed, and relation was closely with the expression of annexinⅡ. It might be the main reason for bleeding and disseminated intravascular coagulation in patients with acute promyelocytic leukemia and acute monocytic leukemia.     

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.     

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.