Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line / Efeito apoptótico da natalensia bulbina e do clorofito comoso na linhagem celular mielogênea da leucemia K562

Abstract Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Resumo Bulbine natalensis Baker e Chorophytum comosum (Thunb.) Jacques são potenciais fontes medicinais para o tratamento de cânceres. A Leucemia Mieloide Crônica (LMC) é um distúrbio das células-tronco hematopoiéticas que é tratado com inibidores da tirosina quinase, mas frequentemente, causa recorrência da leucemia após a interrupção da terapia, portanto, requer um tratamento alternativo. Este estudo determinou o efeito anticancerígeno de extratos metanólicos e aquosos de folha, raiz e bulbo de B. natalensis e C. comosum na linhagem celular de leucemia mieloide humana crônica (K562) por ensaios de MTT, Hoechst bis-benzimida nuclear e anexina V. O extrato metanólico da raiz de B. natalensis e C. comosum apresentou alta citotoxidade de 8,6% e 16,7% respectivamente, na linhagem celular K562 com a concentração de 1,000 μg / ml. Perda morfológica da integridade da membrana celular causando degradação dos núcleos, citoplasma e encolhimento celular foi observada no extrato metanólico da raiz de ambas as plantas. Uma alta apoptose (p <0,0001) foi induzida nas células K562 por extratos de folhas e raízes de C. comosum em comparação com B. natalensis. Este estudo mostrou que ambas as plantas possuem efeito apoptótico contra leucemia mieloide in vitro que contribui para as propriedades anticâncer gerais de B. natalensis e C. comosum para justificar futuras aplicações terapêuticas contra câncer de sangue de LMC.

Effects of miR-144-3p on Proliferation, Cell Cycle and Apoptosis of K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of miR-144-3p on cell proliferation, cell cycle and apoptosis of blast phase chronic myelogenous leukemia (CML) K562 cells.@*METHODS@#K562 cells were cultured in vitro and mimics negative control, hsa-miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor were respectively transfected into K562 cells with transfection reagents. The cells were divided into five groups including blank control, mimics negative control, miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor. After transfection, the cell proliferation activity was detected by CCK-8 assay. The cell cycle distribution and apoptosis were detected by flow cytometry.@*RESULTS@#Compared with the blank control and mimics negative control groups, the proliferation rate of miR-144-3p mimics group was significantly decreased (P<0.05), the proportion of S phase cells was markedly increased (P<0.05), while the proportion of G1 phase cells was obviously decreased (P<0.05), and the apoptosis rate was significantly increased (P<0.05). Compared with the blank control and inhibitor negative control groups, the proliferation rate of miR-144-3p inhibitor group was obviously increased (P<0.05), the proportion of S phase cells was markedly decreased (P<0.05), while the proportion of G1 phase cells was obviously increased (P<0.05), and the apoptosis rate was significantly decreased (P<0.05).@*CONCLUSION@#miR-144-3p can inhibit the proliferation and promote apoptosis of K562 cells, affect the cell cycle, and block K562 cells in S phase, which indicates that miR-144-3p is involved in the cell cycle activity of CML during blastic phase.

Effect of Dichloromethane Extraction Phase of Patrinia Scabiosaefolia Fisch. Stem on Proliferation and Differentiation of K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism.@*METHODS@#MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 μg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry.@*RESULTS@#The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 μg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS.@*CONCLUSION@#DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.

Study on the Relationship between Integrin 2A and Drug Resistance in Chronic Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the expression pattern and clinical significance of Integral membrane protein 2A（ITM2A） in drug resistant patients with chronic myeloid leukemia (CML).@*METHODS@#The expression of ITM2A in CML was evaluated by qRT-PCR, Western blot and immunocytochemistry. In order to understand the possible biological effects of ITM2A, apoptosis, cell cycle and myeloid differentiation antigen expression of CML cells were detected by flow cytometry after over-expression of ITM2A. The nuderlying molecular mechanism of its biological effect was explored.@*RESULTS@#The expression of ITM2A in bone marrow of CML resistant patients was significantly lower than that of sensitive patients and healthy donors（P<0.05）. The CML resistant strain cell K562R was successfully constructed in vitro. The expression of ITM2A in the resistant strain was significantly lower than that in the sensitive strain(P<0.05). Overexpression of ITM2A in K562R cells increased the sensitivity of K562R cells to imatinib and blocked the cell cycle in G2 phase(P<0.05), but did not affect myeloid differentiation. Mechanistically, up-regulation of ITM2A reduced phosphorylation in ERK signaling (P<0.05).@*CONCLUSION@#The expression of ITM2A was low in patients with drug resistance of CML, and the low expression of ITM2A may be the key factor of imatinib resistance in CML.

Effect of Cyr61 on Imatinib Resistance in Chronic Myeloid Leukemia and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Cyr61 on imatinib (IM) resistance in chronic myeloid leukemia (CML) and its mechanism.@*METHODS@#Cyr61 level in cell culture supernatant was determined by enzyme-linked immunosorbent assay. The expression of Cyr61 and Bcl-xL were measured by real-time PCR and Western blot. Cell apoptosis was analyzed using an Annexin V-APC Kit. Expression of signal pathways related proteins was determined by Western blot.@*RESULTS@#The level of Cyr61 obviously increased in K562G cells (IM resistance to CML cell line K562). Down-regulating the expression of Cyr61 decreased the resistance of K562G cells to IM and promoted IM induced apoptosis. In CML mouse model, down-regulating the expression of Cyr61 could increase the sensitivity of K562G cells to IM. The mechanism studies showed that Cyr61 mediated IM resistance in CML cells was related to the regulation of ERK1/2 pathways and apoptosis related molecule Bcl-xL by Cyr61.@*CONCLUSION@#Cyr61 plays an important role in promoting IM resistance of CML cells. Targeting Cyr61 or its related effectors pathways may be one of the ways to overcome IM resistance of CML cells.

Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca 2+ -dependent gap junction intercellular communication / 中华医学杂志(英文版)

BACKGROUND@#Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown.@*METHODS@#Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance.@*RESULTS@#Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments.@*CONCLUSIONS@#Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

The Role and Mechanism of MiR-451 in Multidrug Resistance of Leukemia Cell Line K562/A02 / 中国实验血液学杂志

OBJECTIVE@#To detect the differential expressions of miR-451, ABCB1 and ABCC2 in drug-sensitive leukemia cell line K562 and drug-resistant cell line K562/A02, and explore the regulatory relationship between miR-451 and the expressions of ABCB1 and ABCC2 , and the mechanism of miR-451 involved in drug resistance in leukemia.@*METHODS@#CCK-8 assay was used to detect the drug resistance of K562/A02 and K562 cells. Quantitative Real-time PCR (qRT-PCR) was used to verify the differential expressions of miR-451 in K562 and K562/A02 cells. MiR-451 mimic and negative control (miR-NC), miR-451 inhibitor and negative control (miR-inNC) were transfected into K562 and K562/A02 cells respectively, then qRT-PCR and Western blot were used to detect the expression levels of mRNA and protein of ABCB1 and ABCC2 in K562 and K562/A02 cells and the transfected groups.@*RESULTS@#The drug resistance of K562/A02 cells to adriamycin was 177 times higher than that of its parent cell line K562. Compared with K562 cells, the expression of miR-451 in K562/A02 cells was significantly higher (P <0.001), and the mRNA and protein expression levels of ABCB1 and ABCC2 in K562/A02 cells were significantly higher than those in K562 cells (P <0.001). After transfected with miR-451 inhibitor, the expression of miR-451 was significantly down-regulated in K562/A02 cells (P <0.001), the sensitivity to chemotherapy drugs was significantly enhanced (P <0.05), and the mRNA and protein expressions of ABCB1 and ABCC2 were significantly decreased (P <0.01). After transfected with miR-451 mimic, the expression of miR-451 was significantly upregulated in K562 cells (P <0.001), and the mRNA and protein expressions of ABCB1 and ABCC2 were significantly increased (P <0.01).@*CONCLUSION@#There are significant differences in the expressions of miR-451, ABCB1 and ABCC2 between the drug-sensitive leukemia cell line K562 and drug-resistant cell line K562/A02, which suggests that miR-451 may affect the drug resistance of leukemia cells by regulating the expression of ABCB1 and ABCC2.

Periplaneta americana extract CⅡ-3 induces senescence of leukemia K562 cells via SIRT1/mTOR signaling pathway / 中国中药杂志

This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.

The Relationship between MicroRNA Expression Profiling in Imatinib-Resistant Cell Line K562/G and Potential Mechanism through FOXO3/Bcl-6 Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the drug resistant related FOXO3/Bcl-6 signaling pathway in K562/G cell line and its related microRNA(miRNA) mechanisms.@*METHODS@#The drug resistance potency of imatinib on K562/G was detected by MTT assay. The expression of FOXO3 and Bcl-6 proteins in K562 and K562/G cells was detected by Western blot. Real-time PCR (RT-PCR) was used to detect the expression of FOXO3 and Bcl-6 mRNA. The miRNA expression profiling in K562 and K562/G cells was analyzed by microarray technique, and the miRNA targeted to FOXO/Bcl-6 signaling pathway was identified.@*RESULTS@#The expression of FOXO3 and Bcl-6 protein was significantly increased in K562/G cells as compared with that in K562 cells (P<0.01), the expression level of Bcl-6 mRNA showed no increase in K562/G cells. However, FOXO3 mRNA was up-regulated in K562/G cells (P<0.05). MiRNA microarray results showed that 109 miRNAs were expressed differentially in K562 and K562/G cells. The expression of 81 miRNAs were up-regulated while 28 miRNAs were down-regulated. Through reverse prediction by bioinformatics, miR-6718-5p, miR-5195-5p, miR-4711-3p, miR-4763-5p, miR-4664-5p and miR-3176 were related to FOXO/Bcl-6 signaling pathway.@*CONCLUSION@#The FOXO3/Bcl-6 signaling pathway contributes to imatinib resistance in K562/G cell line, and the miRNA expression profiles showed significant differences between K562/G and K562 cells.

Citotoxicidad in vitro del polipéptido antimicrobiano nisina sobre células tumorales sanguíneas / In vitro cytotoxicity of the antimicrobial polypeptide nisin on blood tumor cells

RESUMEN: Las bacteriocinas son péptidos antimicrobianos de síntesis ribosomal secretadas por bacterias. Dentro de estas destaca nisina que posee potenciales usos en terapias antibióticas, como biopreservante de alimentos y probióticos. También se ha descrito que nisina posee citotoxicidad sobre líneas celulares neoplásicas, pero existe poca información de su efecto sobre células tumorales sanguíneas. Debido al potencial uso que presenta nisina, es relevante determinar la toxicidad que presenta sobre líneas celulares tumorales del tipo sanguíneo. Para esto, se realizaron ensayos de actividad hemolítica sobre eritrocitos humanos y de toxicidad sobre células mononucleares de sangre periférica humanas, determinándose que nisina no posee efecto citotóxico sobre este tipo de células normales humanas sanguíneas. Se realizaron también, ensayos de citotoxicidad con líneas celulares tumorales (K562 y U937), con el fin de determinar dosis, tiempo de exposición y selectividad en el efecto tóxico de nisina sobre las células tumorales humanas. Estos ensayos muestran que nisina presenta actividad citotóxica sobre líneas celulares K562 y U937 a las 72 h de exposición, a una concentración de 40 µg/mL, que corresponde a 100 veces la concentración mínima inhibitoria (MIC) usada para su acción sobre bacterias. Al comparar el efecto de nisina sobre células mononucleares de sangre periférica humanas con las líneas tumorales linfoides y mieloides (K562 y U937 respectivamente), se observa un efecto selectivo de nisina sobre las células tumorales sanguíneas.

SUMMARY: Bacteriocins are antimicrobial peptides of ribosomal synthesis secreted by bacteria. Among these, nisin stands out, which has potential uses in antibiotic therapies, as a food bio preservative and probiotics. Nisin has also been reported to have cytotoxicity on neoplastic cell lines, but there is little information on its effect on blood tumor cells. Due to the potential use that nisin presents, it is relevant to determine the toxicity it presents on tumor cell lines of the blood type. For this, hemolytic activity tests were carried out on human erythrocytes and toxicity on human peripheral blood mononuclear cells, determining that nisin does not have a toxic effect on this type of normal human blood cells. Cytotoxicity tests were also carried out with tumor cell lines (K562 and U937), to determine dose, exposure time and selectivity in the toxic effect of nisin on human tumor cells. These tests show that nisin shows cytotoxic activity on K562 and U937 cell lines at 72 h of exposure, at a concentration of 40 µg / mL, which corresponds to 100 times the minimum inhibitory concentration (MIC) used for its action on bacteria. When comparing the effect of nisin on human peripheral blood mononuclear cells with lymphoid and myeloid tumor lines (K562 and U937 respectively), a selective effect of nisin on blood tumor cells is observed.

Effects of Bacillus firmus e volumine ex muris cellulae 6x on cytolytic activity of natural killer cells in vitro

Natural killer (NK) cells are among the first in defense of the innate immune system by eliminating a variety of abnormal or stressed cells such as cancer cells or virus-infected cells. Individuals who exhibit low cytolytic NK cell activity are believed to be at higher risk of viral infection, tumorigenesis, and various other diseases of the immune system. Therefore, restoration of impaired NK cell function might be an essential step in immunostimulatory therapy of immunocompromised patients. Bacillus firmus is a non-pathogenic gram-positive bacterium of the environment, which possesses various immunomodulatory properties in vitro and in vivo. This retrospective study reports on the effect of B. firmus on the activity of NK cells in vitro. Basal cytolytic NK cell activity against tumor cells among peripheral blood mononuclear cells (PBMCs) of routine patients was determined in a standardized NK cell cytotoxicity assay. The impact of cultivation of PBMCs with B. firmus preparation Bacillus firmus e volumine ex muris cellulae (Bacillus firmus (evc)) 6x on tumor cell killing by NK cells was monitored in relation to basal NK cell activity. This study showed that stimulation of PBMCs with Bacillus firmus (evc) 6x in vitro led to a significant increase in NK cell function. Substantial improvement in cytolytic NK cell activity (more than 1.3-fold of basal activity) was much more pronounced for patients with compromised NK cell function. Due to its immunostimulatory mode of action, Bacillus firmus (evc) may be of particular importance in therapy of patients with NK cell deficiency.

Germacranolide sesquiterpenes from Carpesium cernuum and their anti-leukemia activity / 中国天然药物

In this study, three new germacranolide sesquiterpenes (1-3), together with six related known analogues (4-9) were isolated from the whole plant of Carpesium cernuum. Their structures were established by a combination of extensive NMR spectroscopic analysis, HR-ESIMS data, and ECD calculations. The anti-leukemia activities of all compounds towards three cell lines (HEL, KG-1a, and K562) were evaluated in vitro. Compounds 1-3 exhibited moderate cytotoxicity with IC

Effect of Petroleum Ether Extract of Rhizoma Amorphophalli on Biological Characteristics of K562 Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the role of petroleum ether extract of Rhizoma Amorphophalli (SLG) in inhibiting proliferation and promoting apoptosis and differentiation of leukemia K562 cells.@*METHODS@#K562 cells were processed by SLG and PD98059 which was the ERK signaling pathway blocker. Then cell vitality was tested by MTT. Cell apoptosis rate and positive percentage of antigen expression related with differentiation were detected by flow cytometry. The protein expression levels of ERK1/2 and pERK1/2 were detected by Western blot.@*RESULTS@#The proliferation activity of K562 was reduced by 50, 100, 200 mg/L SLG in a concentration dependent manner (r=0.9997). The apoptosis rate and positive expression rate of CD11b, CD14 and CD42b which were related with differentiation were raised by SLG, as well as the expression of pERK1/2, while PD98059 could reverse the promoting effect of SLG on apoptosis and differentiation partially.@*CONCLUSION@#SLG can inhibit the proliferation and promote apoptosis and differentiation of K562 cells through ERK signaling pathway.

RNA Binding Protein RBM47 Inhibits the K562 Cell Proliferation by Regulating HMGA2 mRNA Expression / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulatory effects of RBM47 on HMGA2 and the function of RBM47 in human chronic myeloid leukemia cell K562.@*METHODS@#K562 cells were transduction by the overexpressed and knockdown RBM47 lentiviral vector. CCK-8 assay was used to detect the effect of RBM47 on the proliferation of K562 cells. Flow cytometry assay was used to detect the effect of RBM47 on the cell cycle progression of K562 cells. RNA immunoprecipitation assay was used to detect the association between RBM47 and HMGA2 mRNA. RT-qPCR was used to detect the effects of RBM47 on the stability of HMGA2 mRNA. Western blot was used to evaluate the effect of RBM47 on HMGA2 protein expression.@*RESULTS@#The overexpressed RBM47 could inhibit the proliferation and cell cycle progression of K562 cells. However, the inhibitation of RBM47 could improve the proliferation and cell cycle progression of K562 cells. RBM47 combined with HMGA2 mRNA could promote the degradation of HMGA2 mRNA. Thus, the overexpressed RBM47 could decrease the expression of HMGA2 protein in K562 cells.@*CONCLUSION@#RNA binding protein RBM47 can inhibit the proliferation of K562 cells by regulating HMGA2 expression.

Effect of DR4 Demethylation to the Proliferation and Apoptosis of Myeloid Leukemia K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of tumor necrosis factor death receptor (DR) 4 demethylation to the proliferation and apoptosis of myeloid leukemia K562 cells.@*METHODS@#The logarithmic phase of K562 cells were treated by desitabine (DCA) at 0, 0.8, 1.6 and 3.2 μmol/L, and the cells were divided into control group, DCA low dose group, DCA medium dose group and DCA high dose group respectively. The cells in control group were treated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 0.5 μg/ml for 24 h, and the cells were divided into TRAIL group. The cells in DCA high dose group were treated by TRAIL 0.5 μg/ml for 24 h, and were divided into DCA high dose + TRAIL group. Methylation-specific polymerase chain reaction (MS-PCR) was used to measure the methylation status of the DR4 gene promoter in the control group and DCA low, medium and high dose groups. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to determine the relative expression of DR4 mRNA and protein in the control group and DCA low, medium and high dose groups. Dime- thylthiazole (MTT) method was used to determine the inhibition rate of cell proliferation of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. Flow cytometry was used to determine the apoptotic rate of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group.@*RESULTS@#The cells in the control group were methylation-positive, the brightness of the methylation bands of the cells in the DCA low, medium, and high dose groups was gradually decreased to disappear, and the DCA high dose group showed negative for methylation. The relative expression of DR4 mRNA and protein in the control group, DCA low, medium and high dose groups was increased sequentially (r=0.624, 0.704). The inhibition rate of cell proliferation of the cells in the control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group was increased sequentially (r=0.653, 0.754, 0.709, 0.725) at 24, 48 and 72 h.@*CONCLUSION@#DCA can reverse the methylation level of DR4 gene promoter in ML K562 cells and up-regulate the expression of DR4, which may enhance the proliferation inhibition and apoptosis promotion effects of TRAIL on K562 cells.

Primary Study on Chronic Myeloid Leukemia in NCG Mice from Qinba Mushroom / 中国实验血液学杂志

OBJECTIVE@#The present study was to evaluate the anti-tumor effects of acidic RNA protein complex (FA-2-b-β) extracted from the wild edible Qinba mushroom in inducing of apoptosis and immunoregulation of tumor cell.@*METHODS@#Cell proliferation inducing rate of FA-2-b-β to K562 cell was measured using CCK-8. Apoptosis rate was detected by using flow cytometry. Chronic myeloid leukemia model was developed by tail vein injection/subcutaneous inoculation of K562 cells in NCG mice. The tumor burden of mice was observed. The general condition of the mice was monitored twice daily. The peripherivcal full blood counts of mice was tested daily. RT-qPCR and Western blot was FA-2-b-β performed to determine involvement of apoptotic-related gene and protenin, Immunofluorescence and immunohistochemistry was used to detected the expression of CD3, CD4 and CD8.@*RESULTS@#The proliferation and apoptosis of K562 cell could be inhibitied and induced by FA-2-b-β, there was 100% successful in the tumor formation in vivo, after treated by drug for 21 days there were significantly increased peripheral leucocytes, but decreased hemoglobin of mice treated by FA-2-b-β as compared with those in control group. The CD3, CD4 and CD8 showed positive in mice, and the propotation was imbalance, but it showed reserved after treated by FA-2-b-β.@*CONCLUSION@#FA-2-b-β is strong anti-leukemia effect in vitro and in vivo, suggesting the traditional Chinese medicine maybe contribute to the anti-cancer and immunoregulation research.

Construction and Identification of Leukemia Cell Line Stably Expressing CD123 and CLL-1 / 中国实验血液学杂志

OBJECTIVE@#To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1.@*METHODS@#The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry.@*RESULTS@#The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×10@*CONCLUSION@#Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.

Costunolide Induces Apoptosis of K562/ADR Cells through PI3K/AKT Pathway / 中国实验血液学杂志

OBJECTIVE@#To explore the effects of costunolide on the proliferation and apoptosis of human chronic myeloid leukemia drug resisitant cell line K562/ADR and its mechanism.@*METHODS@#The proliferation of the cells was assessed by CCK-8 assay, while flow cytometry was used to detect the apoptosis of the cells. The related-proteins were detected by using Western blot.@*RESULTS@#The proliferation of K526/ADR cells was significantly inhibited by costunolide in a dose-dependent manner (r=0.9886) after treated by 0.01, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 μmol/L costunolide for 72 h, and IC@*CONCLUSION@#Costunolide could inhibit the proliferation and apoptosis of K562/ADR cells through regulation of PI3K/AKT pathway.

Silencing DNMT1 Attenuates the Effect of WIF-1 Gene Promoter Methylation on the Biological Behavior of Chronic Myeloid Leukemia K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of silencing DNA methyltransferase 1(DNMT1) to the methylation of the promoter of the tumor suppressor gene wnt-1 (WIF-1) in human chronic myeloid leukemia (CML) cells.@*METHODS@#DNMT1 siRNAi plasmid was constructed and DNMT1 siRNAi was transfected into CML K562 cells. RT-PCR and Western blot were used to detect the expression of DNMT1 gene and related protein, and methylation PCR was used to detect WIF-1 gene promoter methylation level. The trypan blue exclusion and MTT assay were used to detect the cell proliferation, flow cytometry were used to detect the cell apoptosis rate, colony formation assay was used to detect cell colony formation ability. Expression of Wnt/β- catenin and its downstream signaling pathway proteins were detected by Western blot after DNMT1 gene was silenced.@*RESULTS@#The expression level of DNMT1 mRNA and its related protein in the experimental group were significantly lower than those in the control group and negative control group (P<0.05). After 72 hours of successful transfection, the WIF-1 gene in the control group and negative control group were completely methylated, while in the experimental group, the methylation level significantly decreased. The results of MSP showed that the PCR product amplified by the unmethylated WIF-1 primer in the experimental group increased significantly,while by the methylated WIF-1 primer decreased significantly. After 48 h of transfection, the OD value, viable cell number and colony formation of the cells in experimental group were significantly lower than those in the negative control group and the control group (P<0.05). The apoptosis rate of the cells in experimental group was significantly higher than those in the negative control group and control group (P<0.05). The expression levels of β- actin, myc, cyclin D1 and TCF-1 in K562 cells in the experimental group were significantly lower than those in the negative control group and control group (P<0.05).@*CONCLUSION@#Silencing DNMT1 gene can inhibit the proliferation and promote the apoptosis of K562 cells. The mechanism may be related to reverse the hypermethylation level of the WIF-1 gene promoter, thereby inhibit the activity of the Wnt/β- catenin signaling pathway.

Transcriptome Analysis of Chronic Myelogenous Leukemia Cell Line with Imatinib Resistance / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulation of chronic myelogenous leukemia （CML） imatinib resistant genes, in order to improve the therapeutic effect of CML imatinib resistant patients.@*METHODS@#The human CML cell line K562 and imatinib-resistant K562 cells (K562/G01) were collected, and transcriptome of the cells were achieved by RNA-seq. The sequencing data were analyzed by using standard procedures.@*RESULTS@#Compared with K562 cells, 464 genes were significantly changed in K562/G01 cells, including 163 up-regulated and 301 down-regulated genes. The GO function annotation analysis and KEGG pathway analysis results showed that the differentially expressed genes were mainly involved in biological processes such as oxidative phosphorylation, localization to protein organelle, ribonucleoprotein complex biogenesis and so on. Gene Set Enrichment Analysis (GSEA) plots showed that 5 gene-sets were up-regulated in K562/G01 significantly, including the pathway of TGF-beta, mTOR and CML.@*CONCLUSION@#CML imatinib resistance is associated with oxidative phosphorylation, during which the pathway of TGF-beta and mTOR are significantly up-regulated.