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.

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.

Preclinical study of suicide gene as a safety switch to control CAR-T cell cytotoxicity / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate whether AP1903, a small-molecule chemical inducer, can terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vivo and in vitro. Methods: CD19CAR-T cells over-expressing iCasp9 (iCasp9-CD19CAR-T) were constructed and co-incubated with AP1903. Then, the cell phenotype and apoptosis were detected by Flow cytometry, and the iCasp9/CID suicide gene system was verified on K562 and T cells, respectively. The cytotoxicity of iCasp9-CD19CAR-T cells was detected in vivo (survival rate of NCG mice bearing Raji cell transplanted xenograft) and in vitro (cell killing function was detected by Flow cytometry) under the administration of AP1903. Results: Compared with CD19CAR-T cells, iCasp9-CD19CAR-T cells showed in significant difference in proliferation, phenotype and cytotoxicity both in vitro and in vivo (all P>0.05). At 2 h after AP1903 administration, the apoptosis rates of K562 and T cells co-expressing iCasp9 and CD19CAR were (33.8±0.9)% and (27.95±0.35)%, respectively; and at 24 h after AP1903 administration, the apoptosis rates reached 100% in both cell lines. The in vitro cytotoxicity of iCasp9-CD19CAR-T cells induced by AP1903 was significantly lower than that without AP1903 treatment (P<0.01); the 60-day survival rate of mice bearing Raji cell transplanted tumor treated with AP1903-induced iCasp9-CD19CAR-T cells was also significantly lower than those treated with iCasp9-CD19CAR-T cells alone (P<0.01). Conclusion: AP1903 can effectively terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vitro and in vivo.

Knock-down of cytokine induced apoptosis inhibitor 1 gene sensitized leukemia K562 cells to imatinib / 中国肿瘤生物治疗杂志

@#[ [Abstract] ] Objective: To investigate the biological effects and the related mechanisms of cytokine induced apoptosis inhibitor 1 (CIAPIN1) on the sensitivity of K562 chronic myeloid leukemia cells to imatinib. Methods: Specific short hairpin RNA(shRNA) interference vectors targeting CIAPIN1 (CIAPIN1-shRNA) were constructed. Interference efficiency of interference group (K562 cells transfected with CIAPIN1-shRNA) and control group (K562 cells transfected with scramble-shRNA) was evaluated using Real-time PCR, Western blotting and immunofluorescence staining. The interference group and control group were treated by 2 μmol/L imatinib. Cell viability was detected using MTT assay. Colony formation ability was detected using cell colony forming experiment. Cell cycle and apoptosis was detected using Flow cytometry and Western blotting. Results: CIAPIN1 expression was decreased effectively by specific shRNA targeting CIAPIN1. The CIAPIN1 mRNA content in CIAPIN1-shRNA group accounted (29.74±4.03)% of scramble-shRNA group, while the CIAPIN1 protein content in CIAPIN1-shRNA group accounted (21.57±2.18)% of scramble-shRNA group. CIAPIN1 knock-down significantly enhanced the inhibitory activity of imatinib on proliferation and colony forming ability of K562 cells. The colony number and radius of the CIAPIN1-shRNA+imatinib group was (15.60±1.03) and (2.63±0.55) μm, which were all less than those of the scramble-shRNA+imatinib group. The knock down also increased the activity of imatinib to block the cell cycle at G1 phase and to promot apoptosis of cells. The cell ratio at G1 phase of the CIAPIN1-shRNA+imatinib group was obviously increased while the ratio at S phase was obviously decreased compared with those of scramble-shRNA+imatinib group. Hoechst33258 staining and flow cytometry showed that the proportion of apoptotic K562 cells in the CIAPIN1-shRNA+imatinib group increased. The results of Western blotting showed that CIAPIN1 knock-down not only up-regulated the expressions of apoptosis related proteins (p21, Bid and Bim), but also repressed expressions of cell cycle related proteins (Cyclin D1, Bcl-xl, Bcl-2 and Mcl-1), which had synergistic effects with imatinib. Conclusion: CIAPIN1 knock-down significantly sensitized K562 cells to imatinib treatment, and the mechanism might be related with cell cycle arrest and expression of apoptosis-associated proteins.

Over-expression of KLF4 inhibits the proliferation and migration of K562 cell line / 基础医学与临床

Objective To study the effect of over-expression of KLF 4 on the proliferation and migration ability of K562 cells.Methods The K562 cells with KLF4 over expression ( K562-KLF4 cells) were used as experimental group, and paralleled with the vector control ( K562-C1 cells) and blank K562 cell control.Real-time PCR was performed to detect the relative expression quantity of KLF4 mRNA of each group cells respectively;Western blot was performed to detect the level of KLF 4 protein of each group cells respectively .The cell proliferation was tested by MTS assay.The migration ability of K562 cells was detected by Transwell .Results Compared with K562-C1 cells and blank K562 cells, the relative expression of KLF4 mRNA of K562-KLF4 cells was significantly increased (P<0.05).The level of KLF4 protein of K562-KLF4 cells was also significantly increased, by 77.78%(P<0.05).The proliferation ability and migration ability of the K 562 cells with over-expressing KLF4 were inhibited sig-nificantly (P<0.05).Conclusions Over-expression of KLF4 could inhibit the proliferation and migration ability of K562 cells.

The role of aptamer-siRNA nucleic acid compound in promoting the apoptosis of K562 cells / 西安交通大学学报(医学版)

Objective To observe the effect of aptamer-siRNA nucleic acid compound on the apoptosis of K562 cells in human chronic myelogenous leukemia (CML)and explore its acting mechanisms.Methods K562 cells were transfected with different concentrations of aptamer-siRNA solution.The effects of aptamer-siRNA on the proliferation and apoptosis of K562 cells were detected by MTT method and AnnexinV/PI double staining method,respectively.The effects of aptamer-siRNA on the expressions of bcl-2,Bax and casepase-3 at protein and mRNA levels in K562 cells were detected by Western blot and RT-PCR method,respectively.Results Compared with the control group,the proliferation of K562 cells was significantly inhibited,early apoptosis rate of K562 cells increased significantly,the expression levels of bcl-2 protein and mRNA were significantly decreased,while the expression levels of Bax and caspase-3 protein and mRNA were significantly increased after transfection with aptamer-siRNA (P <0.05).Aptamer-siRNA nucleic acid complex at the concentration of 50 -250 μmol/L)had a significant dose-effect relationship on bcl-2,Bax and caspase-3 mRNA.Conclusion Aptamer-siRNA nucleic acid compound can promote the decreased number of bcl-2 gene and the growth of Bax and caspase-3 genes,thus promoting the apoptosis of K562 cells.

Anti-apoptosis of hepatocyte growth factor on CML K562 cells / 中国药学杂志

OBJECTIVE: To investigate the protection of hepatocyte growth factor (HGF) on CML cell line K562 from apoptosis induced by etoposide (VP-16) and its molecular mechanism. METHODS: Quantitative and qualitative analyses on cell morphological change of apoptosis were performed through acridine orange (AO) staining and HE staining, and fluorescent flow cytometry.The test analyzes membrane on the surface of the PS evagination and integrity of cell membrane surface and mitochondrial membrane potential changes were performed through Annexin V-FITC/PI double dyeing and JC-1 cell dyeing tests, and apoptotic factors such as Bcl-2, Bax, Caspase-3 and Caspase-9 were measured by SYBR Green (Takara) qRT-PCR. RESULTS: The HE and AO staining revealed that apoptotic rates in HGF+VP-16 groups were significantly lower than those in VP-16 groups (P<0.05, P<0.05), HGF can inhibit the apoptosis of cells induced by VP-16; FCM (Annexin V-FITC/ PI and JC-1) tests showed that cells apoptotic rates in HGF+VP-16 groups were significantly lower than those in VP-16 groups (P<0.05, P<0.001), indicating that HGF has the anti-apoptosis function. Apoptosis related gene mRNA expression tests found that the Bcl-2 mRNA expression in HGF+VP group was obviously higher than that in the VP-16 group (P<0.001), while Bax mRNA, Caspase-3 mRNA, and Caspase-9 mRNA expressions were significantly lower than those in the VP-16 group (P<0.05, P<0.001, P<0.001), suggesting that HGF possesses antiapoptotic effect through inhibiting apoptosis gene expression and promoting the antiapoptotic gene expression simultaneously. CONCLUSION: HGF can significantly protect K562 cells from apoptosis induced by VP-16 through the HGF/c-Met way to regulate PI3K/AKT pathway.

The role of aptamer-siRNA nucleic acid compound in promoting the apoptosis of K562 cells / 西安交通大学学报(医学版)

Objective To observe the effect of aptamer-siRNA nucleic acid compound on the apoptosis of K562 cells in human chronic myelogenous leukemia (CML)and explore its acting mechanisms.Methods K562 cells were transfected with different concentrations of aptamer-siRNA solution.The effects of aptamer-siRNA on the proliferation and apoptosis of K562 cells were detected by MTT method and AnnexinV/PI double staining method,respectively.The effects of aptamer-siRNA on the expressions of bcl-2,Bax and casepase-3 at protein and mRNA levels in K562 cells were detected by Western blot and RT-PCR method,respectively.Results Compared with the control group,the proliferation of K562 cells was significantly inhibited,early apoptosis rate of K562 cells increased significantly,the expression levels of bcl-2 protein and mRNA were significantly decreased,while the expression levels of Bax and caspase-3 protein and mRNA were significantly increased after transfection with aptamer-siRNA (P <0.05).Aptamer-siRNA nucleic acid complex at the concentration of 50 -250 μmol/L)had a significant dose-effect relationship on bcl-2,Bax and caspase-3 mRNA.Conclusion Aptamer-siRNA nucleic acid compound can promote the decreased number of bcl-2 gene and the growth of Bax and caspase-3 genes,thus promoting the apoptosis of K562 cells.

Assessment of the Cytotoxic and Apoptotic Effects of Chaetominine in a Human Leukemia Cell Line

Chaetominine is a quinazoline alkaloid originating from the endophytic fungus Aspergillus fumigatus CY018. In this study, we showed evidence that chaetominine has cytotoxic and apoptotic effects on human leukemia K562 cells and investigated the pathway involved in chaetominine-induced apoptosis in detail. Chaetominine inhibited K562 cell growth, with an IC50 value of 35 nM, but showed little inhibitory effect on the growth of human peripheral blood mononuclear cells. The high apoptosis rates, morphological apoptotic features, and DNA fragmentation caused by chaetominine indicated that the cytotoxicity was partially caused by its pro-apoptotic effect. Under chaetominine treatment, the Bax/Bcl-2 ratio was upregulated (from 0.3 to 8), which was followed by a decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, and stimulation of Apaf-1. Furthermore, activation of caspase-9 and caspase-3, which are the main executers of the apoptotic process, was observed. These results demonstrated that chaetominine induced cell apoptosis via the mitochondrial pathway. Chaetominine inhibited K562 cell growth and induced apoptotic cell death through the intrinsic pathway, which suggests that chaetominine might be a promising therapeutic for leukemia.

Effect of genetic modified K562 cells on NK cell markers and function / 中国免疫学杂志

Objective:To study the influence of different culture conditions on charcic and inhibition activity of nature killer cells ( NK) ,whether to join the modified K562 cells with IL-6 cytokine.Methods:According to the 5′end of the human IL-6 cDNA sequence,PCR primers designed to amplificate,express and transfect K562 cells cDNA library as a template for DNA.Genetic modified K562 cells as stimulating cells were prepared by expressing IL-6.To extract peripheral blood mononuclear cells( PBMC) from human peripheral blood.PBMC were explanted by genetic modified K562 stimulated.The expansion was initiated by CO-culture of PBMC and irradiate genetic modified K562 cell.The number of NK cell increased by directed induced generation of genetic modified K562 cell.Immunophenotypic analysis of NK cell surface markers was performed by flow cytometry (FCM).51Cr release assay was employed to measure the specific lysis skilling of NK cell target K562 cells.Results:We have constracted genetic modified K562 cells by genetic engineering.As stimulated cell added into the PBMC,an average of 760 ±18 fold expansion of CD56+CD16+CD3-cells was observed after 3 weeks of co-culture system.The NK cells population could proliferated more 91%±2% after expansion comparing with 6%± 0.4%in PBMC before expansion by FCM.The cytotoxical activity of NK cells which was induced by genetic modified K562 cell was the strongest than induced by IL-6 cytokine alone.The expanded NK cells lysed 92%±2% of K562 targets in a 5∶1 effector to target ratio.In this case,the NK cells induced by genetic modified K562 cells against tumor cells was more lethal.Conclusion:PBMC based in vitro expansion of natural killer cells was set up by genetic modified K562 cells.The cytotoxicity of NK cells was the strongest induced by genetic modified K562 cell treated.These results had important guiding significance for the the NK large number of amplification and used in clinical.

Interleukin-15 enhanced the cytotoxicity of Vδ2 γδ T cells against K562 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of interleukin -15 ( IL-15 ) on the cytotoxicity of Vδ2 γδ(Vδ2) T cells against K562 cells.Methods PBMCs were separated and cultured with zoledronate and interleukin-2 ( IL-2) to induce the proliferation of Vδ2 T cells.The obtained Vδ2 T cells were in vitro stimulated with IL-15.Flow cytometry analysis was performed to evaluate the changes of phenotypes of Vδ2 T cells and their cytotoxicity against K562 cells.Results Zoledronate effectively induced the massive prolifer -ation of V2δT cells.The Vδ2 T cells were highly activated and the expression of CD 107a and IFN-γby Vδ2 T cells were upregulated upon IL-15 stimulation.The cytotoxicity of Vδ2 T cells against K562 cells was greatly enhanced by the treatment with IL-15.Conclusion IL-15 could activate the Vδ2 T cells and en-hance their cytotoxicity against K562 cells.

Establishment of the CD19-K562 cell line over-expressing stably CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse / 白血病·淋巴瘤

Objective To construct a MigR1-CD19 recombinant vector which contains CD19 gene, and to establish a CD19-K562 cell line over-expressing stably CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse.Methods The CD19 gene was inserted into the retroviral vector (MigR1) through recombinant DNA technology after transfection into Plat-A packaging cells, and viral supernatant was collected to transduce K562 cell line repeatedly to obtain stable transduction CD19-K562 cell line.Flow cytometry was used to determine the transduction efficiency and RT-PCR was used to confirmed CD19 gene expression.Cell proliferation and apoptosis were detected by cell count and Annexin V/PI, respectively.Then the subcutaneous xenograft subtype of CD19-K562-a cell line was constructed through subcutaneous inoculation and was cultured in vitro and in vivo.Then its subcutaneous xenograft model in NOD-SCID mouse was established.The characteristics of CD19-K562-a cells were detected by RT-PCR, Wright staining and immunohistochemistry.Results MigR1-CD19 recombinant vector was successfully constructed, and the CD19 positive efficiency of K562 cell line was (99.80±0.17) ％ through retrovirus centrifugation transduction.The transduction and passage had no effects on proliferation and apoptosis of CD19-K562 cells.The CD19-K562-a cell line was constructed after CD19-K562 cells were injected subcutaneously and were passaged in vitro and in vivo.The CD19 positive efficiency of the xenograft subtype CD19-K562-a cell line was (99.78± 0.04) ％.CD19-K562-a and CD19-K562 cells were in an undifferentiated state.NOD-SCID subcutaneous xenografts were established through subcutaneous inoculation of CD19-K562-a cells.CD19 in the CD19-K562-a subcutaneous xenografts was positive, while it was negative in its counterparts K562 cells.Conclusion The CD19-K562 cell line over-expressing CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse are successfully established.

Construction of eukaryotic expression vectors of FGFR3 gene and their expressions in human leukemia K562 cell line / 吉林大学学报(医学版)

Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

5-Aza-2 ’-deoxycytidine and 4-phenylbutyric acid exert a cooperative effect on the expression of miR-196 b in chronic myeloid leukemia cells / 解剖学报

Objective To study if 5-Aza-2’-deoxycytidine along or together with 4-phenylbutyric acid could affect miR-196b expression levels in chronic myeloid leukemia cells .Methods K562 cells were treated with DNA methylation inhibitor 5-Aza-2’-deoxycytidine, histone deacetylase inhibitors 4-phenylbutyric acid separately and the combined treatment with both of them, then expression levels of miR-196b were detected using Real-time PCR.Results The half inhibition concentration of 4-phenylbutyric acid was 1.58mmol/L.Comparing with the expression level of miR-196b in normal human bone marrow cells, the expression levels of miR-196b were significantly lower in Aza group , PBA group and negative control cells and nearly consistent among three groups , and as high as normal cells in combined treatment group . Conclusion The expression level of miR-196b in K562 cells could not return to normal treated with 5-Aza-2 ’-deoxycytidine or 4-phenylbutyric acid separately , while could restore normal when treated with both agents , indicating that miR-196b expression level in K562 cells is related with both DNA methylation and histone acetylation .

Enhanced sensitivty of leukemic K562 cell to ursolic acid by miR-203 / 中国药理学通报

Aim To study the influences of miR-203 on ursolic acid (UA ) sensitivity in leukemic K562 cell.Methods In the experimental system,eukaryot-ic expression vector of hsa-miR-203 (PmiR-203 )was transfected into K562 cells using LipofectamineTM 2000.K562 cells were incubated with different con-centration of UA alone or in combination with PmiR-203 .The cell proliferation was analyzed by MTT as-say.The cell apoptosis was measured by double stai-ning with Annexin V and Propidium Iodide.The ex-pression of Bcr/abl protein was detected by Western Blot.Results The miR-203 promoted the sensitivity of UA by up to 1 .55-fold and the IC50 was reduced from 44.3μmol · L-1 to 30.7 μmol · L-1 .The a-mount of apoptotic cells was increased in UA combined with PmiR-203 group (P<0.05).PmiR-203 downreg-ulated the expression of Bcr/abl protein in K562 cells. Conclusion Our results support a substantial role of miR-203 that enhances UA sensitivity in K562 cell and the mechanism appears to be related to the dounregula-tion of Bcr/abl by miR-203 .

Effects of ginsenoside Rg3-induced P27 expression on proliferation of human erythroleukemia K562 cells / 实用医学杂志

Objective To explore the effects of ginsenoside Rg3 on the expression of P27 in human erythrol-eukemia cell line K562 and cell proliferation. Methods Human erythroleukemia cell line K562 cells were cultured to exponential phase, then K562 cells were treated with different concentrations of Rg3 (6.25, 12.5, 25, 50, and 100 μg/mL) as Rg3 group, and cells treated without Rg3 (0 μg/mL) were take as control group. After 3 days, K562 cells were observed by Wright-Giemsa staining with microscopy , the proliferation of K562 cells were examined by tetrazolium salt (MTT) assay, and the expression of P27 mRNA were detected by fluorescent quantitative RT-PCR assay. Results MTT assay showed that after treatment with Rg3,the inhibition rate (IR) of proliferation of cells in Rg3 groups were increased gradually , and the differences were significant compared with the control group (P < 0.05). The results of fluorescent quantitative RT-PCR showed the levels of P27 mRNA expression in 25,50 and 100 μg/mL Rg3 groups were significant higher than that of control group (P < 0.05). Conclusion The ginsenoside Rg3 can inhibit the proliferation of K562 cells by inducing the expression of P27.

Lymphokine activated killer cells from peripheral blood mononuclear cells of endometriosis of patients improve cytotoxicity to endometriosis cell culture.

Background: To assess the increased cellular immunity of Peripheral Blood Mononuclear Cells (PBMC) derived LAK cells from endometriosis patients towards endometriosis cell cultures after stimulation with IL-2. Methods: This study is a quasi-experimental study of pre and post treatment using controls. Phenotype evaluation of CD3+CD4+, CD3+CD8+ and CD56+ effector cells of PBMC from endometriosis patients and controls was performed. Cytotoxicity test of PBMC from endometriosis patients and control towards Daudi, K562 cell line and endometriosis cell cultures using 51Chromium release assay was also carried out. Results: Phenotype evaluation of PBMC from endometriosis patients (n = 10) and controls (n = 6) were done prior to and after IL-2 stimulation. Before IL-2 stimulation, CD3+CD4+, CD56+ from endometriosis group (n = 10) tend to be lower than control (n=6) whereas CD3+CD8+ were higher in endometriosis group than controls. After IL-2 stimulation, CD3+ CD8+, CD56+ of PBMC from endometriosis group were significantly increased (p < 0.05). Cytotoxicity test revealed a significant increase (p < 0.05) in both PBMC’s effector cells from endometriosis and control group towards target cells, Daudi, and K562 cell lines after IL-2 stimulation. PBMC’s effector cells cytotoxicity from both endometriosis and control towards target endometriosis cell cultures were also elevated after IL-2 stimulation. Conclusion: LAK cells derived IL-2 stimulated PBMC from endometriosis patients increased cellular immunity towards endometriosis cell cultures.

Antitumor effect of enhancer binding protein C/EBPα on the leukemic BALB/c nude mice / 白血病·淋巴瘤

Objective To explore the role of tumor inhibition of enhancer binding protein C/EBPα in the leukemic mice. Methods BALB/c nude mice were randomly divided into three groups. Three kinds of cells including pEGFP-C/EBPα-K562 cells, pEGFP-K562 cells and K562 cells as the control were injected into mice separately through the subcutaneous and tail vein, and subcutaneous tumors and leukemic models were formed. The changes of tumors were observed and the apoptosis of cells was detected by TUNEL; The capacity of proliferation of leukemia cells was observed in the bone marrow and the peripheral blood by Wright-Giemsa staining. The expression of genes of related to proliferation was detected by RT-PCR. Results The quality and the max diameter of tumors in the pEGFP-C/EBPα-K562 group were smaller than that of pEGFP-K562 group and K562 control group [(2.4±0.1) g vs (5.1±0.3) g and (5.7±0.4) g, both P ＜0.05; (11+2)mm vs (19+3) mm and (23+3) mm, both P ＜0.05]. More apoptosis cells were found in the pEGFP-C/EBPα-K562 group leukemic cells were found in the peripheral blood of leukemic models, and the proliferation of leukemic cells in the pEGFP-C/EBPo-K562 group were lower than that of other groups, accompany by the conspicuous cell differentiation. p53 was significantly elevated by RT-PCR, while down-regulated of c-myc.Conclusion Enhancer binding protein C/EBPα promote the apoptosis of cells and inhibit the proliferation of leukemia cells in leukemia mice, and further induce the cell differentiation. The inhibition of enhancer binding protein C/EBPα in the leukemia may have effect through the regulation of related genes.

Apoptosis of leukemic cells induced by flavonoids of puerarin and its molecular mechanisms / 白血病·淋巴瘤

Objective To explore the effects and the possible molecular mechanism of flavonoids of puerarin (PR) on chronic myelogenous leukemia (CML) cell line K562 and acute promyelocytic leukemia (APL) cell line NB4 in vitro. Methods MTT assays were used to detect the inhibitory effects of cell proliferation. The apoptosis of K562 and NB4 cells was detected by flow cytometry marked with Annexin V/PI. The expression of bcr-abl, p53, bcl-2, Fas/FasL in K562 cells and JNK, PARP, bcl-2 and Caspase 3 in NB4 cells at protein level was detected by Western blot. Results PR could inhibit the proliferation of K562 and NB4 cells in a time-dose dependent manner. The expression of protein levels of bcr-abl fusion gene declined, while the p53 protein otherwise increased, and both were in a dose-dependent manner (F = 18.74, P <0.05). The application of PR had no effect on bcl-2 and Fas/FasL protein expression in K562 cells. The JNK, PARP and Caspase3 proteins were upregulated in NB4 cells, while bcl-2 was downregulated with the increasing concentrations of PR (F=42.32, P <0.05). Conclusion PR could inhibit leukemic cell proliferation, induce cell cycle block, and increase cell apoptosis through different molecular mechanisms. It suggestes that PR might potentially be a kind of broad spectrum anti-leukemia agent.