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.

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.

Construction of human TCRP1 gene knockout chronic myeloid leukemia K562 cell line using CRISPR/Cas9 technology and its biological function detection / 中国医师杂志

Objective:To select human chronic myeloid leukemia (CML) cell line K562 as the experimental object, and use lentivirus mediated CRISPR/Cas9 gene editing technology to construct a stable CML cell line K562/TCRP1-KO that knocks out the tongue cancer resistance related protein 1 (TCRP1) gene; and through functional tests such as cell proliferation, apoptosis, and drug sensitivity, compare the phenotypic differences between K562/TCRP1-KO and control cells (K562/cas9-CTL), and preliminarily explore the possible mechanism of TCRP1 gene involvement in the pathogenesis of CML.Methods:The small guide RNA (sgRNA) targeting TCRP1 was designed at a specific location. After annealing, the oligonucleotide fragments were recombined with the linearized Cas9 expression vector, and the lentivirus packaging system was transfected into 293T cells. The purified virus was collected and infected with K562 cells. Positive polyclons were screened for puromycin pressure, and monoclonal K562/TCRP1-KO was further screened by limited dilution method. Stable cell lines were successfully knocked out by sanger sequencing and Western blot detection; Simultaneously, K562 cells transfected with lentiCRISPR vector were constructed as control cell lines (K562/cas9-CTL); Using cell counting method, cell counting kit 8 (CCK8) method, imatinib (IM) gradient dilution method, and flow cytometry cell proliferation, drug sensitivity, and apoptosis analysis were performed on K562/TCRP1-KO and K562/cas9-CTL, respectively.Results:The sgRNA-Cas9 recombinant plasmid vector for TCRP1 knockout was successfully constructed, and after transfection into 293T cells, TCRP1 knockout monoclonal cell lines were successfully screened using limited dilution method. Compared with K562/cas9-CTL cells, the proliferation ability of K562/TCRP1-KO cells was significantly reduced, IM drug sensitivity was significantly enhanced, and the process of cell apoptosis was significantly accelerated (all P<0.05). Conclusions:A CML cell line with TCRP1 knockout was successfully constructed using CRISPR/Cas9. TCRP1 may act as a cancer related gene to affect the proliferation, IM resistance, and apoptosis process of CML cells.

Mechanism of berberine reverses drug resistance of K562/A02 cells by down-regulating protein kinase C-alpha / 国际中医中药杂志

Objective:To observe the effect of berberine on leukemia drug-resistant cell strain K562/A02 to Adriamycin resistance and protein kinase C-alpha (PRKCA) and explore its possible mechanism.Methods:The leukemia K562 cells of human chronic myeloid and Adriamycin resistant strain K562/A02 were cultured in vitro with 2.5-50.0 μmol/L doxorubicin to treat thoese cells and drug resistance of K562 and K562/A02 to Adriamycin was detected, the 50% inhibitory concentration (IC 50) of the drug was calculatedthe resistance of K562 and K562/A02 to doxorubicin was detectd , and, K562/A02 cells were treated with doxorubicin solution at a final concentration of 5 μmol/L, and K562/A02 cells were divided into control group, inhibitor group (50 μmol/L PRKCA inhibitor), low dose berberine group, medium dose berberine group and high dose berberine group. Cell counting (CCK-8) method was used to detect the inhibition rate of cell proliferation, the apoptosis was detected by flow cytometry, real-time fluorescent quantitative PCR assay detects PRKCA, MRP, multidrug resistance related genes (MDR1) levels, and the protein expressions of protein kinase C-α (PRKCA), multidrug resistance related protein (MRP), P-glycoprotein (P-gp) were detected by Western blotting. Results:The IC 50 concentration of K562/A02 to Adriamycin was significantly higher than K562. Compared with the control group, the inhibition rate of cell proliferation and the apoptosis rate in the inhibitor group, low-dose berberine group, medium-dose berberine group, and high-dose berberine group were significantly increased ( P<0.05), the expression of PRKCA mRNA (0.45±0.08, 0.92±0.10, 0.57±0.05, 0.35±0.04 vs. 1.00±0.12), MDR1 gene (0.73±0.08, 0.87±0.09, 0.65±0.07, 0.41±0.05 vs. 1.00±0.11) and PRKCA (0.59±0.09, 0.78±0.12, 0.61±0.11, 0.42±0.07 vs. 0.96±0.14), MRP (0.62±0.08, 0.79±0.13, 0.62±0.10, 0.41±0.06 vs. 0.98±0.14), P-gp (0.55±0.08, 0.75±0.12, 0.59±0.09, 0.35±0.06 vs. 0.92±0.15) were significantly reduced ( P<0.05), and berberine was dose-dependent ( P<0.05); Overexpression of PRKCA can inhibit the effect of berberine on reversing the drug resistance of K562/A02 cells. Conclusion:Berberine may reverse the drug resistance of K562/A02 to Adriamycin by down-regulating PRKCA.

Study on the aging of leukemia K562 cells induced by ginsenoside Rg1 / 医学研究生学报

Objective　At present, the main studies of ginsenoside Rg1 are almost on the field of solid tumors and acute leukemias, and few on chronic leukemias. We aims to figure out the role of ATR-Chk1 pathway on cell aging in ginsenoside Rg1-treated leukemia K562 cells.　Methods　K562 cells were treated with ginsenoside Rg1 at different concentrations and divided into a control group (with 50 μL PBS culture solution) and 5 μmol/L ginsenoside group, 10 μmol/L ginsenoside group, 20 μmol/L ginsenoside group, 40 μmol/L ginsenoside group, 80 μmol/L ginsenoside group. CCK-8 assay，colony formation assay and flow cytometry for cell cycle detection were used to determine the effect of ginsenoside Rg1 on the aging of K562 cells. SA-β-Gal staining and Wright’s staining were used to observe the morphological changes of K562 cells’ aging. Real-time quantitative PCR and Western blot were used to detect the changes of ATR and Chk1 expression.　Results　The colony formation rate of K562 cells in the 20 μmol/L ginsenoside group was significantly lower than that in the other groups (P<0.05). CCK-8 test results showed that K562 cell proliferation of ginsenoside Rg1 induced groups was higher than that of the control group at 24, 48, and 72 hours (P<0.05). K562 cell proliferation inhibition rate was the highest in 20 μmol/L ginsenoside group for 48 hours treatment (P<0.05). The rate of SA-β-Gal positive cells [(95.833 ± 1.528) %] in 20 μmol/L ginsenoside-treated K562 cells for 48 h was significantly higher than that of the control group [(3.083 ± 0.764) %]. Cells blocked in G0/G1 phase and entered S and G2/M phases were significantly higher and lower than those in the control group, respectively (P<0.05).The ATR and Chk1 mRNA expression levels [(0.0117 ± 0.0038) %, (0.0120 ± 0.0021) %] were significantly higher than that of the control group ([0.0027 ± 0.0006) %, (0.0058 ± 0.0019) %) (P<0.05). ATR and Chk1 relative protein expression levels [(19.370 ± 0.994) %, (43.520 ± 1.236) %] were significantly increased compared with that of the control group [(17.080 ± 1.274) %, (39.100 ± 0.969) %) (P<0.05).　Conclusion　Ginsenoside Rg1 can induce the aging of K562 cells by regulating the ATR-Chk1 pathway, providing a new target for clinical leukemia treatment.

Inhibitory effect of PI3Kδ inhibitor idelalisib on proliferation of human myeloid leukemia cells and the reversal effect on drug resistance to adriamycin / 中南大学学报(医学版)

OBJECTIVES@#To investigate the effect of adriamycin (ADM), idelalisib or ADM and their combination on cell proliferation and intracellular concentration of ADM, and to explore the reversal effect of idelalisib on drug resistance to ADM.@*METHODS@#The K562 and K562/ADM cells were respectively treated with ADM and idelalisib at different concentrations. The 50% inhibitory concentration (IC@*RESULTS@#The cell survival rates were significantly decreased in a dose-dependent manner when the cells were treated with different doses of ADM (0.001-10.000 mg/L ). The IC@*CONCLUSIONS@#Idelalisib exerts effect on inhibition of the proliferation in myeloid leukemia K562 and K562/ADM cells, which may partially reverse the drug resistance of K562/ADM cells to ADM. The mechanisms for the effect of idelalisib may be related to increasing the accumulation of ADM and inducing the cell apoptosis in the K562 and K562/ADM cells.

Effects of artesunate inhibiting the expression of FOXP3 on proliferation, apoptosis and multidrug resistance of K562/ADR cells / 天津医药

@#Objective To investigate the effect of artesunate inhibiting the expression of FOXP3 on proliferation, apoptosis and multidrug resistance of adriamycin (ADR) -resistant K562/ADR cells in chronic myeloid leukemia (CML), and to explore its mechanism. Methods The expressions of FOXP3 mRNA in K562 and K562/ADR cells were detected by real-time PCR. The expressions of FOXP3 proteins in K562 and K562/ADR cells were detected by Western blot assay. The K562/ADR cells were treated with different concentrations of artesunate (2.5, 5.0, 7.5, 10.0 and 12.5 mg/L) for 24 h. The toxicities of different concentrations of artesunate to K562/ADR cells were detected by CCK-8 assay, and the non-cytotoxic concentrations were screened. The expressions of FOXP3 mRNA and proteins in K562/ADR cells treated by non-cytotoxic concentration of artesunate were detected by RT-PCR and Western blot assay. The changes of toxicities of ADR in K562/ ADR cells were detected by CCK-8 assay. The average fluorescence intensities of ADR were detected by FCM assay. Results The expressions of FOXP3 were higher in K562/ADR cells than those in K562 cells. The mRNA and proteins expressions of FOXP3 were significantly lower in 2.5 mg/L group, 5 mg/L group and 7.5 mg/L group than those in the control group. The toxicities and concentrations of ADR were increased in K562/ADR cells treated by artesunate (both P＜0.05). Conclusion FOXP3 gene is highly expressed in adriamycin-resistant K562/ADR cells in CML. Artesunate can increase the concentrations of ADR and reverse multidrug resistance in K562/ADR cells by inhibiting the expression of FOXP3 in a dose-dependent manner.

Effect of NPM1 mutant A on TGF-β1-induced K562 cell proliferation and AKT phos-phorylation / 中国肿瘤临床

Objective: To investigate the effect of nucleophosmin 1 (NPM1) mutant A on TGF-β1-induced K562 cell proliferation and AKT phosphorylation. Methods: K562 cells were infected with Ad5-NPM1 to create an NPM1 over-expression cell model. NPM1 levels were determined by ELISA and Western blot analysis. The levels of AKT and P-AKT were determined by Western blot. MTT was used to measure the proliferation of K562 cells. Results: NPM1 protein levels in K562 cells increased in an Ad5-NPM1-MOI-dependent manner. Cell proliferation and NPM1 levels in the supernatant were significantly increased in K562 cells infected with Ad5-NPM1-30 and Ad5-NPM1-100 compared to those infected with Ad5-vector-100 (P<0.01). Treatment with (10 ng/mL) TGF-β1 increased P-AKT levels, but not total AKT levels in K562 cells. TGF-β1-induced phosphorylation of AKT was significantly increased by infection of K562 cells with Ad5-NPM1-100. No significant differences were found in total AKT levels among all groups. TGF-β1 (10 ng/mL) treatment also in-creased the proliferation of K562 cells. TGF-β1-induced K562 cell proliferation was significantly increased by infection with Ad5-NPM1-100 (P<0.01). Conclusions: NPM1 improves TGF-β1-induced cell proliferation by up-regulating AKT phosphorylation levels.

Effect of sirolimus on erythropoiesis of K562 cell line and patients with pure red cell aplasia in vitro / 中华血液学杂志

Objective@#To understand the effect of sirolimus on the erythropoiesis of K562 cell line and bone marrow cells from pure red cell aplasia (PRCA) patients and normal controls.@*Methods@#Different concentrations (10, 100, 1 000 nmol/L) of sirolimus were added to the K562 cell line or bone marrow cells from PRCA patients or normal controls and cultured 14 days for BFU-E formation. Meanwhile, sirolimus was also added to the serum treated PRCA bone marrow cells to cultivate for the same priod of time.@*Results@#Neither K562 cells, bone marrow cells from PRCA patients or normal controls showed any difference when sirolimus was added to the culture system for BFU-E. However, BFU-E formation decreased after serum was added in PRCA patients (76.40±22.48 vs 136.33±12.58, t=-4.329, P=0.001) and this suppression of BFU-E was partly corrected by 1 000 nmol/L sirolimus treatment (97.14±15.83 vs 76.40±22.48, P=0.038).@*Conclusions@#Sirolimus may modulate the suppression of erythropoiesis by serum instead of directly stimulate the growth of red blood cells in PRCA patients.

Effect of MCUR1 on proliferation,cell cycle and apoptosis of K562 cells / 军事医学

Objective To investigate the effect of mitochondrial calcium uniporter(MCU)regulator 1(MCUR1)on proliferation,cell cycle and apoptosis of K562 cells and the possible molecular mechanism.Methods Recombinant plasmid vectors containing short hairpin RNAs(shRNAs)targeting MCUR1 were transfected into K562 cells,before the K562 cells stably expressing low MCUR1 were selected with G418.The expression of MCUR1 mRNA was detected by quantitative real-time polymerase chain reaction(qRT-PCR)assays.Western blotting(WB)assays were used to detect the expressions of MCUR1,P53,BAX and BCL2.The proliferation,cell cycle and apoptosis of K562 cells were detected by cell counting kit-8(CCK-8)assays and flow cytometry, respectively.Results The results of qRT-PCR and WB assays revealed that MCUR1 was stably down-regulated at mRNA and protein levels in the K562 cells transfected with shRNAs targeting MCUR1.Knockdown of MCUR1 significantly inhibited the cell proliferation, induced the cell apoptosis, but did not influence the cells cycle.Meanwhile, knockdown of MCUR1 increased the expression of P53 protein and the ratio of protein BAX/BCL2 in K562 cells.Conclusion MCUR1 promotes cell proliferation and inhibits cell apoptosis in K 562 cells.

Antiproliferative and apoptosis effect of atorvastatin on K562 cells / 重庆医学

Objective To explore the effect of atorvastatin on the proliferation and apoptosis of K562 cells andto investigate its mechanisms.Methods The cells were treated by different concentrations of atorvastatin.The CCK-8 assay was employed to detect the cell proliferation.The cell apoptosis was detected by AnnexinV-FITC/PI dual staining;the flow cytometry was used to detect the cellular cycle;the activities of caspase-3,-8,-9 were detected by the colorimetric method;qRT-PCR was employed to measure the mRNA expression levels of Bcl-2 and PDCD5 in K562 cells.Results Atorvastatin could inhibit the proliferation of K562 cells in a time-and dose-dependent manner(P＜0.05);and induced the apoptosis of K562 cells,the percentage of G0/G1 phase cells was increased after atorvastatin treating k562 cells(P＜0.01),while the percentage of S phase cells was decreased(P＜0.01),moreover which showing the concentration dependence(P＜0.01);atorvastatin activated the caspase-3,-8,-9 (P＜0.01);down-regulated Bcl-2 mRNA expression and up-regulated PDCD5 mRNA expressionin a concentration dependence(P＜0.01).Conclusion Atorvastatin can inhibit the proliferation and induce apoptosis in K562 cells.

Lupeol and its esters: NMR, powder XRD data and in vitro evaluation of cancer cell growth

ABSTRACT The triterpene lupeol (1) and some of its esters are secondary metabolites produced by species of Celastraceae family, which have being associated with cytotoxic activity. We report herein the isolation of 1, the semi-synthesis of eight lupeol esters and the evaluation of their in vitro activity against nine strains of cancer cells. The reaction of carboxylic acids with 1 and DIC/DMAP was used to obtain lupeol stearate (2), lupeol palmitate (3) lupeol miristate (4), and the new esters lupeol laurate (5), lupeol caprate (6), lupeol caprilate (7), lupeol caproate (8) and lupeol 3',4'-dimethoxybenzoate (9), with high yields. Compounds 1-9 were identified using FT-IR, 1H, 13C-NMR, CHN analysis and XRD data and were tested in vitro for proliferation of human cancer cell activity. In these assays, lupeol was inactive (GI50> 250µg/mL) while lupeol esters 2 -4 and 7 - 9 showed a cytostatic effect. The XRD method was a suitable tool to determine the structure of lupeol and its esters in solid state. Compound 3 showed a selective growth inhibition effect on erythromyeloblastoid leukemia (K-562) cells in a concentration-dependent way. Lupeol esters 4 and 9 showed a selective cytostatic effect with low GI50 values representing promising prototypes for the development of new anticancer drugs.

Inhibitory effect of polyphyllin D on the proliferation of human chronic myelogenous leukemia cell line K562 and its mechanism / 白血病·淋巴瘤

Objective To explore the inhibitory effect of polyphyllin D on the proliferation of human chronic myelogenous leukemia (CML) cell line K562 and its mechanism. Methods K562 cells were treated with various concentrations of polyphyllin D (0, 0.1, 0.2, 0.4, 0.8, 1.2, 2.4 μmol/L) at 24 h, and cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect the effect of polyphyllin D on the apoptosis, and the cell cycle arrest of K562 cells. The relative proteins were analyzed by using Western blot. Results The polyphyllin D could significantly inhibit the proliferation of K562 cells, and the effective inhibitory concentration (IC50) was (0.9 ± 0.1) μmol/L at 24 h. The results of flow cytometry showed that after treatment with 0.9 μmol/L polyphyllin D at 12 h and 24 h, the apoptotic rate of the cells [(11.46 ±1.51) %, (28.87 ± 2.35) %] were significantly higher than that of the control group [(2.05±0.45) %], and the difference was statistically significant (F= 38.637, P< 0.05). The expressions of bcl-2, CDK1, CyclinB1 and bcr-abl fusion protein were down-regulated by polyphyllin D, and the expressions of Bax, cytochrome C, activated caspase-3 and p21 were up-regulated (all P<0.05). In addition, polyphyllin D could arrest cell-cycle at G2/M phase (F=42.355, P<0.05). Conclusion Polyphyllin D can significantly inhibit the proliferation of human CML cell line K562, and its mechanism could play a role by inducing apoptosis and promoting cell cycle arrest.

Effects of hydroxyl morpholine resistance on K562 cells of chronic myelocytic leukemia / 中国药学杂志

OBJECTIVE: To investigate the inhibitory activity, induced differentiation-inducing activity and apoptosis-inducingactivity of hydroxyl morpholine (QDML-01) on chronic myelocytic leukemia cells line K562. METHODS: The cell growth curve was drawn based on cell counting method. The IC50 value of QDML-01 and positive control medicine to K562 cells were evaluated by methyl thiazolyl tetrazolium (MTT) assay method. Double soft agar assay method was carried out to study the ability of cell proliferation to determine efficacy of phamacognosy. The pathomorphism was analyed by the Wright-Giemsa staining method. The mechanism of cell apoptosis from morphology and gene level were investigated, by AO-EB double-staining method and DNA breakage test. The effect of QDML-01 on K562 cells from the protein level was determined by Western-blot. RESULTS: The growth curves showed the K562 cells had strong cell vitality. They came into logarithmic phase on the third generation. The MTT assay results showed that the IC50 values of QDML-01 and imatinib to K562 cells were 5. 81 and 596.88 nmol ·L-1. Double soft agar colony formation test showed that clone formed at 21 d and the inhibitory rate of QDML-01 was 81.7%. It indicated that K562 cells were sensitive to QDML-01. Morphology test result showed that QDML-01 induced K562 cells to normal cells. The results of AO-EB double-staining method showed that QDML-01 induced the apoptosis of K562 cells. The study of DNA breakage test indicated that QDML-01 can induce the apoptosis of K562 cells to produce DNA banding with step-like. Western-blot analysis result suggested that QDML-01 can downregulated the expression of P210bcr/abl protein. CONCLUSION: QDML-01 has the inhibitory activity on chronic myelocytic leukemia cells line K562 by promoting the apoptosis of K562 cells and inducing differentiation to normal cells.

Ribonucleic acid Ⅱ induces apoptosis in human leukemia cells by up-regulating p53 / 中国药理学通报

Aim To investigate the effect of ribonucleic acidⅡon apoptosis in human leukemia cell lines K562 and KG1 a.Methods Cell counting kit-8(CCK-8)as-say was performed to detect proliferation activity of K562 and KG1 a cells treated with ribonucleic acidⅡ. Apoptosis index was assessed by flow cytometry(FCM) and fluorescent Hoechst 33258 staining was used for observing morphologic changes of apoptosis.Expres-sion levels of p53,Bax,Bcl-2 and cleaved caspase-3 were analyzed by Western blot.Results The prolifera-tion of K562 and KG1 a cells was significantly inhibited by ribonucleic acid Ⅱ treatment for 12 h,24 h,48 h at concentrations of 100~300 mg·L-1 ,which indica-ted the inhibitory effect of ribonucleic acid Ⅱ was in dose-dependent and time-dependent manners.FCM re-sults displayed a dose-dependent increase in cell apop-totic rate.Hoechst 33258 staining showed the typical apoptotic morphology in some leukemic cells treated with ribonucleic acid Ⅱ,including increased nuclear chromatin concentration and edge accumulation.West-ern blot analysis showed the increased expression of p53,Bax,cleaved caspase-3 and decreased expression of Bcl-2 in K562 and KG1 a cells treated with ribonu-cleic acid Ⅱ.Conclusions Ribonucleic acid Ⅱ can induce apoptosis of leukemia K562 and KG1 a cells by up-regulating p53,which mediates Bcl-2/Bax balance and activates caspase-3 .

Effect of Arca subcrenata Lischke anticancer protein on cell proliferation and apoptosis of human myeloid leukemia K562 cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the cytotoxic activity of Arca subcrenata Lischke anticancer protein(ASAP)constituents on human myeloid leukemia K562 cells in vitro and analyze its anticancer mechanisms. METHODS ASAP was extracted by low temperature water and ammonium sulfate precipitation. Protein concentration of ASAP was detected by Bradford method. Morphological changes of cultured K562 cells treated with ASAP were observed under the inverted phase-contrast micro?scope. The cell and nucleus changes were analyzed by Giemsa staining. The cytotoxicity of ASAP on K562 cells was detected by MTT assay. Flow cytometry was used to detect apoptosis and cell cycle of K562 cells treated with ASAP. The expression of apoptosis and cell cycle related proteins procaspase 3, caspase 3,P53 and programmed cell death 4(PDCD4)were analyzed by Western blotting. RESULTS ASAP exhibited significant cytotoxic effect on K562 cells in a time- and concentration-dependent manner. The concentration-effect correlation coefficient of ASAP 50,100 and 200 mg · L-1 on K562 cells for 24, 48 and 72 h was 0.851,0.8977 and 0.8997,respectively. Under an optical microscope,K562 cells showed cytomorphosis,or nuclear fragmentation after treatment with ASAP 200 mg · L-1 for 48 h. Flow cytometry analysis and Giemsa staining assay indicated that apoptotic cells increased and G2/M phase cells accumulated significantly with the increase of ASAP concentration. After treatment with ASAP 200 mg · L-1 for 48 h,the early and late apoptosis cell rate increased to(32.8 ± 0.1)%and(31.2 ± 2.2)%vs control group(3.7 ± 1.1)% and (9.9 ± 0.8)%(P<0.01),respectively,and the G2/M phase cells increased to (55.2 ± 1.7)% vs (15.3 ± 0.8)% in control group(P<0.01). After treatment with ASAP 200 mg · L-1 for 0-40 h,the expression of apoptotic protein procaspase 3 was down-regulated and its active form caspase 3 was significantly up-regulated at 32 h,while PDCD4 and P53 protein expression was down-regulated significantly in 0-40 h. CONCLUSION Apoptosis and cell cycle arrest induced in G2/M phase may account for ASAP cytotoxic activity to K562 cells. K562 cell apoptosis induced by ASAP depends on caspase 3 signal pathway. Down-regulated expression of PDCD4 and P53 proteins may be related to K562 cell apoptosis and cell cycle arrest in G2/M phase by ASAP.

The proteomics research of 4-amino-2-trifluoromethyl-phenyl retinate on human leukemia K562 cells / 中国药理学通报

Aim To explore the proteomics mechanism of the differentiation induction effect of 4-amino-2-trif-luoromethyl-phenyl retinate(ATPR)on human leukemi-a K562 cells. Methods Human leukemia K562 cells were incubated with the same concentration (1 × 10 - 6 mol·L - 1 ) of ATPR or ATRA for 48 hours. The total cell proteins were collected, purified and digested by trypsin, solid phase extraction, and the peptides were detected by ESI-LC-MS / MS. The difference of the pro-tein expression between the cells treated with ATPR and ATRA was compared by using the Discoverer Pro-teome 1. 2 software, and the molecular function, the biological process and other information of those pro-teins were analyzed based on the DAVID, KEGG, STRING databases. Results 120 specific proteins were identified only in the ATPR group, 143 only in the ATRA group, and 422 other proteins in both groups. Results of DAVID analysis showed that ATPR-induced specific proteins were mainly involved in 39 biological processes of proteins and macromolecules metabolism, protein transport and localization and so on. Results of KEGG analysis revealed that ATPR-in-duced proteins participated in signal pathways, mainly metabolic pathways, PI3K-Akt signal pathway, TGF-beta signal pathway and other pathways in cancer. String protein interaction network analysis displayed that ATPR-induced proteins, like EIF3A, EIF6, RPL3, RPL8, RPL13, RPL7A, RPL21, RPS3, RPS14, NACA, BTF3, NHP2L1, PPP2CA proteins had direct interactions with more than or equal to 10 associated proteins. Conclusion The differentiation induction effect of ATPR on K562 cells might be as-cribed to the ATPR-induced proteins interaction net-work and the specific central proteins it induced, which are involved in the regulation of cell prolifera-tion, differentiation and apoptosis.

Icaritin promotes the apoptosis of leukemia K562 cells / 白血病·淋巴瘤

Objective To investigate the effect and mechanism of the icaritin on the human cronic myeloid leukemia K562 cells. Methods The K562 cells at logarithmic growth phase were divided into the control group and the icaritin group. The cells in the control group were normally treated and the cells in the icaritin group were incubated with 8 μmol/L icaritin. Methyl thiazolyl tetrazolium (MTT) method and flow cytometry were used to examine the proliferation and apoptotic changes in the two groups after incubation for 72 h, respectively. Gene expression of p85 and Akt were detected by RT-PCR. The protein changes of p85, Akt, p-p85, p-Akt cleavage-caspase-3 and caspase-3 were detected by Western blot. Results Compared with the control group, the proliferation rate of K562 cells in the icaritin group was significantly decreased (P 0.05). Conclusion Icaritin could induce the proliferation and promote the apoptosis of K562 cell, and its mechanism may be achieved through activating the PI3K-Akt signal transduction pathway.

Influence of AKT inhibitor on Wnt/β-catenin pathway in chronic myeloid leukemia K562 cells / 解放军医学杂志

ObjectiveTo investigate the impact of PI3K-AKT pathway inhibitor (AKTi ) on the acute crisis of chronic myeloid leukemia (CML) and its regulatory effect on PI3K-AKT pathway in Wnt/β-catenin signal pathway. Methods K562 cells were cultured to logarithmic growth phase, and they were treated with 0, 2.5, 5, 10μmol/L AKTi respectiv ely. Cell growth was determined with MTT test. The ability of cell colonization was assessed by colony-forming assay. The protein expression of pAKT (Thr308) and pGSK-3β(Ser9) was determined by Western blotting. The protein expression and mRNA levels of β-catenin and its down-stream targets c-myc and cyclin D1 were analyzed by Western blotting and realtime fluorescence quantitative polymerse chain reaction (Q-PCR), respectively. ResultsCell growth and colony-forming ability of K562 cells were inhibited significantly after treatment with AKTi for 6, 10, 16h, and the best exposure time for K562 cells was 10h. PI3K-AKT pathway was inhibited obviously and the protein level of pAKT (Thr308) was lowered apparently after 2.5, 5, 10μmol/L AKTi treatment. When K562 cells were treated by 5μmol/L AKTi for 10h, pGSK-3β (Ser9) and β-catenin protein levels were lowered significantly, while the mRNA of β-catenin was not affected. The mRNA and protein levels of c-myc and cyclin D1 in the downstream of β-catenin were also decreased obviously after treatment with 5μmol/L AKTi for 10h. ConclusionAKTi can inhibit the proliferation and colony-forming ability of K562 cells in critical stage of CML. The mechanism may be related to down-regulation of expression of Wnt/β-catenin pathway by blocking the signal transduction of cell growth.

Prokaryotic expression of Trx-apoptosis-inducing factor-defected mitochondria localization sign fusion protein and its effect on cell-free system of leukemia K5 62 cells / 肿瘤

Objective: To express the fusion protein-thioredoxin-apoptosisinducing factor-defected mitochondria localization sign (Trx-DMLS-AIF) in prokaryotic cells and to detect its effect on cell-free system of leukemia K562 cells. Methods: The recombinant plasmid expressing Trx-DMLS-AIF fusion protein was established and transformed into E.coli BL21. The expression of Trx-DMLS-AIF fusion protein was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified by Ni-NTA Spin Columns. The cell-free system of leukemia K562 cells was established. The effect of Trx-DMLS-AIF fusion protein on cell-free system of K562 cells was detected by acridine orange (AO) staining. The effect of plasmosin on the ability of Trx-DMLS-AIF fusion protein entering into nuclei of K562 cells was detected by immunofluorescent staining. Results: The expression of Trx-DMLS-AIF fusion protein was successfully induced by IPTG, and the purified Trx-DMLS-AIF fusion protein was obtained. The result of AO staining showed that Trx-DMLS-AIF fusion protein could induce the nucleus apoptosis in cell-free system of K562 cells. The result of immunofluorescent staining showed that the plasmosin of K562 cells could prevent Trx-DMLS-AIF fusion protein from entering into the nuclei of K562 cells. Conclusion: The expression of Trx-DMLS-AIF fusion protein is successfully induced. TrxDMLS-AIF can induce the nucleus apoptosis of K562 cells, and the plasmosin of K562 cells can prevent Trx-DMLS-AIF fusion protein from entering into the nuclei of K562 cells.