Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells

Objective Hydroquinone(HQ),one of the phenolic metabolites of benzene,is widely recognized as an important participant in benzene-induced hematotoxicity.However,there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn’t been fully understood yet.Methods In this study,we treated K562 cells with 40μmol/L HQ for 72 h,examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring(PRM),and performed bioinformatics analysis to identify interaction networks.Results One hundred and eighty-seven upregulated differentially expressed proteins(DEPs)and 279 downregulated DEPs were identified in HQ-exposed K562 cells,which were involved in neutrophilmediated immunity,blood microparticle,and other GO terms,as well as the lysosome,metabolic,cell cycle,and cellular senescence-related pathways.Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways,we constructed the network of protein interactions and determined 6 DEPs(STAT1,STAT3,CASP3,KIT,STAT5B,and VEGFA)as main hub proteins with the most interactions,among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity.Conclusion Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

二烯丙基二硫通过磷酸化Chk1负调控Cdc25C/CyclinB1/CDK1通路阻滞白血病K562细胞G_(2)/M期

目的:研究二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病K562细胞周期阻滞及其分子机制。方法:采用CCK-8、细胞计数及流式细胞术观察DADS对K562细胞增殖与周期阻滞效应。Western Blot检测DADS对K562细胞PCNA、Chk1/2以及下游分子Cdc25C、CyclinB1与CDK1表达的影响。结果:CCK-8检测显示,15μmol/L、30μmol/L、60μmol/L、120μmol/L、240μmol/L DADS处理后,呈浓度依赖性抑制K562细胞增殖,抑制率分别为32.48%、59.34%、66.42%、77.06%、81.05%(P<0.05)。对照组与Tween80组无抑制作用(P>0.05)。细胞计数结果显示,30μmol/L、60μmol/L与120μmol/L DADS处理K562细胞后,群体倍增时间分别为22.71±0.29、36.69±0.93与73.02±0.87呈浓度依赖性增加(P<0.05),而Tween80组与对照组无明显差异(P>0.05)。流式细胞术检测显示,60μmol/L与120μmol/L DADS作用K562细胞24 h与48 h后,G_(2)/M期百分率分别增加到17.6%与28.5%和18.6%与34.4%,较对照组有显著性差异(P<0.05)。60μmol/L DADS作用K562细胞1 h、2 h、4 h、8 h和24 h后,PCNA表达呈时间依赖性表达下调(P<0.05)。p-Chk1表达呈时间依赖性上调(P<0.05),而Chk1、Chk2与p-Chk2表达无明显差异(P>0.05)。并且,Cdc25C、CyclinB1和CDK1分别呈时间依赖性下调(P<0.05),但是,14-3-3蛋白无明显改变(P>0.05)。结论:DADS可磷酸化Chk1通过Cdc25C/CyclinB1/CDK1通路抑制K562细胞增殖与阻滞G_(2)/M期。

钩吻总碱诱导慢性粒细胞白血病K562细胞凋亡的机制分析

目的 探讨钩吻总碱对慢性粒细胞白血病K562细胞的抑杀作用及其机制,为其抗白血病研究提供科学依据。方法 以不同浓度钩吻总碱(25、50、100、200、400μg·mL-1)干预K562细胞,MTT法测定其对K562细胞活力的影响;倒置相差显微镜观察其对K562细胞形态的影响;DAPI染色法观察K562细胞核形态变化;Annexin V FITC/PI流式细胞术检测K562细胞凋亡情况;比色法测定caspase-3活性变化;RT-PCR检测Bax和Bcl-2基因的表达水平。结果 钩吻总碱对K562细胞抑制效果呈时间、剂量依赖性,24 h时IC50为122μg·mL^(-1);TAG干预K562细胞后细胞形态逐渐不规则、胞质不清、胞核皱缩,最终胞膜的完整性破坏;DAPI染色发现细胞体积变小,整体皱缩,胞核固缩、生成典型的凋亡小体;Annexin V FITC/PI流式细胞测定显示K562细胞凋亡以早凋为主,存在量效关系;不同浓度钩吻总碱作用于K562细胞24 h后caspase-3的活性提高;RT-PCR检测发现TAG干预后会下调Bcl-2基因表达、上调Bax基因表达,二者均表现出量效关系。结论 钩吻总碱可能通过上调Bax基因表达、下调Bcl-2基因表达,活化caspase-3,从而诱导慢性粒细胞白血病K562细胞发生早期凋亡,抑制其细胞活力。

牛蒡子苷元对白血病K562/A02细胞耐药性的逆转作用及机制

目的:研究牛蒡子苷元对白血病耐药细胞株K562/A02阿霉素耐药性的影响及其作用机制。方法:体外培养人白血病细胞株K562及阿霉素耐药细胞株K562/A02,使用2.5-50μmol/L阿霉素处理,CCK-8检测细胞生长情况并计算药物半数抑制浓度(IC_(50))。采用不同浓度的牛蒡子苷元(1、2、4、8、16 mmol/L)处理K562/A02细胞,检测牛蒡子苷元对K562/A02细胞的影响,筛选适用浓度用于后续实验。在2 mmol/L牛蒡子苷元处理的K562/A02细胞中加入5μmol/L阿霉素,流式细胞术检测细胞凋亡,Western blot检测P-gp、MRP、cleaved caspase-3、Bax、Bcl-2以及TLR4/NF-κB信号通路蛋白的表达。在牛蒡子苷元和阿霉素共处理的K562/A02细胞中转染TLR4过表达质粒,检测细胞的药物敏感性和凋亡水平。结果:阿霉素对K562/A02细胞的IC_(50)为36.57μmol/L,高于K562细胞(1.30μmol/L)。当牛蒡子苷元浓度≤2 mmol/L时,K562/A02的细胞生长不会受到明显抑制。经2 mmol/L的牛蒡子苷元处理后,阿霉素对K562/A02细胞的IC_(50)明显降低。与对照组相比,牛蒡子苷元组K562/A02细胞凋亡率明显上升,cleaved caspase-3、Bax蛋白的表达明显上调,P-gp、MRP、Bcl-2、TLR4、My D88和p-NF-κB蛋白的表达明显下调,差异均具有统计学意义(P<0.05)。在转染TLR4过表达质粒后,牛蒡子苷元处理的K562/A02细胞对阿霉素的敏感性明显降低(P<0.05),细胞凋亡下降,并显著提升了P-gp、MRP、Bcl-2和TLR4/NF-κB信号通路蛋白的表达(P<0.05),同时降低了cleaved caspase-3和Bax蛋白的表达(P<0.05)。结论:牛蒡子苷元可能通过抑制TLR4/NF-κB信号通路从而逆转人白血病耐药细胞株K562/A02对阿霉素的耐药性。

白花蛇舌草诱导活性氧类物质抑制慢性髓系白血病K562细胞增殖

目的探讨白花蛇舌草(HDW)诱导活性氧类物质对慢性髓系白血病(CML)K562细胞增殖的影响。方法流式细胞法检测HDW作用下K562细胞内活性氧(ROS)水平。将K562细胞分为对照组、HDW组、N-乙酰-L-半胱氨酸组(NAC组)和HDW+NAC组,观察各组细胞ROS、活力、形态、增殖和细胞周期分布情况,Western blotting法检测细胞周期相关蛋白和磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化丝氨酸苏氨酸激酶(p-Akt)蛋白表达情况。结果HDW能提高K562细胞内ROS水平(P<0.05)。对照组、NAC组细胞含大量拒染细胞且形态无明显变化,HDW组、HDW+NAC组拒染细胞减少,着色细胞增多,有染色质聚集和细胞核膜崩解现象。与对照组相比,HDW组细胞内ROS、G1期占比、p21和p53蛋白表达升高,S期占比和细胞周期素D1、细胞周期素依赖蛋白激酶4、p-PI3K、p-Akt蛋白表达降低(P<0.05);NAC干预能部分逆转HDW作用效果(P<0.05)。结论HDW能诱导活性氧类物质升高来降低K562细胞增殖能力,与细胞周期阻滞、调节PI3K/Akt通路活性有关。

利用慢病毒载体构建过表达人TCRP1基因的慢性髓系白血病K562细胞系及其生物学功能检测

目的利用慢病毒载体构建过表达人舌癌耐药相关基因(TCRP1)的慢性髓系白血病(CML)K562细胞系并检测其生物学功能。方法将TCRP1的重组质粒与包装质粒共转染293T细胞,收集病毒液测定滴度后感染K562细胞,使用嘌呤霉素筛选TCRP1过表达的细胞株K562/TCRP1,荧光定量PCR和Western blot方法检测TCRP1的表达,采用连续细胞计数法和CCK-8法分别对K562/TCRP1及其对照细胞株的增殖情况进行检测,并分析2个细胞株对不同浓度的伊马替尼(IM)的药物敏感性。结果TCRP1慢病毒表达载体成功转染进入K562细胞,荧光定量PCR和Western blot结果显示K562/TCRP1细胞株的TCRP1表达在mRNA水平和蛋白水平均显著高于对照组,连续细胞计数法和CCK-8法结果表明K562/TCRP1细胞的增殖能力和细胞活力增强,IM处理K562/TCRP1细胞的IC50值显著高于其对照细胞(P<0.05)。结论利用慢病毒载体成功构建了TCRP1过表达的K562细胞株,并且发现TCRP1的过表达可能增强K562细胞的增殖能力和IM耐药能力,为进一步探讨TCRP1在慢性髓系白血病发病机制中的可能作用提供基础。

Effect on Proliferation and Erythroid Differentiation of K562 Cells by IER3IP1-Knockdown

Objective： To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene. Methods： The shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed. Inhibitory effect was detected by semiquantitative RT-PCR. The impacts on K562 cells by RNAi were studied by MTT assay, benzidine staining, light microscope and electron microscopy observation, cell cycles analysis, colony formation assay and RT-PCR. The expressions of erythroid differentiation correlated genes Gfi-lB, GPA and 7-globin were studied after being exposed to 0.2μmol/L imatinib for two days. Results： The shRNA eukaryotic expression vectors were successfully constructed. The expression of IER3IP1 gene was significantly inhibited with an inhibition efficiency of 76% （P〈0.01）. Compared with the control groups, bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group （P〈0.01）. The proliferation ability was enhanced （P〈0.01） and the proportion of cells at G0/G1 phase decreased but S phase increased （P〈0.05） in K562/shRNA-IER3IP1 group. Under electron microscopy, the amount of euchromatin increased but heterochromatin decreased. There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. The percentage of benzidine staining positive cells and mRNA expression levels of Gfi-1B, GPA and γ-globin were all decreased after being exposed to 0.2 μmol/L STI571 for two days in K562/shRNA-IER3IP1 group （P〈0.01）. Conclusion： IER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells. IER3IP1 may play a role in erythroid differentiation and proliferation of K562 cells.

Effect of Hydroxyapatitc Nanoparticles on K562 Cells in vitro

Stable and single-dispersed hydroxyapatite （HAP） nanoparticles were synthesized with ultrasonic-assisted method. HAP nanoparticles were characterized by dynamic light scattering, XRD （X-ray diffraction） and TEM （Transmission Electron Microscopy）. The effect of HAP nanoparticles on the K562 human myelogenous leukemia cell line was investigated by MTT assay and cell count test, and the mechanism was studied through the changes of cell cycle and ultrastructure. The results showed that HAP nanoparticles inhibited the proliferation of K562 cells dramatically in vitro. HAP nanoparticles entered the cytoplasm of K562 cells and the cells were arrested at G/M phase, thus, the cells died directly.

Lethal Effect of Benzene Nitrogen Mustard Glucoside Derivate on K562 Cells

A new synthesized benzene nitrogen mustard was converted into glycosyl donor-trichloroacetimidate that was glycosylated with p-nitrophenol（glycosyl donors） to form β-lactosyl p-nitrobenzene under the protection of acetyl in a stereoselective manner, was prepared and evaluated for its cytotoxicity towards cultured K562 cell line. Methylthiazoy tetrazolium（MTT） assay, transmission electron microscopy（TEM）, flow cytometry（FCM） and immunohistochemistry were utilized to explore the mechanisms of how the compound arrests the growth of HCT-T cells. This new synthesed benzene nitrogen mustard glucoside derivate（BNMGD） presented a lower toxicity to normal cells, but is significantly more toxic to K562 cells compared with nitrogen mustard, meanwhile it can induce the apoptosis of K562 cells. These results indicate that the new synthesized BNMGD can inhibit the growth of K562 cells and induce the apoptosis, and its cytotoxicity towards cultured K562 cell line is much more effective than that of nitrogen mustard.

Zeylenone promotes apoptosis of chronic myelogenous leukemia-derived K562 cells by a mechanism involving Jak2 and src kinase

OBJECTIVE The present study was designed to investigate anticancer effect of zeylenone(Zey)on K562 cells derived from chronic myelogenous leukemia(CML)both in vitro and in vivo,followed by exploring the underlying mechanisms.METHODS Initially,the effects of Zey on cel viability,proliferation,and apoptosis were measured in K562 cells by MTT,soft agar assay,AO/EB staining,hoechst 33258 staining and flow cytometric analysis after they were treated with Zey for indicated time,the involving signaling pathways were then investigated by JC-1,real-time quantitative polymerase chain reaction(RT-q PCR),Western blotting and immunofluorescence analysis.Furthermore,the in vivo anti-tumoractivity of Zey was assessed with nude xenografts and the involving mechanism was confirmed by immunohistochemical(IHC)and histopathological analysis.RESULTS We identified that Zey dose-dependently decreased cell viability,colony formation and expression of Proliferating Cell Nuclear Antigen(PCNA),and significantly induced K562 cell apoptosis via regulating Bcl-2 family members,decreasing mitochondrial transmembrane potential,and activating caspase-3,caspase-9,and caspase-8(P<0.05 or P<0.01).Further study revealed that Zey significantly inhibited phosphorylation of Jak2 and Src and downregulated their downstream proteins,including stat3,PI3K/AKT/m TOR,and ERK1/2 signaling pathways(P<0.05 or P<0.01).Zey also suppressed tumor growth with low toxicity in mouse xenograft model of K562cells through decreasing expression of Jak2 and Src.CONCLUSION Our data demonstrated that Zey substantially suppressed K562 cells both in vitro and in vivo through Jak2 and Src pathways.These findings suggest the potential of Zey as an effective anticancer agent in CML treatment.

Suppression of Amino Acid Transporter LAT3 Expression on Proliferation of K562 Cells

The activity of the mTOR pathway is frequently increased in acute myeloid leukemia, and is tightly related with cellular proliferation. Leucine is tightly linked to the mTOR pathway and can acti- vate it, thereby stimulating cellular proliferation. LAT3 is a major transporter for leucine, and suppres- sion of its expression can reduce cell proliferation. Here, we show that suppression of LAT3 expression can reduce proliferation of the acute leukemia cell line, K562. We investigated the mRNA and protein expression of LAT3 in several leukemia cell lines and normal peripheral blood mononuclear cells （PBMNCs） using RT-PCR and Western blotting. We also evaluated cell viability using a methyl thia- zolyl tetrazolium （MTT） assay after blocking LAT3 expression with either shRNA targeted to LAT3 or a small molecular inhibitor BCH （2-aminobicyclo-（2,2,1）-heptane-2-carboxylic acid）. LAT3 mRNA and protein expression was detected in leukemia cell lines, but not in normal PBMNCs. Using K562 cells, it was found that cellular proliferation and mTOR pathway activity were significantly reduced when LAT3 was blocked with either shRNA or BCH. Our results suggest that leukemia cell proliferation can be sig- nificantly suppressed by blocking LAT3. This finding may lead to a new strategy to develop clinical therapy for the treatment of acute myeloid leukemia.

INDEPENDENT AND SYNERGIC INHIBITION OF DIPYRIDAMOLE AND RADIATION ON K562-AND K562/ADM CELL LINES IN VITRO

It is first demonstrated that dipyridamole (DP) and radiation were capable of significantly inhibiting, independently and synerglcally, clonogenlc growth in the two kinds of K562 cell lines, adriamycin (ADM) -sensitive and ADM- resistant. DP or radiation alone Increased clonogenlc Inhibition rate (CIR) in the two kinds of cell lines in a dose- dependent fashion. DP potentiated radiosensitivity and radiation increased inhibition of DP in the two kinds of cell lines. K562/ ADM cell lines were higher sensitive to DP. radiation and combination of them than K562 cell lines (P<0. 01). There was stronger synergic inhibition of clonogenlc growth in the two kinds of cell lines when pretreated with DP than when posttreated with DP (P<0. 01).

The modulation of radiation-induced cell death by genistein in K562 cells:Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML)cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization(SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression of TK1 mRNA and TK 1enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition,the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G1/S progression and G2-arrest, and their relationship with TK1 in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.

Expression of Histone H2AX Phosphorylation and Its Potential to Modulate Adriamycin Resistance in K562/A02 Cell Line

DNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy. Histone H2AX phosphorylation （ser139） （γH2AX） occurs rapidly at the onset of DNA double strand break （DSB） and is critical to the regulation of DSB repair. If DNA repair is successful, cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs. In this study, we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy. K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin （ADR） and γH2AX formed. Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR. In order to test the potential of γH2AX to reverse drug resistance, K562/A02 cells were treated with PI3K inhibitor LY294002. It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR. Additionally, the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCA1. These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.

Survivin Antisense Oligodeoxy-Nucleotid Induces Apoptosis in Leukaemia Cell Line K562

OBJECTIVE To investigate the effects of survivin antisense oligodeoxy-nucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562. METHODS Different concentrations of an antisense oligodeoxy-nucleotid and control sequence (scrambled ODN) targeting the survivin gene were transferred into K562 by a lipofectin reagent. The MTT assay was used to measure the growth inhibitory rate, IC50, and to observe the cytotoxicity of survivin ASODN in the K562 cells. The morphologic changes in the nucleus and the apoptotic rate were observed by Hoechst33342/PI staining. Caspase-3 activity was evaluated by a kinase activity assay. The changes of survivin protein expression after transfection were detected by Western blots. RESULTS Eight hours after transfection, fluorescence in the K562 cells was well distributed. Treatment of the cells for 44 h with different concentrations of survivin ASODN produced a IC50 of 800 nmol/L. The growth inhibitory rate with 200, 400, 600 and 1000 nmol/L of survivin ASODN was 15.8±1.6%, 23.8±5.9%, 37.1±5.6% and 77.3±2.5% respectively. After 36 h of of survivin ASODN treatment, distinct morphologic changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst33342/PI staining. Caspase-3 activity increased significantly after treatment of the cells with different concentrations of survivin ASODN(P<0.01)and following treatment with 800 nmol/L survivin ASODN, survivin expression decreased significantly. CONCLUSION Survivin ASODN exerts an anti-cancer effect by inducing apoptosis in K562 leukaemia cells. Up-regulated expression of caspase-3 may play a role in this process.

The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-κB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI double-labeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blo ing.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.

Anti-cancer Effects of Deguelin on Human Leukemia K562 and K562/ADM Cells In Vitro

In order to investigate the anti-cancer effects of deguelin and on K562 and K562/ADM cells in vitro and the underlying molecular mechanism and compare the cytotoxicity of deguelin on K562, K562/ADM cells and human peripheral blood mononuclear cells （PBMCs）. The effects of deguelin on cell proliferation were assessed by MTT assay. Apoptosis were detected by Annexin V/PI double-labeled cytometry. The effects of deguelin on the cell cycle were studied by a propidium iodide method. Our study showed that deguelin inhibited the proliferation of K562 cell and K562/ADM cell in a time- and dose-dependent manner and had minimal effects on normal human peripheral blood mononuclear cells. The ratio of IC50 value of deguelin of 24 h on K562/ADM cells to K562 cells was only 1.27, which was significantly lower than the ratio of IC50 value of ADM （higher than 20）. Deguelin could induce apoptosis of K562 cells and K562/ADM cells. K562 cells were arrested at G2/M phase while K562/ADM cells were arrested at G0/G~ phase. Our results suggested that deguelin was a novel anti-leukemia agents with high efficacy and low toxicity and it is also a promising agent for reversing drug resistance.

The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

The difference of sensitivity to indole- 3-acetic acid （ IAA ） combined with horseradish peroxidase （HRP） in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTF assay. The cell cycle and apoptosis of K562 and BXPC-3 cells were examined by a fluorescence flow cytometer （FCM） and terminal deoxynacleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC-3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.

Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As 2O 3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As 2O 3 induced cell apoptosis, K562 cells were cultured with As 2O 3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As 2O 3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G 2/M phase increased in proportion to As 2O 3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As 2O 3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G 2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As 2O 3-induced apoptosis.

舒尼替尼诱导耐药白血病细胞K562/ADR焦亡的作用及其通路研究

目的:探讨舒尼替尼(SU11248)对耐药白血病细胞K562/ADR死亡的诱导作用及其相关信号通路。方法:使用不同浓度舒尼替尼干预K562/ADR细胞,分别于24、48、72、96 h收集各组细胞,采用MTS法检测舒尼替尼对K562/ADR细胞增殖能力的影响,确定适当的舒尼替尼干预时间和浓度。通过qPCR和Western blot检测舒尼替尼干预后K562/ADR细胞的凋亡相关基因mRNA和蛋白表达水平变化。使用4种不同细胞死亡抑制剂Nec-1、VX-765、CQ、Fer-1检测舒尼替尼干预后K562/ADR细胞的死亡方式。通过qPCR和Western blot检测舒尼替尼干预后K562/ADR细胞的焦亡相关基因mRNA和蛋白表达水平变化。结果:舒尼替尼可明显抑制K562/ADR细胞的增殖,抑制效果呈一定的时间及浓度依赖性(r_(48 h)=0.9579、r_(4μg/ml)=0.9740),其48 h的IC_(50)为(3.96±0.14)μg/ml;舒尼替尼干预后K562/ADR细胞凋亡相关基因Bax、BCL-2、Caspase-3、Caspase-9的mRNA和蛋白表达水平均无明显变化;4种不同细胞死亡抑制剂处理后,仅焦亡抑制剂VX-765能够明显逆转舒尼替尼对K562/ADR细胞的增殖抑制作用(P<0.01);舒尼替尼干预后K562/ADR细胞焦亡相关基因Caspase-1、Caspase-4、Caspase-5、NLRP3、GSDMD、IL-1β的mRNA和蛋白表达水平均明显升高(P<0.01)。结论:舒尼替尼可诱导耐药白血病细胞K562/ADR发生焦亡,深入研究细胞焦亡相关信号通路有望为耐药白血病治疗提供实验依据。