RNA干扰和伊马替尼杀伤K562细胞效果的比较

目的比较RNA干扰（RNAi）和伊马替尼（imatinib）杀伤K562细胞的效果。方法设计有效的bcr—abl干扰shRNA序列，利用基因工程技术插入到干扰载体构建RNAi质粒。测序鉴定正确的RNAi质粒转染K562细胞，48h后荧光显微镜观察转染效率。RT—PCR技术检测K562细胞中bcr—abl表达水平。同时，伊马替尼作用K562细胞。利用凋亡分析、MTT增生实验和磷酸化的酪氨酸蛋白含量检测来评估RNAi和伊马替尼作用效果。结果与伊马替尼一样，RNAi使K562细胞凋亡增强，增生减少，磷酸化的酪氨酸蛋白含量减少。结论RNAi达到伊马替尼杀伤K562细胞的效果，有望在临床用于治疗慢性髓细胞白血病（CML）患者，尤其是那些对伊马替尼等化疗药物不敏感的CML患者。     

慢性粒细胞白血病DNA聚合酶β蛋白研究

目的探讨慢性粒细胞白血病（CML）DNA聚合酶β蛋白表达及其与CML急变机制的关系。方法用Western blotting方法检测62例CML患者及K562细胞DNA聚合酶β蛋白表达,并与23例健康人作对照;对26例异基因外周血干细胞移植（allo-PBSCT）及12例使用伊马替尼治疗的CML患者用RT-PCR、Western blotting分别动态检测bcr-abl mRNA和DNA聚合酶β蛋白表达;伊马替尼体外作用于CML患者MNC及K562细胞后,用Western blotting方法检测DNA聚合酶β表达及bcr-abl融合蛋白磷酸化。结果与健康人比较,CML患者及K562细胞DNA聚合酶β蛋白的表达明显升高（P〈0．05）。allo-PBSCT及伊马替尼治疗后患者DNA聚合酶β蛋白的表达随着bcr-abl mRNA表达的降低而下降。伊马替尼体外作用后,DNA聚合酶β蛋白表达随着bcr-abl融合蛋白酪氨酸磷酸化水平的降低而下降。结论bcr-abl融合基因增加DNA聚合酶β蛋白表达;DNA聚合酶β蛋白增加可能是CML急变机制之一。     

硼替佐米对伊马替尼耐药细胞株K562/G01增殖抑制和诱导凋亡作用的研究

 目的　探讨蛋白酶体抑制剂硼替佐米（BOR）对慢性粒细胞白血病（CML）伊马替尼耐药细胞株K562／G01的增殖抑制和诱导凋亡作用。方法　采用MTT法观察细胞的生长抑制效应;流式细胞术（FCM）检测细胞周期与凋亡。结果　K562／G01细胞对伊马替尼不敏感,伊马替尼对K562／G01和K562细胞的IC50分别是（20.09±1.38）、（0.54±0.13）μmol／L;BOR对K562／G01细胞具有增殖抑制作用,并于48 h时作用效果达高峰,IC50（23.10±2.71）nmol／L,随着BOR浓度和作用时间的增加,FCM检测可见G2／M 期细胞周期阻滞及明显的凋亡峰。结论　BOR对CML伊马替尼耐药细胞株具有增殖抑制和诱导凋亡的作用,其机制可能与细胞周期G2／M 期的阻滞有关。     

敲除CIAPIN1基因提高白血病K562细胞对伊马替尼的敏感性

目的： 探讨靶向干扰细胞因子诱导凋亡抑制因子1 （cytokine induced apoptosis inhibitor 1,CIAPIN1）基因表达后慢性 粒细胞性白血病K562细胞对伊马替尼的敏感性。 方法 ：构建靶向CIAPIN1基因的shRNA干扰载体;使用实时定量PCR、West- ern blotting以及免疫荧光评价CIAPIN1-shRNA组（CIAPIN1-shRNA转染）和scramble-shRNA组（scramble-shRNA转染K562细 胞）干扰效率;伊马替尼（imatinib）处理两组K562细胞后,MTT法检测其增殖能力,集落形成实验检测细胞克隆形成能力,流式细 胞术和Western blotting检测细胞周期、凋亡及凋亡相关蛋白的变化。 结果： shRNA干扰可有效抑制CIAPIN1表达,CIAPIN1- shRNA组的CIAPIN1 mRNA表达量为scramble-shRNA组的(29.74±4.03)%、CIAPIN1蛋白表达量前者为后者的(21.57±2.18)%。 CIAPIN1表达下调能显著抑制伊马替尼对K562细胞的增殖和克隆形成,CIAPIN1-shRNA+imatinib组的细胞克隆形成数为 （15.60±1.03）个/视野,克隆半径为（2.63±0.55）μm,均小于scramble-shRNA+imatinib组（P<0.05或P<0.01）。CIAPIN1-shRNA+ imatinib组的细胞G1期细胞比例比scramble-shRNA+imatinib组明显增多,S期细胞比例较scramble-shRNA+imatinib组明显减少 （均P<0.01）。CIAPIN1-shRNA+imatinib组K562细胞凋亡率明显增加（P<0.01）。敲除CIAPIN1能抑制细胞周期相关蛋白Cyclin D1、Bcl-xl、Bcl-2、Mcl-1表达,诱导细胞内细胞凋亡相关蛋白p21、Bid、Bim表达,且与伊马替尼具有协同作用。 结论： CIAPIN1表 达下调可明显提高K562细胞对伊马替尼的敏感性,该作用主要通过阻滞K562细胞周期及诱导凋亡相关蛋白表达实现。     

高三尖杉酯碱增强慢性粒细胞白血病细胞对于伊马替尼敏感性的研究*

目的: 研究高三尖杉酯碱（HHT）增强慢性粒细胞白血病（CML）细胞对伊马替尼（IM）敏感性的机制。 方法: 采集1例CML患者骨髓血标本,经体外筛选、克隆建立人白血病细胞株NPHA1（初治）和NPHA2（复发）。RNA干扰NPHA2细胞的EphB4蛋白表达,建立NPHA2-EphB4-sh细胞株。 结果: 与NPHA1细胞相比,耐IM的NPHA2细胞中EphB4过表达;在NPHA2-EphB4-sh细胞中,EphB4表达显著低于NPHA1细胞和NPHA2细胞（P<0.001）。NPHA2细胞对IM有明显耐药性（IC₅₀=5.45 mg/L）,但NPHA2-EphB4-sh对IM敏感性增加（IC₅₀=0.93 mg/L,P<0.001）。HHT与IM共处理后,NPHA2细胞对IM的IC50值降至1.17 mg/L（P<0.001）。蛋白磷酸化测定表明HHT抑制了EphB4/RhoA通路表达。 结论: EphB4/RhoA是一个新的IM耐药途径。HHT通过抑制EphB4/RhoA途径,使IM治疗获得优势。      

伊马替尼通过PDGF/PDGFR通路对A549非小细胞肺癌裸鼠移植瘤的抑制作用及机制研究

目的 探讨伊马替尼对A549非小细胞肺癌裸鼠移植瘤生长及肿瘤组织和基质中PDGFB、PDGFRβ蛋白表达的影响，探究伊马替尼的抑瘤机制。方法 建立裸鼠A549非小细胞肺癌移植瘤模型，随机分为对照组（0.9%NaCl）、低、中、高剂量伊马替尼组（50、100、200 mg/（kg·d））。连续灌胃给药28天，观察不同浓度伊马替尼对肿瘤生长的影响；HE染色观察肿瘤组织的病理变化；Western blot法检测移植瘤组织中PDGF/PDGFR通路相关蛋白的表达及AKT、ERK1/2蛋白磷酸化水平；双重免疫荧光染色检测PDGFB、PDGFRβ蛋白在肿瘤基质中的表达。结果 伊马替尼组裸鼠A549非小细胞肺癌移植瘤的生长受到抑制，肿瘤组织中PDGFB的表达降低，PDGFRβ、AKT、ERK1/2的磷酸化水平降低。与对照组相比，给药组的肿瘤基质成纤维细胞中PDGFB、PDGFRβ表达显著减少。结论 伊马替尼对裸鼠非小细胞肺癌A549移植瘤具有显著的抑制作用，其抑瘤机制可能是下调肿瘤基质成纤维细胞中PDGFB和PDGFRβ的表达。     

ERK参与裸鼠体内辛伐他汀诱导K 562细胞凋亡的调控作用

目的：探讨辛伐他汀（simvastatin）作用于裸鼠体内K562细胞后Ras-MAPK信号转导通路胞外信号调节激酶（extracellular signal—regulated protein kinase，ERK）分子水平的变化，以说明ERK参与裸鼠体内辛伐他汀诱导K562细胞凋亡的调控作用。方法：体外培养慢性髓细胞白血病（CML）细胞株K562细胞，构建BALB／c-nu／nu裸鼠的K562细胞移植瘤模型。流式细胞术（FCM）检测对照组和两个辛伐他汀处理组的K562细胞周期变化，TUNEL法检测K562细胞晚期凋亡情况。采用RT-PCR检测K562细胞中Ras-MAPK信号通路N-Ras、ERK1mRNA的差异表达。免疫组织化学标记葡聚糖聚合物（labbled dextran polymer，LDP）法检测P-ERK蛋白水平变化。结果：辛伐他汀能够明显抑制裸鼠K562移植瘤组织的增长，随着辛伐他汀剂量的增加，K562细胞移植瘤体积和质量明显减小（P〈0．05，P〈0．01）。每次注射0．05mg的辛伐他汀能够诱导裸鼠体内K562细胞发生明显的G0／G1期停滞，不同剂量的辛伐他汀能够诱导K562细胞发生明显的凋亡，并随剂量的增加，凋亡率逐渐增高（P〈0．01）；不同剂量的辛伐他汀能够引起N-Ras、ERK1mRNA的表达下调（P〈0．01）。与对照组相比，两个处理组的p-ERK蛋白分别出现表达下调（P=0．01，P〈0．01）。结论：辛伐他汀在体内可能依赖Ras—MAPK信号转导通路ERK基因和蛋白水平的表达下调诱导K562细胞凋亡发生。     

K562多药耐药细胞系中肿瘤干细胞样细胞对伊马替尼耐药机制的初步研究

 目的 进一步阐明一些高表达P-糖蛋白（P-gp）的慢性粒细胞白血病细胞对伊马替尼耐药的机制。方法 经过对K562细胞系长期的足叶乙苷（VP16）诱导和克隆筛选,建立一株耐药细胞系K562／VP16;利用干细胞高效能将Hoechst 33342 荧光染料泵出细胞的特性,采用流式细胞术,从K562／VP16细胞系中分选出一小群细胞,即边缘细胞（SP）,称为K562／VP16 SP细胞,并初步探讨其抗伊马替尼的机制。结果 bcr／abl和abl 蛋白在K562细胞、K562／VP16 SP细胞及非K562／VP16 SP细胞（non-SP K562／VP16）中的表达水平差异无统计学意义;P-gp在K562细胞中不表达,在K562／VP16 SP及non-SP K562／VP16细胞中均高表达且表达水平一致;与non-SP K562／VP16细胞比较,K562／VP16 SP细胞对伊马替尼的耐药性更强,并且这种抗性几乎不能被多种多药耐药逆转剂逆转;另外,体内外实验显示,K562／VP16细胞的致瘤性几乎全部来源于K562／VP16 SP细胞。结论 bcr／abl基因的扩增、过度表达和多药耐药基因及其蛋白表达产物P-gp的高表达,可能不是白血病细胞产生对伊马替尼临床耐药的重要机制;白血病细胞对伊马替尼具有一定的抗性,可能与数量极少的白血病干细胞有直接的关系。因此,这类数量极少的干细胞样的肿瘤细胞应当成为有效治疗肿瘤的靶细胞。     

重组腺病毒Ad5F35-SH2-DED对K562细胞裸鼠致瘤能力的影响

目的:研究融合蛋白SH2-DED(SD)对白血病K562细胞裸鼠皮下移植瘤的影响.方法:建立K562细胞裸鼠皮下移植瘤的重组腺病毒预防和治疗模型.预防模型采用皮下注射经重组腺病毒Ad5F35-SD或Ad5F35-SmD预处理的K562细胞;治疗模型采用皮下注射K562细胞建立皮下移植瘤后,进行Ad5F35-SD或Ad5F35-SmD的瘤体内多点注射.观察裸鼠移植瘤的生长情况以及移植瘤肿瘤细胞的形态学变化.应用TUNEL法和电子显微镜观察移植瘤肿瘤细胞的凋亡情况.结果:治疗模型组中,Ad5F35-SD处理组移植瘤体积明显缩小,肿瘤细胞核浓缩,细胞质染色加深.TUNEL法和电子显微镜检测结果提示,肿瘤细胞发生凋亡,且凋亡相关蛋白caspase-3和caspase-8表达上调.Ad5F35-SD在预防模型组中同样具有抑制移植瘤生长的作用.结论:重组腺病毒Ad5F35-SD能够抑制K562细胞在裸鼠体内的致瘤能力以及裸鼠皮下移植瘤的生长,并促进肿瘤细胞凋亡.     

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.     

Silencing of Suppressor of Cytokine Signaling-3 due to Methylation Results in Phosphorylation of STAT3 in Imatinib Resistant BCR-ABL Positive Chronic Myeloid Leukemia Cells

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulatorgene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinibmesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematologicalremission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patientstreated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profileof SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials andMethods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assayswere performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profileswere investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Geneexpression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1,2 and 3 were examined by Western blotting. Results: The IC50 for imatinib on K562 was 362nM compared to3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing theconcentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation ofthe SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed downregulationof both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but notK562. Conclusions: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells.Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNAmethylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying thedevelopment of imatinib resistant in CML patients.     

Re-Expression of Bone Marrow Proteoglycan-2 by 5-Azacytidine is associated with STAT3 Inactivation and Sensitivity Response to Imatinib in Resistant CML Cells

Background: Epigenetic silencing of tumor suppressor genes (TSG) is involved in development andprogression of cancers. Re-expression of TSG is inversely proportionate with STAT3 signaling pathways.Demethylation of DNA by 5-Azacytidine (5-Aza) results in re-expression of silenced TSG. Forced expression ofPRG2 by 5-Aza induced apoptosis in cancer cells. Imatinib is a tyrosine kinase inhibitor that potently inhibits BCR/ABL tyrosine kinase resulting in hematological remission in CML patients. However, majority of CML patients treatedwith imatinib would develop resistance under prolonged therapy. Methods: CML cells resistant to imatinib weretreated with 5-Aza and cytotoxicity of imatinib and apoptosis were determined by MTS and annexin-V, respectively.Gene expression analysis was detected by real time-PCR, STATs activity examined using Western blot and methylationstatus of PRG2 was determined by pyrosequencing analysis. Result: Expression of PRG2 was significantly higher inK562-R+5-Aza cells compared to K562 and K562-R (p=0.001). Methylation of PRG2 gene was significantly decreasedin K562-R+5-Aza cells compared to other cells (p=0.021). STAT3 was inactivated in K562-R+5-Aza cells which showedhigher sensitivity to imatinib. Conclusion: PRG2 gene is a TSG and its overexpression might induce sensitivity toimatinib. However, further studies are required to evaluate the negative regulations of PRG2 on STAT3 signaling.     

(-)Gossypol and its combination with imatinib induce apoptosis in human chronic myeloid leukemic cells

Chronic myeloid leukemia (CML) is characterized by the presence of chimeric protein BCR-ABL associated with high tyrosine kinase (TK) activity, which leads to cell tumorogenicity, resistance to apoptosis, and differentiation. Gossypol is a natural polyphenolic compound isolated from cottonseed and has antiproliferative activity in a variety of cancer cell lines. (-)Gossypol is proved the potent component. Here we examined the growth inhibitory effect of (-)gossypol and its combination with imatinib in K562 cells. (-)Gossypol inhibited cell growth, promoted apoptosis, induced DeltaPsim loss, and cytochrome C release. Furthermore, (-)gossypol had a synergistic inhibitory effect on growth in K562 cells when combined with imatinib. Enhanced apoptosis, cytochrome C release, and caspase 3 cleavage as well as noticeable decrease of Mcl-1 and Bcl-XL were observed in K562 cells treated with both (-)gossypol and imatinib. These results suggest that (-)gossypol induced apoptosis in K562 cells through a mitochondria pathway and that the combination of imatinib and (-)gossypol might be an effective treatment for CML.     

XIAP and P-glycoprotein co-expression is related to imatinib resistance in chronic myeloid leukemia cells

P-glycoprotein (Pgp) and XIAP co-expression has been discussed in the process of the acquisition of multidrug resistance (MDR) in cancer. Here, we evaluated XIAP and Pgp expression in chronic myeloid leukemia (CML) samples, showing a positive correlation between them. Furthermore, we evaluated the effects of imatinib in XIAP and Pgp expression using CML cell lines K562 (Pgp⁻) and K562-Lucena (Pgp⁺). Imatinib increased XIAP and Pgp expression in K562-Lucena cells, while in K562 cells a downregulation of these proteins was observed, suggesting that imatinib induces an increment of MDR phenotype of CML cells that previously exhibit high levels of Pgp/XIAP co-expression.     

Contribution of BCR–ABL-independent activation of ERK1/2 to acquired imatinib resistance in K562 chronic myeloid leukemia cells

BCR–ABL tyrosine kinase, generated from the reciprocal chromosomal translocation t(9;22), causes chronic myeloid leukemia (CML). BCR–ABL is inhibited by imatinib; however, several mechanisms of imatinib resistance have been proposed that account for loss of imatinib efficacy in patients with CML. Previously, we showed that overexpression of the efflux drug transporter P-glycoprotein partially contributed to imatinib resistance in imatinib-resistant K562 CML cells having no BCR–ABL mutations. To explain an additional mechanism of drug resistance, we established a subclone (K562/R) of the cells and examined the BCR–ABL signaling pathway in these and wild-type K562 (K562/W) cells. We found the K562/R cells were 15 times more resistant to imatinib than their wild-type counterparts. In both cell lines, BCR–ABL and its downstream signaling molecules, such as ERK1/2, ERK5, STAT5, and AKT, were phosphorylated in the absence of imatinib. In both cell lines, imatinib effectively reduced the phosphorylation of all the above, except ERK1/2, whose phosphorylation was, interestingly, only inhibited in the wild-type cells. We then observed that phospho-ERK1/2 levels decreased in the presence of siRNA targeting BCR–ABL, again, only in the K562/W cells. However, using an ERK1/2 inhibitor, U0126, we found that we could reduce phospho-ERK1/2 levels in K562/R cells and restore their sensitivity to imatinib. Taken together, we conclude that the BCR–ABL-independent activation of ERK1/2 contributes to imatinib resistance in K562/R cells, and that ERK1/2 could be a target for the treatment of CML patients whose imatinib resistance is due to this mechanism. ( Cancer Sci 2009)     

Vandetanib drives growth arrest and promotes sensitivity to imatinib in chronic myeloid leukemia by targeting ephrin type‐B receptor 4

The oncogenic role of ephrin type‐B receptor 4 (EPHB4) has been reported in many types of tumors, including chronic myeloid leukemia (CML). Here, we found that CML patients have a higher EPHB4 expression level than healthy subjects. EPHB4 knockdown inhibited growth of K562 cells (a human immortalized myelogenous leukemia cell line). In addition, transient transfection of EPHB4 siRNA led to sensitization to imatinib. These growth defects could be fully rescued by EPHB4 transfection. To identify an EPHB4‐specific inhibitor with the potential of rapid translation into the clinic, a pool of clinical compounds was screened and vandetanib was found to be most sensitive to K562 cells, which express a high level of EPHB4. Vandetanib mainly acts on the intracellular tyrosine kinase domain and interacts stably with a hydrophobic pocket. Furthermore, vandetanib downregulated EPHB4 protein via the ubiquitin‐proteasome pathway and inhibited PI3K/AKT and MAPK/ERK signaling pathways in K562 cells. Vandetanib alone significantly inhibited tumor growth in a K562 xenograft model. Furthermore, the combination of vandetanib and imatinib exhibited enhanced and synergistic growth inhibition against imatinib‐resistant K562 cells in vitro and in vivo. These findings suggest that vandetanib drives growth arrest and overcomes the resistance to imatinib in CML via targeting EPHB4.