Inhibitory effect of norcantharidin on K562 human myeloid leukemia cells in vitro

Mylabris, the dried body of the Chinese blister beetle, has been used as a Chinese medicinal for over 2000 years. Its active constituent, cantharidin, has antitumor properties and causes leukocytosis. Norcantharidin (NCTD), the demethylated form of cantharidin, is easier to synthesize and is less toxic. NCTD irreversibly reduced the clonogenic efficiency of parental and drug-resistant K562 sublines, with drug-resistant sublines showing greater susceptibility to NCTD than parental cells. NCTD inhibited DNA synthesis by blocking cells at the G2/M phase of the cell cycle. The data suggest that NCTD may be suitable in the treatment of drug-resistant leukemia.     

去氢木香内酯对慢性粒细胞白血病K562细胞增殖的影响及其机制

目的探讨去氢木香内酯对慢性粒细胞白血病细胞株K562细胞增殖的影响及其机制。方法用不同浓度去氢木香内酯作用于对数生长期的K562细胞,采用瑞特-吉姆萨染色观察细胞形态,CCK-8法检测细胞增殖情况,流式细胞术检测细胞周期、凋亡情况及细胞表面分化抗原CD14和CD11b表达,蛋白质印迹法检测JAK-STAT通路、细胞凋亡及细胞周期相关蛋白表达。结果不同浓度(4.0、6.0、8.0、10.0、12.0μmol/L)去氢木香内酯作用24h均能抑制K562细胞增殖,与对照组比较,差异有统计学意义(F=109.510,P<0.05)。5.0、10.0μmol/L去氢木香内酯作用24h后,K562细胞凋亡率分别为(16.1±3.8)%、(29.6±4.3)%,较对照组的(3.1±0.5)%升高(F=83.255,P<0.05)。5.0、10.0μmol/L去氢木香内酯作用24h后,K562细胞的G2/M期细胞比例分别为(17.0±3.2)%、(28.8±3.9)%,较对照组的(9.1±2.3)%升高(F=161.598,P<0.05);S期细胞比例分别为(48.1±3.9)%、(61.0±5.4)%,较对照组的(39.6±3.6)%升高(F=192.356,P<0.05)。2.5、5.0μmol/L去氢木香内酯作用72h后,K562细胞的CD14表达率分别为(28.6±3.9)%、(41.1±4.4)%,较对照组的(3.1±0.5)%升高(F=132.811,P<0.05);K562细胞的CD11b表达率分别为(42.4±5.0)%、(61.2±5.7)%,较对照组的(4.2±1.1)%升高(F=179.553,P<0.05)。去氢木香内酯能够降低JAK2、STAT5、cyclinE、CDK2、cyclinA、CDC25C、cyclinB1、CDK1及bcl-2蛋白表达,上调p21及bax蛋白的表达。结论去氢木香内酯能够抑制慢性粒细胞白血病K562细胞增殖,可能是通过细胞周期阻滞、诱导凋亡及分化而实现的。     

DNA damage response in imatinib resistant chronic myeloid leukemia K562 cells

Abstract Resistance to imatinib in patients with chronic myeloid leukemia can lead to advanced disease and blast crisis. Conventional chemotherapy with DNA damaging agents is then used, alone or in combination with other tyrosine kinase inhibitors (TKIs). Our aim was to assess whether imatinib resistant K562 cells were also resistant to DNA damaging agents. After treatment with H(2)O(2) and doxorubicin, but not camptothecin, cell survival was higher in imatinib resistant cells compared to parental cells. DNA damage, measured by comet and γ-H2AX assays, was lower in imatinib resistant cells. mRNA expression levels of 50 genes of the DNA damage response pathway showed increased expression of the base excision repair (BER) genes MBD4 and NTHL1. Knockdown of MBD4 and NTHL1 expression in resistant cells using siRNA decreased cell survival after treatment with H(2)O(2) and doxorubicin. Our results indicate that imatinib resistant cells display cross-resistance to oxidative agents, partly through up-regulation of BER genes. Expression of these genes in imatinib resistant patients was not significantly different compared to sensitive patients. However, the strategy followed in this study could help identify chemotherapeutic agents that are more effective as alternative agents in cases of resistance to TKIs.     

Dihydroartemisinin downregulates vascular endothelial growth factor expression and induces apoptosis in chronic myeloid leukemia K562 cells

Dihydroartemisinin (DHA), a more water-soluble active metabolite of artemisinin derivatives, is safe and the most effective antimalarial analog of artemisinin. In the present investigation, we assessed the effect of DHA on vascular endothelial growth factor (VEGF) expression and apoptosis in chronic myeloid leukemia (CML) K562 cells. The results demonstrated that in addition to its antiproliferation effect on CML cells, DHA was also found to induce K562 cells apoptosis. The percentage of apoptotic cells was increased to 6.9 and 15.8% after being treated with 5 and 10 μmol/l DHA for 48 h, respectively (P<0.001). In order to analyze the effect of DHA on VEGF expression in K562 cells, we assessed the level of VEGF expression by western blot; detected the form of VEGF mRNA by RT-PCR and examined the level of VEGF secreted in conditioned media (CM) by ELISA assay. All these experiments suggested that DHA could inhibit the VEGF expression and secretion effectively in K562 cells, even at a lower concentration (2 μmol/l, P<0.05). Moreover, we further assessed the stimulating angiogenic activity of CM from K562 cells on CAM model. The angiogenic activity was decreased in response to the CM from K562 cells pretreated with DHA in a dose-dependent manner. Taken together, these results from our study together with its known low toxicity make it possible that DHA might present potential antileukemia effect as a treatment for CML therapy, or as an adjunct to standard chemotherapeutic regimens.     

苦参碱通过bcr-abl介导的MEK-ERK通路抑制人慢性粒细胞白血病K562细胞增殖

目的：探讨MEK-ERK通路在苦参碱抑制人慢性粒细胞白血病K562细胞增殖中的分子机制。方法采用Western blot法检测K562细胞内MEK-ERK通路关键分子MEK1、ERK1／2及其上游接头分子Shc、SHP2的总蛋白和磷酸化蛋白的表达;采用反转录聚合酶链反应（RT-PCR）和Western blot检测bcr-abl及有丝分裂原激活的蛋白激酶（MAPK）下游靶蛋白bcl-xL、Cyclin D1、c-myc及p27在转录及蛋白水平的表达。结果苦参碱可明显抑制K562细胞内MEK1、ERK1／2及Shc、SHP2的磷酸化表达,并可在转录及蛋白水平抑制bcr-abl分子表达。同时,RT-PCR和Western blot实验证实,苦参碱处理后,K562细胞内bcl-xL、Cyclin D1、c-myc表达均明显抑制,细胞周期负调控蛋白p27的表达增加。结论苦参碱对K562细胞的抑制效应与bcr-abl介导的MEK-ERK信号通路活性抑制有关,对信号通路中磷酸化蛋白或激酶分子活性的调控可能是其调控MEK-ERK通路活性的重要分子机制。     

Notch signaling inhibits the growth of the human chronic myeloid leukemia cell line K562

Notch signaling functions in the development of some types of leukemia and lymphoma, but the relationship between Notch signaling and chronic myeloid leukemia (CML) remains to be elucidated. In this study, we examined the expression of Notch receptors and ligands in the human CML cell line K562. When the active form of Notch1, the Notch intra-cellular domain (NIC), was over-expressed in K562, the proliferation of K562 was mildly but significantly inhibited, accompanied by increased Hes1 mRNA level. On the other hand, when Notch signaling was attenuated by over-expression of a dominant-negative RBP-J, RBP-J(R218H), in K562 cells, the proliferation of K562 was increased. Moreover, we found that activation of Notch signaling inhibited while repression of Notch signaling promoted the colony-forming activity of K562 cells. We examined cell cycle-related molecules in K562 transfected with NIC or RBP-J(R218H), and found that the protein level of the retinoblastoma gene product (the Rb protein) was induced in K562 expressing NIC, and down-regulated in K562 expressing RBP-J(R218H). These data suggest that the Notch signaling may function as a tumor inhibitor in human CML cells.     

Reproducible metastatic growth of K-562 human myelogenous leukemia cells in nude mice.

A reproducible metastatic growth of K-562 human myelogenous leukemia cells occurred in immunodeficient athymic (nude) mice. Although previous studies have shown that K-562 cells grow as local subcutaneous myelosarcomas which continuously release leukemia cells into the systemic circulation in adult mice, metastases were not observed. However, the subcutaneous "priming" of newborn nude mice resulted in the metastatic proliferation of leukemia cells in the lungs, kidneys, brain, and lymph nodes. Three sc injections of 5 X 10(6) K-562 cells on days 1, 7, and 14 of life produced metastases in 51% of the mice. When the initial series of injections was followed by iv injections on days 35 and 42, the incidence of metastases increased to 67%. Karyotypes demonstrated that the tumor cells retained the same human chromosome markers as those in the human patient and tissue culture. These procedures may provide a model for study of the mechanisms of metastases and for chemotherapeutic and immunotherapeutic trials against metastases of neoplasms of human origin.     

Inhibition of K562 leukemia angiogenesis and growth by expression of antisense vascular endothelial growth factor (VEGF) sequence

Vascular endothelial growth factor (VEGF), a major angiogenic factor, plays a key role in the growth of solid tumor. Recently, expression of VEGF and its receptors has been found on leukemic cells as well as on endothelial cells. VEGF may fulfill a fundamental role in promoting tumor angiogenesis and proliferation by stimulating both endothelial cells and leukemic cells. To investigate the role of VEGF in the angiogenesis and growth of leukemic cell, we used an antisense strategy to downregulate VEGF expression in K562 cells, a human erythroleukemia cell line. Expression of antisense-VEGF in K562 cells reduced the secretion of VEGF protein and inhibited cell survival. The proliferation and migration of human umbilical vein endothelial cells were decreased in response to the conditioned medium (CM) from K562 cells expressed antisense-VEGF, compared to CM from K562 cells transfected with vector control. Moreover, subcutaneous injection of nude mice with antisense-VEGF K562 cells inhibited tumor growth with a reduction of the density of microvessels and an increased apoptosis in those tumors, compared to vector control K562 cells. These results suggest that the efficient downregulation of the VEGF production in leukemic cells using antisense-VEGF may constitute a novel strategy of treatment in leukemia.     

脂质体转染survivin反义寡核苷酸对K562细胞增殖及凋亡的作用

目的:观察脂质体转染survivin反义寡核苷酸(ASODN)对白血病细胞K562的增殖及凋亡的影响。方法:人工合成survivin硫代ASODN,通过脂质体转染K562细胞,倒置显微镜下观察细胞形态学变化;用MTT法观察转染前后对细胞增生的影响;用细胞凋亡检测法(POD)观察细胞凋亡;RT-PCR检测survivin mRNA的表达。结果:(1)survivin ASODN抑制K562细胞增殖呈时间和剂量依赖性;(2)survivin ASODN可诱导K562细胞的凋亡,凋亡指数呈浓度依赖性;(3)survivinASODN处理组K562细胞survivin mRNA的表达水平显著下降。结论:脂质体转染survivin ASODN能够明显抑制K562细胞增殖,且能诱导细胞凋亡。survivin可能成为肿瘤基因治疗的一个新靶点。     

重组人血管内皮生长因子165对K562白血病细胞生物学活性的影响

目的：探讨人重组血管内皮生长的因子165对人K562白血病细胞生物学活性的影响。方法：利用pcDNA3.1（＋）质粒构建人重组含VEGF165的真核表达载体,脂质体转染至K562白血病细胞,并用G418筛选阳性克隆;以RT-PCR测定重组质粒载体VEGF165基因的表达;MTT法测定K562白血病细胞的生长;建立裸鼠皮下移植人K562肿瘤模型,测定肿瘤体积的大小;免疫组化检测肿瘤的微血管密度（MVD）。结果：成功构建pcDNA3.1（＋）-VEGF165表达质粒。K562白血病细胞转染重组质粒后,明显增加K562细胞中VEGF165 mRNA的表达,转染重组质粒的K562细胞增殖明显快于对照组;移植重组pcDNA3.1（＋）-VEGF165质粒转染的K562细胞之后,荷瘤鼠移植瘤生长明显快于对照组;并且移植瘤组织微血管密度明显高于对照组（P〈0.05）。结论：利用pc DNA3.1（＋）真核表达质粒可成功构建含人VEGF165的重组质粒,重组质粒转染K562细胞后,体内外均明显促进K562细胞增殖。     

反义 hTERT 基因对 K562 细胞凋亡及周期的影响简

目的：探讨反义hTERT基因抑制K562细胞端粒酶活性对其细胞周期及增殖的影响。方法：用脂质体介导反义hTERT基因和空载体质粒转染K562细胞株,绘制生长曲线,MTT法检测细胞活性,血清饥饿法同步化细胞周期,流式细胞仪检测细胞周期及细胞凋亡率变化。结果：反义hTERT基因转染后,细胞生长明显受到抑制。细胞同步化后,流式细胞仪检测显示：KA组细胞出现早期凋亡峰,细胞阻滞于S期。但正常培养48h,各组细胞无明显凋亡,周期无显著差异。结论：反义hTERT基因可明显抑制K562的增殖,细胞同步化后具有促凋亡作用。     

水通道蛋白1基因过表达对K562细胞红系分化和增殖的影响

目的:探讨水通道蛋白1(aquaporin-1,AQP1)过表达对人慢性髓细胞白血病(chronic myeloidleukemia,CML) K562细胞红系分化和增殖的影响.方法:以人脑cDNA文库为模板,通过PCR扩增出AQP1基因的编码序列,构建pBABE-puro-AQP1真核表达载体;感染K562细胞,筛选建立稳定过表达AQP1基因的K562细胞株(命名为K562-AQP1);实时荧光定量PCR法、细胞免疫荧光染色法及蛋白质印迹法分别检测AQP1转录和蛋白表达水平.通过MTT法检测细胞生长增殖、实时荧光定量PCR法检测γ珠蛋白表达和分光光度法检测血红蛋白含量,研究AQP1过表达对K562细胞红系分化和增殖的影响.结果:与空载体对照组相比,pBABE-puro-AQP1转染入K562细胞后AQP1 mRNA和蛋白表达水平皆有显著升高(P＜0.01),K562-AQP1细胞中红系分化指标γ珠蛋白和血红蛋白表达水平明显增加,同时细胞生长速度明显降低(P＜0.05).结论:AQP1过表达可以显著促进K562细胞向红系分化,同时抑制细胞增殖.推测AQP1可能成为临床诱导分化治疗CML的基因靶点之一.     

hTERT基因反义核酸对K562细胞端粒酶活性的影响

探讨人类端粒酶逆转录酶（ｈｕｍａｎ ｔｅｌｏｍｅｒａｓｅ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔａｓｅ,ｈＴＥＲＴ）基因的反义寡核苷酸（ａｎｔｉｓｅｎｓ ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,ＡＳＯＮＤ）对Ｋ５６２细胞端粒酶活性的影响。方法：采用多聚酶链反应－酶联免疫测定（ＰＣＲ－ＥＬＩＳＡ）方法检测人工合成的反义脱氧寡核苷酸处理Ｋ５６２细胞前后端粒酶活性的改变,以逆转录ＰＣＲ（ＲＴ－ＰＣＲ）分     

白血病K562细胞XIAP表达RNAi下调对凋亡及化疗敏感性影响的研究

目的:通过RNAi下调K562细胞X染色体连锁的凋亡抑制蛋白(XIAP)的表达,观察XIAP下调后细胞凋亡及对化疗药物敏感性的变化情况。方法:K562细胞分为3组,实验组和阴性对照组分别用XIAP siRNA及阴性对照siR-NA转染,空白对照组不做任何转染处理。用实时荧光定量PCR和蛋白质印迹法检测转染后各组细胞XIAP mRNA及蛋白的表达,CCK-8法检测各组细胞对阿糖胞苷和依托泊苷敏感性的变化,流式细胞仪和Hochest染色法检测各组细胞加入不同化疗药物后细胞的凋亡情况。结果:转染后48h,实验组XIAP mRNA及蛋白表达量较阴性对照组分别下降约70.0%和60.0%,P<0.05;实验组细胞对依托泊苷和阿糖胞苷的敏感性分别提高2.77和2.28倍,P<0.05;转染siR-NA 48h后,实验组与阴性对照组、空白对照组间凋亡率差异无统计学意义,P>0.05;转染siRNA 6h后加入依托泊苷孵育48h,实验组凋亡率(41.3±1.6)%较阴性对照组(34.3±1.9)%和空白对照组(30.6±1.7)%明显增加;加入阿糖胞苷孵育48h,实验组凋亡率(47.4±1.1)%较阴性对照组(37.0±1.8)%和空白对照组(35.5±1.7)%明显增加,P<0.05。结论:XIAP siRNA能特异性下调K562细胞的XIAP mRNA和蛋白质水平的表达,提高K562细胞对依托泊苷和阿糖胞苷的敏感性,同时伴化疗药物诱导的细胞凋亡增强。