三氧化二砷诱导K562/ADM细胞内质网应激反应性凋亡的机制研究

内质网应激反应性凋亡途径是不同于经典的线粒体途径和死亡受体途径的细胞凋亡适路.三氧化二砷(A2O3)对白血病以及其他实体肿瘤均具有良好的抗肿瘤效应,且与常规化疗药物无交叉耐药性[1].     

三氧化二砷经内质网应激反应诱导K562/ADM耐药细胞凋亡

目的　探讨三氧化二砷（As2O3）能否通过内质网应激反应性凋亡途径诱发耐药白血病细胞凋亡。方法　采用细胞形态学和AnnexinV/PI 双染色法检测细胞凋亡,电镜观察凋亡细胞内质网和线粒体形态结构变化;流式细胞术（FCM）测定线粒体跨膜电位（Δψm）、细胞内Ca2+和细胞色素c（Cyt c）含量及caspase-3活性;Western blot法检测GRP78/Bip蛋白表达。结果　2、5 mol/L As2O3诱导K562/ADM细胞发生凋亡过程中,内质网明显扩张和脱颗粒,线粒体内外膜融合,嵴紊乱,肿胀,内膜扩张呈空泡样变。线粒体Δψm降低,细胞质Ca2+和Cyt c水平明显升高, caspase-3活性显著增强,GRP78蛋白表达增高。结论　As2O3可诱导K562/ADM细胞发生内质网应激反应,通过内质网-线粒体途径诱导耐药白血病K562/ADM细胞凋亡。     

三氧化二砷诱导K562/ADM细胞内质网应激反应性凋亡的机制研究

内质网应激反应性凋亡途径是不同于经典的线粒体途径和死亡受体途径的细胞凋亡适路.三氧化二砷(A2O3)对白血病以及其他实体肿瘤均具有良好的抗肿瘤效应,且与常规化疗药物无交叉耐药性[1].     

白血病K562/ADM细胞耐药性与白血病干细胞及耐药蛋白表达的关系

Objective To investigate the drug resistance, proportion of leukemia stem cells (LSC) and expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-sensitive and multidrug-resistant leukemia cell population. Methods The multidrug-resistant leukemia K562/ADM cell and its parental K562 cell were used as the model cells. The drug sensitivity was tested with a MTI" assay. Flow cytometry was employed to detect the immunophenotype of stem celia and the expression of P-gp and BCRP. The serf-renewal and proliferating potential were examined with methylcellulose colony-formlng unit assay. Results K562/ADM cells were highly resistant to adriamycin, daunorubincin and etoposide. The amount of CD34+, CD123+ and CD34+ CD38- cells in K562/ADM cells was much higher than that in K562 cells, and the proportion of CD34+ CD38-CD123+ cells(LSC) in K562/ADM cells was (5.23 ± 0. 21) % versus (1.27 ± 0. 17) % in K562 cells, which was 4. 12-fold higher than that in K562 cells. Both P-gp and BCRP were overexpressed in K562/ADM cells relative to K562 cells, and the K562/ADM cells co-expressing P-gp and BCRP were 11.25-fold higher over K562 cells. The proportion of CD34+ CD38- CD123+BCRP+ and CD34+ CD38- P-gp+ BCRP+cells in K562/ADM cells were (4. 13±0.40)% and (5. 80 ± 1.19)% respectively, which were 3.66- and 11. 37-fold higher than the same cells in K562 cells [(1.13 ± 0. 15) % and (0. 51 ± 0. 01) %]. Furthermore, drug-resistant K562/ADM cells displayed 4. 17-time greater colony-forming ability over the parent K562 cells, corresponding to the proportion of LSCs in K562/ADM cells. Conclusions The ABC transporters-overexpressing LSC population exists in drug-resistant leukemic K562/ADM cells relative to drug-sensitive K562 cells, and the drug-resistant LSCs may be the source of chemotherapeutic resistance of leukemia.     

白血病K562/ADM细胞耐药性与白血病干细胞及耐药蛋白表达的关系

Objective To investigate the drug resistance, proportion of leukemia stem cells (LSC) and expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-sensitive and multidrug-resistant leukemia cell population. Methods The multidrug-resistant leukemia K562/ADM cell and its parental K562 cell were used as the model cells. The drug sensitivity was tested with a MTI" assay. Flow cytometry was employed to detect the immunophenotype of stem celia and the expression of P-gp and BCRP. The serf-renewal and proliferating potential were examined with methylcellulose colony-formlng unit assay. Results K562/ADM cells were highly resistant to adriamycin, daunorubincin and etoposide. The amount of CD34+, CD123+ and CD34+ CD38- cells in K562/ADM cells was much higher than that in K562 cells, and the proportion of CD34+ CD38-CD123+ cells(LSC) in K562/ADM cells was (5.23 ± 0. 21) % versus (1.27 ± 0. 17) % in K562 cells, which was 4. 12-fold higher than that in K562 cells. Both P-gp and BCRP were overexpressed in K562/ADM cells relative to K562 cells, and the K562/ADM cells co-expressing P-gp and BCRP were 11.25-fold higher over K562 cells. The proportion of CD34+ CD38- CD123+BCRP+ and CD34+ CD38- P-gp+ BCRP+cells in K562/ADM cells were (4. 13±0.40)% and (5. 80 ± 1.19)% respectively, which were 3.66- and 11. 37-fold higher than the same cells in K562 cells [(1.13 ± 0. 15) % and (0. 51 ± 0. 01) %]. Furthermore, drug-resistant K562/ADM cells displayed 4. 17-time greater colony-forming ability over the parent K562 cells, corresponding to the proportion of LSCs in K562/ADM cells. Conclusions The ABC transporters-overexpressing LSC population exists in drug-resistant leukemic K562/ADM cells relative to drug-sensitive K562 cells, and the drug-resistant LSCs may be the source of chemotherapeutic resistance of leukemia.     

三氧化二砷诱导K562/ADM细胞内质网应激反应性凋亡的机制研究

内质网应激反应性凋亡途径是不同于经典的线粒体途径和死亡受体途径的细胞凋亡适路.三氧化二砷(A2O3)对白血病以及其他实体肿瘤均具有良好的抗肿瘤效应,且与常规化疗药物无交叉耐药性[1].     

白血病K562/ADM细胞耐药性与白血病干细胞及耐药蛋白表达的关系

目的 观察白血病药物敏感和耐受细胞中白血病干细胞(ISC)、耐药蛋白表达和耐药性的关系.方法 以白血病多药耐药株K562/ADM细胞及其亲本K562细胞为模型,MTT比色法测定细胞的药物耐受性;流式细胞术检测细胞免疫标志、P-糖蛋白(P-gp)和乳腺癌耐药蛋白(BCRP)的表达;甲基纤维素集落形成法检测细胞的自我更新和增殖潜能.结果 K562/ADM细胞对阿霉素、柔红霉素和鬼臼乙叉苷高度耐受.K562/ADM细胞中CD34+、CD123+和CD34+CD38-细胞含量均显著高于K562细胞,LSC细胞相对含量是K562细胞的4.12倍;共表达P-gP和BCRP的K562/ADM细胞比K562细胞高11.25倍,其中CD34+ CD38- CD123+ BCRP+和CD34+ CD38- P-gp+ BCRP+细胞数量分别为K562细胞的3.66倍和11.37倍.K562/ADM细胞集落形成率是K562细胞的4.17倍,与LSC含量相当.结论 白血病K562/ADM耐药细胞中存在的高表达耐药蛋白的耐药性LSC是其多药物耐受性的根源.     

胰岛素抵抗的人肝癌细胞HepG2对多柔比星的敏感性

目的:观察胰岛素抵抗(insulin resistance, IR)的人肝癌细胞对抗癌药物多柔比星(adriamycin,ADM)的敏感性,并探讨其抗药机制.方法:采用5×10-6 moL/L胰岛素诱导(36和48 h)人肝癌细胞HepG2建立HepG2/IR细胞模型,并用盐酸吡格列酮(pioglitazone hydrochloride, PH)逆转HepG2/IR细胞的IR状态.MTT法检测HepG2/IR细胞对ADM的敏感性,FCM检测P糖蛋白(P-glycoprotein, P-gp)的表达水平,实时荧光定量RT-PCR检测多药耐药基因1(multiple drug resistance 1, MDR1)mRNA的表达.结果:HepG2/IR细胞对ADM的敏感性降低(P<0.05),MDR1 mRNA的表达量分别是HepG2细胞的1.62倍(胰岛素诱导36 h)和5.21倍(胰岛素诱导48 h),P-gp蛋白的阳性表达率分别提高了47.50%(胰岛素诱导36 h)和189.05%(胰岛素诱导48 h).PH可逆转HepG2/IR细胞MDR1/P-gp的表达增加以及逆转细胞对ADM的抗药性.结论:胰岛素诱导的人肝癌细胞HepG2/IR可通过增加MDR1/P-gp的表达,使细胞对抗癌药物产生抗药性.     

胰岛素抵抗肝细胞多药耐药基因1的表达

目的体外观察诱导发生胰岛素抵抗（IR）肝细胞中多药耐药基因1（mdr1）的表达。方法采用高浓度胰岛素诱导肝源性细胞HepG2建立IR细胞模型（HepG2/IR细胞）,GOD-POD微量化法测定葡萄糖消耗量,RT-PCR检测HepG2/IR细胞mdr1和胰岛素受体（InsR）基因mRNA的表达,流式细胞术检测P糖蛋白（P-gp）和InsR蛋白水平。结果HepG2/IR细胞葡萄糖消耗量降低10%～45%,InsR基因mRNA表达显著下调,受体表达量降低50.2%～82.9%;mdr1表达显著增强,mRNA转录增高0.7～2.1倍,P-gp表达阳性细胞增加0.6～1.7倍,表达强度增高。结论IR肝细胞mdr1和P-gp的表达显著增强。     

小白菊内酯对白血病K562细胞及其干细胞的作用

目的:研究小白菊内酯(parthenolide,PTL)对白血病K562细胞及其白血病干细胞(leukemia stem cells,LSC)的作用.方法:以白血病K562细胞为靶细胞,四氮唑蓝(MTT)比色法测定细胞增殖活性,Annexin V/PI染色法测定细胞凋亡;流式细胞术检测LSC相对含量,甲基纤维素集落形成法检测细胞的自我更新和增殖能力.结果:PTL显著抑制K562细胞的增殖,24,48,72 h的IC_(50)分别为17.1,8.67,9.42μmol·L~(-1).5,10 μmol·L~(-1)PTL处理48 h,K562细胞的凋亡率分别为(49.56±5.11)%,(71.88±2.12)%.结合干细胞免疫标志分析,K562细胞中LSC样(CD34~+ CD38~-)细胞的凋亡率分别为(52.63±4.14)%,(57.50±4.47)%.K562细胞中LSC的相对含量仅轻度增高,但高浓度(15 μmol·L~(-1))PTL处理,LSC含量则增高15倍.0.5～4.0 μmol·L~(-1)PTL显著抑制K562细胞的集落形成能力,集落数降低24.1%～89.2%;5～15 μmol·L~(-1)PTL预处理,存活K562细胞的集落形成数增高5.0%～50.0%.结论:小白菊内酯可抑制K562细胞及其干细胞的增殖活性,并诱导其凋亡.