自噬在白血病K562/ADM细胞多药耐药性及三氧化二砷抗白血病效应中的作用
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摘要
目的：白血病是造血干细胞的恶性克隆性疾病,近几十年中,得益于化疗药物的联合应用及骨髓移植治疗技术的进展,使其治疗取得了长足的发展,但化学治疗药物诱发产生的白血病细胞多药耐药性(multi-drug resistance, MDR)是影响临床白血病治疗效果的主要障碍,也是影响白血病骨髓移植有效性和成功率的重要因素,目前仍对白血病耐药性缺乏有效而实用的干预措施。研究证实细胞自噬(autophagy)与肿瘤／白血病的发生、发展、预后和治疗效果的关系密切,但细胞自噬活性与肿瘤／白血病对治疗药物的敏感性及耐药性发生的关系尚不清楚。我们选用白血病多药耐药细胞株K562/ADM细胞及其亲本细胞株K562细胞为研究细胞模型,采用营养剥夺(饥饿)的方法诱导白血病细胞发生自噬,比较研究白血病耐药性细胞和敏感细胞的自噬活性的差异以及抑制自噬活性对耐药细胞药物敏感性的影响,探讨细胞自噬活性在白血病细胞多药耐药性中的作用及其调控机制。同时,三氧化二砷因与常规化疗药物无交叉耐受性而应用于临床耐药性白血病的治疗,研究三氧化二砷是否通过细胞自噬途径发挥抗耐药性白血病的作用,对于阐明三氧化二砷治疗耐药性白血病的机制有重要应用价值。
方法：对数生长期的白血病细胞株K562和K562/ADM细胞,以无血清和无氨基酸的磷酸缓冲液(phosphate buffered solution, PB S)代替RPIM-1640+小牛血清培养液培养细胞,培养Oh、3h、6h、9h、12h后收集细胞,透射电镜观察自噬体形态,MDC(单丹磺酰戊二胺)荧光染色、荧光显微镜和流式细胞仪检测自噬体荧光染色；实时荧光定量PCR (RT-qPCR)检测微管相关蛋白1轻链3(microtubule-associated protein-1light chain3, LC3) LC3和自噬相关基因Beclin-1mRNA表达, Western Blotting检测LC3和Beclin-1蛋白的表达以及LC3-Ⅰ向LC3-Ⅱ的转化。同时,采用RT-qPCR法和流式细胞术分别检测细胞自噬过程中多药耐药基因1(mdrl)/P-糖蛋白(P-gp)表达的动态变化。不同浓度阿霉素单独或联合自噬抑制剂3-MA(5mmol/L)作用K562/ADM细胞24h-72h, MIT测定K562/ADM细胞的增殖活性,Annexin V/PI双染、流式细胞术测定K562/ADM细胞的凋亡。透射电镜观察自噬体形态,Western Blotting法检测LC3的表达及LC3-Ⅰ和LC3-Ⅱ的转化。不同浓度三氧化二砷作用K562细胞及K562/ADM细胞不同时间后,透射电镜观察自噬体形态,倒置荧光显微镜及流式细胞仪检测自噬体MDC染色；RT-qPCR法检测Benlin-1与Bcl-2的mRNA表达,Western Blotting分析检测Beclin-1、Bcl-2和LC3蛋白表达以及LC3-Ⅰ向LC3-Ⅱ的转化。不同浓度三氧化二砷单独或联合自噬抑制剂3-MA (5mmol/L)作用K562和K562/ADM细胞24-48h,MTT测定细胞的增殖活性,Annexin V/PI双染、流式细胞术测定细胞的凋亡。
结果：1.与K562细胞相比,K562/ADM耐药细胞具有较高的基础自噬活性；饥饿法诱导K562与K562/ADM细胞发生自噬,在诱导的早期(3h) K562/ADM耐药细胞的自噬活性即迅速增高,而敏感的K562细胞的自噬活性发生较晚(9h),提示耐药白血病细胞早期而快速发生的自噬活性更有利于应对外界环境改变、保持细胞稳定性；自噬相关基因Beclin-1参与自噬的调节,K562/ADM细胞的表达高于K562细胞,与自噬活性的变化一致。有趣的是,K562/ADM细胞的mdrl/P-gp的表达随细胞自噬活性增强而表达增高,3-MA抑制自噬活性其表达相应降低。提示P-gp可能参与耐药性白血病细胞自噬活性的调控。
2.K562/ADM细胞对阿霉素诱导的凋亡效应有很强的抵抗性。10μmol/L的阿霉素处理K562/ADM细胞48h,电镜下可见明显的自噬形态改变, Western Blotting检测可见LC3-Ⅰ向LC3-Ⅱ转化增强,说明阿霉素处理可诱导K562/ADM耐药细胞发生较强的自噬。若用3-MA处理可显著减低K562/ADM细胞的自噬活性,阿霉素诱导的K562/ADM细胞的凋亡显著增高、存活细胞率明显减少。提示抑制细胞自噬活性可提高K562/ADM细胞对阿霉素的敏感性、降低其耐药性。
3.K562细胞和K562/ADM细胞均对三氧化二砷具有较高的敏感性,在不同浓度三氧化二砷诱导K562细胞和K562/ADM细胞增殖抑制和凋亡过程中,两种细胞均发生显著的自噬活性改变,自噬活性差异并不明显,但K562/ADM细胞自噬的出现较K562细胞为早。同时,三氧化二砷可增高K562和K562/ADM细胞Beclin-1基因mRNA及蛋白的表达,而显著降低Bcl-2基因mRNA及蛋白的表达,Beclin-1可能通过与Bcl-2的相互作用参与三氧化二砷诱导白血病细胞凋亡和自噬过程。3-MA抑制细胞自噬活性,可提高三氧化二砷诱导的K562细胞和K562/ADM细胞的死亡率,但增高的程度均并不十分显著。综合分析结果提示三氧化二砷在K562细胞和K562/ADM细胞中诱发的自噬活性,早期主要为应激性的保护反应,但随三氧化二砷作用时间的延长和诱导凋亡作用的增强,K562/ADM耐药细胞的自噬活性反而有所降低,提示三氧化二砷主要诱导耐药性白血病细胞发生细胞凋亡而非自噬性死亡,细胞自噬可能参与三氧化二砷的抗白血病机制但非主要因素。
结论：1.白血病K562/ADM耐药细胞较其亲本K562敏感细胞具有更高的基础自噬活性,营养剥夺可引发白血病耐药性细胞发生早期而快速的自噬活性增强。Beclin-1参与自噬的调节,抑制细胞自噬活性可明显增高K562/ADM耐药细胞对阿霉素的敏感性、降低药物抵抗性,证明细胞自噬是参与白血病K562/ADM细胞多药耐药性形成的机制之一。
2.营养剥夺诱导K562/ADM耐药细胞发生自噬过程中mdr1/P-gp的表达随细胞自噬活性增强而表达增高,细胞自噬活性被自噬抑制剂3-MA抑制其表达相应降低。mdr1/P-gp的表达变化与耐药细胞自噬活性的变化高度一致,提示P-gp可能参与耐药性白血病细胞自噬活性的调控,具体机制有待进一步探讨。
3.三氧化二砷可诱发K562细胞和K562/ADM细胞发生自噬反应,Beclin-1及Bcl-2参与了其自噬形成机制,但随三氧化二砷作用时间的延长和诱导凋亡作用的增强,耐药细胞的自噬活性反而有所降低,抑制自噬活性白血病细胞对三氧化二砷的敏感性有所增强但不显著。提示三氧化二砷在耐药性白血病中诱发的早期自噬活性主要为应激性的保护反应,三氧化二砷主要诱导白血病细胞发生细胞凋亡而非自噬性死亡,细胞自噬可能参与三氧化二砷的抗白血病机制但非主要因素。
Objective:Leukemia is a malignant clonal disease of hematopoietic stem cells, characterized by abnormal primitive and naive cells increased in the bone marrow. The treatment was improved due to the use of combination of chemotherapy drugs and bone marrow transplantation recent decades, but the overall prognosis remains still poor. The major obstacle to clinical outcomes of AML is multidrug resistance of leukemia cells induced by chemotherapy drug, which also is the main factor to affect the effectiveness and success rate of leukemic bone marrow transplantation. Clinical leukemia is resistance to the lack of effective interventions. There are still no effective interventions to multidrug resistance of AML currently. Autophagy is very important for the pathogenesis and development of tumor/leukemia. Then the role of autophagy in Hematological malignancies and its relationship with drug-induced resistance mechanism is not yet fully understood. Therefore, the multidrug-resistant acute myeloid leukemia cell line K562/ADM and its parental cell line K562were used to investigate the change of autophagic activity induced by starvation and drugs, the relationship between autopahagy and the multi-drug resistance of K562/ADM cells, and the role of autophagy in the effect of arsenic trioxide in leukemia cells.
Methods:We induced human multidrug-resistant leukemia K562/ADM cells and its drug-sensitive parental K562cells by starvation (0h,3h,6h,9h and12h) in vitro. Autophagic vacuoles were observed by electron microscope, the increased MDC (Monodansylcadaverin)-positive cells were detected by Fluorescence microscope and Flow cytometry; Furthermore, the expression of LC3mRNA and Beclin-1mRNA were detected by real-time quantitative PCR (RT-qPCR), the expression of LC3, Beclin-1and the result of LC3-Iconverted to LC3-Ⅱ were detected byWestern Blotting.The expression of mdrl mRNA was detected by RT-qPCR, P-glycoprotein of K562/ADM was detected by flow cytometry.The proliferation of K562/ADM cells treated with different concentrations of doxorubicinand in combination with3-MA for24-72h were detected with MTT assay, the apoptotic rates of K562/ADM treated with different concentrations of doxorubicinand in combination with3-MA for24-72h were detected with flow cytometry in order to explore the relationship between autophagy and apoptosis.Adriamycin inducedautophagic changes in K562/ADM cells, autophagic vacuoles were observed by electron microscope,and the result of LC3-Iconverted to LC3-II were detected by Western blotting. Autophagic vacuoles were observed in As2O3-treated K562and K562/ADM cells using electron microscope. MDC-labeled autophagic vacuoles were observed by fluorescent inverted phase contrast microscopy in As2O3-treated cells. The changes of LC3protein expression were detected byWestern Blotting. Furthermore, real-time quantitative PCR and Western blotting revealed that the expression of Beclin-1and Bcl-2. The apoptotic rates of K562/ADM treated with different concentrations of AS2O3and in combination with3-MA were detected with flow cytometry in order to study the possible molecular mechanism of leukemia cell death induced by arsenic trioxide.
Results:1. Compared to K562cells, the autophagy activity of K562/ADM cells was higher under normal circumstances, Starvation could induce the changes of autophagic activity of K562and K562/ADM cells, the autophagic activity of K562/ADM cells occurred faster and earlier than K562cells to environmental changes in order to maintain the stability of cells; Beclin-1was involved in the regulation of autophagy, the expression of Beclin-1gene mRNA and protein in control K562/ADM cells were significantly higher than that in control K562cells, Moreover, we found that the expression of mdrl mRNA and P-gp in K562/ADM cells incresed as enhanced autophagic activity, after inhibited the autophagic activity of K562/ADM cells with3-MA, the expression of mdrl mRNA and P-gpdecreased. That suggested P-gp may be involved in the regulation of autophagic activity in K562/ADM cells.
2. K562/ADM cell was resistant to doxorubicin. K562/ADM cells treated with10μmol/L of Adriamycin for48hours, obvious autophagic vacuoles were observed under electron microscope, enhanced expression of LC3-II protein was detected by Western Blotting, electron microscope and Western Blotting also revealed that doxorubicin could induce increased autophagic activity.3-MA, as an inhibitor of autophagy, could enhance the inhibited effect of K562/ADM cells induced by doxorubicin, the mortality rate of K562/ADM cells treated with3-MA and doxorubicin detected by flow cytometry was higher than treated with doxorubicin alone.
3. After treated with different concentrations As2O3, The autophagic activity of K562and K562/ADM cells raised,the autophagic activity of K562/ADM cells increased earlier than K562cells treated with As2O3. The expression levels of Beclin-1, which play key roles in autophagy, increased in As2O3treated samples than in controls. The expression level of Bcl-2, an anti-apoptotic molecule, decreased in As2O3treated samples than in controls, Western Blotting also showed that As2O3up-regulated Beclin-1and LC3-Ⅱ protein and down-regulated Bcl-2protein, the mortality rates of K562and K562/ADM cells treated with3-MA and As2O3detected by flow cytometry were higher than treated with As2O3alone.The autophagic activity of K562/ADM cells decreased with prolonged duration with As2O3, suggested that the apoptosis is the main form of death of leukemia cells of anti-leukemia effect of arsenic trioxide but not autophagic death, autophagy may be involved in the anti-leukemia mechanism of arsenic trioxide, but not the main factor.
Conclusion:1.The autophagic activity of K562/ADM cells is higher than K562cells; Under starvation condition, the autophagic activity of K562/ADMincreased earlier than K562cells, Beclin-1may be involved in the autophagic Molecular mechanisms; The inhibitedautophagic activity of K562/ADM cells with3-MA,couldenhancethe sensitivity of K562/ADM cells to doxorubicin, taken together, these results suggest that K562/ADM cells have more autophagy activity than K562cells. Autophagy may contribute to the mechanism of multi-drug resistance in K562/ADM cells.
2. The expression of mdrl mRNA and P-gp in K562/ADM cells increased as enhanced autophagic activity under starvation, and decreased as inhibited autophagic activity by3-MA; The ability of multidrug-resistant in K562/ADM cells is increased after starvation, suggested that p-gp may be involved in the autophagicmolecular mechanisms of K562/ADM cells.
3. Arsenic trioxide could induce autophagy in K562and K562/ADM cells, Beclin-1and Bcl-2may be involved in the autophagicmolecular mechanisms of K562and K562/ADM cells treated with As2O3, the changes of autophagic activity of leukemia cells are opposite to the apoptosis, inhibiting autophagy with3-MA, the mortality rate of K562and K562/ADM cells increased but not significantly, suggesting that this kind of autophagy is a protective mechanism and reduces the inhibited effect of AsaO3on leukemia cells, the main form of death of leukemia cells of anti-leukemia effect of arsenic trioxide is apoptosis. Autophagy may be involved in the anti-leukemia mechanism of arsenic trioxide, but not the main factor.
方法：对数生长期的白血病细胞株K562和K562/ADM细胞,以无血清和无氨基酸的磷酸缓冲液(phosphate buffered solution, PB S)代替RPIM-1640+小牛血清培养液培养细胞,培养Oh、3h、6h、9h、12h后收集细胞,透射电镜观察自噬体形态,MDC(单丹磺酰戊二胺)荧光染色、荧光显微镜和流式细胞仪检测自噬体荧光染色；实时荧光定量PCR (RT-qPCR)检测微管相关蛋白1轻链3(microtubule-associated protein-1light chain3, LC3) LC3和自噬相关基因Beclin-1mRNA表达, Western Blotting检测LC3和Beclin-1蛋白的表达以及LC3-Ⅰ向LC3-Ⅱ的转化。同时,采用RT-qPCR法和流式细胞术分别检测细胞自噬过程中多药耐药基因1(mdrl)/P-糖蛋白(P-gp)表达的动态变化。不同浓度阿霉素单独或联合自噬抑制剂3-MA(5mmol/L)作用K562/ADM细胞24h-72h, MIT测定K562/ADM细胞的增殖活性,Annexin V/PI双染、流式细胞术测定K562/ADM细胞的凋亡。透射电镜观察自噬体形态,Western Blotting法检测LC3的表达及LC3-Ⅰ和LC3-Ⅱ的转化。不同浓度三氧化二砷作用K562细胞及K562/ADM细胞不同时间后,透射电镜观察自噬体形态,倒置荧光显微镜及流式细胞仪检测自噬体MDC染色；RT-qPCR法检测Benlin-1与Bcl-2的mRNA表达,Western Blotting分析检测Beclin-1、Bcl-2和LC3蛋白表达以及LC3-Ⅰ向LC3-Ⅱ的转化。不同浓度三氧化二砷单独或联合自噬抑制剂3-MA (5mmol/L)作用K562和K562/ADM细胞24-48h,MTT测定细胞的增殖活性,Annexin V/PI双染、流式细胞术测定细胞的凋亡。
结果：1.与K562细胞相比,K562/ADM耐药细胞具有较高的基础自噬活性；饥饿法诱导K562与K562/ADM细胞发生自噬,在诱导的早期(3h) K562/ADM耐药细胞的自噬活性即迅速增高,而敏感的K562细胞的自噬活性发生较晚(9h),提示耐药白血病细胞早期而快速发生的自噬活性更有利于应对外界环境改变、保持细胞稳定性；自噬相关基因Beclin-1参与自噬的调节,K562/ADM细胞的表达高于K562细胞,与自噬活性的变化一致。有趣的是,K562/ADM细胞的mdrl/P-gp的表达随细胞自噬活性增强而表达增高,3-MA抑制自噬活性其表达相应降低。提示P-gp可能参与耐药性白血病细胞自噬活性的调控。
2.K562/ADM细胞对阿霉素诱导的凋亡效应有很强的抵抗性。10μmol/L的阿霉素处理K562/ADM细胞48h,电镜下可见明显的自噬形态改变, Western Blotting检测可见LC3-Ⅰ向LC3-Ⅱ转化增强,说明阿霉素处理可诱导K562/ADM耐药细胞发生较强的自噬。若用3-MA处理可显著减低K562/ADM细胞的自噬活性,阿霉素诱导的K562/ADM细胞的凋亡显著增高、存活细胞率明显减少。提示抑制细胞自噬活性可提高K562/ADM细胞对阿霉素的敏感性、降低其耐药性。
3.K562细胞和K562/ADM细胞均对三氧化二砷具有较高的敏感性,在不同浓度三氧化二砷诱导K562细胞和K562/ADM细胞增殖抑制和凋亡过程中,两种细胞均发生显著的自噬活性改变,自噬活性差异并不明显,但K562/ADM细胞自噬的出现较K562细胞为早。同时,三氧化二砷可增高K562和K562/ADM细胞Beclin-1基因mRNA及蛋白的表达,而显著降低Bcl-2基因mRNA及蛋白的表达,Beclin-1可能通过与Bcl-2的相互作用参与三氧化二砷诱导白血病细胞凋亡和自噬过程。3-MA抑制细胞自噬活性,可提高三氧化二砷诱导的K562细胞和K562/ADM细胞的死亡率,但增高的程度均并不十分显著。综合分析结果提示三氧化二砷在K562细胞和K562/ADM细胞中诱发的自噬活性,早期主要为应激性的保护反应,但随三氧化二砷作用时间的延长和诱导凋亡作用的增强,K562/ADM耐药细胞的自噬活性反而有所降低,提示三氧化二砷主要诱导耐药性白血病细胞发生细胞凋亡而非自噬性死亡,细胞自噬可能参与三氧化二砷的抗白血病机制但非主要因素。
结论：1.白血病K562/ADM耐药细胞较其亲本K562敏感细胞具有更高的基础自噬活性,营养剥夺可引发白血病耐药性细胞发生早期而快速的自噬活性增强。Beclin-1参与自噬的调节,抑制细胞自噬活性可明显增高K562/ADM耐药细胞对阿霉素的敏感性、降低药物抵抗性,证明细胞自噬是参与白血病K562/ADM细胞多药耐药性形成的机制之一。
2.营养剥夺诱导K562/ADM耐药细胞发生自噬过程中mdr1/P-gp的表达随细胞自噬活性增强而表达增高,细胞自噬活性被自噬抑制剂3-MA抑制其表达相应降低。mdr1/P-gp的表达变化与耐药细胞自噬活性的变化高度一致,提示P-gp可能参与耐药性白血病细胞自噬活性的调控,具体机制有待进一步探讨。
3.三氧化二砷可诱发K562细胞和K562/ADM细胞发生自噬反应,Beclin-1及Bcl-2参与了其自噬形成机制,但随三氧化二砷作用时间的延长和诱导凋亡作用的增强,耐药细胞的自噬活性反而有所降低,抑制自噬活性白血病细胞对三氧化二砷的敏感性有所增强但不显著。提示三氧化二砷在耐药性白血病中诱发的早期自噬活性主要为应激性的保护反应,三氧化二砷主要诱导白血病细胞发生细胞凋亡而非自噬性死亡,细胞自噬可能参与三氧化二砷的抗白血病机制但非主要因素。
Objective:Leukemia is a malignant clonal disease of hematopoietic stem cells, characterized by abnormal primitive and naive cells increased in the bone marrow. The treatment was improved due to the use of combination of chemotherapy drugs and bone marrow transplantation recent decades, but the overall prognosis remains still poor. The major obstacle to clinical outcomes of AML is multidrug resistance of leukemia cells induced by chemotherapy drug, which also is the main factor to affect the effectiveness and success rate of leukemic bone marrow transplantation. Clinical leukemia is resistance to the lack of effective interventions. There are still no effective interventions to multidrug resistance of AML currently. Autophagy is very important for the pathogenesis and development of tumor/leukemia. Then the role of autophagy in Hematological malignancies and its relationship with drug-induced resistance mechanism is not yet fully understood. Therefore, the multidrug-resistant acute myeloid leukemia cell line K562/ADM and its parental cell line K562were used to investigate the change of autophagic activity induced by starvation and drugs, the relationship between autopahagy and the multi-drug resistance of K562/ADM cells, and the role of autophagy in the effect of arsenic trioxide in leukemia cells.
Methods:We induced human multidrug-resistant leukemia K562/ADM cells and its drug-sensitive parental K562cells by starvation (0h,3h,6h,9h and12h) in vitro. Autophagic vacuoles were observed by electron microscope, the increased MDC (Monodansylcadaverin)-positive cells were detected by Fluorescence microscope and Flow cytometry; Furthermore, the expression of LC3mRNA and Beclin-1mRNA were detected by real-time quantitative PCR (RT-qPCR), the expression of LC3, Beclin-1and the result of LC3-Iconverted to LC3-Ⅱ were detected byWestern Blotting.The expression of mdrl mRNA was detected by RT-qPCR, P-glycoprotein of K562/ADM was detected by flow cytometry.The proliferation of K562/ADM cells treated with different concentrations of doxorubicinand in combination with3-MA for24-72h were detected with MTT assay, the apoptotic rates of K562/ADM treated with different concentrations of doxorubicinand in combination with3-MA for24-72h were detected with flow cytometry in order to explore the relationship between autophagy and apoptosis.Adriamycin inducedautophagic changes in K562/ADM cells, autophagic vacuoles were observed by electron microscope,and the result of LC3-Iconverted to LC3-II were detected by Western blotting. Autophagic vacuoles were observed in As2O3-treated K562and K562/ADM cells using electron microscope. MDC-labeled autophagic vacuoles were observed by fluorescent inverted phase contrast microscopy in As2O3-treated cells. The changes of LC3protein expression were detected byWestern Blotting. Furthermore, real-time quantitative PCR and Western blotting revealed that the expression of Beclin-1and Bcl-2. The apoptotic rates of K562/ADM treated with different concentrations of AS2O3and in combination with3-MA were detected with flow cytometry in order to study the possible molecular mechanism of leukemia cell death induced by arsenic trioxide.
Results:1. Compared to K562cells, the autophagy activity of K562/ADM cells was higher under normal circumstances, Starvation could induce the changes of autophagic activity of K562and K562/ADM cells, the autophagic activity of K562/ADM cells occurred faster and earlier than K562cells to environmental changes in order to maintain the stability of cells; Beclin-1was involved in the regulation of autophagy, the expression of Beclin-1gene mRNA and protein in control K562/ADM cells were significantly higher than that in control K562cells, Moreover, we found that the expression of mdrl mRNA and P-gp in K562/ADM cells incresed as enhanced autophagic activity, after inhibited the autophagic activity of K562/ADM cells with3-MA, the expression of mdrl mRNA and P-gpdecreased. That suggested P-gp may be involved in the regulation of autophagic activity in K562/ADM cells.
2. K562/ADM cell was resistant to doxorubicin. K562/ADM cells treated with10μmol/L of Adriamycin for48hours, obvious autophagic vacuoles were observed under electron microscope, enhanced expression of LC3-II protein was detected by Western Blotting, electron microscope and Western Blotting also revealed that doxorubicin could induce increased autophagic activity.3-MA, as an inhibitor of autophagy, could enhance the inhibited effect of K562/ADM cells induced by doxorubicin, the mortality rate of K562/ADM cells treated with3-MA and doxorubicin detected by flow cytometry was higher than treated with doxorubicin alone.
3. After treated with different concentrations As2O3, The autophagic activity of K562and K562/ADM cells raised,the autophagic activity of K562/ADM cells increased earlier than K562cells treated with As2O3. The expression levels of Beclin-1, which play key roles in autophagy, increased in As2O3treated samples than in controls. The expression level of Bcl-2, an anti-apoptotic molecule, decreased in As2O3treated samples than in controls, Western Blotting also showed that As2O3up-regulated Beclin-1and LC3-Ⅱ protein and down-regulated Bcl-2protein, the mortality rates of K562and K562/ADM cells treated with3-MA and As2O3detected by flow cytometry were higher than treated with As2O3alone.The autophagic activity of K562/ADM cells decreased with prolonged duration with As2O3, suggested that the apoptosis is the main form of death of leukemia cells of anti-leukemia effect of arsenic trioxide but not autophagic death, autophagy may be involved in the anti-leukemia mechanism of arsenic trioxide, but not the main factor.
Conclusion:1.The autophagic activity of K562/ADM cells is higher than K562cells; Under starvation condition, the autophagic activity of K562/ADMincreased earlier than K562cells, Beclin-1may be involved in the autophagic Molecular mechanisms; The inhibitedautophagic activity of K562/ADM cells with3-MA,couldenhancethe sensitivity of K562/ADM cells to doxorubicin, taken together, these results suggest that K562/ADM cells have more autophagy activity than K562cells. Autophagy may contribute to the mechanism of multi-drug resistance in K562/ADM cells.
2. The expression of mdrl mRNA and P-gp in K562/ADM cells increased as enhanced autophagic activity under starvation, and decreased as inhibited autophagic activity by3-MA; The ability of multidrug-resistant in K562/ADM cells is increased after starvation, suggested that p-gp may be involved in the autophagicmolecular mechanisms of K562/ADM cells.
3. Arsenic trioxide could induce autophagy in K562and K562/ADM cells, Beclin-1and Bcl-2may be involved in the autophagicmolecular mechanisms of K562and K562/ADM cells treated with As2O3, the changes of autophagic activity of leukemia cells are opposite to the apoptosis, inhibiting autophagy with3-MA, the mortality rate of K562and K562/ADM cells increased but not significantly, suggesting that this kind of autophagy is a protective mechanism and reduces the inhibited effect of AsaO3on leukemia cells, the main form of death of leukemia cells of anti-leukemia effect of arsenic trioxide is apoptosis. Autophagy may be involved in the anti-leukemia mechanism of arsenic trioxide, but not the main factor.
引文
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