三氧化二砷诱导白血病细胞株自噬性死亡及其分子机制研究
|
摘要
国内外临床研究已经证实三氧化二砷(Arsenic trioxide，As_2O_3)可有效地治疗复发和／或初发的急性早幼粒细胞白血病(Acute Promyelocytic Leukemia，APL)，并能达到完全缓解。由于As_2O_3和化疗药物作用的靶点不同，临床应用时亦没有明显的骨髓抑制；且没有化疗药物的其他严重毒副反应。因此，国内外研究人员试图将As_2O_3应用于其他恶性肿瘤的治疗。在血液肿瘤方面，许多体外实验研究表明：As_2O_3可诱导髓系、淋巴系和浆细胞等恶性血液病细胞株凋亡。在临床研究方面，As_2O_3已经进入治疗慢性粒细胞性白血病、急性粒细胞性白血病、多发性骨髓瘤和骨髓增生异常综合征(MDS)等疾病的临床Ⅰ期和Ⅱ期研究。但是As_2O_3治疗淋巴细胞白血病的研究报道尚少，其对淋巴细胞恶性肿瘤和MDS的作用机制尚不清楚。
本研究采用MTT(3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltelra-zoliumbromide)比色法观察As_2O_3对T细胞白血病细胞株Molt-4和MDS细胞株Mutz-1细胞的生长抑制作用。结果发现，As_2O_3可显著抑制两种白血病细胞株的生长，并呈时间—剂量依赖性。药物作用48小时对Molt-4细胞的半数抑制浓度(IC50)为4.68μmol／L，对Mutz-1细胞的IC50为1.79μmol／L。用碘化丙啶(PI)和AnnexinV对As_2O_3处理的白血病细胞株双标记，流式细胞仪(FACS)检测结果显示Annexin V~+、PI~-细胞(即凋亡细胞)呈剂量依赖性增加，提示As_2O_3导致了白血病细胞凋亡。同时，FACS分析结合TUNEL检测法表明：As_2O_3诱导的死亡细胞一部分是TUNEL阴性的。荧光显微镜下观察细胞在细胞膜没有破坏的情况下PI能否染色细胞，结果显示：药物处理48h后，细胞被PI所标记，提示药物处理能够导致白血病细胞发生非凋亡形式的死亡。Western-Blot分析结果显示：药物作用Molt-4细胞后，启动Caspase蛋白，即Caspase-8和Caspase-9被激活，呈时间依赖性；Bid也被激活，提示Caspase-9可能通过Bid的激活而被激活。执行Caspase蛋白中，Caspase-7呈时间依赖性激活，而Caspase-3、Caspase-6没有显著变化。Caspase抑制剂实验结果显示：Pan-caspase抑制剂能部分抑制As_2O_3诱导的Molt-4细胞凋亡，而Caspase-3、Caspase-7和Caspase-8抑制剂均不能抑制As_2O_3诱导的Molt-4细胞死亡。应用透射电镜观察药物作用前后白血病细胞的超微结构。实验结果显示，药物作用后，白血病细胞的细胞膜不完整，胞浆中出现大量囊泡。提示As_2O_3诱导的Molt-4和Mutz-1细胞发生了自噬性细胞死亡，有别于细胞凋亡。磷脂酰肌醇激酶3(Phosphatidylinositol 3-kinase，PI3K／AKT)通路的抑制剂(3-MA)预育后再和As_2O_3共同作用于白血病细胞株(4μM As_2O_3作用于Molt-4细胞48h，2μM As_2O_3作用于Mutz-1细胞48h)，流式细胞仪(PI和FSC参数)分析3-MA对As_2O_3诱导的非凋亡细胞的抑制作用，结果表明3-MA显著抑制了非凋亡细胞的产生，同时3-MA显著增加了细胞凋亡。用Western-blot分析结果显示用三氧化二砷作用后的Molt-4细胞磷酸化的AKT上调，而用3-MA作用后的磷酸化的AKT下调，进一步证明As_2O_3诱导了白血病细胞发生自噬性死亡。
近年来研究发现，自噬性细胞死亡作为非细胞凋亡死亡的一种主要方式在恶性肿瘤的发生和发展中起着十分重要的作用。但目前对血液肿瘤的自噬性细胞死亡及其机制尚不清楚。Jia等报道肿瘤坏死因子α诱导T淋巴细胞白血病细胞株发生凋亡前有自体吞噬现象发生。动物实验研究表明，Beclin-1—人类Bcl-2相互作用蛋白基因敲除的小鼠容易发生上皮细胞和血液系统恶性肿瘤。Saeki等报道应用Beclin-1反义寡核苷酸处理HL-60细胞导致了自噬性细胞死亡。2003年Kanzawa等的研究表明As_2O_3可诱导神经胶质瘤肿瘤细胞发生自噬性细胞死亡，但作用机制还不清楚。这些研究和我们的实验结果均提示自体吞噬和自噬性细胞死亡在血液肿瘤的发生、发展中起重要作用，对血液肿瘤细胞的自噬性死亡及其信号途径研究将有可能为血液肿瘤的治疗提供新的靶点。
我们应用Western-blot和定量实时RT-PCR研究了As_2O_3对白血病细胞株Beclin-1表达的影响。结果表明：在蛋白水平，As_2O_3显著上调了白血病细胞株Beclin-1的表达，呈时间和剂量依赖性。而在mRNA水平，As_2O_3对Beclin-1的表达无影响。用蛋白合成抑制剂(CHX)能显著抑制As_2O_3对白血病细胞株Beclin-1表达的上调作用，提示As_2O_3对白血病细胞株Beclin-1表达的影响是发生在转录后水平的；As_2O_3可能通过使Beclin-1表达增加导致细胞发生自体吞噬，进一步引起自噬性细胞死亡。由于Bcl-2家族与自体吞噬的发生有密切关系，我们进一步研究了As_2O_3对白血病细胞株Bcl-2家族蛋白表达的影响。结果表明：药物作用后，Molt-4细胞Bcl-2蛋白表达明显下调，呈时间依赖性。Bax蛋白表达呈时间—剂量依赖性下调。而Bak蛋白没有明显受到影响。为了了解As_2O_3影响Molt-4细胞Bax和Bak表达的机制，我们用定量实时RT-PCR检测了药物作用后Bcl-2家族相关基因mRNA水平的变化；结果显示：在mRNA水平，Bax表达和Bak表达没有受到影响。提示As_2O_3对Bax的调控发生在转录后水平。
综上所述，我们的研究结果表明：(1)在临床有效血药浓度范围内，As_2O_3显著抑制Molt-4细胞(2μmol／L-6μmol／L)和Mutz-1细胞(1μmol／L—3μmol／L)的增殖；As_2O_3对这两种白血病细胞株的生长抑制作用呈时间和剂量依赖性。药物作用48小时，Molt-4细胞的IC50大于4μmol／L；而药物作用48小时后Mutz-1细胞的IC50为1.79μmol／L。表明Mutz-1细胞株对三氧化二砷的敏感性高于Molt-4细胞株。(2)As_2O_3对两种白血病细胞株有诱导凋亡作用，但也有非凋亡死亡。药物作用后有Caspase-8和Caspase-9的激活，Bid也被激活，提示Caspase-9可能通过Bid的激活而被激活。执行Caspase蛋白中，Caspase-7呈时间依赖性激活，而Caspase-3、Caspase-6没有显著变化。Pan-caspase抑制剂能部分抑制三氧化二砷诱导的Molt-4细胞凋亡，而Caspase-3、Caspase-7和Caspase-8抑制剂均不能抑制三氧化二砷诱导的Molt-4细胞死亡。(3)Rhodamin123染色表明，药物作用后细胞的线粒体膜电位没有显著变化；细胞超微结构和磷脂酰肌醇激酶3抑制剂(3-MA)研究结果显示As_2O_3可诱导Molt-4和Mutz-1
细胞发生自噬性细胞死亡。Western blot分析提示As_2O_3可能通过使Beclin-1蛋白表达增加导致细胞发生自体吞噬，进一步引起自噬性细胞死亡。(4)As_2O_3对白血病细胞株Bcl-2家族蛋白表达也有影响。三氧化二砷作用后，Molt-4细胞Bcl-2蛋白表达明显下调，呈时间依赖性。Bax蛋白表达也呈时间—剂量依赖性下调。而Bak蛋白没有受到影响。用定量实时RT-PCR证明：这种调节发生在转录后水平。As_2O_3对Bcl-2家族蛋白表达的影响可能与其诱导自噬性死亡有关。
Arsenic trioxide (As_2O_3) has been used successfully in the treatment of patients with newly diagnosed acute promyelocytic leukaemia (APL) and those with relapsed or refractory APL without severe marrow suppression. Up to now, it has been reported that the effects of As_2O_3 are not confined to APL cells but can also be observed in a variety of malignant myeloid, lymphoid, megakaryocytic, and plasma cells. These studies lead to the pre-clinical and Phase I/II evaluation of As_2O_3 as a potential therapy for many kinds of haematological malignancies and solid tumours. Although it has been demonstrated that As_2O_3 is capable of inducing cell death via cell cycle arrest and apoptosis in both APL cells and non-APL cells, the mechanisms of As_2O_3 -mediated cell death are not fully understood.
In this study, the growth inhibition of As_2O_3 for leukaemia cell lines was evaluated by the MTT assay. The results showed that As_2O_3 induced a dose-and time-dependent proliferation inhibit. The 50% inhibitory concentration (IC50) after 3 days of As_2O_3 treatment for Mutz-1 cells was 1.79 umol/1, and for Molt-4 cells greater than 4 umol/1. we next performed in vitro the dual staining of cells with
annexin V and PI analyzed by flow cytometry (FACS) to know whether As_2O_3 treatment could induce the apoptosis both in Molt-4 and Mutz-1 cells. FACS analysis showed that leukaemia cells treated with As_2O_3 underwent apoptosis defined as cells with annexin V~+ and PI~- in a dose-dependent manner. As_2O_3-induced apoptosis further was confirmed by TUNEL assay. It was shown a dose-dependent increase of TUNEL-positive cells in Molt-4 cells treated with As_2O_3. The percentage of TUNEL-positive cells was consistent with those of annexin V~+ and PI~- cells induced by same dose of As_2O_3, suggesting that As_2O_3-induced cell death are both TUNEL-positive and negative. We next performed the cells staining with PI, which was used to evaluate the cell death related to etoposide-induced autophagy. The high levels of PI stain which indicate the disruption of the plasma membrane had been observed in Molt-4 cells exposed to 4 umol/1 As_2O_3 for 48 h. FACS analysis using forward-angle light scattering (FSC) and PI staining revealed that the R1 cells induced by As_2O_3 were increased by 27.36% and 37.47% respectively in Molt-4 and Mutz-1 cells comparing with untreated control. The R2 cells, smaller cells with less PI stains which indicate that plasma membrane integrity is better maintained, were also induced by As_2O_3. Taken together, our findings suggested that As_2O_3 induced not only apoptosis but also non-apoptotic cell death in leukaemia cells.
To characterize mechanism of As_2O_3-induced cell death, western blotting of lysates obtained from Molt-4 cells exposure to As_2O_3 was performed using antibodies. After treatment with As_2O_3, pro-caspase-3 level decreased and cleaved caspase-3 were not observed in Molt-4 cells, although PARP cleavage occurred. Next, we performed western blotting to detect other two kind of downstream caspase (i.e., caspase-6, and -7). It was shown that caspases-7 but not caspase-6 were proteolytically cleaved in Molt-4 cells. A time-dependent activation of caspase-8 and -9 was observed in Molt-4 cells, although caspase-9 was less active. To further link caspase activation to apoptosis of Molt-4 cells, three different caspase blocking
peptides cells were employed. Neither caspase-8 nor caspase-7 inhibitor (both in 50 μM) abrogated As_2O_3 -induced apoptosis in Molt-4 cells, whereas pan-caspase inhibitor (10 μM) was able to import partial protection against cytotoxicity of As_2O_3. These data suggest that caspase inhibition is not sufficient to sustain viability in As_2O_3 -treated Molt-4 cells.
The ultrastructural information on the morphology of As_2O_3 -induced leukaemia cell death was observed using TEM. After 48 h treatment of As_2O_3, cells were collected and analyzed. Numerous large cytoplasmic inclusions that were membrane-bound vacuoles were observed in the cytoplasm of As_2O_3-treated leukaemia cell lines. TEM also revealed that the treatment of As_2O_3 resulted in the vacuoles present the morphology of multilamellar bodies (MLBs) in Mutz-1 cells. These ultrastructural features seen in As_2O_3-treated cells are consistent with autophagy as described previously. To elucidate the involvement of the autophagic process in non-apoptotic death of leukaemia cells, we studied if 3-MA, which inhibits autophagic vacuoles in many cell types, could abrogate As_2O_3-induced autophagic cell death. Addition of 3-MA (10 mmol/1), R1 population decreased from 32.73% to 13.85% (p <0.001) in Molt-4 cells and decreased from 42.52% to 22.5% (p <0.05) in Mutz-1 cells. In contrast, apoptotic cells (R2 population) increased from 19.55% to 58.16% (p <0.001) in Molt-4 cells and increased from 26.61% to 41.22% (p <0.05) in Mutz-1 cells. TEM analysis revealed that vauolated cells reduced significantly after adding 3-MA. Next, we studied the phenotypic changes of cells treated with 3-MA by TUNEL assay. After 3-MA treatment, the apoptotic cells increased from 32.71% to 83% in Molt-4 cells. TEM also showed the apoptotic features. These data indicated that 3-MA inhibited significantly autophagic cell death and sequential induced apoptosis in As_2O_3-treated leukaemia cells.
To elucidate the mechanism of As_2O_3-induced autophagic cell death, we performed western blot assay to evaluate the effect of As_2O_3 on expression of
Beclin-1 protein, which plays a key role in autophagy, in leukaemia cells. The results showed that As_2O_3 induced a time-dependent increase in Beclin-1 protein expression of Molt-4 cells and Mutz-1 cells. To gain insights into the mechanism of how Beclin-1 is elevated, we performed real-time RT-PCR to quantify the mRNA level of Beclin-1. The results showed that beclin-1 mRNA did not increase significantly after As_2O_3 treatment. Next, we supplemented the medium with 1 μg/ml or 2 μg/ml of the protein synthesis inhibitor, cycloheximide (CHX), to see if As_2O_3 can inhibit the degradation of Beclin-1. The results showed that CHX abrogated the effect of As_2O_3 on the up-regulation of Beclin-1 protein, suggesting that protein synthesis may be required for the up-regulation of Beclin-1.
Of particular interest was the relationship between the role of Bcl-2 family proteins and the onset of autophagic cell death. Bcl-2, Bax and Bak proteins expression was also detected using western blot. As_2O_3-treated Molt-4 cells exhibited a time-dependent decrease in both Bcl-2 and Bax proteins expression. In contrast, Bak did not exhibit down-regulation. Because Bax is a key component for cellular induced apoptosis through mitochondrial stress, we studied whether As_2O_3 affected the mitochondrial function by assessing its membrane potential (△Ψm). There was no loss of △Ψm in Molt-4 cells after exposure to As_2O_3 for 12h and 24h respectively, indicating that the mitochondrial function is well maintained in As_2O_3-treated Molt-4 cells. Finally, the mRNA levels of Bcl-2 family genes in Molt-4 cells treated with As_2O_3 (4 μmol/1) were analyzed by the real time RT-PCR. Our data showed that no significant changes of Bax and Bcl-2 mRNA expression were observed in As_2O_3-treated Molt-4 cells, suggesting that the effects of As_2O_3 on Bax and Bcl-2 are independent of transcription of mRNA.
In conclusion, our results showed that As_2O_3 significantly inhibited the proliferation of Molt-4 and Mutz-1 cells in dose- and time-dependent manner. Autophagic cell death (programmed cell death type II) and apoptosis (programmed
cell death type I) were activated together in leukaemia cell lines after exposed to As_2O_3. Numerous large cytoplasmic inclusions and vacuoles were observed in As_2O_3-treated cells using electron microscope. Furthermore, 3-methyladenine (an autophagy inhibitor) significantly reduced autophagic cell death and sequential induced apoptosis. Finally, leukaemia cells treated with 4 μM As_2O_3 showed a considerable up-regulation of Beclin-1 (a Bcl-2-interacting protein) expression, which was independent of transcription of mRNA and required protein synthesis. In addition, Molt-4 cells treated with As_2O_3 exhibited the down-regulation of Bax protein expression suggesting that Bax may be involved in accumulating of Beclin-1 and triggering autophagic cell death in As_2O_3-treated leukaemia cells. These results may lead to a better understanding of the unique mechanism of action of As_2O_3, and provide a suggestion that As_2O_3 may be of therapeutic value for the treatment of patients with human T lymphocytic leukaemia and myelodysplastic syndrome.
本研究采用MTT(3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltelra-zoliumbromide)比色法观察As_2O_3对T细胞白血病细胞株Molt-4和MDS细胞株Mutz-1细胞的生长抑制作用。结果发现，As_2O_3可显著抑制两种白血病细胞株的生长，并呈时间—剂量依赖性。药物作用48小时对Molt-4细胞的半数抑制浓度(IC50)为4.68μmol／L，对Mutz-1细胞的IC50为1.79μmol／L。用碘化丙啶(PI)和AnnexinV对As_2O_3处理的白血病细胞株双标记，流式细胞仪(FACS)检测结果显示Annexin V~+、PI~-细胞(即凋亡细胞)呈剂量依赖性增加，提示As_2O_3导致了白血病细胞凋亡。同时，FACS分析结合TUNEL检测法表明：As_2O_3诱导的死亡细胞一部分是TUNEL阴性的。荧光显微镜下观察细胞在细胞膜没有破坏的情况下PI能否染色细胞，结果显示：药物处理48h后，细胞被PI所标记，提示药物处理能够导致白血病细胞发生非凋亡形式的死亡。Western-Blot分析结果显示：药物作用Molt-4细胞后，启动Caspase蛋白，即Caspase-8和Caspase-9被激活，呈时间依赖性；Bid也被激活，提示Caspase-9可能通过Bid的激活而被激活。执行Caspase蛋白中，Caspase-7呈时间依赖性激活，而Caspase-3、Caspase-6没有显著变化。Caspase抑制剂实验结果显示：Pan-caspase抑制剂能部分抑制As_2O_3诱导的Molt-4细胞凋亡，而Caspase-3、Caspase-7和Caspase-8抑制剂均不能抑制As_2O_3诱导的Molt-4细胞死亡。应用透射电镜观察药物作用前后白血病细胞的超微结构。实验结果显示，药物作用后，白血病细胞的细胞膜不完整，胞浆中出现大量囊泡。提示As_2O_3诱导的Molt-4和Mutz-1细胞发生了自噬性细胞死亡，有别于细胞凋亡。磷脂酰肌醇激酶3(Phosphatidylinositol 3-kinase，PI3K／AKT)通路的抑制剂(3-MA)预育后再和As_2O_3共同作用于白血病细胞株(4μM As_2O_3作用于Molt-4细胞48h，2μM As_2O_3作用于Mutz-1细胞48h)，流式细胞仪(PI和FSC参数)分析3-MA对As_2O_3诱导的非凋亡细胞的抑制作用，结果表明3-MA显著抑制了非凋亡细胞的产生，同时3-MA显著增加了细胞凋亡。用Western-blot分析结果显示用三氧化二砷作用后的Molt-4细胞磷酸化的AKT上调，而用3-MA作用后的磷酸化的AKT下调，进一步证明As_2O_3诱导了白血病细胞发生自噬性死亡。
近年来研究发现，自噬性细胞死亡作为非细胞凋亡死亡的一种主要方式在恶性肿瘤的发生和发展中起着十分重要的作用。但目前对血液肿瘤的自噬性细胞死亡及其机制尚不清楚。Jia等报道肿瘤坏死因子α诱导T淋巴细胞白血病细胞株发生凋亡前有自体吞噬现象发生。动物实验研究表明，Beclin-1—人类Bcl-2相互作用蛋白基因敲除的小鼠容易发生上皮细胞和血液系统恶性肿瘤。Saeki等报道应用Beclin-1反义寡核苷酸处理HL-60细胞导致了自噬性细胞死亡。2003年Kanzawa等的研究表明As_2O_3可诱导神经胶质瘤肿瘤细胞发生自噬性细胞死亡，但作用机制还不清楚。这些研究和我们的实验结果均提示自体吞噬和自噬性细胞死亡在血液肿瘤的发生、发展中起重要作用，对血液肿瘤细胞的自噬性死亡及其信号途径研究将有可能为血液肿瘤的治疗提供新的靶点。
我们应用Western-blot和定量实时RT-PCR研究了As_2O_3对白血病细胞株Beclin-1表达的影响。结果表明：在蛋白水平，As_2O_3显著上调了白血病细胞株Beclin-1的表达，呈时间和剂量依赖性。而在mRNA水平，As_2O_3对Beclin-1的表达无影响。用蛋白合成抑制剂(CHX)能显著抑制As_2O_3对白血病细胞株Beclin-1表达的上调作用，提示As_2O_3对白血病细胞株Beclin-1表达的影响是发生在转录后水平的；As_2O_3可能通过使Beclin-1表达增加导致细胞发生自体吞噬，进一步引起自噬性细胞死亡。由于Bcl-2家族与自体吞噬的发生有密切关系，我们进一步研究了As_2O_3对白血病细胞株Bcl-2家族蛋白表达的影响。结果表明：药物作用后，Molt-4细胞Bcl-2蛋白表达明显下调，呈时间依赖性。Bax蛋白表达呈时间—剂量依赖性下调。而Bak蛋白没有明显受到影响。为了了解As_2O_3影响Molt-4细胞Bax和Bak表达的机制，我们用定量实时RT-PCR检测了药物作用后Bcl-2家族相关基因mRNA水平的变化；结果显示：在mRNA水平，Bax表达和Bak表达没有受到影响。提示As_2O_3对Bax的调控发生在转录后水平。
综上所述，我们的研究结果表明：(1)在临床有效血药浓度范围内，As_2O_3显著抑制Molt-4细胞(2μmol／L-6μmol／L)和Mutz-1细胞(1μmol／L—3μmol／L)的增殖；As_2O_3对这两种白血病细胞株的生长抑制作用呈时间和剂量依赖性。药物作用48小时，Molt-4细胞的IC50大于4μmol／L；而药物作用48小时后Mutz-1细胞的IC50为1.79μmol／L。表明Mutz-1细胞株对三氧化二砷的敏感性高于Molt-4细胞株。(2)As_2O_3对两种白血病细胞株有诱导凋亡作用，但也有非凋亡死亡。药物作用后有Caspase-8和Caspase-9的激活，Bid也被激活，提示Caspase-9可能通过Bid的激活而被激活。执行Caspase蛋白中，Caspase-7呈时间依赖性激活，而Caspase-3、Caspase-6没有显著变化。Pan-caspase抑制剂能部分抑制三氧化二砷诱导的Molt-4细胞凋亡，而Caspase-3、Caspase-7和Caspase-8抑制剂均不能抑制三氧化二砷诱导的Molt-4细胞死亡。(3)Rhodamin123染色表明，药物作用后细胞的线粒体膜电位没有显著变化；细胞超微结构和磷脂酰肌醇激酶3抑制剂(3-MA)研究结果显示As_2O_3可诱导Molt-4和Mutz-1
细胞发生自噬性细胞死亡。Western blot分析提示As_2O_3可能通过使Beclin-1蛋白表达增加导致细胞发生自体吞噬，进一步引起自噬性细胞死亡。(4)As_2O_3对白血病细胞株Bcl-2家族蛋白表达也有影响。三氧化二砷作用后，Molt-4细胞Bcl-2蛋白表达明显下调，呈时间依赖性。Bax蛋白表达也呈时间—剂量依赖性下调。而Bak蛋白没有受到影响。用定量实时RT-PCR证明：这种调节发生在转录后水平。As_2O_3对Bcl-2家族蛋白表达的影响可能与其诱导自噬性死亡有关。
Arsenic trioxide (As_2O_3) has been used successfully in the treatment of patients with newly diagnosed acute promyelocytic leukaemia (APL) and those with relapsed or refractory APL without severe marrow suppression. Up to now, it has been reported that the effects of As_2O_3 are not confined to APL cells but can also be observed in a variety of malignant myeloid, lymphoid, megakaryocytic, and plasma cells. These studies lead to the pre-clinical and Phase I/II evaluation of As_2O_3 as a potential therapy for many kinds of haematological malignancies and solid tumours. Although it has been demonstrated that As_2O_3 is capable of inducing cell death via cell cycle arrest and apoptosis in both APL cells and non-APL cells, the mechanisms of As_2O_3 -mediated cell death are not fully understood.
In this study, the growth inhibition of As_2O_3 for leukaemia cell lines was evaluated by the MTT assay. The results showed that As_2O_3 induced a dose-and time-dependent proliferation inhibit. The 50% inhibitory concentration (IC50) after 3 days of As_2O_3 treatment for Mutz-1 cells was 1.79 umol/1, and for Molt-4 cells greater than 4 umol/1. we next performed in vitro the dual staining of cells with
annexin V and PI analyzed by flow cytometry (FACS) to know whether As_2O_3 treatment could induce the apoptosis both in Molt-4 and Mutz-1 cells. FACS analysis showed that leukaemia cells treated with As_2O_3 underwent apoptosis defined as cells with annexin V~+ and PI~- in a dose-dependent manner. As_2O_3-induced apoptosis further was confirmed by TUNEL assay. It was shown a dose-dependent increase of TUNEL-positive cells in Molt-4 cells treated with As_2O_3. The percentage of TUNEL-positive cells was consistent with those of annexin V~+ and PI~- cells induced by same dose of As_2O_3, suggesting that As_2O_3-induced cell death are both TUNEL-positive and negative. We next performed the cells staining with PI, which was used to evaluate the cell death related to etoposide-induced autophagy. The high levels of PI stain which indicate the disruption of the plasma membrane had been observed in Molt-4 cells exposed to 4 umol/1 As_2O_3 for 48 h. FACS analysis using forward-angle light scattering (FSC) and PI staining revealed that the R1 cells induced by As_2O_3 were increased by 27.36% and 37.47% respectively in Molt-4 and Mutz-1 cells comparing with untreated control. The R2 cells, smaller cells with less PI stains which indicate that plasma membrane integrity is better maintained, were also induced by As_2O_3. Taken together, our findings suggested that As_2O_3 induced not only apoptosis but also non-apoptotic cell death in leukaemia cells.
To characterize mechanism of As_2O_3-induced cell death, western blotting of lysates obtained from Molt-4 cells exposure to As_2O_3 was performed using antibodies. After treatment with As_2O_3, pro-caspase-3 level decreased and cleaved caspase-3 were not observed in Molt-4 cells, although PARP cleavage occurred. Next, we performed western blotting to detect other two kind of downstream caspase (i.e., caspase-6, and -7). It was shown that caspases-7 but not caspase-6 were proteolytically cleaved in Molt-4 cells. A time-dependent activation of caspase-8 and -9 was observed in Molt-4 cells, although caspase-9 was less active. To further link caspase activation to apoptosis of Molt-4 cells, three different caspase blocking
peptides cells were employed. Neither caspase-8 nor caspase-7 inhibitor (both in 50 μM) abrogated As_2O_3 -induced apoptosis in Molt-4 cells, whereas pan-caspase inhibitor (10 μM) was able to import partial protection against cytotoxicity of As_2O_3. These data suggest that caspase inhibition is not sufficient to sustain viability in As_2O_3 -treated Molt-4 cells.
The ultrastructural information on the morphology of As_2O_3 -induced leukaemia cell death was observed using TEM. After 48 h treatment of As_2O_3, cells were collected and analyzed. Numerous large cytoplasmic inclusions that were membrane-bound vacuoles were observed in the cytoplasm of As_2O_3-treated leukaemia cell lines. TEM also revealed that the treatment of As_2O_3 resulted in the vacuoles present the morphology of multilamellar bodies (MLBs) in Mutz-1 cells. These ultrastructural features seen in As_2O_3-treated cells are consistent with autophagy as described previously. To elucidate the involvement of the autophagic process in non-apoptotic death of leukaemia cells, we studied if 3-MA, which inhibits autophagic vacuoles in many cell types, could abrogate As_2O_3-induced autophagic cell death. Addition of 3-MA (10 mmol/1), R1 population decreased from 32.73% to 13.85% (p <0.001) in Molt-4 cells and decreased from 42.52% to 22.5% (p <0.05) in Mutz-1 cells. In contrast, apoptotic cells (R2 population) increased from 19.55% to 58.16% (p <0.001) in Molt-4 cells and increased from 26.61% to 41.22% (p <0.05) in Mutz-1 cells. TEM analysis revealed that vauolated cells reduced significantly after adding 3-MA. Next, we studied the phenotypic changes of cells treated with 3-MA by TUNEL assay. After 3-MA treatment, the apoptotic cells increased from 32.71% to 83% in Molt-4 cells. TEM also showed the apoptotic features. These data indicated that 3-MA inhibited significantly autophagic cell death and sequential induced apoptosis in As_2O_3-treated leukaemia cells.
To elucidate the mechanism of As_2O_3-induced autophagic cell death, we performed western blot assay to evaluate the effect of As_2O_3 on expression of
Beclin-1 protein, which plays a key role in autophagy, in leukaemia cells. The results showed that As_2O_3 induced a time-dependent increase in Beclin-1 protein expression of Molt-4 cells and Mutz-1 cells. To gain insights into the mechanism of how Beclin-1 is elevated, we performed real-time RT-PCR to quantify the mRNA level of Beclin-1. The results showed that beclin-1 mRNA did not increase significantly after As_2O_3 treatment. Next, we supplemented the medium with 1 μg/ml or 2 μg/ml of the protein synthesis inhibitor, cycloheximide (CHX), to see if As_2O_3 can inhibit the degradation of Beclin-1. The results showed that CHX abrogated the effect of As_2O_3 on the up-regulation of Beclin-1 protein, suggesting that protein synthesis may be required for the up-regulation of Beclin-1.
Of particular interest was the relationship between the role of Bcl-2 family proteins and the onset of autophagic cell death. Bcl-2, Bax and Bak proteins expression was also detected using western blot. As_2O_3-treated Molt-4 cells exhibited a time-dependent decrease in both Bcl-2 and Bax proteins expression. In contrast, Bak did not exhibit down-regulation. Because Bax is a key component for cellular induced apoptosis through mitochondrial stress, we studied whether As_2O_3 affected the mitochondrial function by assessing its membrane potential (△Ψm). There was no loss of △Ψm in Molt-4 cells after exposure to As_2O_3 for 12h and 24h respectively, indicating that the mitochondrial function is well maintained in As_2O_3-treated Molt-4 cells. Finally, the mRNA levels of Bcl-2 family genes in Molt-4 cells treated with As_2O_3 (4 μmol/1) were analyzed by the real time RT-PCR. Our data showed that no significant changes of Bax and Bcl-2 mRNA expression were observed in As_2O_3-treated Molt-4 cells, suggesting that the effects of As_2O_3 on Bax and Bcl-2 are independent of transcription of mRNA.
In conclusion, our results showed that As_2O_3 significantly inhibited the proliferation of Molt-4 and Mutz-1 cells in dose- and time-dependent manner. Autophagic cell death (programmed cell death type II) and apoptosis (programmed
cell death type I) were activated together in leukaemia cell lines after exposed to As_2O_3. Numerous large cytoplasmic inclusions and vacuoles were observed in As_2O_3-treated cells using electron microscope. Furthermore, 3-methyladenine (an autophagy inhibitor) significantly reduced autophagic cell death and sequential induced apoptosis. Finally, leukaemia cells treated with 4 μM As_2O_3 showed a considerable up-regulation of Beclin-1 (a Bcl-2-interacting protein) expression, which was independent of transcription of mRNA and required protein synthesis. In addition, Molt-4 cells treated with As_2O_3 exhibited the down-regulation of Bax protein expression suggesting that Bax may be involved in accumulating of Beclin-1 and triggering autophagic cell death in As_2O_3-treated leukaemia cells. These results may lead to a better understanding of the unique mechanism of action of As_2O_3, and provide a suggestion that As_2O_3 may be of therapeutic value for the treatment of patients with human T lymphocytic leukaemia and myelodysplastic syndrome.
引文
1. Chen GQ, Shi XG, Tang W, Xiong SM, Zhu J, Cai X, Han ZG, Ni JH, Sh GY, Jia PM, Liu MM, He KL, Niu C, Ma J, Zhang P, Zhang TD, Paul P, Naoe T, Kitamura K, Miller W, Waxman S, Wang ZY, de The H, Chen SJ, Chen Z. Use of arsenic trioxide As2O3 in the treatment of acute promyelocytic leukemia (APL). I. As2O3 exerts dose-dependent dual effects on APL cells. Blood 1997; 89: 3345-3353.
2. Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, Zhu J, Tang W, Sun GL, Yang KQ, Chen Y, Zhou L, Fang ZW, Wang YT, Ma J, Zhang P, Zhang TD, Chen SJ, Chen Z, Wang ZY. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL), II: clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997;89: 3354-3360.
3. Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ, Corso D,DeBlasio A, Gabrilove J, Scheinberg DA, Pandolfi PP, Warrell RP Jr. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med 1998; 339:1341-1348.
4. Sternsdorf T, Puccetti E, Jensen K, Hoelzer D, Will H, Ottmann OG, Ruthardt M. PIC-1/SUMO-1-modified PML-retinoic acid receptor a mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia. Mol Cell Biol 1999; 19:5170-5178.
5. Chen YC, Lin-Shiau SY, and Lin JK. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 1998; 177:324-333.
6. Ling Y, Jiang J, James F. Holland, Perez-soler Roman. Arsenic Trioxide Produces Polymerization of Microtubules and Mitotic Arrest before Apoptosis in Human Tumor Cell Lines. Mol Pharmacol 2002;62:529-538.
7. Mathas S, Lietz A, Janz M, Janz M, Hinz M, Jundt F, Scheidereit C, Bommert K, Dorken B. Inhibition of NF-kB essentially contributes to arsenic-induced apoptosis. Blood 2003, 102:1028-1034.
8. Seol JG, Park WH, Kim ES, et al. Potential role caspase-3 and -9 in arsenic trioxide-mediated apoptosis in PCI-1 head and neck cancer cells. Int J Oncol 2001; 18:249-455.
9. Kitamura K, Minami Y, Yamamoto K, et al. Involvement of CD95-independent caspase 8 activation in arsenic trioxide-induced apoptosis. Leukemia 2000;14:1743-1750.
10. Miller WH Jr, Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide. Cancer Res 2002;62:3893-3903.
11. Huang XJ, Wiernik PH, Klein RS, Gallagher RE. Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases. Med Onco 1999;16:58-64.
12. Li YM, Broome JD. Arsenic targets tubulins to induce apoptosis in myeloidleukemia cells. Cancer Res 1999; 59:776-780.
13. Zhu XH, Shen YL, Jing YK, Cai X, Jia PM, Huang Y, Tang W, Shi GY, Sun YP, Dai J, Wang ZY, Chen SJ, Zhang TD, Waxman S, Chen Z, Chen GQ. Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations. J Natl Cancer Inst 1999;91: 772-778.
14. Zhang W, Ohnishi K, Shigeno K, Fujisawa S, Naito K, Nakamura S, Takeshita K, Takeshita A, Ohno R. The induction of apoptosis and cell cycle arrest by arsenic trioxide in lymphoid neoplasms. Leukemia 1998; 12:1383-1391.
15. Hu XM, Hirano T, Oka K. Arsenic trioxide induces apoptosis equally in T lymphoblastoid leukemia MOLT-4 cells and P-gp-expressing daunorubicin-resistant MOLT-4 cells. Cancer Chemother Pharmacol 2003; 51: 119-126
16. Lu M, Levin J, Sulpice E, Sequeira-Le Grand A, Alemany M, Caen JP, Han ZC.Effect of arsenic trioxide on viability, proliferation and apoptosis in human megakaryocytic leukemia cell lines. Exp Hematol 1999; 27:845-852.
17. Hayashi T, Hideshima T, Akiyama M, Richardson P, Schlossman RL, Chauhan D, Munshi NC, Waxman S, Anderson KC. Arsenic trioxide inhibits growth of human multiple myeloma cells in the bone marrow microenvironment. Mol Cancer Ther 2002; 1:851-860.
18. McCafferty-Grad J, Bahlis NJ, Krett N, Aguilar TM, Reis I, Lee KP, Boise LH. Arsenic trioxide uses caspase-dependent and caspase-independent death pathways in myeloma cells. Mol Cancer Ther 2003; 2: 1155-1164.
19. Liu Q, Hilsenbeck S, Gazitt Y. Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL. Blood 2003; 101: 4078-4087.
20. Guimaraes C. A., Linden R., Programmed cell death: apoptosis and alternative deathstyles, Eur J Biochem 2004; 271: 1638-1650.
21. Klionsky D. J., Emr S. D. Autophagy as a regulated pathway of cellular degradation, Science, 2000; 290: 1717-1721.
22. Shimizu S., Kanaseki T., Mizushima N., Mizuta T., Arakawa-Kobayashi S., Thompson C. B., Tsujimoto Y., Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes, Nature cell biology, 2004; 6: 1221-1228.
23 倪万茂，钱文斌．三氧化二砷诱导T淋巴细胞白血病细胞株凋亡及机理研究．浙江医学，2004；12：905-907
24. Ono K., Kim S. O., Han J., Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death, Mol Cell Biol, 2003; 23: 665-676.
25. Yu L., Alva A., Su H., Dutt P., Freundt E., Welsh S., Baehrecke E. H., Lenardo M. J., Regulation of an ATG7-beclin-1 program of autophagic cell death by caspase-8, Science, 2004; 304: 1500-1502.
26. Klapper W., Qian W., Schulte C., Parwaresch R., DNA damage transiently increases TRF2 mRNA expression and telomerase activity, Leukemia, 2003; 17: 2007-2015.
27. Bazarbachi A., Ghez D., Lepelletier Y., Nasr R., The H. de, El-Sabban M. E., Hermine O., New therapeutic approaches for adult T-cell leukaemia, Lancet Oncology, 2004; 5: 664-672.
28 McCafferty-Grad J., Bahlis N. J., Krett N., Aguilar T. M., Reis I., Lee K. P., Boise L. H., Arsenic trioxide uses caspase-dependent and caspase-independent death pathways in myeloma cells, Mol Cancer Ther, 2003; 2: 1155-1164.
29. Kanzawa T., Kondo Y., Ito H., Kondo S., Germano I., Induction of autophagic cell death in malignant glioma cells by arsenic trioxide, Cancer Res, 2003;63:2103-2108.
30. Jia L., Dourmashkin R.R., Allen P.D., Gray A.B., Newland A.C., Kelsey S.M., Inhibition of autophagy abrogates tumour necrosis factor α-induced apoptosis inhuman T-lymphoblastic leukaemia cells, Br J Haematol, 1997;98:673-685.
31. Qu X., Yu J., Bhagat G., Furuya N., Hibshoosh H., Troxel A., Rosen J., Eskelinen E.L., Mizushima N., Ohsumi Y., Cattoretti G., Levine B., Promotion of tumorigenesis by heterozygous disruption of the beclin-1 autophagy gene, J Clin Invest, 2003; 112:1809-1820.
32. Yue Z., Jin S., Yang C., Levine, A.J. Heintz N., Beclin-1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor, Proc Natl Acad Sci U S A, 2003;100:15077-15082.
33. Saeki K., You A., Okuma E., Yazaki Y., Susin SA., Kroemer G., Takaku F., Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells, Cell Death Differ, 2000;7 :1263-1269.
34. Kihara A., Kabeya Y., Ohsumi Y., Yoshimori T., Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network, EMBO Rep, 2001;2:330-335.
35. Liang X.H., Jackson S. Seaman M., Brown K., Kempkes B., Hibshoosh H.,Levine B., Induction of autophagy and inhibition of tumorigenesis by beclin-1, Nature, 1999;402:672-676.
36. Liang X.H., Yu J., Brown K., Levine B., Beclin-1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function, Cancer Res, 2001;61:3443-3449.
37. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider M D, Levine B. Bcl-2 antiapoptotic proteins inhibit Beclin 1-Dependent autophagy. Cell ,2005, 122:927-939
38. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism, Oncogene, 2004,23:2891-2906
39. Martin DN, Baehrecke EH. Caspase function in autophagic programmed cell death in Drosophila. Development, 2003,131(2):275-284
1. Edinger AL, Thompson CB. Defective autophagy leads to cancer. Cancer Cell. 2003;4:422-424.
2. Strasser A, O'Connor L, Dixit VM.Apoptosis signaling. Annu Rev Biochem.2000;69:217-245.
3. Leist M, Jaattela M.Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol. 2001;2:589-598.
4. Francis Ka-Ming Chan, Joanna Shisler, Jacqueline G. Bixby, Martin Felices, Lixin Zheng, Michael Appel, Jan Orenstein, Bernard Moss, Michael J. Lenardo.A Role for Tumor Necrosis Factor Receptor-2 and Receptor-interacting Protein in Programmed Necrosis and Antiviral ResponsesJ. Biol. Chem., 2003; 278: 51613 - 51621.
5. Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J.Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000; 1:489-495.
6. Grace Ng, Jingxiang Huang . The significance of autophagy in cancer.Mol Carcinog. 2005;43:183-187.
7. Clarke PG.Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl). 1990;181:195-213.
8.Devrim Gozuacikl , Adi Kimchi.Autophagy as a cell death and tumor suppressor mechanism, Oncogene 2004 ;23.2891-2906.
9. Liang, X. H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., and Levine, B. Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature (Lond.), 1999.;402: 672-676,
10. Vincent M. Aitaa, Xiao Huan Liangb, V. V. V. S. Murtyc, David L. Pincusb, Weiping Yub, Eftihia Cayanisd, Sergei Kalachikovd, T. Conrad Gilliamd, a and Beth Levineb, Cloning and Genomic Organization of Beclin 1, a Candidate Tumor Suppressor Gene on Chromosome 17q21.GENOMICS. 1999;59: 59-65 .
11. Xiao Huan Liang, Saadiya Jackson, Matthew Seaman, Kristy Brown, Bettina Kempkes§, Hanina Hibshoosh Beth Levine.Induction of autophagy andinhibition of tumorigenesis by beclin 1 .NATURE VOL1999 ;402 :672-676.
12. Xiao Huan Liang, Jie Yu, Kristy Brown, Beth Levine , Beclin 1 Contains a Leucine-rich Nuclear Export Signal That Is Required for Its Autophagy and Tumor Suppressor Functionl, CANCER RESEARCH 2001;61:3443-3449.
13. Devrim Gozuacikl Adi Kimchi.Autophagy as a cell death and tumor suppressor mechanism Oncogene, 2004; 23 :2891-2906 .
14. Daisuke Furuya, Naoki Tsuji, Atsuhito Yagihashi, Naoki WatanabeT.Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity, Experimental Cell Research 2005;307 : 26— 40.
15.Li Yu,Ajjai Alva,Helen Su,Parmesh Dutt,Eric Freundt,Sarah Welsh,Eric H.Baehrecke,Michardo.Regulation of ATG7-beclinl Program of Autophagic Cell Death by Caspase-8.Science, 2004;304: 1500-1502
16.Shimizu S,Kanaseki T,Mizushima N,et al.Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes.nature cellbiology 2004; 6:1221-1228.
17. M. Gajewskal, B. Gajkowska, T. Motyl.Apoptosis And Autophagy Induced By Tgf-B1 In Bovine Mammary Epithelial Bme-Uv1 Cells Journal Of Physiology And Pharmacology 2005; 56, Supp 3:159-179.
18. Sophie Pattingre, Amina Tassa, Xueping Qu, Rita Garuti, Xiao Huan Liang, Noboru Mizushima, Milton Packer, Michael D. Schneider, and Beth Levine. Bcl-2 Antiapoptotic Proteins Inhibit Beclin 1-Dependent Autophagy, Cell, 2005; 122:927-939,
19. Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ and Codogno P. Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem. 2000;275:992-998.
20.Mizushima N, Yamamoto A, Hatano M, Kobayashi Y,Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y Yoshimori T. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells .J Cell Biol. 2001; 152:657-668.
21. Per O. Seglen and Paul B. Gordon .3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes,Proc Natl Acad Sci U S A. 1982; 79: 1889-1892
22. Kim J, Huang WP, Stromhaug PE, Klionsky DJ. Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation. J Biol Chem. 2002;277:763-773.
23. Stack JH, DeWald DB, Takegawa K, Emr SD .Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J Cell Biol. 1995;129:321-334.
24. Akio Kihara,Yukiko Kabeya,Yoshinori Ohsumi ,Tamotsu Yoshimori.Beclin— phosphatidylinositol 3-kinase complex functions at trans-Golgi nerwork,EMBOreports,2001 ; 2 : 330-335.
25. Panaretou C, Domin J, Cockcroft S, Waterfield MD. Characterization of p150, an adaptor protein for the human phosphatidylinositol (Ptdlns) 3-kinase. Substrate presentation by phosphatidylinositol transfer protein to the p150.Ptdins 3-kinase complex. J Biol Chem. 1997;272:2477-2485.
26. Cohen O, Kimchi A. DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer.Cell Death Differ. 2001;8(1):6-15.
27. Jang CW, Chen CH, Chen CC, Chen JY, Su YH, Chen RH. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase.Nat Cell Biol. 2002;4(1):51-58.
28. Kogel D, Prehn JH, Scheidtmann KH. The DAP kinase family of pro-apoptotic proteins: novel players in the apoptotic game.Bioessays. 2001 Apr;23(4):352-358.
29. Kogel D, Prehn JH, Scheidtmann KH. The DAP kinase family of pro-apoptotic proteins: novel players in the apoptotic game.Bioessays. 2001 Apr;23(4):352-358.
30. Inbal B, Bialik S, Sabanay I, Shani G, Kimchi A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.J Cell Biol. 2002 Apr 29;157(3):455-68
31 .Gunn JM, Clark MG, Knowles SE, Hopgood MF, Ballard FJ, Reduced rates of proteolysis in transformed cells. Nature. 1977;266(5597):58-60
32. Lockwood TD, Minassian IA, Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation .Biochem J. 1982 Aug 15;206(2):251-258.
33. Cockle SM, Dean RT. Distinct proteolytic mechanisms in serum-sufficient and serum-restricted fibroblasts. Transformed 3T3 cells fail to regulate proteolysis in relation to culture density only during serum-sufficiency. Biochem J. 1984 1;221(1):53-60.
34. Kisen GO, Tessitore L, Costelli P, Gordon PB, Schwarze PE, Baccino FM, Seglen PO. Reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells.Carcinogenesis. 1993;14(12):2501-2505.
35. Canuto RA, Tessitore L, Muzio G, Autelli R, Baccino FM. Tissue protein turnover during liver carcinogenesis.Carcinogenesis. 1993 Dec;14(12):2581-2587.
36. Schwarze PE, Seglen PO. Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats. Exp Cell Res. 1985;157(1):15-28.
37. Yucel T, Ahlberg J, Glaumann H. Overall proteolysis in perfused and subfractionated chemically induced malignant hepatoma of rat: effects of amino acids.Exp Mol Pathol. 1989 Feb;50(1):38-49.
38. Ahlberg J, Yucel T, Eriksson L, Glaumann H. Characterization of the proteolytic compartment in rat hepatocyte nodules.Virchows Arch B Cell Pathol Incl Mol Pathol. 1987;53(2):79-88.
39. Toth S, Nagy K, Palfia Z, Rez G., Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation. Cell Tissue Res. 2002;309(3):409-416.
40. Rez G, Toth S, Palfia Z. Cellular autophagic capacity is highly increased in azaserine-induced premalignant atypical acinar nodule cells. Carcinogenesis.1999;20(10):1893-1898.
2. Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, Zhu J, Tang W, Sun GL, Yang KQ, Chen Y, Zhou L, Fang ZW, Wang YT, Ma J, Zhang P, Zhang TD, Chen SJ, Chen Z, Wang ZY. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL), II: clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997;89: 3354-3360.
3. Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ, Corso D,DeBlasio A, Gabrilove J, Scheinberg DA, Pandolfi PP, Warrell RP Jr. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med 1998; 339:1341-1348.
4. Sternsdorf T, Puccetti E, Jensen K, Hoelzer D, Will H, Ottmann OG, Ruthardt M. PIC-1/SUMO-1-modified PML-retinoic acid receptor a mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia. Mol Cell Biol 1999; 19:5170-5178.
5. Chen YC, Lin-Shiau SY, and Lin JK. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 1998; 177:324-333.
6. Ling Y, Jiang J, James F. Holland, Perez-soler Roman. Arsenic Trioxide Produces Polymerization of Microtubules and Mitotic Arrest before Apoptosis in Human Tumor Cell Lines. Mol Pharmacol 2002;62:529-538.
7. Mathas S, Lietz A, Janz M, Janz M, Hinz M, Jundt F, Scheidereit C, Bommert K, Dorken B. Inhibition of NF-kB essentially contributes to arsenic-induced apoptosis. Blood 2003, 102:1028-1034.
8. Seol JG, Park WH, Kim ES, et al. Potential role caspase-3 and -9 in arsenic trioxide-mediated apoptosis in PCI-1 head and neck cancer cells. Int J Oncol 2001; 18:249-455.
9. Kitamura K, Minami Y, Yamamoto K, et al. Involvement of CD95-independent caspase 8 activation in arsenic trioxide-induced apoptosis. Leukemia 2000;14:1743-1750.
10. Miller WH Jr, Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide. Cancer Res 2002;62:3893-3903.
11. Huang XJ, Wiernik PH, Klein RS, Gallagher RE. Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases. Med Onco 1999;16:58-64.
12. Li YM, Broome JD. Arsenic targets tubulins to induce apoptosis in myeloidleukemia cells. Cancer Res 1999; 59:776-780.
13. Zhu XH, Shen YL, Jing YK, Cai X, Jia PM, Huang Y, Tang W, Shi GY, Sun YP, Dai J, Wang ZY, Chen SJ, Zhang TD, Waxman S, Chen Z, Chen GQ. Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations. J Natl Cancer Inst 1999;91: 772-778.
14. Zhang W, Ohnishi K, Shigeno K, Fujisawa S, Naito K, Nakamura S, Takeshita K, Takeshita A, Ohno R. The induction of apoptosis and cell cycle arrest by arsenic trioxide in lymphoid neoplasms. Leukemia 1998; 12:1383-1391.
15. Hu XM, Hirano T, Oka K. Arsenic trioxide induces apoptosis equally in T lymphoblastoid leukemia MOLT-4 cells and P-gp-expressing daunorubicin-resistant MOLT-4 cells. Cancer Chemother Pharmacol 2003; 51: 119-126
16. Lu M, Levin J, Sulpice E, Sequeira-Le Grand A, Alemany M, Caen JP, Han ZC.Effect of arsenic trioxide on viability, proliferation and apoptosis in human megakaryocytic leukemia cell lines. Exp Hematol 1999; 27:845-852.
17. Hayashi T, Hideshima T, Akiyama M, Richardson P, Schlossman RL, Chauhan D, Munshi NC, Waxman S, Anderson KC. Arsenic trioxide inhibits growth of human multiple myeloma cells in the bone marrow microenvironment. Mol Cancer Ther 2002; 1:851-860.
18. McCafferty-Grad J, Bahlis NJ, Krett N, Aguilar TM, Reis I, Lee KP, Boise LH. Arsenic trioxide uses caspase-dependent and caspase-independent death pathways in myeloma cells. Mol Cancer Ther 2003; 2: 1155-1164.
19. Liu Q, Hilsenbeck S, Gazitt Y. Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL. Blood 2003; 101: 4078-4087.
20. Guimaraes C. A., Linden R., Programmed cell death: apoptosis and alternative deathstyles, Eur J Biochem 2004; 271: 1638-1650.
21. Klionsky D. J., Emr S. D. Autophagy as a regulated pathway of cellular degradation, Science, 2000; 290: 1717-1721.
22. Shimizu S., Kanaseki T., Mizushima N., Mizuta T., Arakawa-Kobayashi S., Thompson C. B., Tsujimoto Y., Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes, Nature cell biology, 2004; 6: 1221-1228.
23 倪万茂，钱文斌．三氧化二砷诱导T淋巴细胞白血病细胞株凋亡及机理研究．浙江医学，2004；12：905-907
24. Ono K., Kim S. O., Han J., Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death, Mol Cell Biol, 2003; 23: 665-676.
25. Yu L., Alva A., Su H., Dutt P., Freundt E., Welsh S., Baehrecke E. H., Lenardo M. J., Regulation of an ATG7-beclin-1 program of autophagic cell death by caspase-8, Science, 2004; 304: 1500-1502.
26. Klapper W., Qian W., Schulte C., Parwaresch R., DNA damage transiently increases TRF2 mRNA expression and telomerase activity, Leukemia, 2003; 17: 2007-2015.
27. Bazarbachi A., Ghez D., Lepelletier Y., Nasr R., The H. de, El-Sabban M. E., Hermine O., New therapeutic approaches for adult T-cell leukaemia, Lancet Oncology, 2004; 5: 664-672.
28 McCafferty-Grad J., Bahlis N. J., Krett N., Aguilar T. M., Reis I., Lee K. P., Boise L. H., Arsenic trioxide uses caspase-dependent and caspase-independent death pathways in myeloma cells, Mol Cancer Ther, 2003; 2: 1155-1164.
29. Kanzawa T., Kondo Y., Ito H., Kondo S., Germano I., Induction of autophagic cell death in malignant glioma cells by arsenic trioxide, Cancer Res, 2003;63:2103-2108.
30. Jia L., Dourmashkin R.R., Allen P.D., Gray A.B., Newland A.C., Kelsey S.M., Inhibition of autophagy abrogates tumour necrosis factor α-induced apoptosis inhuman T-lymphoblastic leukaemia cells, Br J Haematol, 1997;98:673-685.
31. Qu X., Yu J., Bhagat G., Furuya N., Hibshoosh H., Troxel A., Rosen J., Eskelinen E.L., Mizushima N., Ohsumi Y., Cattoretti G., Levine B., Promotion of tumorigenesis by heterozygous disruption of the beclin-1 autophagy gene, J Clin Invest, 2003; 112:1809-1820.
32. Yue Z., Jin S., Yang C., Levine, A.J. Heintz N., Beclin-1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor, Proc Natl Acad Sci U S A, 2003;100:15077-15082.
33. Saeki K., You A., Okuma E., Yazaki Y., Susin SA., Kroemer G., Takaku F., Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells, Cell Death Differ, 2000;7 :1263-1269.
34. Kihara A., Kabeya Y., Ohsumi Y., Yoshimori T., Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network, EMBO Rep, 2001;2:330-335.
35. Liang X.H., Jackson S. Seaman M., Brown K., Kempkes B., Hibshoosh H.,Levine B., Induction of autophagy and inhibition of tumorigenesis by beclin-1, Nature, 1999;402:672-676.
36. Liang X.H., Yu J., Brown K., Levine B., Beclin-1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function, Cancer Res, 2001;61:3443-3449.
37. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider M D, Levine B. Bcl-2 antiapoptotic proteins inhibit Beclin 1-Dependent autophagy. Cell ,2005, 122:927-939
38. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism, Oncogene, 2004,23:2891-2906
39. Martin DN, Baehrecke EH. Caspase function in autophagic programmed cell death in Drosophila. Development, 2003,131(2):275-284
1. Edinger AL, Thompson CB. Defective autophagy leads to cancer. Cancer Cell. 2003;4:422-424.
2. Strasser A, O'Connor L, Dixit VM.Apoptosis signaling. Annu Rev Biochem.2000;69:217-245.
3. Leist M, Jaattela M.Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol. 2001;2:589-598.
4. Francis Ka-Ming Chan, Joanna Shisler, Jacqueline G. Bixby, Martin Felices, Lixin Zheng, Michael Appel, Jan Orenstein, Bernard Moss, Michael J. Lenardo.A Role for Tumor Necrosis Factor Receptor-2 and Receptor-interacting Protein in Programmed Necrosis and Antiviral ResponsesJ. Biol. Chem., 2003; 278: 51613 - 51621.
5. Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J.Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000; 1:489-495.
6. Grace Ng, Jingxiang Huang . The significance of autophagy in cancer.Mol Carcinog. 2005;43:183-187.
7. Clarke PG.Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl). 1990;181:195-213.
8.Devrim Gozuacikl , Adi Kimchi.Autophagy as a cell death and tumor suppressor mechanism, Oncogene 2004 ;23.2891-2906.
9. Liang, X. H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., and Levine, B. Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature (Lond.), 1999.;402: 672-676,
10. Vincent M. Aitaa, Xiao Huan Liangb, V. V. V. S. Murtyc, David L. Pincusb, Weiping Yub, Eftihia Cayanisd, Sergei Kalachikovd, T. Conrad Gilliamd, a and Beth Levineb, Cloning and Genomic Organization of Beclin 1, a Candidate Tumor Suppressor Gene on Chromosome 17q21.GENOMICS. 1999;59: 59-65 .
11. Xiao Huan Liang, Saadiya Jackson, Matthew Seaman, Kristy Brown, Bettina Kempkes§, Hanina Hibshoosh Beth Levine.Induction of autophagy andinhibition of tumorigenesis by beclin 1 .NATURE VOL1999 ;402 :672-676.
12. Xiao Huan Liang, Jie Yu, Kristy Brown, Beth Levine , Beclin 1 Contains a Leucine-rich Nuclear Export Signal That Is Required for Its Autophagy and Tumor Suppressor Functionl, CANCER RESEARCH 2001;61:3443-3449.
13. Devrim Gozuacikl Adi Kimchi.Autophagy as a cell death and tumor suppressor mechanism Oncogene, 2004; 23 :2891-2906 .
14. Daisuke Furuya, Naoki Tsuji, Atsuhito Yagihashi, Naoki WatanabeT.Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity, Experimental Cell Research 2005;307 : 26— 40.
15.Li Yu,Ajjai Alva,Helen Su,Parmesh Dutt,Eric Freundt,Sarah Welsh,Eric H.Baehrecke,Michardo.Regulation of ATG7-beclinl Program of Autophagic Cell Death by Caspase-8.Science, 2004;304: 1500-1502
16.Shimizu S,Kanaseki T,Mizushima N,et al.Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes.nature cellbiology 2004; 6:1221-1228.
17. M. Gajewskal, B. Gajkowska, T. Motyl.Apoptosis And Autophagy Induced By Tgf-B1 In Bovine Mammary Epithelial Bme-Uv1 Cells Journal Of Physiology And Pharmacology 2005; 56, Supp 3:159-179.
18. Sophie Pattingre, Amina Tassa, Xueping Qu, Rita Garuti, Xiao Huan Liang, Noboru Mizushima, Milton Packer, Michael D. Schneider, and Beth Levine. Bcl-2 Antiapoptotic Proteins Inhibit Beclin 1-Dependent Autophagy, Cell, 2005; 122:927-939,
19. Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ and Codogno P. Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem. 2000;275:992-998.
20.Mizushima N, Yamamoto A, Hatano M, Kobayashi Y,Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y Yoshimori T. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells .J Cell Biol. 2001; 152:657-668.
21. Per O. Seglen and Paul B. Gordon .3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes,Proc Natl Acad Sci U S A. 1982; 79: 1889-1892
22. Kim J, Huang WP, Stromhaug PE, Klionsky DJ. Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation. J Biol Chem. 2002;277:763-773.
23. Stack JH, DeWald DB, Takegawa K, Emr SD .Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J Cell Biol. 1995;129:321-334.
24. Akio Kihara,Yukiko Kabeya,Yoshinori Ohsumi ,Tamotsu Yoshimori.Beclin— phosphatidylinositol 3-kinase complex functions at trans-Golgi nerwork,EMBOreports,2001 ; 2 : 330-335.
25. Panaretou C, Domin J, Cockcroft S, Waterfield MD. Characterization of p150, an adaptor protein for the human phosphatidylinositol (Ptdlns) 3-kinase. Substrate presentation by phosphatidylinositol transfer protein to the p150.Ptdins 3-kinase complex. J Biol Chem. 1997;272:2477-2485.
26. Cohen O, Kimchi A. DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer.Cell Death Differ. 2001;8(1):6-15.
27. Jang CW, Chen CH, Chen CC, Chen JY, Su YH, Chen RH. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase.Nat Cell Biol. 2002;4(1):51-58.
28. Kogel D, Prehn JH, Scheidtmann KH. The DAP kinase family of pro-apoptotic proteins: novel players in the apoptotic game.Bioessays. 2001 Apr;23(4):352-358.
29. Kogel D, Prehn JH, Scheidtmann KH. The DAP kinase family of pro-apoptotic proteins: novel players in the apoptotic game.Bioessays. 2001 Apr;23(4):352-358.
30. Inbal B, Bialik S, Sabanay I, Shani G, Kimchi A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.J Cell Biol. 2002 Apr 29;157(3):455-68
31 .Gunn JM, Clark MG, Knowles SE, Hopgood MF, Ballard FJ, Reduced rates of proteolysis in transformed cells. Nature. 1977;266(5597):58-60
32. Lockwood TD, Minassian IA, Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation .Biochem J. 1982 Aug 15;206(2):251-258.
33. Cockle SM, Dean RT. Distinct proteolytic mechanisms in serum-sufficient and serum-restricted fibroblasts. Transformed 3T3 cells fail to regulate proteolysis in relation to culture density only during serum-sufficiency. Biochem J. 1984 1;221(1):53-60.
34. Kisen GO, Tessitore L, Costelli P, Gordon PB, Schwarze PE, Baccino FM, Seglen PO. Reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells.Carcinogenesis. 1993;14(12):2501-2505.
35. Canuto RA, Tessitore L, Muzio G, Autelli R, Baccino FM. Tissue protein turnover during liver carcinogenesis.Carcinogenesis. 1993 Dec;14(12):2581-2587.
36. Schwarze PE, Seglen PO. Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats. Exp Cell Res. 1985;157(1):15-28.
37. Yucel T, Ahlberg J, Glaumann H. Overall proteolysis in perfused and subfractionated chemically induced malignant hepatoma of rat: effects of amino acids.Exp Mol Pathol. 1989 Feb;50(1):38-49.
38. Ahlberg J, Yucel T, Eriksson L, Glaumann H. Characterization of the proteolytic compartment in rat hepatocyte nodules.Virchows Arch B Cell Pathol Incl Mol Pathol. 1987;53(2):79-88.
39. Toth S, Nagy K, Palfia Z, Rez G., Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation. Cell Tissue Res. 2002;309(3):409-416.
40. Rez G, Toth S, Palfia Z. Cellular autophagic capacity is highly increased in azaserine-induced premalignant atypical acinar nodule cells. Carcinogenesis.1999;20(10):1893-1898.