Autophagic Cell Death Induced By Arsenic Trioxide In Leukemia Cell Lines And Its Molecular Mechanisms

Arsenic trioxide (As₂O₃) 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 As₂O₃ 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 As₂O₃ as a potential therapy for many kinds of haematological malignancies and solid tumours. Although it has been demonstrated that As₂O₃ is capable of inducing cell death via cell cycle arrest and apoptosis in both APL cells and non-APL cells, the mechanisms of As₂O₃ -mediated cell death are not fully understood.In this study, the growth inhibition of As₂O₃ for leukaemia cell lines was evaluated by the MTT assay. The results showed that As₂O₃ induced a dose-and time-dependent proliferation inhibit. The 50% inhibitory concentration (IC50) after 3 days of As₂O₃ 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 withannexin V and PI analyzed by flow cytometry (FACS) to know whether As₂O₃ treatment could induce the apoptosis both in Molt-4 and Mutz-1 cells. FACS analysis showed that leukaemia cells treated with As₂O₃ underwent apoptosis defined as cells with annexin V⁺ and PI^- in a dose-dependent manner. As₂O₃-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 As₂O₃. The percentage of TUNEL-positive cells was consistent with those of annexin V⁺ and PI^- cells induced by same dose of As₂O₃, suggesting that As₂O₃-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 As₂O₃ for 48 h. FACS analysis using forward-angle light scattering (FSC) and PI staining revealed that the R1 cells induced by As₂O₃ 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 As₂O₃. Taken together, our findings suggested that As₂O₃ induced not only apoptosis but also non-apoptotic cell death in leukaemia cells.To characterize mechanism of As₂O₃-induced cell death, western blotting of lysates obtained from Molt-4 cells exposure to As₂O₃ was performed using antibodies. After treatment with As₂O₃, 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 blockingpeptides cells were employed. Neither caspase-8 nor caspase-7 inhibitor (both in 50 μM) abrogated As₂O₃ -induced apoptosis in Molt-4 cells, whereas pan-caspase inhibitor (10 μM) was able to import partial protection against cytotoxicity of As₂O₃. These data suggest that caspase inhibition is not sufficient to sustain viability in As₂O₃ -treated Molt-4 cells.The ultrastructural information on the morphology of As₂O₃ -induced leukaemia cell death was observed using TEM. After 48 h treatment of As₂O₃, cells were collected and analyzed. Numerous large cytoplasmic inclusions that were membrane-bound vacuoles were observed in the cytoplasm of As₂O₃-treated leukaemia cell lines. TEM also revealed that the treatment of As₂O₃ resulted in the vacuoles present the morphology of multilamellar bodies (MLBs) in Mutz-1 cells. These ultrastructural features seen in As₂O₃-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 As₂O₃-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 As₂O₃-treated leukaemia cells.To elucidate the mechanism of As₂O₃-induced autophagic cell death, we performed western blot assay to evaluate the effect of As₂O₃ on expression ofBeclin-1 protein, which plays a key role in autophagy, in leukaemia cells. The results showed that As₂O₃ 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 As₂O₃ treatment. Next, we supplemented the medium with 1 μg/ml or 2 μg/ml of the protein synthesis inhibitor, cycloheximide (CHX), to see if As₂O₃ can inhibit the degradation of Beclin-1. The results showed that CHX abrogated the effect of As₂O₃ 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. As₂O₃-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 As₂O₃ affected the mitochondrial function by assessing its membrane potential (△Ψm). There was no loss of △Ψm in Molt-4 cells after exposure to As₂O₃ for 12h and 24h respectively, indicating that the mitochondrial function is well maintained in As₂O₃-treated Molt-4 cells. Finally, the mRNA levels of Bcl-2 family genes in Molt-4 cells treated with As₂O₃ (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 As₂O₃-treated Molt-4 cells, suggesting that the effects of As₂O₃ on Bax and Bcl-2 are independent of transcription of mRNA.In conclusion, our results showed that As₂O₃ 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 (programmedcell death type I) were activated together in leukaemia cell lines after exposed to As₂O₃. Numerous large cytoplasmic inclusions and vacuoles were observed in As₂O₃-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 As₂O₃ 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 As₂O₃ 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 As₂O₃-treated leukaemia cells. These results may lead to a better understanding of the unique mechanism of action of As₂O₃, and provide a suggestion that As₂O₃ may be of therapeutic value for the treatment of patients with human T lymphocytic leukaemia and myelodysplastic syndrome.