The Molecular Mechanism Of Arsenic Trioxide Induced APL Cell Apoptosis

Acute promyelocytic leukemia (APL) is one of the most common cancers in blood system, which mechanism is not fully understood. The major pathological changes is that promyelocytic leukemia cells lose the ability to differentiate and maturate, and are stagnated in the promyelocytic state. Arsenic trioxide(As₂O₃), which has a successful clinical application to enable the APL patients get an overall clinical remission, has become an effective strategy for APL clinical treatment. Studies showed that the efficient induction of APL cell differentiation and apoptosis by arsenic trioxide is one of the important mechanism. Current studies revealed that the expression change of many genes in the nuclear genome is involved in apoptosis of promyelocytic leukemia cells by As₂O₃. But there is few reports on the DNA microarray study of apoptosis related gene expression in the apoptosis course of APL cells by As₂O₃. It has been shown that some genes, which expression has effects on the outer membrane of mitochondria, are involved in regulation of cancer cell apoptosis by As₂O₃. And mitochondria plays an important role in cell apoptosis. But there is no reports on expression of mitochondria genome in the course, and the role and status of mitochondria genome in apoptosis. It is of great importance for the elucidation of As₂O₃ mechanism to explore the mitochondria genome expression in As₂O₃ induced NB4 cell apoptosis course.The present study, in order to understand the apoptosis induction and growth inhibition effects inducing by As₂O₃ on NB4 cells fluorescent microscopy, electronic microscopy, flow cytometry, mitochondria membrane potential detection, RT-PCR were been used; design mitochondria genome primers, and study the differential expression of mitochondria genome in the course at gene and protein level; study the expression of 1384 apoptosis related gene using DNA microarray in As₂O₃ induced NB4 cell apoptosis. This study is to provide powerful clues for further discussion of the molecular mechanism of As₂O₃ induced APL cells apoptosis, and to provide basis for As₂O₃ application in APL and other cancer treatment. The major results were as follows:1. NB4 cells were showed typical apoptosis morphological changes after inducing by As₂O₃. After As₂O₃ 48h treatment on the NB4 cells, the fluorescent microscopy assays showed condensed and heavily stained nuclear, solidified and shrunk chromatin, and even fragmentation and debris, transmission electronic microscopy assays showed shrunk nuclear, irregular shape, marginal gathered chromatin. There was no morphological change in the control cells.2. It was shown a significant growth inhibitory effect after NB4 cells were treated with As₂O₃, in the concentration ranging from 0.5umol/L to 8umol/L, the inhibitory effects were increase when the concentration of As₂O₃ were been raised on NB4 cells at a fixed time point, the growth inhibitory curve were an S shape, an obvious quantity-effect effect was presenting. The inhibitory effects were increase with time when the concentration of As₂O₃ was been fixed on NB4 cells, presenting an obvious time-effect effect. NB4 cells were treated with 8umol/L As₂O₃, The growth inhibitory ratio were 78.8%, 91.2% and 100% respectively at 24h, 48h and 72h.3. It was shown that the NB4 cells apoptosis ratio was 5.02%,6.40%,28.40%,33.34% respectively with As₂O₃ treatment at concentration of 0.5umol/L,1umol/L,2umol/L,3umol/L. NB4 cells apoptosis ratio increase with As₂O₃ concentration by flow cytometry analysis, which showed a quantity-effect relation.4. It was shown that after NB4 cells were treated with As₂O₃ of 0.5umol/L,1umol/L,2umol/L,4umol/L,8umol/L at 48h, the mitochondria potential of NB4 cells decreased 12.8%,21.6%,66.9%,83.7%,83.8% respectively by flow cytometry analysis.5. RT-PCR assays was used to detect the expression of 13 genes in mitochondria genome in NB4 cell apoptosis by As₂O₃. The expression of COX2 gene was shown a down-regulated in this course, The expression of another 12 genes showed no significant changes. Western blot analysis also demonstrated a down- regulated protein expression of COX2.6 The mRNA expression of 1384 apoptosis related genes were profiled by oligonucleotide DNA microarray. The differential expression of 16 genes were observed during NB4 cells treated with 2umol/L As₂O₃. The expression of MT2A, SST, NFAT5 and SERPINB5 were up-regulated, The expression of EIF4G2, FDXR, CCT5, TFDP1, PRG1, P2RY6, MPO, ANP32A, CEPBA, HLA_B, YWHAE and TXNDC5 were down-regulated. The expression of 4 genes that showed most obvious expression change were detected by RT-PCR for confirmation. The RT-PCR result was in accordance with that of DNA microarray.The results indicated that:1. It was shown that there was a significant apoptosis and growth inhibition effect of As₂O₃ on acute promyelocytic leukemia cells (NB4 cells), these effects showed a relationship of quantity-effect and time-effect in a certain concentration range.2 The apoptosis effect inducing by As₂O₃ on NB4 was tightly related with the mitochondria membrane potential. The mitochondria membrane potential was decreased while cell apoptosis ration increasing.3 The expression change of the mitochondria gene COX2 was involved in the As₂O₃ induced apoptosis of NB4 cells.4 The up-regulated of MT2A, SST, NFAT5 and SERPINB5 genes and down-regulated of EIF4G2, FDXR, CCT5, TFDP1, PRG1, P2RY6, MPO, ANP32A, CEPBA, HLA_B, YWHAE and TXNDC5 genes were involved in the course of NB4 cells apoptosis inducing by As₂O₃. The genes mentioned above were tightly related with NB4cells apoptosis .