[Mechanisms of arsenic trioxide induced tumor cell apoptosis in myelodysplastic syndrome mice model in vivo].
OBJECTIVE
To explore the possible mechanisms of cell apoptosis induced by arsenic trioxide (ATO) alone or in combination with thalidomide (THAL) on SCID mice model transplanted with human myelodysplastic syndrome (MDS) cell line MUTZ-1 in vivo.


METHODS
In a previous study by the authors, a Hum-MDS mice model was successfully established and the results showed that ATO alone or in combination with THAL had markedly inhibitory effect on the tumors growth in the MDS mice model of the treated groups with higher-rates of cell apoptosis and longer-survival periods than control groups (P < 0.01). In the present study, 48 Hum-MDS mice were randomly grouped and ATO groups had 32 mice which were treated with ATO [5.0 microg or 7.5 microg/(g.d), 5 d a week, intraperitoneal injection (i.p.) x 3 weeks alone or in combination with THAL 8 microg/(g.d) x 3 weeks] and the control groups had 16 mice [treated with 0.9% NaCl 10 microl/(g.d) (i.p) or untreated]. The expressions of the proteins and the genes related to apoptosis were detected by using immunohistochemical (IHC) method, Western blot, reverse transcription-polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA) with [gamma-32P] ATP oligonucleotide probe end labeling.


RESULTS
(1) By IHC, compared to controls, the expressions of the pro-apoptosis protein Bax (F = 1080.150, P < 0.01) and the ratio of Bax/Bcl-2 were higher, but Bcl-2 (F = 2777.978, P < 0.01) was lower in the treated groups. (2) Western-blot results showed that the pro-apoptosis protein expressions of mitochondrial Smac/DIABLO and cytochrome C (IHC) were strikingly up-regulated and pro-caspase 9, 7, 6, 3 and full PARP were down-regulated and cleaved caspase-6, 7, 9 and PARP were clearly observed in the treated groups. The expressions of apoptosis protein inhibitors of cIAP1 and survivin mRNA (RT-PCR) were markedly down-regulated in the treated groups. But no evidences of cleavage of BID and caspase-8 had been observed in any group. (3) The expression of IkappaB-alpha protein [nuclear factor-kappa B (NF-kappaB) inhibitor] was up-regulated (Western-blot) and the activity of NF-kappaB were sharply down-regulated in ATO-treated groups by EMSA.


CONCLUSIONS
These results suggest that mitochondria are the primary intracellular target of ATO. At least, two pathways, namely, mitochondrial mediated-caspase dependent cell apoptosis pathway and NF-kappaB signal conduct pathway were involved in the mechanisms of ATO inducing cell apoptosis on MDS mice model in vivo. The mechanisms seem to be a complicated network regulated by multi-signal pathways, multi-layers, and multi-factors instead of a single pathway.
Lu
Jie
J
Department of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Jin
Jie
J
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D015153:Blotting, Western; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009190:Myelodysplastic Syndromes / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010087:Oxides / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013792:Thalidomide / Q000494:pharmacology
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D001152:Arsenicals; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D010087:Oxides; D000072000:NF-KappaB Inhibitor alpha; D013792:Thalidomide; D020169:Caspases; D000077237:Arsenic Trioxide