The Establishment Of A Human MDS Mice Model And The Study Of Anti-tumor Effect And Mechanisms Of Arsenic Trioxide Alone Or In Combination With Thalidomide For Therapy On Hum-MDS Mice Model

Myelodysplastic syndromes(MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and a variable risk of acute myelocytic leukaemia(AML) transformation. Until recently the mechanisms of MDS were ambiguous and treatment options beyond supportive care were limited. So, new treatment are also urgently needed. Arsenic trioxide (ATO)had been used for treating the relapsed/refractory acute promyelocytic leukaemia(APL) for many years with a higher rate of the clinic continuance complete remission (CCR). The curative effects of ATO on MDS were vary and mechanism unclear. The satisfacted anti-tumor effects of thalidomide(THAL) were observed onmultiple myeloma. Heretofore, there was no report of the successful establishing human MDS animal model and the study of anti-tumor mechanism of ATO in combination with THAL for treating on MDS-SCID mice model. We try to establish a SCID mice model transplanted with human MDS cell line MUTZ-1 cells and investigate the anti-tumor effect and the possible mechanism of ATO alone or in combination with THAL for therapy on MDS mice model in vivo . There are three sections in our research.Section 1: The establishment of a SCID mice model transplanted with human MDS cell line MUTZ-1 cellsAnimal model is one of the best carriers for studying the disorder pathogenesis and medication filtration. Heretofore , there was no report of the human MDS animal model successful established. Our purpose is to establish a SCID mice model transplanted with human MDS cell line MUTZ-1 cells for investigating the pathogenesis of MDS and the probable effect mechanisms of ATO alone or in combination with THAL for therapy on Hum-MDS-SCID mice model in vivo.In this experiment, 75 SCID mice and 10 BALB/CA-node mice were studied. The human MDS cell line MUTZ-1 cells were cultured in vitro andQmade for mono-cell suspension (with 1 xlO /ml cell density and in exponential growth behavior) and subcutancously implanted on 4~ 6-week-old First-Generation SCID mice and BALB/CA-node mice respectively. The tumor formation and the latent period of the tumor formation were observed.Subsequently, the biological characteristics of the subcutaneous tumor cells were evaluated by the methods of cell morphology, histopathology, immunology by flow cytometer, Chromosome analysis and immunohistochemistry (IHC) in SCID mice model.The results showed that CD10: 50.37%> CD 19: 92.50%> CD38: 97.97%> HLADR: 68.77% were higher and CDll^ CD^ CD14, > CD34> CD33 lower by immunologic analysis by flow cytometry. The chromosome del(5q)> der(15)^ t(5;15) andder (7), +8, +9, +10, +11 were observed. The over-expressions of Survivin mRNA (RT-PCR) and P53 protein were detected in tumor cells of MDS-SCID mice model. Our results showed that the biological characteristics of the tumor cells of MDS-SCID mice model were anthropo- source and consistent with human MDS cell line MUTZ-1 and completely different from that of normal SCID mice.In order to establish the Hum-MDS-SCID mice model with stable biological characteristics, the tumor cells from First-Generation Hum-MDS-SCID mice model were respectively subcutaneously implantedin Second- Generation 61 SCID mice and 8 BALB/CA-node mice. The rate of the tumor formation and the latent period of the tumor formation were observed and tumor size was measured by mean tumor diameters(MTD) twice a week in Second-Generation SCID mice and BALB/CA-node mice.The results showed that the rate of the subcutaneous tumor formation was 98.4% (60/61) in SCID mice and 62.5% (5/8) in BALB/CA-nude micerespectively (P=0.0027) . The latent period of the tumor formation were significantly longer (23~28d, median 26d) in BALB/CA-node mice than that in SCID mice (Z=4.605, P<0.001) .The biological characteristics of the subcutaneous tumor cells of MDS-SCID mice model were evaluated by using the multi-parameter analysis methods of cell morphology, histopathology, immunology by flow cytometry, chromosome analysis, immunohistochemistry.In order to observe the drug effect on tumor formation in vivo , we do the experiment with arsenic trioxide (ATO) (with 7.5|tig/g mouse/d x 3d ) injecting into abdominal cavity in 10 SCID mice. The result showed that the latent period of the tumor formation were significantly longer (26~-3 Id , median 29d) in the treated group by ATO than that of group of without injected ATO(10~17d,median 12d) (Z=5.339, P<0.001).Summary: (1) A Hum-MDS-SCID mice model was successfully established, which could be used for studying pathogenesis of MDS andexploring the mechanism of drug treating MDS in vivo. (2) ATO had a marked inhibition effect on the subcutaneous tumors formation and growth of MDS mice model.Section 2: Experimental study of arsenic trioxide alone or in combination with thalidomide for therapy on MDS mice modelArsenic trioxide (ATO) is purified component of a traditional Chinese medicine and extracted from a arsenic. For many years, it had been used for treating the relapsed/refractory acute promyelocytic leukaemia(APL) with a higher rate of the clinic continuance complete remission (CCR). The curative effects were different and the mechanisms still intangibly of ATO on MDS patients . The satisfacted anti-tumor effects of thalidomide(THAL) were observed on multiple myeloma.Heretofore, there was no report of the successful establishing human MDS animal model and anti-tumor mechanism of ATO in combination with THAL for treating on the MDS-SCID mice model. We try to investigate the anti-tumor effect and the possible mechanisms of ATO alone or in combination with THAL for therapy on MDS mice model in vivo.In vitro, the inhibition effect of cell growth and apoptosis of MUTZ-1 cells induced by ATO solution of different concentrations werestudied with cell morphology, MTT and Annexin V/PI by flow cytometry. The results showed the treatment by ATO l~8umol/L for 12~48h had marked inhibition effect on MUTZ-1 cells growth, which showed a dose and time dependent manner (r= — 0.962, P=0.009;r=—1.000, P=0.004). MUTZ-1 cells presented typical features of apoptosis by observed under the microscope.In order to exactly analyze the MUTZ-1 cell apoptosis quantitatively and qualitatively, the flow cytometry was also used. The results showed the higher-rates of cell apoptosis were observed by Annexin V/PI in ATO 8umol/L for 12h than other groups. 50% growth inhibition (IC50) at 12h, 24h and 48h in MUTZ-1 cells occurred at about 5.147umol/L, 4.25umol/L and 1.25 umol/L respectively.In vivo, 56 Hum-MDS-SCID mice model were randomly grouped. 40 cases served as treated groups by ATO( 5.0|a.g or 7.5ug/g /d i.p. 5d a week, intraperitoneal injection [i.p.]x3 week) alone or in combination with THAL 8(j,g/g /d x3 week or THAL alone) and 16 cases as the control groups(with 0.9%NaCL lOuI/g /d i.p or untreated).The mean tumor diameters (MTD) of subcutaneous tumors were measured with slide gauge and the curative effects and the survival period and the rates of survival were evaluated and cell apoptosis detected by the methods of histopathology, immunohistochemistry (IHC) , Microvesseldensity count (MVD), DNA ladder, TUNEL and PI with flow cytometry.The results showed that marked inhibition effect on the subcutaneous tumors growth (F=146.94, P=0.000)and the higher-rates of cells apoptosis were showed in treated groups by ATO 5.0|ig or 7.5jj.g alone or in combination with thalidomide (THAL) than control groups (F=30A0, P=0.000) .The longer-survival periods (F=25.11, P<0.01) with lower toxicity were only observed in lower-dose group of ATO 5.0\ig than other treated groups and control groups.THAL alone group had a mild inhibition effect on the tumors growth (>2 weeks) with a longer-survival period and higher-rate of survival than controls, but had no events of cell apoptosis.In order to investigate the possible mechanism of THAL. The expressions of vascular endothelial growth factor (VEGF) protein and CD34 protein and MVD were detected and the results showed the expressions of VEGF and CD34 protein markedly down-regulated by THAL compared with control group (P<0.0\), suggesting that the mechanisms of the inhibition effect on tumor growth by THAL related to inhibiting vascular endothelial growth.In our study, we had also observed that the legs paralysis in two MDS-SCID mice model had been observed after treated by THAL alone(1 Id ,15d respectively ) , which were considered as the side-effect of THAL associated with deep venous thrombotic (DVT) events and the mechanism for these events is unclear.We had also detected the tumor cell apoptosis by using DNA ladder , TUNEL, PI by flow cytometry in all groups of MDS-SCID mice model. The results showed the higher-rates of cells apoptosis were showed in treated groups by ATO 5.0|ng or 7.5u.g alone or in combination with thalidomide (THAL) than control groups (F=30.10,ZM).000) .We first investigated the curative effects of ATO in combination with THAL(ATO+THAL) for treating the Hum-MDS-SCID mice model. The results showed that more intense markedly inhibition effect on the subcutaneous tumors growth and the higher-rates of cells apoptosis in ATO+THAL groups than other groups, but the final curative effects was unsatisfied due to the more intense toxicity and the shorter-survival periods than other treated groups (P<0.001) .The results had not supported the preconceived hypothesis of a synergistic effect by ATO in combination with THAL for therapy in MDS mice model due to the more intense toxicity and the shorter-survival periods in ATO7.5(a,g +THAL groups.Summary : ATO had marked inhibition effect both on the subcutaneous tumors growth in MDS-SCID mice model in vivo and on MUTZ-1 cells proliferation in vitro. The results of experimental study of arsenic trioxidealone or in combination with thalidomide for therapy on MDS mice model showed (l)The marked inhibition effect on the subcutaneous tumors growth and the higher-rates of cells apoptosis were observed in treated groups by ATO 5.0ug or 7.5ug alone or in combination with thalidomide (THAL) than control groups. (2)The longer-survival periods and higher-rates of survival with lower toxicity were observed in lower-dose ATO 5.0ug alone than other groups. (3)The mechanisms of the anti-tumor effect of ATO were considered as related to inducing cells apoptosis, but THAL related to inhibiting vascular endothelial growth. (4)Our results had not supported the preconceived hypothesis of a synergistic effect by ATO in combination with THAL for therapy in MDS mice model due to the more intense toxicity and the shorter-survival periods in ATO7.5ug +THAL groups. Whether or not this will translate into clinically relevant effect of the ATO or THAL in MDS patients deserves further consideration.Section 3: Study of mechanisms of arsenic trioxide alone or in combination with thalidomide inducing tumor cell apoptosis on MDS mice model in vivoIn order to deeply explore the possible mechanisms and signal conduct pathway of cell apoptosis by induced with ATO alone or in combination withthalidomide (THAL) in human MDS-SCID mice model in vivo, the expressions of the proteins and the genes related to apoptosis were detected by using the multi-parameter analysis methods of the immunohistochemistry (IHC), Western-blot, RT-PCR, Electrophoretic mobility shift assay (EMSA) with [y-32P]ATP oligonucleotide probe end labeling.In order to explore the connection of the mechanisms of induced cell apoptosis by the ATO alone or in combination with THAL on MDS mice model related to Bcl-2 family and mitochondria, we had detected the expressions of the proteins and genes related to cell apoptosis.The results showed (l)By IHC, compared to controls, the expressions ofthepro-apoptosis protein Bax of Bcl-2 family (F= 1080.150, P <0.01) and the ratio of Bax/Bcl-2 were up-regulated, but of the the expression of anti-apoptosis protein Bax Bcl-2 down-regulated (F =2111.91%, P <0.01) in the treated groups with a dose dependent manner. (2)Western-blot showed that the protein expressions of mitochondrial SMAC/DIABLO and CytC (IHC) were strikingly up-regulated in treated groups, suggesting mitochondria considered as the impotent target of ATO. (3)In other level, We had further detected the expressions of inhibitor of apoptosis proteins cIAPl (IHC) and Survivin mRNA (RT-PCR). The results showed the expressions of cIAPl and Survivin mRNA were markedly down-regulated in treated groups with ATO alone or in combination with THAL, suggestingone of mechanisms of ATO inducing cell apoptosis on MDS-mice model by down-regulated the expressions of IAP1 and Survivin mRNA resulting in the inhibit effect wiped off for Caspases. (4) In order to explore the possible mechanisms and the signal conduction pathway of the cell apoptosis induced by ATO alone or in combination with THAL, we using western blot to detect the protein expressions change of caspase-9, -8,-7,-6,-3 and PARP. The results showed caspase pathway were started and Pro-Caspase 9, 7, 6, 3 and Full PARP were down-regulated with a dose dependent manner (ordinal, ATO 5.0ug,ATO 5.0ug +THAL>ATO 7.5ug ,ATO 7.5ug+THAL). Cleaved caspase-6, 7, 9 and Cleaved PARP were clearly observed in the treated groups. (5) But, no evidences of cleavage of BID and caspase-8 had been observed in our study, suggesting the ATO inducing cell apoptosis mechanisms in MDS mice model were independent on caspase-8 and BID. (6) In order to investigate the possible signal conduct pathway of the tumor cells apoptosis by inducing ATO in MDS-SCID mice model, the activity of nuclear factor-kappaB ( NF-kB ) and activating protein l(APl)and transcription factor SP1 were assessed by Electrophoretic mobility shift assay with [y- P] ATP oligonucleotide probe end labeling (EMSA) and the expression of IkB-o, protein (a NF-kB inhibitor) was detected by Western-blot. The result showed that the activity of NF-kB, API and SP1 were sharply down- regulated in treated groups with a dose dependentmanner (ordinal, ATO 5.0ug> ATO 5.0ug +THAL> ATO 7.5|ig . ATO 7.5u,g+THAL)by EMS A, but the IkB-oi expression were up-regulated in the treated groups, suggesting NF-kB signal conduct pathway were involved in the mechanisms .Summary: Our results showed ATO alone or in combination with thalidomide (THAL) had the marked effects of inducing cell apoptosis in MDS-SCID mice model in vivo. Mitochondria have been considered as the primary intracellular target of ATO. At least, two pathways, namely, Mitochondrial mediated-caspase dependent cell apoptosis pathway and NF-kB signal conduct pathway were involved in the mechanisms of ATO inducing cell apoptosis on MDS mice model in vivo. The two pathways were relatively self-governed and related to each other in a way in the apoptosis processes. The mechanisms were considered as a complicated network effect regulated by multi-signal pathways, multi-layers, and multi-factors, but not any single pathway.