The Study Of Uroacitides On The Proliferation Of Myelodysplastic Syndrome (MDS) Cells And Its Mechanism

Myelodysplastic syndrome(MDS)is a heterogeneous group of clonal bone marrow disorders characterized by varying degrees of cytopenias with ineffective hematopoiesis.It is now still considered to be uncurable disease.The WHO instituted the IPSS for MDS according to the percentage of the blast cells,the karyotype of chromosome and the extent of cytopenias.The MDS patients are devided into three groups according to the IPSS,including low-risk group(score 0),middle-risk group (score 0.5-2.0)and high-risk group(score more than 2.5).The blast cells of the middle-risk and high-risk patients are hyperplasia,with severe cytopenias.Because the bone marrow compensation is poor in these patients,it is very difficult to treat them.Most patients need frequently for blood transfusion.Over time,there is worsening bone marrow fail or progression to acute myeloid leukemia(AML).The advanced age of the majority of MDS patients limits the therapeutic strategies often to supportive care.The patients can' t suffer the standard chemotherapy and bone marrow transplantation.A number of cytidine analogs such as 5-azacytidine(Aza C) and 5-Aza-2' -deoxycitidine(decitabine,DAC)have been approved by the FDA in the United States for the treatment of MDS.The drugs have intensive bone marrow suppression,which limits their clinical use.So there is a tremendous need for novel approaches which have better effect and no bone marrow toxicity in the management of middle-risk and high-risk MDS patients.Uroacitides,another name CDA-Ⅱ(cell differentiation agentⅡ),is a mixture product and isolated from healthy human urine in China.It is characterized as a novel molecular targeting agent for cancer therapy.The human urine is acidified during collection and pass through an ultrafiltration process to remove molecules with molecular weight larger than 10,000 daltons.Multiple active components,such as peptides(MW 400-2800),organic acids,pigments and phenylacetylglutamine,with different mechanisms of action act concurrently to contribute to the anticancer effect of CDA-Ⅱ.Because CDA-Ⅱis healthy human urine extracts,it has no medullary and extramedullary toxicities,which makes it a suitable candidate for the treatment of MDS patients.However,there has been no report that clearly describes the mechanisms of CDA-Ⅱin MDS cells.In the present study,we evaluate both in-vitro and in-vivo anticancer activities and the mechanisms of action of CDA-Ⅱon MDS model, which might provide the experimental therapeutic data to expand its indications in clinical trials.There are three sections in our research.Section 1:Uroacitides(CDA-Ⅱ)induces MDS cells into apoptosis via cell survival signaling pathway.Objective:The aim of this study was to investigate the antitumor activity of CDA-Ⅱagainst MDS cells in vitro and in vivo and to determine if CDA-Ⅱ-induced apoptosis of MDS cells was associated with PI3K/Akt and NF-κB survival signaling pathways.Methods:We used MTT assay to investigate the effects of CDA-Ⅱon the MDS,AML and lymphoma cell lines and normal bone marrow mononuclear cells(BMMCs)growth. MDS-RAEB cell line MUTZ-1 was used to examine the effects of CDA-Ⅱon the induction of growth arrest and apoptosis.Apoptosis was measured by flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer' s instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.The cell-cycle analysis was done according to the PI staining by flow cytometry.Apoptotic proteins including Caspase-9,8,3,Bcl-2 family,IAPs family and FLIP were studied by western-blot. Phosphoinositide 3-kinase(PI3K)/Akt and NF-κB survival signaling pathways were also examined using western-blot analysis.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.PI3K inhibitor LY294002 and NF-κB inhibitor PDTC were used to examine the involvement of PI3K/Akt and NFκB signaling pathways.TNF-αwas used to determine the inhibition of CDA-Ⅱon the translocation of NF-κB(p65).The PIK3CA gene,which expressed the PI3Kp110 a protein,was examined by using RT-PCR to identify the target of CDA-Ⅱ- induced apoptosis.MUTZ-1 cell xenografted SCID mice were used for in-vivo study.Results:①CDA-Ⅱcould inhibit growth of MUTZ-1 cells and human leukemia cell lines.The sensitivity of malignant lymphoid cell lines to CDA-Ⅱwas lower than those of MUTZ-1 cells and myeloid leukemia cells.MUTZ-1 cell line was most sensitive to CDA-Ⅱand cell growth was inhibited in a time- and dose-dependent manner, corresponding to the reduced cell viability.In contrasts,CDA-Ⅱdid not induce cytotoxicity in BMMCs from three normal volunteers(p＜0.01).②CDA-Ⅱinduced apoptosis of MUTZ-1 cells in a dose-dependent manner.③CDA-Ⅱinduced G1 arrest, accompanied with a concomitant reduction of Cyclin D1 and CDK4 expression in a dose-dependent manner.④Treatment with CDA-Ⅱinduced marked activation of caspase-9,3 in a dose-dependent manner,however,caspase-8 was activated only at CDA-Ⅱconcentrations greater than 4 mg/mL.Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following CDA-Ⅱtreatment.⑤Bcl-2,Mcl-1 and p-Bad proteins were decreased,however,Bax and Smac proteins were increased in a dose-dependent manner after CDA-Ⅱtreatment for 24h;Bid protein was cleavaged only at CDA-Ⅱconcentrations greater than 4 mg/mL.⑥The XIAP,CIAP1 and Survivine proteins were down-regulated in a dose-dependent manner after CDA-Ⅱtreatment,and not of CIAP2 protein.⑦MUTZ-1 cell line was high expression of FLIP_L and it was inhibited at CDA-Ⅱconcentrations greater than 4mg/mL.⑧CDA-Ⅱinhibited both intrinsic and TNF-α-induced IκBαphosphorylation and prevented NFκB (p65)nuclear translocation in a dose-dependent manner;CDA-Ⅱcombined with PDTC significantly inhibited the expression of XIAP,CIAP1,Survivine,FLIP_L,BCl-2 as well as Cyclin D1 proteins.⑨CDA-2 significantly reduced expression of PI3Kp110αas well as p-Akt in a dose-dependent manner;the results were further confirmed by PIK3CA gene at mRNA level in a time-and dose-dependent manner;CDA-Ⅱcombined with LY294002 significantly inhibited the expression of p-IκBα,p-Bad,Mcl-1 proteins and up-regulated the expression of Bax protein as well as activated caspase-9.⑩CDA-Ⅱinhibited tumor growth in MUTZ-1 xenografted SCID mice in a dose-dependent manner and improved the survival time of the animal.There was no significant body weight change in SCID mice during the experiment.Conclusions:CDA-Ⅱcould induce growth arrest and apoptosis of MUTZ-1 cells in vitro and in vivo.The main mechanisms were related to the inhibition of PI3Kp110αexpression at transcriptional level,which inactivated the phosphorylation of Akt involving prevention NF-κB nuclear translocation,downregulation of IAP family and FLIP_L protein as well as involvement of Bcl-2 family and triggered the mitochondrial pathway mediated caspase-3-dependent apoptosis.Section 2:The effects of Uroacitides(CDA-Ⅱ)on the abnormal hypermethylation patterns of PTEN and p151NK4B genes in MDS cells.Objective:The aim of this study was to investigate the methylation patterns of PTEN gene in MDS and MDS transformed AML in order to determine the relationship between the PTEN gene and the development of MDS.Another purpose of this study was to identify the effects of CDA-Ⅱon the abnormal high expressions of DNMT1,3A and 3B as well as on the abnormal hypermethylation patterns of PTEN and p15INK4B genes in MDS cells.Methods:We used western-blot to investigate the expressions of DNMT1,3A and 3B as well as PTEN and p15INK4B proteins on the MUTZ-1 cell line,low-risk MDS, high-risk MDS and MDS transformed AML cells as well as normal BMMCs.RT-PCR and western-blot were used to determine the effects of CDA-Ⅱon the mRNA and protein expressions of DNMT1,3A and 3B as well as PTEN and p15INK4B.The methylation patterns of PTEN and p15INK4B genes in MUTZ-1 and AML cell lines,low-risk MDS,high-risk MDS and MDS transformed AML cells as well as normal BMMCs were examined using methylation specific PCR(MSP).MSP was also used to identify the effects of CDA-Ⅱon the abnormal hypermethylation patterns of PTEN and p15INK4B genes in MUTZ-1 cells. The demethylating agent decitabine was used as positive control.Results:①The high expressions of DNMT1,3A and 3B proteins were found in MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells;The expression level of DNMT3B was higher than that of DNMT1 and 3A.The low-risk MDS and nomal BMMCs expressed DNMT1,3A and 3B proteins only at low levels.②Treatment with CDA-Ⅱinduced marked inhibition of DNMT1,3A and 3B in a time- and dose-dependent manner. Among them,DNMT1 was the most effective in CDA-Ⅱmanagement.③The absence of PTEN gene expression in protein level was found in MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells,however,the low-risk MDS and normal BMMCs cells all expressed the PTEN protein.④We used MSP to determine if the absence of PTEN gene expression was due to the abnormal hypermethylation pattern of it.We found that MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells were PTEN completely methylation,the AML cell line Kasumi-1 was PTEN partly methylation,however,the KG1 and K562 cell lines,low-risk MDS and normal BMMCs were PTEN completely unmethylation,which indicated that the tumor suppressor gene PTEN abnormal hypermethylation was associated with the development of MDS and it might be a new target in MDS therapy.⑤CDA-Ⅱcould downreglate the abnormal hypermethylation pattern of PTEN gene in a time- and dose-dependent manner,resulting in the upregulation of PTEN mRNA and protein expressions and triggering inhibition of PI3K/Akt survival signaling pathway,which induced MUTZ-1 cells apoptosis.⑥CDA-Ⅱcould downreglate the abnormal hypermethylation pattern of p15INK4B gene in a timeand dose-dependent manner,resulting in the upregulation of p15INK4B mRNA and protein expressions and triggering inhibition of CDK4 expression,which induced MUTZ-1 cells G1 arrest.Conclusions:That MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells were PTEN hypermethylation,however,low-risk MDS and normal BMMCs were PTEN completely unmethylation indicated that PTEN abnormal hypermethylation was associated with the development of MDS and it might be a new target in MDS therapy. CDA-Ⅱcould downreglate the abnormal hypermethylation patterns of PTEN and p15INK4B genes in a time- and dose-dependent manner,resulting in the upregulation of PTEN and p15INK4B expressions.The high expression of PTEN triggered inhibition of PI3K/Akt survival signaling pathway,which induced MUTZ-1 cells apoptosis.The high expression of p15INK4B triggered inhibition of CDK4 protein,which induced MUTZ-1 cells G1 arrest.Section 3:The study of Uroacitides(CDA-Ⅱ)on the telomerase activity and hTERT expression in MDS cells and its mechanism.Objective:The aim of this study was to investigate the effects of CDA-Ⅱon the telomerase activity and hTERT expression in MDS cells and its mechanism.We wanted to identify if the WT1 gene and PI3K/Akt/NF-κB survival signaling pathway were involved in the effect of CDA-Ⅱon the expression of hTERT in MDS cells.Methods:TRAP method was used to investigate the effect of CDA-Ⅱon the telomerase activity in MDS cells.We used real-time RT-PCR and western-blot to determine the effects of CDA-Ⅱon the mRNA and protein expressions of hTERT.RT-PCR was used to study the effect of CDA-Ⅱon the expression of WT1 gene on transcriptional level.The PI3K inhibitor LY294002 and the NF-κB inhibitor PDTC were used to identify the involvement of PI3K/Akt/NF-κB survival signaling pathway in the effect of CDA-Ⅱon the expression of hTERT in MUTZ-1 cells.Results:①CDA-Ⅱcould significantly inhibited the telomerase activity in a time- and dose-dependent manner,which was accordance with the CDA-Ⅱ-induced apoptosis.It suggested that the inhibition of telomerase activity might be the caspase-3 independent mechanism involved in CDA-Ⅱ-induced apoptosis.②CDA-Ⅱ exerted substantial inhibition of hTERT on transcriptional and protein levels in a time- and dose-dependent manner,which was concordance with that of telomerase activity in MDS cells.Therefore hTERT might be a new target for CDA-Ⅱtreatment.③Treatment with CDA-Ⅱinduced marked inhibition of WT1 gene expression in a timeand dose-dependent manner,suggesting that CDA-Ⅱcould inhibit hTERT through downregulation of WT1 gene expression.④CDA-Ⅱcombined with LY294002 and PDTC could significantly inhibit the expression of hTERT protein,which indicated that CDA-Ⅱmight inhibit hTERT expression and triggered caspase-3 independent apoptosis through PI3K/Akt/NF-κB survival signaling pathway in MDS cells.Conclusions:CDA-Ⅱcould significantly inhibit the telomerase activity as well as the expressions of hTERT on transcriptional and protein levels,which might be a new target for CDA-Ⅱtreatment.CDA-Ⅱcould inhibit hTERT expression and telomerase activity as well as triggered caspase-3 independent apoptosis through PI3K/Akt/NF-κB survival signaling pathway and inhibition of WT1 gene in MDS cells.Summary:CDA-Ⅱcan induce growth arrest and apoptosis of MUTZ-1 cells in vitro and in vivo.CDA-Ⅱdoes not induce cytotoxicity in normal BMMCs,nor does it show any toxicity on the SCID mice.CDA-Ⅱis an effective medicine with multiple targets, including involvement of PI3K/Akt/NF-κB survival signaling pathway directly, downreglation of the abnormal hypermethylation patterns of PTEN and p15INK4B genes and inhibition of hTERT expression and telomerase activity.Different mechanisms of action acted concurrently to contribute to the CDA-Ⅱ-induced MDS cell apoptosis (See Figure 3-4).This research work may provide new insights for the treatment of high-risk MDS.