title: Possible involvement of activation of P53/P21 and demethylation of RUNX 3 in the cytotoxicity against Lovo cells induced by 5-Aza-2'-deoxycytidine.
abstract: OBJECTIVE
  In our model, we aimed to explore the cytotoxicity of 5-Aza-2'-deoxycytidine (5-Aza-CdR) against the colorectal cell line, Lovo, and further characterize the possible mechanisms.
  
  
  METHODS
  After Lovo cells were treated with 5-Aza-CdR at different concentrations for different periods of time, the cell viability was examined using an MTT assay and apoptosis was examined using both flow cytometry and DNA laddering. To examine the mechanisms by which Lovo cells respond to 5-Aza-CdR, we measured both caspase 3 activity as well as DNA damage. Western blotting and RT-PCR assays were used to assess the changes in the expression levels of P53, P21(Waf1/Cip1), runt-related transcription factor 3 (RUNX 3), DNA methyltransferases (DNMTs) and matrix metalloproteinases (MMPs). Additionally, we performed gelatin zymography to examine the effects of 5-Aza-CdR on metastasis.
  
  
  RESULTS
  We observed that the growth and survival advantages of Lovo cells were overcome with 5-Aza-CdR treatment at limited concentrations. Mechanistic exploration demonstrated that 5-Aza-CdR was incorporated into the DNA to induce DNA damage in Lovo cells, which was evidenced by activation of P53, P21(Waf1/Cip1) and a caspase-independent cell apoptosis pathway. Also, further experiments preliminarily suggested that 5-Aza-CdR results in the deletion of DNMT 3a and DNMT 3b, but not DNMT 1, which reactivates the expression of RUNX 3. Finally, our data revealed that 5-Aza-CdR potentially reduces the activity and expression of MMP 2.
  
  
  CONCLUSIONS
  These data greatly enhance our understanding of how human cancer cells respond to 5-Aza-CdR and also reveal a new role for 5-Aza-CdR in improving patient outcome in human colorectal cancer.
authors.affiliation: Department of Gastroenterology, Renmin Hospital of Wuhan University, No 238 Jiefang Road, Wuhan, Hubei 430064, China. dengtao790601@yahoo.com.cn
authors.forename: Tao
authors.initials: T
authors.lastname: Deng
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D000090205:DNA Methyltransferase 3A; D015254:DNA Modification Methylases / Q000378:metabolism; D000077209:Decitabine; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000964:Antimetabolites, Antineoplastic; D050677:Core Binding Factor Alpha 3 Subunit; D050759:Cyclin-Dependent Kinase Inhibitor p21; C000717967:DNMT3A protein, human; D004791:Enzyme Inhibitors; C493729:Runx3 protein, human; D016159:Tumor Suppressor Protein p53; D000077209:Decitabine; D015254:DNA Modification Methylases; D000090205:DNA Methyltransferase 3A; D053148:Caspase 3; D020778:Matrix Metalloproteinase 2; D001374:Azacitidine