title: Metallothionein 2A an interactive protein linking phosphorylated FADD to NF-κB pathway leads to colorectal cancer formation.
abstract: BACKGROUND
  The rapid increase in the incidence rate of colorectal cancer has led to the search and identification of biomarkers that can predict risk for and future behavior of this malignancy and management. To study the biological role of the phosphorylated Fas associated death domain (pFADD) gene in colorectal cancer, we performed a GAL4-based yeast two-hybrid screening of a human heart cDNA library.
  
  
  METHODS
  A series of two yeast hybrid method was used to identification of protein-protein interaction. It was confirmed by glutathione S-transferase (GST) pull down assay and co-immunoprecipitation (co-IP). Three channeled fluorescence microscopy further confirmed the interaction in cellular level. Xenograft in vivo model was developed and knockdown relevant genes by RNAi techniques and confirmed the relationship which leads to colorectal cancer.
  
  
  RESULTS
  Using the FADD cDNA as bait, we identified six putative clones as associated proteins. The interaction of pFADD and metallothionein 2A (MT2A) was confirmed by GST pull-down assays in vitro and co-IP experiments in vivo. FADD co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by three channel fluorescence microscopy. Co-transfection of pFADD with MT2A gene inhibited cell apoptosis and induced cell proliferation in colorectal cancer cells compared with control groups. When we used antisense MT2A and pFADD which is serine 194 in the C terminal of FADD gene that has been reported to be phosphorylated to interdict the effect of respective genes the inhibition of cell proliferation and induction of apoptosis were significantly enhanced in animal model.
  
  
  CONCLUSIONS
  Further in this study we identify non-canonical nuclear factor-κB (NF-κB) signaling up regulated and it was directly linked with the tumor necrosis with MT2A and pFADD genes. pFADD with MT2A can inhibit the apoptosis and promote proliferation, of colorectal cancer cells, and antisense sequence of MT2A and pFADD approaches which might swell the combination of deregulated proliferation and suppressed apoptosis.
authors.affiliation: Molecular Biology Unit, Sir John Kotelawala Defence University, Kandawala Estate, Ratmalana, Sri Lanka. faiz.marikar@fulbrightmail.org.
authors.forename: Faiz M M T
authors.initials: FM
authors.lastname: Marikar
authors.affiliation: Department of Basic Medical Sciences, Xiamen University, Xiamen 361005, China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Jin
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, College of Life Science and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, Nanjing 210004, China.
authors.forename: Wang
authors.initials: W
authors.lastname: Sheng
authors.affiliation: Center of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Dingyuan
authors.initials: D
authors.lastname: Ma
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, College of Life Science and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, Nanjing 210004, China.
authors.forename: Zichun
authors.initials: Z
authors.lastname: Hua
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D053200:Fas-Associated Death Domain Protein / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D014408:Biomarkers, Tumor; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; C491053:MT2A protein, human; D016328:NF-kappa B; D008668:Metallothionein
keywords: Fas associated death domain (FADD); GAL4 two yeast hybrid; Metallothionein (MT); RNAi; antisense; cancer; colorectal; nuclear factor-κB (NF-κB)