טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGochman Einat
SubjectInvolvement of Reactive Nitrogen Species (RNS) in Activation
of Transcription Factor NF-kB in Colon Cancer
Cells In Vitro and in Human Colon
Sections Ex Vivo
DepartmentDepartment of Medicine
Supervisors Professor Abraham Reznick
Dr. Jamel Mahajna
Full Thesis textFull thesis text - English Version


Abstract

The reactive nitrogen species peroxynitrite (ONOO?) is a product of the reaction of nitric oxide (NO?) with superoxide (O2??).
This powerful oxidant (ONOO?) has been proposed to contribute to the pathology of a large variety of diseases associated with increased inflammation and cancer development by its ability to oxidize and nitrate a variety of macromolecules. Peroxynitrite was also found to activate the transcription factor NF-κB in the non-canonical or aberrant pathway. 
NF-κB is a predominant player in inflammatory and carcinogenic processes and has been found to play a major role in the transition from chronic inflammation to cancer initiation.
The involvement of peroxynitrite in NF-κB activation in cancer cells and in cancer development is unclear. Recently, several studies have demonstrated specific interactions between the MAPK pathways, especially p38, and the NF-κB pathway. Therefore, the aim of the present study was to elucidate the molecular mechanisms by which NF-κB is activated by peroxynitrite and whether there is interaction with the MAPK's pathway under nitrative stress in a human colon cancer cell line. In addition, ex-vivo studies have tried to support the in vitro results of NF-κB activation and inflammatory and proliferation markers under its regulation in healthy, colitis and in human colon cancer sections of patients.
In the in vitro studies, in HT-29 colon cancer cells we observed that the dynamic, kinetic, and intensity activation of NF-κB caused by ONOO?,  lead to an aberrant and belated pathway compared to the canonical activation by TNF-α.
We also observed that peroxynitrite activates the NF-κB pathway in HT-29 in a simultaneous dual mechanism: IKK dependent by which phosphorylation on IκBα occurs and translocation of p65 occurs after 60 min and IKK independent by which direct nitration of IκBα occurs as well as lowered nuclear translocation. It is also important to note that nitration of IκBα occurs at the expense of its phosphorylation and vice versa. We also showed that p38 is involved in IKK dependent NF-κB activation under peroxynitrite treatment. Finally, in the clinical studies we found that higher protein nitration, which is product of peroxynitrite presence, was also predominant in the histological sections. In addition, higher activation of NF-kB, as well as activation of its target genes, was observed in colitis and colon cancer sections compared to controls which strengthens our in vitro findings.