טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentCohen Shiri
SubjectThe Synergistic Effect of Cigarette Smoke and Saliva on the
Translocator Protein (TSPO) in an In Vitro Model
of Lung Cancer
DepartmentDepartment of Medicine
Supervisors Professor Rafael M. Nagler
Professor Emeritus Moshe Gavish
Full Thesis textFull thesis text - English Version


Abstract

A synergistic effect of cigarette smoke (CS) and saliva was recently demonstrated, being based on the reaction between redox active metals in saliva and low reactive free radicals in CS, which results in production of highly active hydroxyl free radicals. Previously, it was suggested that the Translocator Protein (TSPO) might protect cells from damage induced by free radicals. TSPO is involved in essential functions, which are related to tumorigenicity. The purpose of the current study was to examine the synergistic effect of CS and saliva on epithelial H1299 lung cancer cells. Moreover, we wanted to explore the possible mediatory role of redox active metals, by applying antioxidants or metal chelators to the cell medium prior to the exposure to CS. We also examined whether TSPO binding and expression were influenced by the synergistic effect of CS and saliva. For these goals, we exposed the H1299 cells to CS in the presence, as well as in the absence of saliva. We found that CS exposure alone for 120 min resulted in a 37% survival loss, while following exposure to CS in the presence of saliva, more than 57% of the cells died (p<0.001).  Moreover, H1299 cells being exposed to CS alone demonstrated a significant accumulation of protein carbonyls, an increase of CL oxidation, and mitochondrial potential reduction. These were also accompanied by reduction in the specific binding of [3H]PK 11195 to TSPO, as well as reduction in the 72kDa TSPO protein expression levels and induction of its associated protein, VDAC. CS exposure in the presence of saliva resulted in an additional increase of protein carbonylation, and CL oxidation. The most potent protecting agents found in our system were the cupper chelator Penicillamine (PenA), the iron chelator Desferal (DES), and the thiol Glutathione. While DES and GSH protected against the lethal effect of CS alone, PenA significantly protected the cells from the lethal synergistic effect of CS and saliva.

In summary, our results demonstrate that CS and more so in the presence of saliva is lethal to the cells and deleterious to the TSPO. These effects are mediated by free radicals and can be prevented by antioxidants and metal chelators. Moreover, redox active metals, which are present in saliva and in the pleural fluid in the lung, may have a possible role in the high worldwide incidence of oral and lung cancer, which is mostly related to CS.