|Ph.D Student||Zeineh Nidal|
|Subject||Cigarette Smoke, TSPO, Oral and Respiratory Diseases|
|Department||Department of Medicine||Supervisors||ASSOCIATE PROF. Rafael M. Nagler|
|PROFESSOR EMERITUS Moshe Gavish|
|Full Thesis text|
The 18 kDa translocator protein (TSPO) plays an important role in the initiation of the mitochondrial apoptosis cascade, including apoptosis induced by cigarette smoke (CS) and cobalt chloride (CoCl2). Both CS and CoCl2 provoke alterations in TSPO expression as well as upregulation of its related functions such as mitochondrial membrane potential (ΔΨm) and reactive oxygen species generation (ROS), which are associated with the commence of the programmed cell death process.
In the first part of this project, we investigated the mechanism via which CS exert it’s cytotoxic effects by exposing the H1299 lung cancer cell line to different exposure times of CS (30 minutes, 60 minutes, and 120 minutes). In addition, we examined the protective capacity of our TSPO ligands MGV-1 and 2-Cl-MGV-1 against the CS-induced damages following 60 minutes of CS exposure of lung cells, and following 30 minutes of CS exposure of cardiomyocytes. In the second part of the project, we investigated the effect of CoCl2 on TSPO-related mitochondrial processes in lung cancer cells (H1299), and the role of high affinity TSPO ligand (CB86; Ki=1.6 nM) and a low affinity TSPO ligand (CB204; Ki=117.7 nM) in protection against the CoCl2-induced cytotoxicity. In the third part of the project, we examined the impact of the circulating CS extracts (CSE) in the blood on neuroinflammation, by investigating the impact of CSE on the endothelial cells in the in vitro model of the blood brain barrier (BBB) and on brain cells in the same model.
Our results showed that CS induces alterations in TSPO and TSPO-associated mitochondrial processes in a time-dependent manner. The ligands alone did not cause adverse effects in our cellular models. The protective capacity of 2-Cl-MGV-1 was superior to MGV-1 at a concentration of 25 µM, in prevention of the CS-induced alterations in TSPO expression levels, TSPO binding, ADP/ATP ratio, oxidative stress, mitochondrial membrane potential (ΔΨm) depolarization, cAMP levels, and cell death levels. In the second model, the results demonstrated the superiority of the low affinity ligand (CB204) over the high affinity ligand (CB86) in protection against the CoCl2-induced damage. In the third model, it was demonstrated that CSE can, at least partially, penetrate the BBB and induce inflammatory responses on both sides of the BBB, as well as effect on the MAPK pathway and cell death in glial cells.
To conclude, CS and CoCl2 exposure induces upregulation of TSPO expression and TSPO-related cell death processes. This suggests that TSPO may be involved in the development of CS- and CoCl2- induced tissue damage. Our findings demonstrate the protective capacity of our TSPO ligands (MGV-1 and 2-Cl-MGV-1) in prevention of CS-related lung diseases, but not oral diseases. Furthermore, it appears that the low affinity for TSPO target enhances the potency of TSPO ligands in the protection from hypoxic cell death. These findings suggest that the low affinity ligands may be candidates for the treatment of various CS-induced and hypoxia-induced diseases, such as pulmonary ischemic diseases and chronic obstructive diseases (COPD).