טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAmara Rula
SubjectHypoxia, TSPO, and Lung Cancer
DepartmentDepartment of Medicine
Supervisors ? Rafael M. Nagler
Professor Emeritus Moshe Gavish
Full Thesis textFull thesis text - English Version


Abstract

The elucidated and yet to be conclusively determined functions of TSPO indicate a possible role in the pathogenesis of peripheral and brain diseases such as cancer, neurodegeneration, and hypoxic conditions. Previous studies of our lab have shown that the hypoxic-mimicking agent cobalt chloride (CoCl2) led to alterations in several TSPO-related mitochondrial processes. Moreover, TSPO was identified as a modulator of the apoptotic pathway involving reactive oxygen species (ROS) generation, mitochondrial membrane potential (Δψm) collapse, activation of caspases and eventually initiation of the apoptotic process.

This study aims to evaluate the protective capacity of the classical TSPO ligand PK 11195 in the protection from CoCl2-induced cellular toxicity in peripheral (lung cancer) and brain (microglial) cell lines by assessment of various mitochondrial processes.

In this in vitro study, H1299 lung cells and BV-2 microglial cells were exposed to the toxic CoCl2 agent with or without PK 11195. Several assays were conducted to examine the alterations in cell viability including XTT assay, TSPO levels, ROS generation using both NAO and Cellular ROS/superoxide assay, mitochondrial membrane potential depolarization, apoptosis, and apoptotic markers.

Exposing the H1299 cells to 0.5 mM CoCl2 for 24 hours resulted in significant decreases in cell viability (63%, p<0.001), significant elevation of cardiolipin peroxidation levels (38%, p<0.05), mitochondrial membrane potential depolarization (13%, p<0.001), and apoptotic cell death(117%, p<0.05). Pretreatment with PK 11195 at a concentration of 25 µM exhibited significant protective capacity against CoCl2-induced alterations in the previously mentioned processes.

Exposure of BV-2 cells to increasing concentrations of CoCl2 (0.3, 0.5, 0.7 mM) for 4 and 24 hours resulted in alterations in cellular processes at various degrees. These alterations were obtained in a dose-dependent manner, that include significant alterations in cellular viability, TSPO levels, and oxidative stress levels. These dose-dependent alterations were not accompanied with in changes in   caspases 3 and 9.

Our findings have shown that CoCl2 causes cellular damage in both peripheral and brain cell lines, and PK 11195 exhibited protective capacity against CoCl2-induced cellular damage. However, PK 11195 by itself and CoCl2 at low concentrations (0.3 mM) resulted in significant reduction in some mitochondrial-related processes. Our findings indicate that TSPO ligands may offer new therapeutic opportunities for the treatment of lung and brain diseases which fail to respond to conventional therapies.