Pharmacological studies of a novel inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway [Elektronische Ressource] / from Samy Abd El-Raouf Fahim Khalafalla Morad

Morad Mousli, Ama Samy

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

From the
Department of Veterinary Sciences
Faculty of Veterinary Medicine
LudwigMaximiliansUniversity of Munich
Prof. Dr. Hermann Ammer


Performed under the supervision of
Prof. Dr. Thomas Simmet
Institute of Pharmacology of Natural Products & Clinical Pharmacology
Faculty of Medicine, Ulm University


Pharmacological Studies of a Novel Inhibitor of the Mammalian
Target of Rapamycin (mTOR) Signaling Pathway


Inaugural Thesis
for the
Doctor Degree in Veterinary Medicine
Faculty of Veterinary Medicine
Ludwig Maximilians University of Munich


From
Samy Abd ELRaouf Fahim Khalafalla Morad
from Qena, Egypt



Munich 2010

Printed with the permission of the Faculty of Veterinary Medicine
Ludwig Maximilians University of Munich






Dekan: Univ.Prof. Dr. Braun
Bericterstatter: Univ.Prof. Dr. Ammer
Korreferent/en: Univ.Prof. Dr. Gabius
Univ.Prof. Dr. Stangassinger
Univ.Prof. Dr. Hirschberger
Univ.Prof. Dr. Wanke




Defence date: July 24, 2010

























This work is dedicated to
my parents, my wife, my son, my daughter,
and the spirit of Abd El Mageed Foud










C O N T E N T S

Table of Contents
ABBREVIATIONS..................................................................................................... IV
1. INTRODUCTION .............................................................................................. 1
1.1 APOPTOSIS ......................................................................................................... 1
1.1.1 Historical perspective ........................................................................................................ 1
1.1.2 Natural occurrence of apoptosis ........................................................................................ 3
1.1.3 Morphological features of apoptosis.................................................................................. 4
1.1.4 Apoptosis versus necrosis ................................................................................................. 5
1.1.5 Biochemical features of apoptosis..................................................................................... 7
1.1.6 Apoptosis pathways (induction of apoptosis) .................................................................... 7
1.1.6.1 Extrinisic (death receptor) pathway .......................................................................... 8
1.1.6.2 Intrinsic (mitochondrial) pathway .............................................................................. 9
1.1.6.3 Perforin/granzyme pathway ...................................................................................... 9
1.2 Mammalian target of rapamycin (mTOR) signaling ......................................... 10
1.2.1 The mTOR proteins ......................................................................................................... 10
1.2.2 Regulation of mTOR Activity ........................................................................................... 12
1.2.2.1 Activation of mTOR by the PI3K signaling pathway ............................................... 12
1.2.2.2 Inhibition of mTOR by the LKB1/AMPK/TSC2 signaling pathway .......................... 13
1.2.2.3 Inhibition of mTOR by the tuberous sclerosis complex (TSC1/TSC2) ................... 14
1.2.2.4 Regulation of pathways downstream of mTOR ...................................................... 15
1.2.2.5 Regulation of p70S6K activation ............................................................................ 15
1.2.2.6 Regulation of the 4EBP1/eIF4E complex .............................................................. 16
1.2.2.7 Regulation of protein serine/threonine phosphatase .............................................. 18
1.2.3 mTOR related diseases and the challenges associated with targeting mTOR ............... 21
1.2.3.1 Tuberous sclerosis complex (Hamartomas) ........................................................... 21
1.2.3.2 Hamartoma related syndromes .............................................................................. 22
1.2.3.3 Polycystic kidney disease ....................................................................................... 22
1.2.3.4 Neurodegenerative disorders ................................................................................. 23
1.2.3.5 Cancer .................................................................................................................... 24
1.2.3.6 Other disorders and drug resistances .................................................................... 27
1.3 Apoptosis and mTOR signaling ........................................................................ 28
1.4 Aims of the study .............................................................................................. 29
2. MATERIALS and METHODS ........................................................................ 30

I
C O N T E N T S

2.1 Synthesis of 32cinnamoyl2112ketoββββ22boswellic acid (C2KβBA) ....................... 30
2.2 Cell culture ......................................................................................................... 31
2.3 Antiproliferative effect of C2KβBA in vitro ...................................................... 33
2.3.1 Cell proliferation assay (XTT assay) ............................................................................... 33
2.3.2 Clonogenic survival assay ............................................................................................... 34
2.3.3 Antiproliferative effect of CKβBA in vivo ........................................................................ 35
2.4 Analysis of apoptosis parameters .................................................................... 36
2.4.1 Expression of phosphatidylserine on the cell surface ..................................................... 36
2.4.2 Measurement of caspase activity .................................................................................... 38
2.4.3 Measurement of DNA fragmentation ............................................................................... 40
2.5 Cell cycle analysis ............................................................................................. 41
2.6 Protein phosphatase assay .............................................................................. 43
2.7 Analysis of protein expression ......................................................................... 45
2.7.1 Preparation of samples ................................................................................................... 46
2.7.2 Protein determination ...................................................................................................... 47
2.7.3 SDSPAGE ...................................................................................................................... 48
2.7.4 Western blotting and detection of proteins ...................................................................... 49
2.8 FKHR (FOXO1) ELISA ........................................................................................ 53
2.9 Statistical analysis............................................................................................. 53
3. RESULTS ...................................................................................................... 54
3.1 Synthesis of C2KβBA ......................................................................................... 54
3.2 Antiproliferative effect of C2KβBA ................................................................... 57
3.3 C2KβBA induces cell cycle arrest .................................................................... 60
3.4 C2KβBA triggers apoptosis in vitro .................................................................. 61
3.4.1 CKβBA induces phosphatidylserine expression on the cell surface .............................. 61
3.4.2 CKβBA induces caspase3 activation ............................................................................ 61
3.4.3 CKβBA induces DNA laddering ..................................................................................... 63
3.5 C2KβBA inhibits growth, proliferation, and triggers apoptosis in vivo .......... 64
3.6 C2KβBA inhibits the mTOR signaling pathway ............................................... 65
3.7 C2KβBA inhibits the mTOR signaling pathway independent from upstream
kinases ............................................................................................................... 70

II
C O N T E N T S

3.8 C2KβBA inhibits the mTOR signaling pathway independent from the LKB12
AMPK signaling pathway ..............................................................