Title Page
Contents
CHAPTER I. Mechanism for cancer cell specific ROS increase by metformin 9
INTRODUCTION 10
MATERIALS and METHODS 13
1. Reagents 13
2. Cell culture 13
3. MTT assay 14
4. Immunofluorescence 14
5. ROS Measurement 15
6. Western blots analysis 15
9. Statistical analysis 16
RESULTS 17
1. Metformin differentially regulates cellular ROS and ATP levels in normal and cancer cells. 17
2. Metformin causes nuclear localization of p-AMPK (Thr172) and p-FOXO3a (Ser413) by activating the AMPK/FOXO3a/MnSOD pathway in normal cells but not in cancer cells 17
3. Transfection with wild type FOXO3a (wt-FOXO3a) and si-FOXO3a RNA suggests that FOXO3a activation plays a key role in determining cellular ROS levels 18
DISCUSSION 23
CHAPTER II. Combination of metformin/efavirenz/fluoxetine exhibits profound anticancer activity via a cancer cell-specific ROS amplification 25
INTRODUCTION 26
MATERIALS and METHODS 30
1. Cell culture and treat 30
2. Transfection of cells with si-FOXO3a and wt-FOXO3a 30
3. MTT assays 30
4. Cell cycle analysis 31
5. ROS measurements 31
6. Measurements of mitochondrial membrane potential and mitochondrial electron transport chain complex I, II, III and IV activities 32
7. Western blot analysis 33
8. In vivo test of anticancer activity 33
9. Statistical analysis 34
RESULTS 35
1. Metformin induces cancer cell-specific ROS increase via the AMPK/FOXO3a/MnSOD pathway 35
2. Combination of metformin, efavirenz and fluoxetine amplifies cellular ROS and induces cell death in HCT116 human colon cancer cells 36
3. Combination of metformin/efavirenz/fluoxetine (M+E+F) induces ROS amplification via synergistic inhibition of mitochondrial membrane potential and mitochondrial electron transport complex I and III activities only in HCT116 cancer cells 37
4. Effect of drug combination M+E+F on the levels of DNA damage-induced apoptosis-, autophagy- and necroptosis-related proteins in HCT116 and HDF cells 38
5. Combination of metformin/efavirenz/fluoxetine (M+E+F) effectively reduces tumor growth in an in vivo model 39
DISCUSSION 49
REFERENCES 55
국문 초록 64
Abstract 66
Chapter I. Mechanism for cancer cell specific ROS increase by metformin 6
Figure 1. Effects of metformin on cellular ROS levels in HEF, MDA-MB231, SkBr3, MCF-7, T47D, and ZR-75 cells. Cellular ROS production was detected by CellROX... 19
Figure 2. Levels of AMPK, p-AMPK (Thr172), FOXO3a, p-FOXO3a (Ser413) and MnSOD proteins were measured in metformin-treated HEF, MDA-MB231, SkBr3,... 20
Figure 3. Effect of metformin on FOXO3a, p-FOXO3a, AMPK, p-AMPK, MnSOD, ATP, and ROS levels in HCT116 and HDF cells. (A) HCT116 and HDF cells were... 21
Figure 4. A hypothetical diagram depicting mechanism of cancer cell death induced via cancer cell specific ROS amplification by metformin. 22
Chapter II. ROS amplification through mitochondrial complex I and III inhibition by combination of mitochondrial binding compounds 6
Figure 1. Effect of metformin on FOXO3a, p-FOXO3a, AMPK, p-AMPK, MnSOD, ATP, and ROS levels in HCT116 and HDF cells. (A) HCT116 and HDF cells were... 40
Figure 2. Changes in cell survival, cellular ROS levels, and cell death by the combination of metformin/efavirenz/fluoxetine in HDFs and HCT116 cells. (A)... 42
Figure 3. Effect of metformin, efavirenz, fluoxetine, metformin/efavirenz, metformin/fluoxetine, efavirenz/fluoxetine or metformin/efavirenz/fluoxetine... 43
Figure 4. Changes in protein levels for DNA damage and apoptosis-related factors in HDFs and HCT116 cells treated with metformin/efavirenz/fluoxetine... 44
Figure 5. Changes in protein levels for apoptosis-related factors in HDF and HCT116 cells treated with the combination of metformin/efavirenz/fluoxetine.... 45
Figure 6. Changes in the protein levels for autophagy and necroptosis-related factors in HDFs and HCT116 cells treated with the combination of... 46
Figure 7. Effect of metformin, efavirenz, fluoxetine, metformin/efavirenz, metformin/fluoxetine, efavirenz/fluoxetine or metformin/efavirenz/fluoxetine... 47
Figure 8. A hypothetical diagram depicting mechanism of cancer cell death induced via cancer cell specific ROS amplification by combination of... 48