Metabolic Assessment of Metformin Treatment in Colon Cancer Cells

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ABSTRACT ... 2

ACKNOWLEDGMENTS ... 3

CONTRIBUTIONS ... 4

TABLE OF CONTENTS ... 5

LIST OF ABBREVIATIONS ... 8

LIST OF TABLES ... 10

LIST OF FIGURES ... 11

CHAPTER 1. LITERATURE REVIEW ... 12

1.1 INTRODUCTION ... 12

1.2 COLORECTAL CANCER ... 13

1.2.1 Epigenetics, Modifiable Risk Factors and Cellular Metabolism Crosstalk in CRC ... 14

1.3 CANCER CELL METABOLISM ... 17

1.3.1 Regulators of Cell Energetics ... 19

1.3.2 Oxidative Phosphorylation dysfunction in cancers... 21

1.3.3 Genetic implications of the impaired cell signalling and altered metabolism in CRC. ... 24

1.4 THE VALUE OF BIOMARKERS IN CRC. ... 25

1.4.1 Biomarkers of Interest in CRC Metabolic Interpretation and Diagnosis ... 25

1.5 THERAPEUTIC AGENTS ... 26

1.5.1 Metformin use in Cancer Treatment ... 28

1.5.2 Metformin’s Dual Modes of Action ... 29

CHAPTER 2. RESEARCH AIMS AND HYPOTHESIS ... 33

2.1 RATIONALE ... 33

2.2 SPECIFIC AIMS ... 34

2.2.1 Characteristics of SW948 Cell line ... 34

2.2.2 In vitro approach ... 35

2.3 RESEARCH IMPACT ... 35

CHAPTER 3. METHODS ... 36

3.1 CELL LINE AND CELL CULTURE... 36

3.1.1 Differential culturing conditions: ... 36

3.2 MANUAL PROLIFERATION ASSAY UNDER DIFFERENTIAL CULTURING CONDITIONS ... 36

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3.3 MTSCELL VIABILITY ASSAY ... 37

3.3.1 Uniform Sample Settings ... 38

3.4 GLUT-1PROTEIN ISOLATION AND IMMUNOBLOTTING ... 38

3.4.1 Preparation of Protein Lysates ... 38

3.4.2 Determination of total protein concentrations, SDS Gel and Western Blotting ... 39

3.5 IMMUNOSTAINING ... 40

3.5.1 GLUT-1 immunofluorescence and confocal imaging ... 40

3.6 RNAISOLATION, REVERSE TRANSCRIPTION AND QPCR ... 40

3.6.1 RNA extraction and Reverse transcription ... 40

3.6.2 Primers Efficiency and qPCR ... 41

3.7 FLOW-CYTOMETRIC ASSAYS ADDRESSING PROLIFERATION,PROTEIN EXPRESSION, AND MITOCHONDRIAL CONTENT AMID METFORMIN TREATMENT ... 41

3.7.1 Sample preparation. ... 41

3.7.2 Flow Cytometric Proliferation Assessments and cell sorting. ... 42

3.7.3 Estimation of mitochondrial content using flow cytometer. ... 42

3.7.4 Flow cytometric analysis of GLUT-1 expression ... 42

3.7.5 Data analysis: ... 42

3.8 STATISTICAL ANALYSIS ... 43

CHAPTER 4. RESULTS ... 44

4.1 PHYSIOLOGICAL GLUCOSE CONDITIONS PROLONGS THE DOUBLING TIME OF THE HIGHLY GLYCOLYTIC SW948 COLORECTAL CANCER CELL LINE ... 44

4.2 METFORMIN INHIBITS SW948PROLIFERATION DIFFERENTLY UNDER HIGH AND PHYSIOLOGICAL GLUCOSE... 45

4.3 PHYSIOLOGICAL GLUCOSE CONCENTRATIONS ENFORCE HIGHER GLUT-1EXPRESSION FURTHER INCREASED WITH METFORMIN, WHILE DOWNREGULATED WHEN CULTURED IN HIGH GLUCOSE CONCENTRATIONS ... 48

4.4 METFORMIN REDUCE THE SURFACE EXPRESSION OF GLUT-1 IN NON-PERMEABILIZED SW948CELLS ... 50

4.5 GENE EXPRESSION ANALYSIS ... 52

4.5.1 GLUT-1 mRNA levels regulated inversely in high and physiological glucose conditions .. 52

4.5.2 SW948 responds to metformin treatment by reducing OCT-1 expression ... 53

4.5.3 Elevated UCP2 and PDK2 expression in physiological glucose is antagonized by higher concentrations of metformin ... 53

4.6 FLOW-CYTOMETRIC ANALYSIS ... 55

4.6.1 Flow cytometric assay confirmed the restraining effect of metformin on proliferation . 55 4.6.2 Cell size and accumulation affects the estimation of GLUT-1 ... 56

4.6.3 Metformin induced an increase in mitochondrial content in high and physiological glucose conditions ... 57

CHAPTER 5. DISCUSSION ... 58

5.1 DISCUSSION OF RESULTS ... 58

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5.2 INCORPORATING METFORMIN IN CRC TREATMENT STRATEGY ... 62

CHAPTER 6. FUTURE RESEARCH PROSPECTS AND FINAL REMARKS ... 66

6.1 RESEARCH PROSPECTS ... 66

6.1.1 Improvments on the current reaserch ... 66

6.1.2 Enhancing metformin’s activity ... 66

6.2 CONCLUSIONS ... 67

REFERENCES ... 69

APPENDIX ... 74

SUPPLEMENTARY TO SECTION 3.4(WESTERN BLOTS)... 78

(3.4.1 annex) BCA results ... 78

(3.4.2 annex) Band Acquisition from Lane profile ... 78

(3.4.2 annex) Stain Free Normalization ... 79

SUPPLEMENTARY TO SECTION 3.6(QPCR)... 79

(3.6.1 annex) RNA purity results ... 79

(3.6.2 annex) Primers efficiency results ... 80

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