EFFECT OF ANTIOXIDANT-DIETARY FIBER MIXTURES ON CANCER GROWTH IN ...

261 Pages·2013·4.26 MB·English

EFFECT OF ANTIOXIDANT-DIETARY FIBER
MIXTURES ON CANCER GROWTH IN COLORECTAL
CANCER-INDUCED RATS
By
Samsu Udayana Nurdin
A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
AT
THE SCHOOL OF BIOLOGICAL SCIENCES
FLINDERS UNIVERSITY

MARCH 2013
i

TABLE OF CONTENTS

TABLE OF FIGURES .................................................................................... vi
TABLE OF TABLES ..................................................................................... viii
ABBREVIATIONS USED IN THIS THESIS .................................................. xv
ABSTRACT..................................................................................................xvii
DECLARATION ............................................................................................ xx
PREFACE .................................................................................................... xxi
ACKNOWLEDGEMENT ..............................................................................xxii
PUBLSHED WORK ....................................................................................xxiv
1. GENERAL INTRODUCTION .................................................................... 1
1.1. Biology of Colorectal cancer .............................................................. 3
1.2. Rodent models for CRC prevention ................................................... 8
1.3. Effect of Diet on Colorectal Cancer Risk.......................................... 10
1.4. Dietary fiber and colorectal cancer .................................................. 13
1.4.1. Inulin ......................................................................................... 14
1.4.2. Pectin ........................................................................................ 23
1.4.3. Cellulose ................................................................................... 26
1.5. Antioxidants and Colorectal cancer development ............................ 28
1.5.1. Ascorbic acid ............................................................................. 29
1.5.2. Vitamin E ................................................................................... 30
1.5.3. Carotenoids ............................................................................... 30
1.5.4. Polyphenols .............................................................................. 31
ii

1.6. Green cincau (Premna oblongifolia Merr.) ....................................... 33
1.7. Study Hypothesis ............................................................................. 35
1.8. Aim of my PhD ................................................................................. 35
2. SCFA MODULATION OF PROLIFERATION, DIFFERENTIATION AND
APOPTOSIS DID NOT DEPEND ON THE pH ............................................. 38
2.1. Abstract ........................................................................................... 38
2.2. Introduction ...................................................................................... 39
2.3. Material and Methods ...................................................................... 41
2.3.1. Cell culture ................................................................................ 41
2.3.2. SCFA stock solutions in pH adjusted media ............................. 41
2.3.3. Proliferation assay..................................................................... 43
2.3.4. Alkaline phosphatase (AP) activity assay .................................. 44
2.3.5. Caspase 3-7 and lactate dehydrogenase (LDH) assay ............. 45
2.3.6. Statistical analysis ..................................................................... 45
2.4. Results ............................................................................................. 46
2.4.1. Effect of SCFA and pH on Caco-2 cell proliferation .................. 46
2.4.2. Effect of SCFA and pH on Caco-2 cell differentiation................ 50
2.4.3. Effect of SCFA and pH on Caspase 3/7 activity ........................ 52
2.4.4. Role of apoptosis mechanism in cell death induced by SCFA. . 55
2.5. Discussion ....................................................................................... 58
2.6. Conclusion ....................................................................................... 62
iii

3. EXAMINING THE EFFECTS OF COMBINATIONS OF NON-
DIGESTIBLE CARBOHYDRATE SOURCES ON SHORT CHAIN FATTY
ACID PRODUCTION USING AN in vitro FECAL FERMENTATION SYSTEM
AND CACO-2 CELLS AS A MODEL OF THE HUMAN COLON................... 64
3.1. Abstract ........................................................................................... 64
3.2. Introduction ...................................................................................... 65
3.3. Material and Methods ...................................................................... 68
3.3.1. In vitro fermentation of dietary fiber. .......................................... 68
3.3.2. Green Cincau Leave materials .................................................. 69
3.3.3. Cell Culture ............................................................................... 70
3.3.4. SCFA analysis .......................................................................... 70
3.3.5. Proliferation assay. .................................................................... 71
3.3.6. Alkaline Phosphatase (AP) activity assay ................................. 72
3.3.7. Caspase 3-7 and LDH Assay .................................................... 72
3.3.8. Statistical analysis ..................................................................... 73
3.4. Results ............................................................................................. 73
3.4.1. SCFA content of dietary fiber fermentation supernatant ........... 73
3.4.2. Effect of dietary fiber FS on Caco-2 cell proliferation. ............... 76
3.4.3. Effect of dietary fiber FS on cell differentiation. ......................... 77
3.4.4. Effect of dietary fiber FS on caspase 3/7 activity. ..................... 79
3.4.5. Mechanism of cell death induced by FS containing SCFA ........ 80
3.5. Discussion ....................................................................................... 83
3.6. Conclusion ....................................................................................... 90
iv

4. PROTECTIVE EFFECTS OF PHENOLIC ANTIOXIDANTS ON
COLORECTAL CANCER-INDUCED RATS ARE DEPENDENT ON
DIETARY FIBER SOURCE IN THE DIET .................................................... 91
4.1. Abstract ........................................................................................... 91
4.2. Introduction ...................................................................................... 92
4.3. Material and Methods ...................................................................... 96
4.3.1. Green Cincau Leave materials .................................................. 96
4.3.2. Animals and diet........................................................................ 96
4.3.3. Determination of SCFA composition and pH of fecal and cecal
samples .................................................................................................. 99
4.3.4. Measuring ACF number and multiplicity .................................. 100
4.3.5. Measuring proliferative activities ............................................. 100
4.3.6. Measuring lipid peroxidation in rat liver ................................... 101
4.3.7. Measuring fecal bacterial community ...................................... 101
4.3.8. Statistical methods .................................................................. 103
4.4. Results ........................................................................................... 105
4.4.1. Body weight and daily intake ................................................... 105
4.4.2. SCFA Concentration and pH ................................................... 107
4.4.3. The number of aberrant crypt foci (ACF) ................................. 108
4.4.4. Cell proliferation in distal colon ............................................... 111
4.4.5. Lipid peroxidation product ....................................................... 113
4.4.6. Microbial profile of the colon digesta ....................................... 113
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4.5. Discussion ..................................................................................... 117
4.6. Conclusion ..................................................................................... 123
5. CONCLUSIONS AND FUTURE DIRECTIONS .................................... 124
6. APPENDIX ........................................................................................... 134
6.1. Analysis of Varian (Anova) and LSD Tables .................................. 134
6.2. Dipeptidyl peptidase IV (DPIV) activity .......................................... 213
6.3. The effects of Dietary fiber sources and antioxidant on average
weights of rats during 13-week experiment ............................................. 215
6.4. Composition of chemicals used in dietary fiber fermentation ......... 216
7. REFERENCES ..................................................................................... 218
vi

TABLE OF FIGURES
Figure 1-1. A genetic model for colorectal tumorigenesis (from Fearon and
Vogelstein, 1990) ............................................................................................ 5
Figure 1-2. New model of adenoma-carcinoma(from Harrison and Benziger,
2011) .............................................................................................................. 6
Figure 1-3. Chemical structure of inulin-type fructans. .................................. 15
Figure 1-4. Chemical structure of pectin (Khotimchenko et al., 2007) .......... 24
Figure 1-5. Chemical structure of cellulose (Colebrook 2012) ...................... 27
Figure 1-6. Green cincau plant (Premna oblongifolia Merr.) ......................... 34
Figure 2-1. Effect of SCFA on Caco-2 cell proliferation in media (A) pH 6.0,
(B) pH 6.5, (C) pH 7.0 and (D) pH 7.5. ......................................................... 48
Figure 2-2. Effects of SCFA on alkaline phosphatase (AP) activity of Caco-2
cells cultured at pH 6.0 (A) and pH 7.5 (B) media. ........................................ 50
Figure 2-3. Effects of SCFA on caspase 3/7 activity of Caco-2 cells cultured
in pH 6.0 (A) and pH 7.5 (B) media. .............................................................. 53
Figure 2-4. Effects of butyrate on the caspase 3/7 (A), LDH (B) activity and
Caco 2 cell proliferation (C) with or without caspase inhibitor. ...................... 56
Figure 3-1. Effect of dietary fiber on the concentration of SCFA total (A),
acetate (B), propionate (C), and butyrate of fermentation supernatants. ...... 74
Figure 3-2. Effect of dietary fiber FS on the proliferation of Caco-2 cells. ..... 76
Figure 3-3. Effect of dietary fiber FS on AP enzyme levels. .......................... 78
Figure 3-4. Effects of dietary fiber FS on caspase 3/7 activity. ..................... 79
Figure 3-5. Effect of dietary fiber FS on caspase 3/7 activity (A), LDH release
(B) and Caco 2 cell proliferation (C) with or without caspase inhibitor. ......... 81
vii

Figure 4-1. The effects of dietary fiber sources and antioxidant on PCNA
labelling index (dark bar) and the number of PCNA positive cells (light bar) in
the mucosa of distal colon of AOM‑treated rats. ........................................ 112
Figure 4-2. The effects of dietary fiber sources and antioxidant on MDA levels
in the livers of AOM‑treated rats................................................................. 113
Figure 4-3. UPGMA dendrogram of 16S rDNA based DGGE profiles of
digesta bacterial communities. Scale refers to similarity index .................. 114
Figure 4-4. DGGE analysis of 16S rRNA gene fragment of total bacterial
population from digesta of AOM-induced rats fed different dietary fiber and/or
EGCG ......................................................................................................... 115
Figure 6-1. Effect of SCFA on the Dipheptidyl peptidase IV (DP IV) activity in
(A) pH 6.0 and (B) pH 7.5 media. ............................................................... 213
Figure 6-2.Effect of dietary fiber FS on the Dipheptidyl peptidase IV (DP IV)
activity. ........................................................................................................ 214
Figure 6-3. The effects of Dietary fiber sources and antioxidant on average
weights of rats during 13-week experiment ................................................ 215
viii

TABLE OF TABLES
Table 2-1. Final concentration of SCFA in media ......................................... 43
Table 2-2. P* values of one way ANOVA test for effect of SCFA and pH on
cell proliferation, differentiation and apoptosis. ............................................. 47
Table 3-1. Dietary fiber/dietary fiber mixture and their ratio .......................... 68
Table 3-2. Composition of dried green cincau extract ................................... 70
Table 4-1. Composition of experimental diets (per 1 Kg) .............................. 98
Table 4-2. The effects of dietary fiber sources and antioxidant on body weight
gain, liver weight, water and food intake of azoxymethane (AOM)‑induced
rats .............................................................................................................. 106
Table 4-3. The effects of dietary fiber sources and antioxidants on SCFA
concentration in the digesta and feces (µmol/g)a of AOM‑induced rats ..... 109
Table 4-4. The effects of dietary fiber sources and antioxidant on AOM-
induced ACF ............................................................................................... 110
Table 4-5. Closest relatives of band sequences excised from the acrylamide
gel in Fig. 4.4. Samples are from digesta of AOM-induced rats fed different
dietary fiber and/or EGCG. ......................................................................... 116
Table 6-1. Anova of Effect of SCFA and pH on Caco-2 cell proliferation .... 134
Table 6-2. LSD of Effect of pH on Caco-2 cell proliferation ........................ 136
Table 6-3. LSD of Effect of SCFA on Caco-2 cell proliferation.................... 137
Table 6-4. Anova of Effect of SCFA and pH on Caco-2 cell differentiation
(Alkaline phosphatise activity)..................................................................... 139
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Table 6-5. LSD of Effect of SCFA on Caco-2 cell differentiation (Alkaline
phosphatise activity) ................................................................................... 140
Table 6-6. Anova of Effect of SCFA and pH on Caspase 3/7 activity ......... 141
Table 6-7. LSD of Effect of SCFA on Caspase 3/7 activity ......................... 142
Table 6-8. Anova of Effect of SCFA and Caspase inhibitor on Caspase 3/7
activity ......................................................................................................... 144
Table 6-9. LSD of Effect of SCFA and Caspase inhibitor on Caspase 3/7
activity ......................................................................................................... 145
Table 6-10. Anova of Effect of SCFA and Caspase inhibitor on LDH release
.................................................................................................................... 146
Table 6-11. LSD of Effect of SCFA and Caspase inhibitor on LDH release 147
Table 6-12. Anova of Effect of SCFA and Caspase inhibitor on cell
proliferation (% growth). .............................................................................. 148
Table 6-13. LSD of Effect of SCFA and Caspase inhibitor on cell proliferation
(% growth). ................................................................................................. 149
Table 6-14. Anova effect of dietary fiber on Acetate content of fermentation
supernatant ................................................................................................. 150
Table 6-15. Anova effect of dietary fiber on Acetate content of fermentation
supernatant ................................................................................................. 151
Table 6-16. Anova of dietary fiber on Propionate content of fermentation
supernatant ................................................................................................. 153
Table 6-17. LSD of dietary fiber on Propionate content of fermentation
supernatant ................................................................................................. 154
Table 6-18. Anova of dietary fiber on Butyrate content of fermentation
supernatant ................................................................................................. 156