Chemopreventive potential of Se-enriched broccoli and kale sprouts in vitro and in a human prostate cancer xenograft model [Elektronische Ressource] / vorgelegt von Jung-Hyun Kim

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Chemopreventive potential of Se-enriched broccoli and kale sprouts in vitro and in a human prostate cancer xenograft model German Cancer Research Center, Heidelberg (Division of Epigenomics and Cancer Risk Factor) University of Heidelberg (Biology) DISSERTATION Inaugural-Dissertation zur Erlangung der Doktorwürde der Ruprecht-Karls-Universität Heidelberg 2010 vorgelegt von Jung-Hyun KIM 1 2 Presented by Jung-Hyun KIM References: Prof. Dr. Thomas Rausch Prof. Dr. Bernard Watzl 3 4 Jesus Christ ―A man's heart deviseth his way: but the LORD directeth his steps‖ KING JAMES (PROVERBS 16:9)5 6 TABLE OF CONTENTS I. TABLE OF CONTENTS I TABLE OF CONTENTS ........................................................................................................ 7 II. LIST OF ABBREVIATIONS ............................. 11 1. INTRODUCTION ............................................................................................................... 13 1.1 Prostate cancer................ 13 1.1.1 Prostate cancer incidence ......................................................................................... 13 1.1.2 Multistep process of prostate carcinogenesis .......................... 13 1.1.

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Chemopreventive potential of Se-enriched broccoli and kale sprouts
in vitro and in a human prostate cancer xenograft model


German Cancer Research Center, Heidelberg
(Division of Epigenomics and Cancer Risk Factor)
University of Heidelberg
(Biology)



DISSERTATION


Inaugural-Dissertation zur
Erlangung der Doktorwürde der Ruprecht-Karls-Universität
Heidelberg







2010









vorgelegt von


Jung-Hyun KIM
1

2































Presented by
Jung-Hyun KIM





References: Prof. Dr. Thomas Rausch
Prof. Dr. Bernard Watzl

3

4








Jesus Christ





































―A man's heart deviseth his way: but the LORD directeth his steps‖
KING JAMES (PROVERBS 16:9)
5

6
TABLE OF CONTENTS
I. TABLE OF CONTENTS

I TABLE OF CONTENTS ........................................................................................................ 7
II. LIST OF ABBREVIATIONS ............................. 11
1. INTRODUCTION ............................................................................................................... 13
1.1 Prostate cancer................ 13
1.1.1 Prostate cancer incidence ......................................................................................... 13
1.1.2 Multistep process of prostate carcinogenesis .......................... 13
1.1.3 AR signaling in prostate cancer progression ........................... 14
1.1.4 Prostate cancer cell lines .......................................................................................... 16
1.2 Chemoprevention ........................................................................................................... 16
1.2.1 Chemoprevention of prostate cancer ....... 19
1.2.2 Chemopreventive agents of prostate cancer ............................................................ 19
1.3 Selenium-enriched broccoli................................ 27
1.4 Kale sprouts – a potential dietary agent for prostate cancer prevention ........................ 29
1.5 Inhibition of angiogenesis as a promising target mechanism of cruciferous vegetables31
1.5.1 The process of angiogenesis .................................................................................... 31
1.5.2 Vascular normalization as an anti-angiogenic approach point ................................ 32
1.5.3 ITCs as anti-angiogenic agents ................ 34
1.6 Epigenetic regulation of gene expression through HDAC inhibition ............................ 35
1.6.1 Regulation of histone acetylation ............................................................................ 35
1.6.2 Histone acetylation in prostate cancer ..... 36
1.6.3 Effects of ITCs on histone acetylation .................................................................... 37
2. AIM OF THESIS ................................................. 38
3 MATERIALS AND METHODS .......................................................................................... 40
3.1 Instruments ..................... 40
3.2 Disposables................................ 40
3.3 Materials ......................................................................................... 40
3.3.1 Chemicals ................ 40
3.3.2 Ready to use kit ....................................................................................................... 42
3.3.3 Antibodies ................ 42
3.3.4 PCR reagents ........... 42
3.4 Cell culture ..................................................................................................................... 43
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TABLE OF CONTENTS
3.4.1 Cell lines .................................................................................................................. 43
3.4.2 Cell culture reagents ................................ 43
3.4.3 Cell culture condition .............................................................................................. 43
3.5 Animals .......................................................... 44
3.5.1 Animal strain ........................................................................................................... 44
3.5.2 Animal diet .............. 44
3.5.3 LNCaP xenograft model .......................................................................................... 45
3.6 Methods – Effects of Se-enriched broccoli .................................... 45
3.6.1 Fertilization of Se-enriched broccoli (Prepared by HIP) ......... 45
3.6.2 Determination of enzyme activities in mouse liver tissue ....... 46
3.6. 3 Determination of antioxidant enzymes protein expression by western blot ........... 49
3.6.4 Determination of sulforaphane metabolites in mouse plasma, urine and feces
(Prepared by Johanna Hauder, DFA) ............................................................................... 50
3.6.5 Elemental analysis of total selenium (Prepared by Fu-Chou Hsu, HIP) ................. 51
3.7 Methods- Anti-angiogenic potential of kale sprouts ...................... 51
3.7.1 RNA isolation, reverse transcription and quantitative real time PCR ..................... 51
3.7.2 Detection of AR, TNF , angiostatin by western blotting ....................................... 52
3.7.3 Chromatin Immunoprecipitation- PSA promoter .................... 52
3.7.4 Quantification of PSA levels by ELISA (prepared by Achim Bub, BfEL) ............. 53
3.7.5 Measurement of Hb concentration in tumor xenografts .......................................... 53
+3.7.6 Immunohistochemistry-CD31 , SMA, AR (Prepared by Mattthias Wieland,
DKFZ) .............................................................................................................................. 53
3.7.7 Immunohistochemistry- AR .................... 54
3.7.8 Gelatin zymography ................................ 54
3.8 Histone modification by kale sprouts ............................................. 55
3.8.1 Determination of mitotic cells by immunohistochemistry ...... 55
3.8.2 Immunoblotting ....................................................................... 55
3.8.3 Detection of histone acetylation .............................................. 55
3.8.4 Nuclear protein extraction and PACF expression assay .......... 56
3.8.5 HDAC activity measurement (Prepared by Julia Wagner, University of Friebrug) 56
3.8.6 RNA isolation, reverse transcription and quantitative real time PCR ..................... 56
3.8.7 Chromatin Immunoprecipitation – p21, PCAF promoter ........................................ 57
4 RESULTS ............................................................................................................................. 58
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TABLE OF CONTENTS
4.1 Se-enriched broccoli as a functional food for prostate cancer prevention (Induction of
detoxifying and antioxidant enzymes) ................................................................................. 58
4.1.1 Combination of Se and broccoli extract in vitro...................... 58
4.1.2 Effects of Se-enriched broccoli sprouts in vitro 61
4.1.3 Activity of Se-enriched broccoli florets in vitro ...................................................... 63
4.1.4 Dietary Se-enriched broccoli and functional food in LNCaP xenograft model ...... 65
4.1.5 Sulforaphane metabolites in mouse plasma, urine and feces after the interventions.
.......................................................................................................................................... 71
4.1.6 Summary .................. 73
4.2 Anti-angiogenic potential of kale sprouts ...... 75
4.2.1 Influence on tumor growth, necrosis and hemorrhage ............................................ 75
4.2.2 Induction of vascular maturation ............................................. 79
4.2.3 Inhibition of pro-angiogenic factors and induction of anti-angiogenic factors ....... 81
4.2.4 Kale sprouts prevent down-regulated PSA secretion .............................................. 84
4.2.5 Expression of AR mediated by TNF  ..................................... 88
4.2.6 Summary .................................................. 90
4.3 Epigenetic modification by kale sprouts ........................................ 92
4.3.1 Induction of histone acetylation but, no HDAC inhibition ..... 92
4.3.2 Acetylation on each lysine residue and HDACs expression ... 93
4.3.3 Induction of HAT expression .................................................................................. 95
4.3.4 p21 gene expression mediated by histone modification .......... 96
4.3.5 Mitotic arrest............................................ 97
4.3.6 Human intervention study- induction of histone acetylation in PBMC .................. 99
4.3.7 Summary – histone modification by kale sprouts ................................................. 100
5 DISCUSSIONS ................................................................................... 101
5.1 Potential of Se-enriched broccoli as a chemopreventive agent in prostate cancer ....... 102
5.2 Anti-angiogenic potential of kale sprouts .................................................................... 106
5.3 Histone modification by kale sprouts ........... 110
5.4 Out-look ....................................................................................................................... 112
6. SUMMARY 113
7 ZUSAMMENFASSUNG ................................................................................................... 116
8 COLLABORATORS .......... 119
8.1 Potential of Se-enriched broccoli as a chemopreventive agent in prostate cancer ....... 119
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TABLE OF CONTENTS
8.2 Anti-angiogenic potential of kale sprouts .................................................................... 119
8.3 Histone modification by kale sprouts ........... 120
9 REFERENCES ................................................................................................................... 121
V PUBLICATIONS ............... 138
V.I Papers ........................................................................................................................... 138
V.II Poster presentations .................................... 138
VI. ACKNOWLEDGEMENTS ............................. 140
VII. LIST OF ACADEMIC REFERENCES ......................................... 142
VIII. CURRICULUM VITAE ............................................................... 143
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