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Contribution to the elucidation of the anti-inflammatory activity of sesquiterpene lactones [Elektronische Ressource] / vorgelegt von Alfonso J. García Piñeres

227 pages
Contribution to the Elucidation of the Anti-inflammatory Activity of Sesquiterpene Lactones Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Chemie, Pharmazie und Geowissenschaften der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Alfonso J. García Piñeres aus San José, Costa Rica April 2003 Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes Dekan: Prof. Dr. P. Gräber Leiterin der Arbeit: Prof. Dr. I. Merfort Referentin: Prof. Dr. I. Merfort Koreferent: Prof. Dr. A. Bechthold Dritter Prüfer: Prof. Dr. P. Gräber Tag der Verkündigung des Prüfungsergebnisses: 15. Mai 2003 Teile der Arbeit wurden in den folgenden Publikationen, Posterpräsentationen und Kurzvorträgen veröffentlicht: Publikationen: - García-Piñeres, A.J., Castro, V., Mora, G., Schmidt, T.J., Strunck, E., Pahl, H.L., Merfort, I. (2001). Cysteine 38 in p65/NF-κB plays a crucial role in DNA binding inhibition by sesquiterpene lacontes. Journal of Biological Chemistry 276, 39713-39720. - Zorn, B., García-Piñeres, A.J., Castro, V., Murillo, R., Mora, G., Merfort, I. (2001). 3-desoxyanthocyanidins from Arrabidaea chica. Phytochemistry 56, 831-835. - Schorr, K., García-Piñeres, A.J., Siedle, B., Merfort, I., Da Costa, F.B. (2002). Guaianolides from Viguiera gardneri inhibit the transcription factor NF-κB.
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Contribution to the Elucidation of the Anti-inflammatory
Activity of Sesquiterpene Lactones





Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Fakultät für Chemie, Pharmazie und Geowissenschaften
der Albert-Ludwigs-Universität
Freiburg im Breisgau




vorgelegt von
Alfonso J. García Piñeres
aus San José, Costa Rica



April 2003
























Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes

Dekan: Prof. Dr. P. Gräber
Leiterin der Arbeit: Prof. Dr. I. Merfort
Referentin: Prof. Dr. I. Merfort
Koreferent: Prof. Dr. A. Bechthold
Dritter Prüfer: Prof. Dr. P. Gräber




Tag der Verkündigung des Prüfungsergebnisses: 15. Mai 2003



Teile der Arbeit wurden in den folgenden Publikationen, Posterpräsentationen und
Kurzvorträgen veröffentlicht:
Publikationen:
- García-Piñeres, A.J., Castro, V., Mora, G., Schmidt, T.J., Strunck, E., Pahl, H.L., Merfort,
I. (2001). Cysteine 38 in p65/NF-κB plays a crucial role in DNA binding inhibition by
sesquiterpene lacontes. Journal of Biological Chemistry 276, 39713-39720.
- Zorn, B., García-Piñeres, A.J., Castro, V., Murillo, R., Mora, G., Merfort, I. (2001). 3-
desoxyanthocyanidins from Arrabidaea chica. Phytochemistry 56, 831-835.
- Schorr, K., García-Piñeres, A.J., Siedle, B., Merfort, I., Da Costa, F.B. (2002).
Guaianolides from Viguiera gardneri inhibit the transcription factor NF-κB.
Phytochemistry 60, 733-740.
* *
- Humar, M. , García-Piñeres, A.J. , Castro, V., Merfort, I. Effect of sesquiterepene
lactones on the expression of the activation marker CD69 and of IL-2 in T-lymphocytes in
whole blood. Biochemical Pharmacology. In Press.
* Both authors contributed equally to the results of this work.
- Petrovic, S.D, Dobric, S., Bokonjic, D., Niketic, M., García-Piñeres, A.J., Merfort, I. Anti-
inflammatory and gastric anti-ulcer activity of Tanacetum larvatum (Griseb. Ex Pant.)
Kanitz. Ethnopharmacology. Eingereicht.
- García-Piñeres, A.J., Lindenmeyer, M., Merfort, I. Cysteine 38 of p65/NF-κB: a key
amino acid for the regulation of NF-κB function. Biochemical Pharmacology.
Eingereicht.
Posterpräsentationen:
- García-Piñeres, A.J., Schmidt, T.J., Strunck, E., Merfort, I., Pahl, H.L. (2000).
“Sesquiterpene lactones selectively inhibit the transcription factor NF-κB by alkylating
cysteine residues in its p65 subunit”. 48. Tagung der Gesellschaft für Arzneipflanzen,
Zürich, Schweiz.
- Zorn, B., García-Piñeres, A.J., Castro, V., Mora, G., Pahl, H.L., Merfort, I. (2000).
“Arrabidaea chica – New 3-desoxyanthocyanidins and the transcription factor NF-κB”.
48. Tagung der Gesellschaft für Arzneipflanzen, Zürich, Schweiz.
- Murillo, R., Castro, V., Mora, G., Araya, M., García-Piñeres, A.J., Klaas, C., Merfort, I.
(2000). “Estudio de lactonas sesquiterpénicas aisladas de la Decachaeta thieleana y su
actividad anti-inflamatoria, usando los factores de transcripcion NF-κB y NF-AT como
dianas moleculares”. IX Congreso Italo-Latinoamericano de Etnomedicina, Urbino,
Italien.
- García-Piñeres, A.J., Strunck, E., Pahl, H.L., Merfort, I. (2001). “The sesquiterpene
lactone parthenolide inhibits NF-κB DNA binding by alkylating cysteine 38 in its p65
subunit”. International NF-κB Symposium, Gent, Belgien.
- Petrovic, S.D., Dobirc, S., Bokonjic, D., García-Piñeres, A.J., Merfort, I. (2001). “Anti-
inflammatory and anti-ulcer activity of Tanacetum larvatum”. 49. Tagung der
Gesellschaft für Arzneipflanzen, Erlangen.
Kurzvorträge:
- García-Piñeres, A.J., Merfort, I. (2001). “Transcription factor NF-κB – an interesting
target for the development of anti-inflammatory drugs”. Workshop: Einsatz tierischer
Zellkultursysteme in der Phytopharmaka-Forschung, Berlin.
- ., Strunck, E., Pahl, H.L., Merfort, I. (2001). “The sesquiterpene
lactone parthenolide inhibits NF-κB DNA binding by alkylating cysteine residues on its
p65 subunit”. International Symposium of the Phytochemical Society of Europe,
Lausanne, Schweiz.
- García-Piñeres, A.J., Merfort, I. (2002). “Análisis de la actividad anti-inflamatoria y del
mecanismo molecular de acción de las lactones sesquiterpénicas”. Seminar of the School
of Chemistry, University of Costa Rica, San José, Costa Rica.
- García-Piñeres, A.J., Merfort, I. (2002). “Cysteine 38 in p65/NF-κB plays a crucial role in
DNA binding inhibition by sesquiterpene lactones”. Freiburg Arteriogenesis Lunch
Seminar, ZKF, Freiburg.


A mis padres
Index I
A. Introduction and Objectives............................................................................................ 1
B. Literature Review............................................................................................................. 7
1 Activation and regulation of the transcription factor NF-κB ........................................ 7
1.1 The Rel/NF-κB and IκB protein families............................................................... 7
1.2 NF-κB activation pathways.................................................................................... 8
1.3 The IκB-kinase....................................................................................................... 9
1.4 Cellular mechanisms to achieve specificity in gene activation............................ 10
1.4.1 Receptor-coupled events..............................................................................10
1.4.2 Differential subunit activation...................................................................... 12
1.4.3 Nuclear import of NF-κB............................................................................. 15
1.4.4 Post-translational modification of NF-κB.................................................... 16
1.4.5 Chromatin modification...............................................................................17
1.4.5.1 NF-κB-mediated chromatin acetylation................................................... 17
1.4.5.2 NF-κB-independent, signal-induced chromatin modification ................. 19
1.4.6 Promoter architecture...................................................................................19
1.4.7 Post-transcriptional events...........................................................................22
2 Cyclopentenone prostaglandins as anti-inflammatory mediators. .............................. 23
C. Results ............................................................................................................................. 27
1 Studies on the anti-inflammatory mechanism of sesquiterpene lactones..................... 27
38 38,1201.1 DNA binding of NF-κB/p65 Cys !Ser and Cys !Ser mutants is inhibited
at higher SL concentrations than wild type p65............................................................... 30
1.2 Further evidence that the SL parthenolide also directly inhibits the p65 subunit of
NF-κB .............................................................................................................................. 37
1.3 Sesquiterpene lactones partially inhibit IκB degradation .................................... 40
1.4 Parthenolide does not effectively inhibit the IKK................................................ 44
1.5 Comparison of the inhibition mechanism of parthenolide in different cell types 46
1.5.1 Parthenolide directly targets NF-κB in all cell lines tested.......................... 46
1.5.2 Parthenolide partially inhibits IκB degradation in all cell lines tested......... 49
1.6 Analysis of the inhibitory effect of N-ethyl-maleimide (NEM) .......................... 51
1.7 Purification of p65 from SL-treated cells, in order to obtain direct evidence of
p65 alkylation................................................................................................................... 52 II Index
2 Analysis of the reactivity of SLs with further amino acids........................................... 54
2.1 Isolation and structure of the parthenolide-arginine adduct................................. 56
2.2 Iscture ofN-t-BOC-lysine adduct .................... 61
3 An attempt to find out the identity of the unspecific band in NF-κB-EMSA analyses . 66
4 Establishment of a densitometric method for the quantification of EMSA analyses ... 69
4.1 Determination of the inhibition curve of SLs using densitometry....................... 70
4.2 Quantification of the IC of structurally different SLs, using the EMSA data ... 72 50
5 Analysis of the effect of sesquiterpene lactones on markers of the T-lymphocyte
activation in whole blood ..................................................................................................... 76
5.1 Effect of SLs on T-cell activation measured by the expression of CD69 ............ 77
5.2 Effect of SLs on the IL-2 expression in CD4 and CD8 positive T-lymphocytes. 84
5.3 SLs show low cytotoxic effects on blood cells at the concentrations tested........ 87
5.4 Effect of Arnica tinctures on the expression of CD69 and IL-2 in CD4 and CD8
positive T-lymphocytes .................................................................................................... 90
5.5 Bifunctional SLs may bind serum protein at a higher extent than monofunctional
SLs .............................................................................................................................. 96
6 Analysis of the effect of sesquiterpene lactones on the toxic activity of myotoxin II from
snake venom ......................................................................................................................... 98
6.1 Analysis of the effect of SLs on the phospholipase activity of myotoxin II........ 99
6.2 Analysis of the effect of SLs on the toxicity of myotoxin II.............................. 100
κB by different compounds ....................... 103 7 Inhibition of the transcription factor NF-
7.1 Sesquiterpene lactones of different classes ........................................................ 103
7.1.1 Inhibition of NF-κB by sesquiterpene lactones from Elephantopus mollis103
7.1.2 IF-κB by sesquiterpene lactones from Decachaeta thieleana...
....................................................................................................................105
7.1.3 Inhibition of NF-κB by sesquiterpene lactones from Viguiera gardneri... 107
7.1.4 Inhibition of NF-κB by various hypocretenolides, guaianolides, and a
germacranolide. .......................................................................................................... 108
7.1.5 Inhibition of NF-κB by isohelenin and dihydrohelenalin tiglinate ............ 111
7.1.6 Adducts of sesquiterpene lactones, as prodrugs for targeted drug delivery113
7.2 Other compounds...............................................................................................118
7.2.1 Inhibition of NF-κB by celecoxib .............................................................. 118 Index III
7.2.2 Diterpenes...................................................................................................120
D. Discussion...................................................................................................................... 123
E. Summary ....................................................................................................................... 141
F. Experimental Procedures ............................................................................................ 145
1 Instruments................................................................................................................. 145
2 Chemicals and materials 146
2.1 Antibodies, enzymes and polypeptides .............................................................. 149
2.2 Plasmids.............................................................................................................150
2.3 Site directed mutagenesis ................................................................................... 151
2.3.1 PCR............................................................................................................151
2.3.2 DNA Ligation.............................................................................................152
2.3.3 Digestion with restriction enzymes ............................................................
2.3.4 DNA purification from gel......................................................................... 152
2.3.5 Transformation of competent cells............................................................. 153
2.3.6 In-colony PCR............................................................................................154
2.3.7 Sample preparation for sequencing
2.3.8 Isolation of Plasmid DNA.......................................................................... 155
2.4 Oligonucleotide sequences.................................................................................157
2.5 Tested substances...............................................................................................159
2.6 Sample preparation.............................................................................................162
2.7 Preparation of Arnica Tinctures ......................................................................... 162
2.8 Cell culture.........................................................................................................163
2.8.1 Cell lines.....................................................................................................163
2.8.2 Cell culture media and conditions..............................................................
2.8.2.1 Handling of suspension cell lines:.......................................................... 163
2.8.2.2 of adherent cell lines: ............................................................. 163
2.8.2.3 Detachment of HeLa cells:..................................................................... 164
2.8.2.4 Freezing/Thawing of cell stocks: ........................................................... 164
3 Analysis of the DNA binding activity of the transcription factor NF-κB................... 164
3.1 In vivo experiment .............................................................................................. 165
3.2 Modified NF-κB in vivo experiment.................................................................. 165
3.3 Protein extraction...............................................................................................166 IV Index
3.3.1 Whole cell protein extracts......................................................................... 166
3.3.2 Cytosolic and Nuclear protein extracts ...................................................... 166
3.4 Analysis of NF-κB DNA binding ...................................................................... 167
3.4.1 Oligonucleotide probe labeling .................................................................. 167
3.4.2 Electrophoretic Mobility Shift Assay (EMSA).......................................... 168
3.4.3 Modified DOC experiment......................................................................... 169
4 Transient transfections............................................................................................... 170
5 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ............................................. 171
6 Staining of SDS-PAGE gels........................................................................................ 173
6.1 Coomassie blue staining..................................................................................... 173
6.2 Silver staining.....................................................................................................173
7 Western Blot analysis................................................................................................. 174
7.1 Blotting (Semi-dry electroblotting procedure)................................................... 174
7.2 Immunologic detection of antigens .................................................................... 175
8 Immunoprecipitation and IκB-Kinase Assay ............................................................. 177
8.1 Immunoprecipitation..........................................................................................177
8.2 Kinase Assay......................................................................................................179
8.3 Western Blot.......................................................................................................179
9 Determination of cell viability.................................................................................... 179
9.1 Trypan blue method ........................................................................................... 179
9.2 Lactate dehydrogenase method .......................................................................... 180
10 FACS measurements .............................................................................................. 181
10.1 Determination of CD69 expression in whole blood T-lymphocytes.................. 182
10.2 Determination of IL-2 expression in whole blood T-lymphocytes .................... 183
10.3 Determination of apoptosis and necrosis in whole blood T-lymphocytes ......... 184
11 Statistical analysis.................................................................................................. 185
12 Determination of plasma protein binding levels of SLs in whole blood ................ 185
13 Immunoaffinity chromatography............................................................................ 186
13.1 Column derivatization........................................................................................187
13.2 Affinity purification of proteins using the derivatized column.......................... 188
14 Preparation of the purified p65 sample for MS analysis ....................................... 189

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