ANALYSIS OF THE ANTI-FIBROTIC EFFECTS OF HOP BITTER ACIDS ON HEPATIC STELLATE CELLS AND THEIR ANTI- TUMORIGENIC EFFECTS ON HEPATOCELLULAR CARCINOMA CELLS DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT CHEMIE UND PHARMAZIE DER UNIVERSITÄT REGENSBURG vorgelegt von Michael Sebastian Saugspier aus Deggendorf im Jahr 2012 Promotionsgesuch eingereicht: Juli 2012 Die Arbeit wurde angeleitet von: Herrn Prof. Dr. Claus Hellerbrand Prüfungsausschuss: Vorsitzender: Herr Prof. Dr. Gerhard Franz 1. Gutachter (1. Prüfer): Herr Prof. Dr. Jörg Heilmann 2. Gutachter (2. Prüfer): Herr Prof. Dr. Claus Hellerbrand 3. Prüfer: Frau Prof. Dr. Charlotte Wagner Für meine Eltern I Table of Contents 1 Summary......................................................................................................... 1 2 Introduction..................................................................................................... 3 2.1 Botany...................................................................................................... 3 2.2 Hop Chemistry......................................................................................... 5 2.2.1 Lupulin ingredients............................................................................ 5 2.2.2 Hop bitter acids................................................................................. 6 2.3 Biological effects of hop bitter acids......................................................... 8 2.3.1 Anti-bacterial effects......................................................................... 8 2.3.2 Anti-inflammatory effects .................................................................. 9 2.3.3 Anti-cancer effects.......................................................................... 12 2.3.4 Effects on lipid and carbohydrate metabolism ................................ 14 2.3.5 Other effects................................................................................... 16 2.4 Toxicology.............................................................................................. 16 2.5 Effects of hop bitter acids on the liver.................................................... 18 2.6 Liver diseases........................................................................................ 20 2.6.1 Definition and development ............................................................ 20 2.6.2 Liver fibrosis.................................................................................... 20 2.6.3 Liver cancer.................................................................................... 21 2.7 Aim of the thesis..................................................................................... 23 3 Materials and Methods.................................................................................. 24 3.1 Chemicals and Reagents....................................................................... 24 3.2 Laboratory expendables......................................................................... 25 3.3 Laboratory instruments.......................................................................... 25 3.4 Cell culture............................................................................................. 26 3.4.1 Cell culture medium........................................................................ 26 3.4.2 Cultivation of cells........................................................................... 27 3.4.3 Determination of cell number and viability ...................................... 27 3.4.4 Cryopreservation of cells ................................................................ 28 3.4.5 Human hepatocellular carcinoma cell lines..................................... 28 3.4.6 Isolation of primary human hepatic stellate cells............................. 28 3.4.7 Isolation of primary murine hepatic stellate cells............................. 29 3.5 Hop bitter acid extracts.......................................................................... 31 Table of Contents II 3.6 Enrichment of hop bitter acids................................................................ 32 3.7 Preparation of a hop bitter acid-supplemented chow for oral application32 3.8 Hop bitter acid-recovery from the hop bitter acid-supplemented chow .. 33 3.9 Thin layer chromatography .................................................................... 33 3.10 RNA Analysis......................................................................................... 34 3.10.1 RNA Isolation.................................................................................. 34 3.10.2 Reverse transcription PCR ............................................................. 34 3.10.3 Quantitative real time PCR ............................................................. 35 3.11 Protein Analysis..................................................................................... 36 3.11.1 Isolation of whole cell proteins........................................................ 36 3.11.2 Determination of protein concentration........................................... 37 3.11.3 Western Blot Analysis..................................................................... 37 3.11.3.1 SDS PAGE gel electrophoresis............................................... 37 3.11.3.2 Protein blot and detection........................................................ 38 3.11.4 Immunocytochemistry..................................................................... 40 3.11.5 Quantification of ERK 1/2 and NFκB............................................... 40 3.11.6 AP-1 reporter gene assay............................................................... 41 3.12 Analysis of cell culture supernatants...................................................... 41 3.13 Functional assays.................................................................................. 42 3.13.1 Proliferation assay (XTT)................................................................ 42 3.13.2 Migration assay............................................................................... 42 3.14 Statistical analysis.................................................................................. 43 4 Results.......................................................................................................... 44 4.1 Effects of hop bitter acids on hepatic stellate cells................................. 44 4.1.1 Rationale......................................................................................... 44 4.1.2 In vitro activation of HSC ................................................................ 45 4.1.3 Proliferation and cell viability........................................................... 46 4.1.4 NFκB activity and pro-inflammatory gene expression..................... 49 4.1.5 Summary ........................................................................................ 50 4.2 Effects of hop bitter acids on hepatocellular carcinoma cells................. 51 4.2.1 Rationale......................................................................................... 51 4.2.2 Cell viability..................................................................................... 51 4.2.3 Proliferation and migratory potential............................................... 53 4.2.4 ERK1/2, AP-1 and NFκB activity..................................................... 54 Table of Contents III 4.2.5 Comparison of bitter acids and remnant lipophilic compounds....... 57 4.2.6 Summary ........................................................................................ 57 4.3 BA-recovery experiments from rodent chow supplemented BA-extract. 58 5 Discussion..................................................................................................... 61 5.1 Hop bitter acids and hepatic inflammation and fibrosis.......................... 61 5.2 Hop bitter acids and hepatocellular carcinoma ...................................... 63 5.3 Poor recovery of hop bitter acids in animal chow supplemented with a bitter acid-extract.............................................................................................. 64 5.4 Conclusion............................................................................................. 66 6 References.................................................................................................... 68 7 Abbreviations................................................................................................ 77 8 Appendix....................................................................................................... 80 8.1 Curriculum Vitae..................................................................................... 80 8.2 Advanced training courses..................................................................... 81 8.3 Publications............................................................................................ 81 8.4 Poster presentations.............................................................................. 82 8.5 Danksagung........................................................................................... 83 8.6 Eidesstattliche Erklärung........................................................................ 85 1 1 Summary Bitter acids (BA) from the hop plant Humulus lupulus L. exhibit multiple beneficial biological properties. However, no studies are available regarding the effects of hop bitter acids on liver cells or chronic liver diseases, respectively. Chronic liver diseases are accompanied by chronic hepatic inflammation which can lead to liver fibrosis. If left untreated, liver fibrosis can progress to cirrhosis, which can cause liver failure. Importantly, cirrhosis is also the main risk factor for the development of hepatocellular carcinoma (HCC). Both cirrhosis and HCC are associated with high morbidity and mortality, and up until now, no effective treatment options are available. The aim of this thesis was to assess the effects of BA on hepatic stellate cells, the central mediators of liver fibrosis, and on hepatocellular carcinoma cells. First, the effects of hop bitter acids on hepatic stellate cells (HSC) were analyzed. HSC were isolated from murine and human liver tissues and were incubated with a standardized hop bitter acid extract. At a concentration of 25 µg/ml hop bitter acids (BA) started to induce lactate dehydrogenase leakage. Already at lower concentrations BA led to a dose dependent inhibition of HSC proliferation and inhibited IκB-α-phosphorylation and nuclear p65 translocation in a dose dependent way. Accordingly, the same doses of BA inhibited the expression of pro- inflammatory and NFκB-regulated genes, but did not affect expression of genes not related to NFκB signaling. In addition to these effects on activated HSC, BA inhibited the in vitro activation of non-activated HSC as evidenced by delayed expression of collagen I and α-sma mRNA and protein. Together, these findings indicate that BA inhibit NFκB activation, and herewith, the activation and development of pro-fibrogenic phenotype of HSC in vitro. Next, we analyzed the effects of BA on hepatocellular carcinoma (HCC) cells. Here, we used two different standardized BA extracts enriched for either α-acids Summary 2 or β-acids to get a first insight whether biological activity vary between these two groups of bitter acids. At a concentration of 25 µg/ml, only the β-acid rich extract started to induce aspartate aminotransferase (AST) release as marker for cell injury, whereas at a dose of 50 µg/ml both extracts led to a significant increase of AST liberation. Already at lower concentrations both extracts dose-dependently inhibited proliferation and migration of HCC cells. Analysis of different signaling pathways revealed an inhibition of ERK1/2 phosphorylation, down-regulation of AP-1 activity and an alleviation of NFκB activity in HCC cells in vitro. Hereby, the β-acid rich extract showed more pronounced effects. Furthermore, the stability of BA in a rodent chow supplemented with an α-acid rich extract was assessed applying high pressure liquid chromatography diode array detection technology. Mimicking feasible storage conditions we observed a very poor recovery, indicative of a strong degradation of BA. This prompted us not to proceed with application of this particular BA supplemented chow in rodent models, since in addition to imprecise BA-concentration also potential ill-defined degradation products would not have allowed a sound interpretation of such studies. In conclusion, hop bitter acids alleviate the pro-fibrogenic phenotype of hepatic stellate cells and inhibit central signaling pathways, which are known to play important roles in tumor development and progression, in hepatocellular carcinoma cells. These data suggest the potential use of hop bitter acids as functional nutrient or therapeutical agent for both prevention and treatment of chronic liver diseases. Further studies are needed to verify the beneficial effects in experimental models of liver injury in vivo. However, analysis of a newly designed BA-supplemented experimental chow for rodents revealed strong degradation processes even under optimal storage conditions. Therefore, special care has to be taken when planning long term BA-application experiments. 3 2 Introduction 2.1 Botany The hop plant Humulus lupulus L. is a member of the small family Cannabaceae which consists only of two genera Humulus and Cannabis. The genus Humulus comprises three species, Humulus lupulus Lineus, Humulus japonicus Siebold & Zucc. and Humulus yunnanensis Hu. Originally, this genus belonged to the order Urticales but in 2003 it was incorporated to Rosales (Van Cleemput et al., 2009a; Zanoli and Zavatti, 2008). Humulus lupulus L. is a perennial herb that typically grows in the moderate climatic zones of the Northern and Southern hemispheres. During spring the plant regrows from the primary root with her succulent rhizome generating twining stems which can reach a height of about 10 meters. This climbing property was determinative involved in its naming as the Roman naturalist Plinius described the plant as “the wolf of the willow” (“lupus salictarius”) (Van Cleemput et al., 2009a). For him, wild hop grow behaviour was comparable with a wolf who raptures a sheep. The hop plant is dioecious and almost all seen cultivated hop are female individuals of the species Humulus lupulus L. because the female inflorescences (strobiles, cones) contain the lupulin powder which has been traditionally used as a preservative and flavouring agent in beer (Milligan et al., 2002). The selection of plants with specific organoleptic properties for a long time caused the existence of hundreds of named cultivars and recognized chemotypes (Zanoli and Zavatti, 2008). Harvesting time is from August till September when the amount of lupulin has reached its maximum level. Cultivation technique and a flowering female hop plant with inflorescences are shown in Figure 2.1. Introduction 4 Figure 2.1 (A) Cultivation of hop in long rows with supporting aids to allow vertical growth. (B) Female plant rich in inflorescences. The fine yellow secreted resinous powder (lupulin) is synthesized in the lipophilic lupulin glands, a peltate-type of glandular trichomes, which are located on the abaxial surfaces of the cone bracts and leaves with the highest affinity to the basal part of the bracteols as seen in Figure 2.2 A. The lupulin glands consist of four basal cells, four stalk cells and one large glandular head cell with a typical diameter of 100 – 200 µm (Oliveira et al., 2008). They originate from single epidermis cells after multiple cell divisions and lupulin is secreted in the cuticle- enclosed cavity as seen in Figure 2.2 B (Wärtgen, 1990). Figure 2.2 (A) A single bract with many trichomes, mainly lupulin glands. (B) Close-up view on lupulin glands.