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Isolated ginsenosides aPPD and aPPT induced cytochrome P450 1 A 1 mRNA expression Zhao, Yang 2006

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I S O L A T E D G I N S E N O S I D E S A P P D A N D A P P T I N D U C E D C Y T O C H R O M E P450 1 A l M R N A E X P R E S S I O N by Y A N G Z H A O B . Med. , Shenyang Medical College, 2000 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Surgery) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A July 2006 © Yang Zhao, 2006 A B S T R A C T Introduction: Ginseng is commonly used in herbal preparations for traditional Chinese medicine. It contains over twenty ginsenosides amongst which aglycon protopanaxadiol (aPPD) and aglycon protopanaxatriol (aPPT) have been shown to be the primary circulating metabolites with potent medicinal properties. To evaluate the prospect of metabolic drug interactions the effects of aPPD and aPPT on expression and function of human cytochromes P450 C Y P 1 A 1 was assessed. Methods: Real Time R T - P C R and western blotting were used to measure C Y P 1 A 1 m R N A and protein expression in human HepG-2 and Caco-2 cells treated with aPPD or aPPT for 12 and 24 hours. A P450-Glo™ C Y P 1 A 1 assay was used to measure C Y P 1 A 1 activity. Gudluc 1.1 was luciferase labeled and used as the C Y P 1 A 1 promoter constructs. It was co-transfected with A r y l hydrocarbon receptor (AhR) and p R L - T K (control) plasmids to examine C Y P 1 A 1 m R N A induction via A h R . p G L 3 B - C Y P l A l plasmid was constructed containing longer C Y P 1 A 1 promoter sequence (-2425, +352) than Gudluc 1.1 (-1301, -819) and used to investigate the induction mechanism outside of A h R pathway. Results: There was a dose-dependent induction of C Y P 1 A 1 m R N A expression in both HepG-2 and Caco-2 cells dosed for 12h and 24h with either aPPD or aPPT. The result was statistically significant at concentrations of 5 u M and above; however, this was not correlated with increased protein levels. P450-Glo™ reporter assay produced a significant increase in C Y P 1 A 1 activity only after treatment with 80 and 160 u M i i aPPD. N o effects were found at lower concentrations of aPPD and all concentrations of aPPT. Induction of C Y P 1 A l m R N A by aPPD and aPPT was independent of A h R activation. Functional sequence was outside of the region from -2425 to +352. Conclusions: Overall, our results suggest that aPPD and aPPT exert a significant inductive effect on C Y P 1 A 1 m R N A level, while also activating C Y P 1 A 1 protein activity only with high concentrations of aPPD. This induction of m R N A level is not likely to be regulated by A h R . i i i T A B L E O F C O N T E N T S A B S T R A C T ii T A B L E OF CONTENTS iv LIST OF TABLES vii LIST O F FIGURES vii A C K N O W L E D G E M E N T S viii C H A P T E R 1 INTRODUCTION 1 1.1 Ginseng 1 1.1.1 Ginseng History 1 1.1.2 Ginseng Classification and Composition 2 1.1.3 Ginsenoside Structure 3 1.1.4 Ginsenoside Nomenclature 3 1.1.5 Functions and Properties of Ginseng 4 1.1.6 Functions and Properties of Ginsenoside 5 1.1.7 Clinical use of ginseng 6 1.1.8 Metabolism o f Ginsenosides 7 1.2 Cytochrome P450s 8 1.2.1 Discovery, Nomenclature of Cytochrome P450s 8 1.2.2 Cytochrome P450 Structure 9 1.2.3 Oxidative Function of Cytochrome P450s 11 1.2.4 Induction and Inhibition of C Y P s 12 1.2.5 Cytochrome P450 1A1 14 1.2.6 Ginsenosides and Cytochrome P450s 15 1.3 Hypothesis and Objectives 17 iv C H A P T E R 2 MATERIALS AND M E T H O D S 25 2.1 Cell Culture 25 2.2 Growth Factor and Drug Treatment 25 2.2.1 Time from C Y P 1 A l m R N A to Protein Determination 26 2.2.2 M T T Procedure 26 2.2.3 Induction Study in Both Transcriptional and Translational Levels 27 2.3 Cell Lysis 27 2.3.1 Total R N A Extraction 27 2.3.2 R N A Concentration Determination 28 2.3.3 Total Protein Extraction 28 2.3.4 Protein Concentration Determination 28 2.4 Genomic DNA Digestion in RNA Sample 29 2.5 Reverse Transcription 30 2.6 Conventional PCR 30 2.7 Real Time PCR 31 2.8 Western Blotting 32 2.8.1 Electrophoresis 32 2.8.2 Transfer of Separated Proteins to Nitrocellulose Membrane 33 2.8.3 Ponceau Red Stain 33 2.8.4 Blocking 34 2.8.5 Primary Antibody Incubation 34 2.8.6 Secondary Antibody Incubation and Detection 34 2.9 P450 Glo CYP1 A l Activity Assay 35 2.10 Plasmid 37 v 2.11 CYP1A1 Promoter Plasmid Cloning 37 2.11.1 Conventional P C R 37 2.11.2 T O P O T A Cloning 38 2.11.3 Restriction Digestion 38 2.11.4 T4 Ligation and S equencing 39 2.12 Transfection 40 2.12.1 Optimization Experiment 40 2.12.2 Transfection with Gudluc Plasmid 41 2.12.3 Transfection with p G L 3 B - C y P / ^ / Plasmid 41 2.13 Luciferase Assays 42 C H A P T E R 3 RESULTS 44 3.1 Study of CYP1 A l Induction at The Transcriptional and Translational Level..44 3.1.1 Determination of The Time Required for C Y P 1 A 1 m R N A to Translate Protein 44 3.1.2 Cytotoxicity Study 45 3.1.3 Induction Study in Transcriptional Level 45 3.2 Effects of Ginsenoside Treatment on Protein Translation 46 3.3 CYP1 A l Metabolic Activity 47 3.4 Mechanistic Studies of CYP1 A l 47 3.4.1 Gudluc 1.1 plasmid, and Induction of CYP1A1 by aPPD and aPPT with A h R 47 3.4.2 p G L 3 B - C T P L 4 7 Plasmid and Induction of CYP1A1 by aPPD and aPPT ..48 C H A P T E R 4 DISCUSSION 56 vi R E F E R E N C E 67 L I S T O F T A B L E S Table 1.1 Ginseng species 19 L I S T O F F I G U R E S Figure 1.1 The ginseng saponins of protopanaxatriol 20 Figure 1.2 The ginseng saponins of protopanaxadiol 21 Figure 1.3 Thin-layer chromatograms of the saponins of Panax ginseng roots 22 Figure 1.4 The proposed catalytic cycle of cytochrome P450 for hydroxylation reactions 23 Figure 1.5 General CYP1A1 induction model 24 Figure 2.1 Structure of the PGL3B-CYP1 A l plasmid 43 Figure 3.1 Determination of the time required for CYP1A1 mRNA to translate protein 49 Figure 3.2 Cytotoxicity study result for aPPD and aPPT in HepG-2 and Caco-2 cell lines 50 Figure 3.3 Induction study of CYP1A1 by aPPD and aPPT in transcriptional level.51 Figure 3.4 The induction study of CYP1 A l after aPPD and aPPT 24h treatment at translational level 52 Figure 3.5 CYP1A1 metabolic activity study after aPPD and aPPT treatment 53 Figure 3.6 CYP1A1 induction mechanism study related to AhR using Gudluc 1.1 plasmid 54 Figure 3.7 pGL3B-CFPL41 plasmid function test and the use of this construct in CYP1A1 induction mechanism study 55 vn A c k n o w l e d g e m e n t s First and foremost, I would like to express my sincere appreciation to my two supervisors: Dr. Emma Guns and Dr. Wi l l iam Jia. They provided me not only with techniques and knowledge in research, but also with strong moral support. I feel very fortunate to have benefited from their immense knowledge and kindness. I would also like to thank Dr. Marcel Bally, who offered many useful suggestions throughout my project. A special thank you to Dr. Simon Cowel l , who was a post-doctoral fellow in Dr. Guns' laboratory. He was very patient and gave me an endless supply o f ideas. He guided me through the initial challenges at the beginning of my research career. M y deepest thanks to L ing Tian, who was a visiting scholar in Dr. Jia's laboratory. He saved me a great deal of time by teaching and assisting me with cloning techniques. M y infinite gratitude to Andy Eberding and Catherine Wood. They were always very patient and helpful. I greatly appreciate their help with the early drafts of this thesis. M y English improved a great deal from their word-by-word revision. Finally, warmest thanks to my family, especially my mom, for their unconditional love and invaluable spiritual support throughout my Masters program. CHAPTER 1 INTRODUCTION 1.1 G i n s e n g 1.1.1 Ginseng History Ginseng has been used as a popular herbal remedy in As i a for over 5500 years (Yun 2001a). People in northern China who struggled with hunger and disease used it as food. They discovered that it had healing properties and this knowledge was passed down through generations. Many myths evolved regarding its medicinal and healing abilities to restore homeostasis (Hu 1977). The first written account of ginseng was in a document describing plants and herbs, 'Shennong Bencao Jing' by Tao Hongjing during the Liang Dynasty, 502-557 A D (Yun 2001a). The translation of this work describes ginseng as follows: "Ginseng is also called Jen-hsien, or Kuei-kai. It tastes sweetish, and its property is slightly cooling. It grows in the gorges of the mountains. It is used for repairing the five viscera, quieting the spirit, curbing the emotion, stopping agitation, removing noxious influence, brightening the eyes, enlightening the mind, and increasing the wisdom. Continuous use leads one to longevity with lightweight. " Shiu-ying Hu , a researcher from Harvard University, translated it in late 1970s, to make it accessible to people in the scientific and academic world outside of China (Hu 1977). Currently, ginseng is still widely used as a health tonic, not only in Asia , but 1 throughout the world. In 1993, it was reported in the U S A that 34% o f the adults in the U S use at least one alternative therapy (Astin 1998). O f these alternative therapies, herbal medicines are the most prevalent. In 1998, total sales in the U . S . A . for herbal remedies approached 4 bi l l ion U S dollars. A t the same time, annual sales o f ginseng were $98 mil l ion U S dollars with an annual growth rate of 26% (Yun 2001a). Due to increasing consumption, the role of ginseng as a medicine has drawn the attention of numerous researchers. 1.1.2 Ginseng Classification and Composition There are two types of ginseng commercially available that differ only by method of preparation: white ginseng and red ginseng. White ginseng is prepared by drying the root after peeling off the skin, while red ginseng is prepared by steaming and drying the root with the skin still intact (Shibata 2001). The ginseng family contains many species (Table 1.1), and most herbalists recognize three species as having medicinal properties: Panax ginseng C A . Meyer (Chinese and Korean ginseng), Panax japonicus C A . Meyer (Japanese ginseng), and Panax quinquefolius (American ginseng) (Bahrke & Morgan 2000). Several classes of compounds have been isolated from ginseng root. These include triterpene saponins, essential oil-containing polyacetylenes and sesquiterpenes, polysaccharides, peptidoglycans, nitrogen-containing compounds, and various ubiquitous compounds such as fatty acids, carbohydrates, and phenolic compounds (Sticher 1998). The most active o f these compounds found in all species o f ginseng are considered to be the triterpene saponins, which are also called ginsenosides. To 2 date, thirty-five ginsenosides have been isolated from fresh, white, or red ginseng, of which 22 are protopanaxadiols, 12 are protopanaxatriols and one, Ro, is an oleanane (Yun 2003). These ginsenosides vary in content and relative proportions among different species o f ginseng (Kitts & H u 2000). 1.1.3 Ginsenoside structure Ginsenosides are believed to be the main pharmacologically active components in ginseng. Their fundamental skeleton structure is a dammarane-type tetracyclic triterpene (Figure 1.1 and Figure 1.2). They are categorized in three groups depending on their aglycones: protopanaxadiol, protopanaxatriol and oleanolic acid-type saponins (Sticher 1998). Nearly all dammarane ginsenosides isolated from white ginseng root are derivatives of 20S protopanaxatriol and 20S protopanaxadiol. Ginsenosides isolated from white ginseng are also found in red ginseng; however, some ginsenosides (20R Rg2, 20R R h i , R h 2 , R s i , Rs2, Q - R i , and N G - R i ) are characteristic saponins found only in red ginseng(Sticher 1998). 1.1.4 Ginsenoside Nomenclature Ginsenosides are named according to their R f values, and are designated Rx , where x=0, a-1, b-1, b-2, b-3, c, d, e, f, 20-gluco-f, g-1, g-2, h-1, etc, starting from lowest R f to highest, as shown in Figure 1.3. R f is determined by thin layer chromatography R f values of the different ginsenosides. 3 1.1.5 Functions and Properties of Ginseng Based on the fact that ginsenoside composition is different in each species, it is logical to suggest that different species w i l l not have the same pharmacological properties. In fact different species have been suggested to have distinguishing factors throughout historical folklore, for example, i f we consider the ancient Asian concept of the complementary forces o f yin and yang, it is claimed that North American ginseng provides yin, or a cooling effect to offset stress; while Panax ginseng C A . Meyer provides yang, or a warming effect to counter-balance stress (Kitts & H u 2000). Most often, the simple title ginseng is generally considered to mean the Panax ginseng C A . Meyer. Ginseng has been reported to prevent aging, fatigue, headaches, amnesia, tuberculosis, diabetes and maladies of the liver, heart, and kidneys, as well as nervous disorders (A. 1966; Bittles A H 1979; Popov I M 1973). It has been used to treat anaemia, anxiety, shortness of breath and perspiration, continuous thirst, lack of sexual desire, dyspepsia, heart pain and nausea (Bahrke & Morgan 1994). Ginseng has been reported to possess non-organ specific preventive effects against various cancers (Yun 2001b). Chronic consumption is thought to decrease the incidence o f cancers such as lip, oral cavity and pharynx, larynx, lung, gastric, liver, pancreas, ovarian and colorectal tumors (Shin et al. 2000; Yun 2001b; Yun 2003). A n in vivo study suggested that Panax ginseng may reduce cell damage, especially D N A damage, caused by gamma- rays ( K i m et al. 1993). One study reports that American ginseng extract inhibits breast cancer cell growth in vitro (Duda et al. 1999). Generally, as was described in 'Shennong Bencao Jing' , ginseng has been assumed to have low toxicity (Helms 2004) and to possess a variety of beneficial 4 properties, including anti-inflammatory, antioxidant, and anti-cancer activities, as well as psychological and immune function improvement (Bahrke & Morgan 2000). 1.1.6 Functions and Properties of Ginsenoside Among all the ginsenosides extracted from ginseng, R g 3 , R h 2 , and Rhi have been well studied in recent decades. It was reported that Rg3 caused cell cycle arrest in the G l phase and inhibited cell growth through a caspase-3-mediated apoptosis mechanism (L iu et al. 2000). RJ12 was shown to inhibit in vitro proliferation of 3 L L lung cancer cells (murine), Morris liver cancer cells (rat), B-16 melanoma cells (murine), and Hela cervical cancer cells (human) by causing cells to arrest in G i phase (Han 1994; Nakata et al. 1998; Shibata 2001). Rh i did not inhibit cancer cell proliferation but activated adenyl cyclase and promoted melanin synthesis in melanoma cells, which might be related to reverse transformation o f cancer cells (Shibata 2001). In M a y 2000, a new anti-cancer drug, 'Rg3 Shenyi Jiaonang', appeared on the Chinese market. Its clinical application was to inhibit tumor angiogenesis and prevent tumor cell adhesion, invasion, and metastasis. N o obvious side effects or toxicity have been reported (Shibata 2001). Previous studies conducted in our laboratory have demonstrated that 50 mg/kg of orally administered Rh2 produces significant growth inhibition of a subcutaneous L N C a P (human prostate cancer metastasis to lymph node) xenograft tumor in mice (Guns ES 2004). *Rh2 and paclitaxel were found to act synergistically in cultured L N C a P cells to lower both ED50 and ED75 values and produce a significant decrease in both tumor growth and serum P S A (Xie et al. 2006). 5 1.1.7 Clinical use of ginseng Clinical trials have been conducted with ginseng in several different patient populations. To assess the time-dependent effects of Panax ginseng on health-related quality o f life ( H R Q O L ) , a randomized controlled trial was conducted using a general health status questionnaire (Ellis & Reddy 2002). The improvement in overall health-related quality of life cannot conclusively be attributed to Panax ginseng despite some positive results. A double-blind crossover study was conducted by Hong et al. in 2002 to investigate the efficacy of Korean red ginseng for erectile dysfunction. In this study, 60% of the patients answered that Korean red ginseng improved erectile function (p <0.01) (Hong et al. 2002). The effect of eight popular ginseng types was investigated for postprandial plasma glucose (PG) and insulin (PI) indices were studied by Sievenpiper et al in 2004. The outcome of this study suggests that some benefit to taking ginseng may be obtained by diabetics and would be attributable to the PPD:PPT-ginsenoside ratio. Other unmeasured ginsenoside or non-ginsenoside components may also be important. (Sievenpiper et al. 2004). I have highlighted only a sample of some of the clinical trials which have been conducted with ginseng and numerous others include the evaluation o f its use for fatigue (Elam et al. 2006), neurodegenerative disorders (Radad et al. 2006) and ergogenic properties ( K i m et al. 2005). C O L D - f X (CVT-E002), a commercial natural health product manufactured in Canada by C V Technologies, is used to prevent respiratory infections. It is a proprietary extract of the roots of North American ginseng (Panax quinquefolium). Poly-furanosyl-pyranosyl-saccharides are the main content and the contents of polysaccharides and ginsenoside levels are 6 lower and differ significantly from other Asian and American ginseng products (McElhaney et al. 2006). Ginseng is widely consumed worldwide, highlighting the importance of understanding its biological effects, safety and herb-drug interactions. In recent years, several papers reported that cranberry juice had substantial interaction with warfarin (Grant 2004; Rindone & Murphy 2006; Suvarna et al. 2003). Pomelo juice was also discovered to increase the bioavailability of cyclosporine, possibly by inhibiting C Y P 3 A or P-gp activity (or both) in the gut wall (Grenier et al. 2006). These examples emphasize the importance of also studying the metabolism of highly consumed ginseng products and their potential interactions with other drugs. 1.1.8 Metabolism of Ginsenosides Ginseng is usually administered orally; therefore, a study o f the metabolism of ginsenosides in the digestive system is important. It was reported that Rbi ,Rb2 , and Rc were converted into Rg3 under acidic conditions such as those found in the stomach. Rg3 has been shown to be transformed to 20(S) protopanaxadiol (aPPD) via Rh2 by bacteria (including Bacteroides sp., Eubacterium sp., and Bifidobacterium sp.) in human intestine (Bae et al. 2002). However, Fusobactrium sp. has only been found to metabolize Rg3 to RJ12 with no further conversion to aPPD (Bae et al. 2002). A similar process converts Rg i to protopanaxatriol (aPPT) via the ginsenoside Rhi (Shibata 2001). These intestinal bacterial metabolites, including compound K , protopanaxadiol, and protopanaxatriol are easily absorbed after deglycosylation in the stomach and the small intestine and circulate in the blood as aglycone sapogenins (Hasegawa 2004). 7 Considering that aglycone ginsenosides are the circulating metabolites of Ginseng products, it is important to investigate their biological activity, as wel l as their effect on drug metabolism enzymes, such as cytochrome P450s. 1.2 C y t o c h r o m e P450s A n array of foreign chemicals (xenobiotics) confront us daily, including environmental contaminants, drugs, carcinogens, etc. These compounds are often lipophilic, which facilitates their passage through biological membranes. They may accumulate to toxic levels unless they are metabolized to polar, water-soluble products that can be readily excreted from the body. This biotransformation is often catalyzed by a group o f enzymes called cytochrome P450s (Denison & Whitlock 1995). 1.2.1 Discovery, nomenclature of Cytochrome P450s Cytochrome P450 enzymes ( C Y P ) were originally discovered by Klingenberg in rat liver microsomes in 1958. These enzymes constitute a large super-family of haem-thiolate proteins (Sato 1964). They are present in all eukaryotic organisms, and some prokaryotes. In eukaryotic organisms, they bind to the endoplasmic reticulum or mitochondrial inner membranes, whereas in most bacteria they remain dissolved in the cytosol (Omura 1999). C Y P enzymes are characterized by an intense spectral absorbance peak at 450 nm after being reduced by carbon monoxide and their name derives from this particular characteristic (Sato 1964). Cytochrome P450 is easily converted to another solubilized form with an optical absorption peak at 420 nm by anaerobic treatment of microsomes with snake venom or deoxycholate (Omura 1999; 8 Sato 1964). A standard system of nomenclature was determined by the P450 Nomenclature Committee ( ,, based on the level of amino acid sequence identity, phylogenetic association and gene organization (Danielson 2002). The root for all cytochrome P450 genomic and c D N A sequence names is an italicized C Y P . A n Arabic numeral presents for an individual family, and a letter presents for the subfamily, such as CYP1A (Danielson 2002). The same nomenclature is used for the m R N A and protein sequences except that the designation are not italicized (e.g., C Y P 1 A 1 ) (Danielson 2002). Members of the same family exhibit about 40% amino acid sequence homology, and members of the same subfamily possess greater than 55% homology (Nelson et al. 1993). 1.2.2 Cytochrome P450 structure Cytochrome P450 enzymes have been found in all l iving organisms and in most tissue types. Their primary function is to modify drugs and other xenobiotics into more soluble forms, prior to excretion in urine. Needless to say, such an abundant and active enzyme system has garnered a great deal of interest in the pharmaceutical industry. Generally, eukaryotic cytochrome P450s range in size from approximately 480 to 560 amino acids. A l l members o f the cytochrome P450 superfamily share a common globular to triangular structural framework, which consists o f 2 halves, the carboxy-terminal rich in alpha helices and the amino-terminal rich in beta sheets (Charles A Hasemann 1995). P 4 5 0 c a m (CYP101) of Pseudomonas putida was the first 9 purified and crystallized P450. It is a water-soluble bacterial P450, isolated by Dus et al. in 1970 (K. Dus 1970) and crystallized in 1974 (Yu & Gunsalus 1974). To date, high-resolution crystal structures have been determined for several of the cytosolic bacterial cytochrome P450s such as C Y P 1 0 1 A 1 , C Y P 1 0 2 A 1 , C Y P 1 0 7 A 1 , CYP108 , C Y P 1 1 1 A 1 , C Y P 1 9 A 1 , C Y P 1 2 1 A 1 , CYP152 , and C Y P 1 7 5 A 1 (Danielson 2002). Eukaryotic P450s have similar structures, except they possess membrane-anchoring regions. However, there are many aggregation products formed in X-ray crystallography o f eukaryotic C Y P proteins, and it is difficult to obtain single crystals of them (Guengerich 1993). In recent decades, research has been performed to determine crystal structures of other prokaryotic P450s, and complexes with other compounds. In 1987, Poulos determined by crystal structure that P450 c a r n complexes with camphor (Thomas L . Poulos 1987). Raag determined X-ray crystal structures of complexes formed by ferric (Fe ) cytochrome P450 c a m and different substrates and inhibitors, as well as the ferrous (Fe ) carbon monoxide and camphor bound forms (Raag 1991b; Raag 1989a; Raag 1989b; Raag 1991a). With this information regarding the 3-dimensional structure of C Y P enzymes, P450BM3 (CYP102) was deemed a good model for membrane bound forms of the enzymes, and aided the construction of computer models for numerous microsomal C Y P s including members o f the C Y P 1 A , C Y P 2 A , C Y P 2 B subfamilies and C Y P 3 A 4 (Lewis 1999; Lewis & Lake 1995; Lewis et al. 1999a; Lewis et al. 1999b). In 2000, the mammalian C Y P 2 C 5 protein was crystallized by Williams (Williams et al. 2000). The crystal structure o f human C Y P 2 C 9 was subsequently determined in 2003 (Pamela A . Williams 2003). In 2004, Williams et al. identified an unexpected peripheral binding site using three crystal 10 structures of C Y P 3 A 4 : unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. This active binding site is located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors (Williams et al. 2004). 1.2.3 Oxidative function of cytochrome P450s Human cytochrome P450 enzymes are found in most organs throughout the body, but the highest concentrations are found in the liver (hepatocytes) and small intestine. The first three C Y P families are the main C Y P families participating in the metabolism of xenobiotics. They are highly expressed within the liver, but are also expressed in other tissues (Kaminsky 2003; L i n & L u 1998). Members o f the C Y P families 2 and 3 are present in relatively high concentrations in small intestinal epithelium (Obach et al. 2001; Peters et al. 1991; Zhang Q Y 1999). C Y P 1 A 1 is primarily expressed in extrahepatic tissues, such as lung, small intestine, and placenta (Gonzalez et al. 1992). C Y P enzymes are essential phase I oxidative enzymes (34) involved in the metabolism of fatty acids, steroids, prostaglandins, and environmental pollutants; and the conversion of procarcinogens and promutagens to deleterious genotoxic compounds (Bernhardt 2006). In 1996, Coon et al. identified more than 40 different types of reactions catalyzed by C Y P s , primarily hydroxylations but also including deaminations, desulfurations, dehalogenations, epoxidations, N - , S-, and O-dealkylations, N-oxidations, peroxidations, and sulfoxidations (Coon et al. 1996; Danielson 2002; Omura 1999). A representative model generally accepted for the hydroxylation reaction is shown in Figure 1.4. and may be simplified using the 11 equation: N A D ( P ) H + H + + O2 + R H —> N A D ( P ) + + H 2 0 + R O H (Danielson 2002) This reaction involves a series o f electron transfer steps: ferric P450 reduction, molecular O2 activation, and the making and breaking of covalent bonds (Guengerich 1993). In these monooxygenation reactions, nicotinamide adenine dinucleotide phosphate ( N A D P H ) is an electron donor to both microsomal and mitochondrial eukaryotic P450s, whereas nicotinamide adenine dinucleotide ( N A D H ) donates electrons to most bacterial P450s (Omura 1999). In endoplasmic reticulum, cytochrome P450 reductase includes two subcomponents: flavin adenine dinucleotide (FAD) and flavin mononucleotide ( F M N ) , which facilitate the direct transfer of reducing equivalents from N A D P H to the cytochrome P450 heme iron (Danielson 2002). In the mitochondria, electrons from N A D P H are transferred via ferredoxin reductase to ferredoxin and then to P450 (Nelson 2003). 1.2.4 Induction and inhibition of CYPs Drug-drug interactions are a major concern in pharmacotherapy. Whenever two or more drugs are administered over close or overlapping time periods, the possibility o f drug interactions exists. Interactions may be pharmacokinetic and pharmacodynamic in nature, pharmacokinetic interactions being more common. Induction and inhibition of C Y P enzymes are the most common cause of documented 12 drug interactions (L in & L u 2001). Inhibition of C Y P enzymes can be classified grossly into reversible and irreversible inhibition based on the enzymatic mechanism (Lin & L u 1998), with reversible inhibition indicated as the most common mechanism in drug-drug interactions (Yan & Caldwell 2001). A drug with a high affinity for an enzyme can greatly raise the plasma concentration o f any low affinity drugs metabolized by the same enzyme and thereby enhance pharmacological and toxicological effects of the low affinity drug (Hollenberg 2002). Enzyme inhibition occurs quickly and produces almost immediate effects (Hollenberg 2002; L i n & L u 2001). A number of factors must be considered with regards to enzyme inhibition. One of the most important considerations is the therapeutic index o f the drug. Patients who receive drugs with a narrow therapeutic index, anticoagulants, antidepressants or cardiovascular drugs, for instance, are at a much greater risk for drug interactions than patients receiving other types of drugs (Lin & L u 1998). In drug metabolism research, the term 'induction' means that a substance stimulates the synthesis o f an enzyme whose metabolic capacity is thereby increased. Induction occurs either due to increased transcription or translation or as a result of stabilization o f the enzyme, and is a slow regulatory process compared to inhibition. It may take days or even weeks for the full effects to manifest (Hollenberg 2002; L i n & L u 2001). Enzyme induction may attenuate the plasma concentration and pharmacological effect of a drug, which is a substrate of C Y P enzymes. Most C Y P enzymes, including human C Y P 1 A l / 2 , 2A6, 2C9, 2C19, 2E1, and 3A4 are inducible (56), but the extent of induction is variable. Depending on, for instance, which C Y P enzyme is being induced and the compounds that the individual is exposed to, induction may enhance or decrease the toxicity o f the 13 compound. If the induced C Y P catalyzes the metabolism o f a toxin or carcinogen, the induction of the C Y P w i l l increase the capability for metabolic detoxification and elimination, thus is considered a valuable part of the defense system against exposure to xenobiotics. Although induction of the C Y P s may be advantageous in most cases, it can have a variety of pharmacological consequences including alterations in drug efficacy, drug-drug interactions, and increases in the metabolic activation of procarcinogens. Consequently, induction of C Y P s can be viewed as a 'double-edged sword' for the organism involved (Hollenberg 2002). 1.2.5 Cytochrome P450 1A1 C Y P 1 A 1 has a more significant involvement in generation o f carcinogenic metabolites than other C Y P enzymes. Even though it is expressed at a very low level in many tissues, such as liver, skin, kidney, lung, it metabolizes a large number of xenobiotic chemicals to cytotoxic and/or mutagenic derivatives (Denison & Whitlock 1995; Dickins 2004). These compounds include polycyclic aromatic hydrocarbons (PAH), which are ubiquitous in cigarette smoke, city smog and charcoal-cooked foods (Miners JO 2000). The current literature suggests that, the classical mechanism of C Y P 1 A 1 induction is by activation cascade of the aryl hydrocarbon receptor (AhR) (Ma 2001) A number of PAHs , including 3-methylcholanthrene, 3,4-benzo(a)pyrene, and 2,3,7,8-tetrachloro-(p)-dioxin ( T C D D ) were shown to have high affinity for the A h R and also be potent inducers o f C Y P 1 A 1 (Dickins 2004). Alpha-naphthoflavone (a-NF) is a classic C Y P 1 A 1 inhibitor (Taura et al. 2004). A h R is normally present in cytosol as an inactive form associated with two heat shock Hsp90 proteins and another 14 not well characterized protein. After the ligand binding, this complex dissociates and A h R is activated. After its activation, A h R is able to translocate in the nucleus, where it dimerizes with the A h R nuclear translocator. This new complex binds specifically to enhancer D N A sequences within the CYP1A1 promoter called xenobiotic responsive elements (XREs) and stimulates transcription of the target gene (Delescluse et al. 2000; Dickins 2004; Seree E 2004). There are exceptions of the general rule o f C Y P 1 A 1 induction via the A h R pathway (Delescluse et al. 2000). The mechanism needs to be explored and w i l l be discussed further as part of this thesis. 1.2.6 Ginsenosides and Cytochrome P450s Many studies have been conducted to evaluate the influence of ginseng on cytochrome P450 enzymes. The natural ginsenosides, including R b i ; R b 2 , Rc,Rd,Re,Rf, or Rg i , were not found to inhibit the metabolic activity o f P450 enzymes, such as C Y P 3 A 4 , C Y P 2 D 6 , C Y P 2 C 9 , C Y P 2 A 6 , and C Y P 1 A 2 (Chang et al. 2002; He & Edeki 2004; Henderson et al. 1999; L i u et al. 2006b). Rhi competitively inhibited the activity o f C Y P 3 A 4 and slightly stimulated C Y P 2 E l ( L i u et al. 2006a). A Panax ginseng extract ( G i l 5 ) and North American ginseng extract ( N A G E ) decreased human recombinant C Y P 1 A 1 , C Y P 1 A 2 , and C Y P I B 1 activities in a concentration-dependent manner (Henderson et al. 1999). Rb i ,Rb2, Rc.Rd, and Rf inhibited C Y P 1 activities at a concentration of 50 jig/ml (Chang et al. 2002). The intestinal bacterial metabolites, including Compound K , 20(S)-protopanaxadiol (aPPD), 20(S)-protopanaxatriol (aPPT) all exhibited moderate inhibition against C Y P 2 C 9 activity, and aPPD and aPPT also exhibited potent competitive inhibition against C Y P 3 A 4 activity (L iu et al. 2006b). 15 This result was confirmed in our laboratory: C Y P 3 A 4 , 2C9, 2C19 and 2B6 were noticeably inhibited by aPPD and aPPT, which have minimal inhibitory effects on C Y P 1 A 2 or C Y P 2 D 6 . Further work is needed to verify the effect of aPPD and aPPT on C Y P 1 A 1 expression and activity. This experiment is important for clarifying the relationship between C Y P 1 A 1 inducibility and susceptibility to chemical carcinogenesis in ginseng consuming populations. 16 1.3 H y p o t h e s i s a n d Objectives Hypothesis Aglycone ginsenoside aPPD and aPPT can affect C Y P 1 A 1 activity in human hepatocytes and intestinal cells. This regulation can be at the transcriptional, translational and protein levels. Objective 1: To determine the time required for CYP1A1 mRNA to translate CYP1A1 protein Significance: It is important to know the time required for C Y P 1 A 1 m R N A to be translated into protein. Drug incubation time course w i l l be determined based on this result for the subsequent experiments. Objective 2: To determine if aPPD and aPPT have a cytotoxic effect on HepG-2 and Caco-2 cell lines in vitro Significance: It is well recognized that animal data is inadequate to predict C Y P induction in humans, because both the extent and pattern of C Y P induction may differ markedly between species. To this end, there have been significant advances in human in vitro methods to assess enzyme induction in man (Dickins 2004). This work w i l l be conducted in an effort to determine the proper concentrations of drug for treatment. 17 Objective 3: To study the impact of aPPD and aPPT on human cytochrome P450 1A1 Aim 1: To determine whether aPPD and aPPT induce or decrease CYP1A1 gene expression in HepG-2 and Caco-2 cell lines Aim 2: To examine the impact of aPPD and aPPT on translation of CYP1 A l Aim 3: To observe effects of aPPD and aPPT on CYP1 A l activity in human liver microsomes Significance: It was found that C Y P 1 A 1 has a more significant involvement in generation of carcinogenic metabolites from xenobiotics than other C Y P enzymes. aPPD and aPPT are the major active metabolites of ginseng formed in the human gut. It is anticipated that when these three aims are accomplished, the impact o f isolated ginsenosides aPPD and aPPT on C Y P 1 A 1 transcription and translation, as well as protein activity w i l l be determined. Objective 4: To explore the mechanism of CYP1A1 induction on a transcriptional level. Significance: A common model of C Y P 1 A 1 induction in literature is based on aryl hydrocarbon receptor (AhR) activation initiating a cascade which results in the induction of C Y P 1 A 1 (Ma 2001). The objective is to determine whether aPPD and aPPT induce CYP1A1 gene expression via the A h R pathway or by some other means. 18 Table 1.1: Ginseng species. Taken from Yun, T. K . 2001 1. Panax ginseng C . A . Meyer (Korean ginseng) 2. Panax japonicus C . A . Meyer (Japanese ginseng) 3. Panax major Ting 4. Panax notoginseng (Burkill) F. H . Chen (Sanchi ginseng) 5. Panax omeiensis J. Wen 6. Panax pseudoginseng Wallich 7. Panax quinquefolius L . (American ginseng) 8. Panax sinensis J. Wen 9. Panax stipuleanatus H . T. Tsai & K . M . Feng 10. Panax trifolius L . (Dwarf ginseng) 11. Panax wangianus Sun 12. Panx zingiberensis C .Y. W u & K M . Feng 13. Panax vietnamensis H a et Grushv. (Vietnamese ginseng) 19 Saponin/Sapogenin R i R 2 Re -Glc-Rha -Glc R f -Glc-Glc - H glc-Rc -Glc-Glc -Glc Rg i -Glc -Glc Rg2 -Glc-Rha - H N G - R i - G l c - X y l -Glc Rh i -Glc - H aPPT - H - H Figurel.l: The ginseng saponins of protopanaxatriol. Source reference: Sticher, O. 1998 (Sticher 1998). 20 Saponin/Sapogenin R i R 2 Rb, Glc -Glc Glc -Glc R b 2 Glc-Glc Glc-Ara(p) R c Glc -Glc Glc-Ara(f) R d Glc -Glc -Glc mRbi G l c - G l c - M a Glc -Glc mRb2 G l c - G l c - M a Glc-Ara(p) mRc G l c - G l c - M a Glc-Ara(f) Rg3 Glc-Glc - H R h 2 Glc - H aPPD - H - H Figure 1.2: The ginseng saponins of protopanaxadiol. The variation in the structure of saponins is shown in Figure 1.1 and 1.2. A l l compounds listed are 20S configuration except R g 3 , which is a mixture of S and R. A comprehensive list of all saponins is too large to include. Source reference: Sticher, O. 1998 (Sticher 1998) 21 Ro • o • R a , • O R a g - O Rag • o R b , • o R b 2 • O Rbg • O 0 - R t • O R s , • O Rs? - O Ro • o Rd • o Re • 0 N G - R f 0 R! • O glo-R1- O Rg« • o • o R h , • o • o « o O O o O Ro R a R b 2 Re Rf Rfofte Rd Ro • o • Ra j • o Rag • o R a a • o R b , • o R b g » o Rbg • 0 Q - R , • 0 R s , • o Rs^, • o R c * • o Rd • o Re • a N G - R f o R l • o g)c-R1" o R g , • o R g 2 • 0 R b , • o o e> O ° ° o» Q • R o R a R b i Rc RdReRI R g 1 R b 2 R g 2 C H C I ; j - C H s O H - H a 0 (14/&('1) B U O H - A C O C S H S - M J J O (4/1/2. upper layer) Figure 1.3: Thin-layer chromatograms of the saponins of Panax ginseng roots. The crude saponin fraction was analyzed on a plate of silica gel 100F254 (Merck) with solvents as indicated. Image taken from Sticher et al. 1998 (Sticher 1998). 22 nativa hexacoor dinata • ferric form (low-spin) Product Release / d - c ^ r Substrate Binding BOH £^>*\S BH oxyforryl intermodiato ** (tow-spin) pentacoordinatG ferric complex (hlgh-apln) 'Cys e* First electron roduction o-o 'Cys ferric peroxycomptex (low-spin) pontacoordlnato ferrous complex (Mgh*ptn) Second electron reduction i" hoxacoordinate forrous-02 adduet (low-spin) WW hexacoordlnato CyS fcrrou»-CO Inhibitor complex (tow-spin) Figure 1.4: The proposed catalytic cycle of cytochrome P450 for hydroxylation reactions. Image taken from Danielson, P. B . 2002 (Danielson 2002). 23 Figure 1.5: General CYP1A1 induction model also known as the AhR signaling transduction pathway. Image was derived from Delescluse, C . et al. 2000 (Delescluse et al. 2000). 24 CHAPTER 2 M A T E R I A L S A N D M E T H O D S 2.1 C e l l C u l t u r e HepG-2 hepatoma cell line ( A T C C # HB-8065™, Manassas, V A , U S A ) and Caco-2 human colonic carcinoma cell line ( A T C C # HTB-37™, Manassas, V A , U S A ) were cultured in Dulbecco's Modification of Eagle's Medium ( D M E M ; Invitrogen), supplemented with 10% fetal bovine serum (FBS; G I B C O ™ , Grand Island, N .Y . ) at 37 °C in a 5% CO2 environment. Growth medium was changed every three days. A t 80% confluence by experience, cells were trypsinized in 0.25% trypsin with E D T A 4Na (Invitrogen, Burlington, O N , Canada), and 10% of the cells were subcultured. 2.2 G r o w t h F a c t o r a n d D r u g T r e a t m e n t After trypsinizing and re-suspending in growth medium, cells were counted using a haemocytometer. In order to maintain the same confluence in the experiments, cells were seeded at approximately 30% confluence for both cell lines 24 h prior treatment. Both aPPD and aPPT were received as gifts from Panagin Pharmaceuticals Company*, and were dissolved in 100% ethanol or D M S O . The concentrations of aPPD/aPPT tested ranged from 0 to 80 u M in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( M T T ) assay and from 0 to 40 u M in other experiments. The concentration range was decided using other literature based reports of ginsenoside use in in vitro assay (Kitts. 2004). During * Purity of the compounds was confirmed by Panagin Pharmaceuticals Company using H P L C . 25 treatment with aPPD or aPPT, cells were incubated in D M E M , supplemented with 2% F B S (Ota T 1991). 2.2.1 To determine the time required for CYP1A1 mRNA to be translated into CYP1A1 protein 2,3,7,8-tetrachloro-(p)-dioxin ( T C D D ) was obtained from Chromatographic Specialties Inc (Brockvi l le ,ON, Canada). HepG-2 cells were treated with 10 n M of T C D D for 0, 2, 4, 6, 8, and 12 hours in duplicate for R N A extraction, while for protein extraction, the treatment duration range was 0, 4, 8, 12, 18, and 24 hours. This experiment was repeated two times. 2.2.2 M T T procedure Both cell lines were seeded at 2.5 x 10 4 cells per well in 96-well plates. Popovich and Kitts determined the cytotoxicity of aPPD and aPPT in Caco-2 cell line in 2004 with reported LC 5 ns of 50 u M and 300 u M respectively (Kitts. 2004). Therefore concentrations lower than 80 u M were used for both cell lines. Cells were treated in duplicate with a final concentration at 0, 5, 10, 20, 40, 80 u M of aPPD and aPPT in D M E M media supplemented with 2% F B S . The duration of exposure to aPPD/aPPT was 12 and 24 hours. After 12 or 24 hours, the treated media was removed and 100 u L of 0.5 mg/ml M T T in serum free media was added, followed by a four-hour period incubation at 37 C in 5% CO2. To solubilize the formazan crystals after incubation, 100 pi of lysis buffer (20% SDS, 50% N , N-dimethylformamide, 0.4% glacial acetic acid) were added to each well . The optical density was read at 562 nm 26 absorbance in a microplate reader and relative survival determined compared to untreated wells. These experiments were repeated three times. 2.2.3 Study of the induction of the CYP1A1 at both a transcriptional and translational level For both cell lines, 6 x l 0 5 cells were plated into each well in 6-well plates. The duration of treatment for transcriptional studies was 12 h and 24 h, and 24 h for translational studies. The concentrations o f aPPD and aPPT used for treatment were 0, 1, 5, 10, 20, and 40 u M . Transciptional level experiments were repeated three times no replicates each time, but technical triplicate in Real Time P C R step (n=3). Translational level experiments were repeated two times with biological triplicate each time. 2.3 Cell lysis 2.3.1 Total RNA extraction TRIzoi®Reagent (Invitrogen, Carlsbad, C A ) was used to lyse cells directly in a culture dish by adding 1 ml of T R I Z O i ® Reagent per well using a 6-well plate. The lysed samples were incubated at room temperature (RT) for 5 min. After adding 0.2 ml of chloroform, tubes were capped securely. The tubes were vigorously agitated by hand and then incubated at room temperature for 2 to 3 min. After incubation, samples were centrifuged at 12,000 x g for 15 min at 4 C. The aqueous phase was then transferred to a fresh tube. To precipitate R N A , 0.5 ml of isopropyl alcohol was added into each sample to the aqueous phase, followed by incubation at RT for 10 min. The sample was 27 again centrifuged at 12,000 x g for lOmin at 4 °C. The supernatant was removed and the precipitated R N A samples were washed with 1 ml of 75% ethanol in H2O by vortexing for 10 sec at low speed. A final centrifugation was performed at 7,500 g for 5 min at 4 C. The R N A pellet was briefly dried in the open air at RT for 5 to 10 min, and then dissolved in D E P C treated H2O by passing the solution through a pipette tip. 2.3.2 RNA concentration determination Nanodrop-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, Delaware U S A ) was used according to the protocol provided with the instrument. 2.3.3 Total protein extraction Harvested cells were washed with ice-cold Phosphate Buffered Saline (PBS) and were detached gently from the plate with a rubber scraper on ice into another 0.5 m l of cold P B S . The cells were pelleted by centrifuging at 4000 rpm for 4 min. The pellets were resuspended in 100 uL of RIPA buffer (NaCl 150 m M , NP-40 1%, N a Deoxycholate 0.5%, SDS 0.1%, Tris (Base) 50mM) supplemented with l x Complete Protease Inhibitor (Roche, Penzberg, Germany). 2.3.4 Protein concentration determination Before quantitation, protein samples were sonicated on ice twice, for 10 sec, at power level 3 using 550 Sonic Dismembrator (Fisher Scientific). There was an interval o f at least 1 min between each sonication. During this interval, samples were cooled on ice. B C A ™ Protein Assay K i t (P IERCE, Rockford, IL) was used. Diluted 28 B S A (bovine serum albumin) standards were prepared at the following concentrations from a 2.0 mg/ml B S A stock standard: 25, 50, 125, 250, 500, 750, 1000, 1500, and 2000 pg/ml. To prepare the B C A working reagent (WR), 50 parts o f B C A Reagent A and 1 part of B C A Reagent B were mixed, followed by a brief vortex to mix. In order to be within the range of the standard curve, all protein samples were diluted 1/10 prior to protein determination. 25 uL of each standard and unknown sample was pipetted into a flat bottomed 96-well plate in duplicate. 200 uL of the W R was then added to each well , and samples were shaken for 30 s before being covered and incubated at 37 °C for 30 min. After the incubation, the plate was cooled to RT. Absorbance was measured at or near 562 nm on a plate reader (Power Wave, Bio-Tek Instruments, INC) 2.4 Genomic DNA digestion in RNA sample Deoxyribonuclease I, Amplification Grade was purchased from Invitrogen, Carlsbad, C A . Throughout the experiment, DEPC-treated H 2 0 and RNase-free microcentrifuge tubes were used. Duplicate tubes were prepared for each sample. One set was used for the treatment, while the other was used as a negative control (no superscript II enzyme during RT step). R N A mass was balanced between each sample at 1 [ig by calculation. The total sample volume was 10 pL . Initially, R N A sample, D E P C treated H2O, 10 x reaction buffer and 1 p L of DNase I were added into the sample and control tubes, followed with an incubation at RT for 15 min. 1 uL of 25 m M E D T A solution was added into each reaction to inactivate the DNase and then samples were heated for 10 min at 65 C. The R N A samples were ready to use in reverse transcription. 29 2.5 Reverse Transcription Superscript 1 M II RT kit and RNaseOUT were purchased from Invitrogen, Carlsbad, C A . Each sample had a no-RT control during RT process. The total reaction volume was 20 uL. After mixing and a 10 sec centrifuge at 5000 rpm for each component, RNA/pr imer mixture was added to sterile 0.5 m l tubes as follows: 1 ug o f total R N A , 1 uL of dNTP mix (10 m M each), 1 uL of Oligo (dT) 12-18(0.5 ug/uL), and using D E P C treated H2O to create a volume of 12 u.L. The mixture was incubated at 65 C for 5 min, and then placed on ice for at least 1 min. A reaction mixture was prepared as follows: 2 uL of lOx RT buffer, 2 uL of 50 m M M g C b , 2 uL of 0.1 M DTT, and 1 uL of RNaseOUT™. 7 uL of this reaction mixture was added into each RNA/pr imer mixture followed by a 10 s vortex and a 30 s centrifugation at 5000 rpm for collection. The final mixture was incubated at 42 °C for 2 min. Following incubation, 1 uL of Superscript II RT was added into each sample tube, while the no RT controls had 1 uL D E P C treated water added to each tube, followed by mixing and incubation at 42 C for 50 min. The reaction was terminated by heating to 70 °C for 15 min and then chilled on ice. 2.6 Conventional PCR The purity o f R T step c D N A samples and negative controls were tested by conventional P C R . Platinum Taq D N A polymerase was purchased from Invitrogen (Carlsbad, C A ) . dNTPs were from Superscr ip t™ II R T kit used in R T step. The following primers were used in the experiment: 30 Beta actin: 5 ' - C G T A C C A C T G G C A T C G T G A T - 3'(Forward) Beta actin: 5'- G T G T T G G C G T A C A G G T C T T T G - 3'(Reverse) The P C R protocol included incubation for 2 min at 94 °C; followed by 35 cycles of 94 °C for 15 s, 58 °C for 30 s, and 68 C for 30 s, with an additional 10 min for elongation at 68 °C after the last cycle. 2.7 Real Time PCR S Y B R Green P C R Master M i x was purchased from Applied Biosystems, Warrington, U K . The primer sets described below were used for the real time P C R experiments: CYP1A1: 5'- C C T T C G T C C C C T T C A C C A T - 3Xforward) CYP1A1: 5'- G T A A A A G C C T T T C A A A C T T G T G T C T C T -3'(reverse) Beta actin: 5'- G C T C T T T T C C A G C C T T C C T T -3'(forward) Beta actin: 5'- C G G A T G T C A A C G T C A C A C T T -3' (reverse). c D N A samples from RT step were diluted 1/5. 2 p L of the diluted c D N A template was added into each wel l o f a Mic roAmp Optical 96-well Reaction Plate (Applied Biosystems, Foster, C A ) with triplicates of both CYP1A1 and beta actin gene for each sample. For the 25-pL-reaction volume, the primer master mix for each reaction contained 12.5 u L of S Y B R Green Master M i x , 1 uL of forward primer, 1 uL of reverse primer and 8.5 p L of dFkO. 23 uL of the primer master mix was added in each well . The plate was sealed with Optical Adhesive Covers (Applied Biosystem, Foster, C A ) . A brief centrifugation was conducted to collect the samples. Following 2 min at 50 °C 31 and 10 min at 95 C, the amplification was carried out with 40 cycles o f 15 sec at 95 C and l m i n at 60 °C. This amplification was conducted using a 7900HT Fast Real-Time P C R System (Applied Biosystems, Foster, C A ) . Real time P C R results were calculated using C T values. Delta C T equals the value o f the C T value for C Y P 1 A 1 gene minus the C T value for beta actin. Delta delta C T equals the value of delta C T for a given sample minus the control. 2A-delta delta C T was 2 power minus delta delta C T value. Fold change was calculated by determining the ratio o f 2Adelta delta C T value for a gievn samples over the control. Standard deviation was calculated among replicates. 2.8 Western blotting 2.8.1 Electrophoresis Total protein was standardized to a loading volume of 30 jag per lane. To ensure a final volume of approx 40 uL, the protein sample mixture contained 26 uL of protein lysate/H 2 0, 4 u L of N u P A G E Reducing Agent (10x) (Invitrogen, Carlsbad, C A ) , and 10 uL of N u P A G E L D S Sample Buffer (4*) (Invitrogen, Carlsbad, C A ) . The samples were denatured at 100 C for 5 min followed by a brief spin at 4 °C. N u P A G E ™ 10% Bis-Tris Gels (Invitrogen, Carlsbad, C A ) were used. After the comb was removed from the gel, lanes were rinsed with 1 x NuPAGE® M O P S SDS Running Buffer (50 m M M O P S , 50 m M Tris base, 0.1% SDS, and I m M E D T A , p H 7.7). The chamber was filled with 800 m l running buffer to submerge the whole system. PageRuler ™ Prestained Protein ladder (Fermentas, Burlington, Ontario) was used as a marker, and 25 ul o f protein samples were loaded in each well . The gel was 32 run at 200 V for 50 minutes in Invitrogen X C e l l SureLock gel apparatus. 2.8.2 Transfer of Separated Proteins to Nitrocellulose Membrane One nitrocellulose membrane ( B I O - R A D , Hercules, C A ) (6 cm x 9 cm), two fiber pads, and four pieces of filter paper (7 cm x 9 cm) were prepared to make the transferring sandwich for each gel. A large container was filled with l x transfer buffer (with 20% methanol) to a depth of approx. 5 cm. One o f the transferring cassettes was opened and placed in the container with the black panel touching the bottom. From the bottom to the top, the sequence of the contents of the sandwich was as follows: a fiber pad, two pieces o f filter paper, gel, a nitrocellulose membrane, two pieces o f filter paper and a fiber pad. A l l materials were saturated in transfer buffer before being placed into the cassette and all bubbles were removed from between the nitrocellulose membrane and the gel. After the cassette was closed, it was placed in one of the transferring boxes (black side of the cassette facing the black side o f the box). The transferring box was placed into a transferring container with a stirring bar and an ice pack inside. Transfer buffer was added so that the membranes are covered. The transfer was run at 100 v for 1 hour at room temperature or run at 30 v overnight at 4 o C. 2.8.3 Ponceau Red Stain The membrane was removed from the transferring apparatus, followed by washing in P B S twice for 10 min, on a shaker. Ponceau Red Stain was used to detect proteins and ensure a consistent transfer. The membrane was covered by Ponceau and 33 placed on a shaker for 5 min. After detection, stain was removed with a destain solution (1% acetic acid) which was applied for 5min. Proteins lower than 40 kDa was cut off and the remaining membrane was washed twice in P B S on a shaker for 5 min. 2.8.4 Blocking To minimize non-specific binding, Odyssey Blocking Buffer ( L I - C O R Biosciences, Lincoln, Nebraska) was used with 1:1 dilution in P B S . The diluted blocking buffer was applied to completely cover the membrane and placed on the shaker for 1 hour at room temperature. 2.8.5 Primary Antibody Incubation A 1:1 solution of 1 x T B S - T (0.2% Tween-20) and Odyssey Blocking Buffer was prepared to cover the membrane, and primary antibody C Y P 1 A 1 (B-4) (Santa Cruz, Santa Cruz, California), a mouse monoclonal antibody, was added at a dilution of 1:300. The membrane was incubated for one hour at room temperature and then overnight at 4 °C. Vincul in (Sigma, Oakville, Ontario), a monoclonal anti-mouse antibody, was used as an internal standard primary antibody with a dilution of 1:5000. This was incubated at room temperature for one hour. The membrane was washed in 1 x T B S - T (0.2% Tween-20) 4 times for 5 min. 2.8.6 Secondary Antibody Incubation and Detection Fluorescent secondary antibody, Alexa Fluor® 680 goat anti-mouse IgG (H+L) 34 (Molecular Probes, Eugene, Oregon) was used at 1:5000 dilution with 0.02% SDS, and incubated at room temperature for one hour. The membrane was washed in 1 x T B S - T (0.2% Tween-20) 4 times for 5 min. The membrane was scanned using an Odyssey™ Infrared Imaging System ( L I - C O R Biosciences, Lincoln, Nebraska). 2.9 P450 Glo™ CYP1A1 activity assay P450-Glo™ C Y P 1 A 1 Assay kit was purchased from Promega, Madison, WI, U S A . A n N A D P H regenerating system was used (BD Gentest™, Oakville, Ontario) and it included two solutions: solution A containing 26.1 m M N A D P + , 66 m M Glucose-6-phosphate, and 66 m M M g C l 2 in H 2 0 ( N A D P + and GIC-6-PO4) and solution B containing 40 U / m l Glucose-6-phosphate dehydrogenase (G6PDH) in 5 m M sodium citrate. The experiment was repeated twice with technical triplicates each time. Human liver microsomes, containing a mixture of C Y P proteins, were used instead o f cell lines. The P450-Glo™ C Y P 1 A 1 assay kit consists of three components, a specific luminogenic substrate (Luciferin Chloroethyl Ether - Lucifer in-CEE), a lyophilized Luciferin Detection Reagent and its reconstitution buffer (P450-Glo™ Buffer). To assay cytochrome P450 1A1 activity, the substrate was incubated with human liver microsomes to generate the luciferin reporter. T C D D and alpha-naphthoflavone (ot-NF), a C Y P 1 A 1 protein inducer and a C Y P 1 A 1 activity inhibitor respectively, were used as controls throughout the experiment. Higher concentrations, 80 and 160 u M , were used, because cytotoxicity was not a consideration due to the use of human liver microsomes. Reconstituted Luciferin Detection Reagent was prepared by equilibrating the 35 P450-Glo Buffer and Luciferin Detection Reagent to room temperature and transferring the contents o f one bottle of P450-Glo Buffer (10 ml) to a bottle containing the lyophilized luciferin Detection Reagent. A homogeneous solution was obtained by mixing thoroughly. In each reaction, 4 X Cytochrome P450/KPCVSubstrate Reaction Mixture contained 20 p:g of human liver microsome ( H L M ) , 400mM of KPO4, and 100 u M of Lucifer in-CEE. The concentration o f all materials was optimized by Promega Corporation (Technical Bulletin No.325). H L M and C Y P 1 A 1 substrate Lucifer in-CEE were thawed in a 3 7 C water bath quickly and then kept on ice. For each reaction, 12.5 ul o f 4 X Cytochrome P450/KPO4/Substrate Reaction Mixture was added per well in a 96-well plate. For negative C Y P control reactions, 4 X Control/KPOVSubstrate Reaction Mixture was prepared using an equivalent amount of a preparation that lacked the C Y P 1 A l substrate Lucifer in-CEE. Stock solutions of aPPD/aPPT/a-NF in D M S O were added at a final concentration of 0, 1, 5, 10, 20, 40, 80, 160 u M ; while T C D D was added at concentrations of 0, 1, 5, 10, 20, 40, 80, 160 n M . D M S O volume was balanced between each sample. 10.5 ul of d H 2 0 was added to bring the volume to 12.5 ul per well in a 96-well plate. For sample reactions, 12.5 ul of the 4x microsome/KPOVSubstrate Reaction Mixture was added in each wel l and mixed gently. For 'minus substrate' control reaction, 12.5 uL of the 4x microsome/KP04 Mixture was added instead. 100 uL of d H 2 0 was added into wells designated as blanks, for signal o background subtraction. The plate was pre-incubated at 37 C for 15 min. 2x N A D P H regenerating system was made by mixing 4x solution A and 4x 36 solution B . The reaction was started by adding 25 uL of 2x N A D P H regenerating system to both the sample and the control wells. The plate was gently shaken for 30 s o and then incubated at 37 C for 30 min. After the incubation, the same amount of substrate was added into the minus substrate control reactions. To stop the reaction, 50 p L of reconstituted Luciferin Detection Reagent was added into both the sample and the control reactions, but not added into the blank wells. After shaking for 30 s, the plate was incubated at room temperature for 20 min to stabilize the luminescent signal. The luminescence was recorded using E G & G B E R T H O L D Microplate Luminometer L B 96 V (Fisher Scientific). 2.10 Plasmid Three plasmids: A h R , p R L - T K and Gudluc 1.1 were obtained as gifts from Dr. Colleen C . Nelson (University of British Columbia). The Gudluc 1.1 plasmid contains the firefly luciferase gene under control of a portion of the upstream promoter region (from -1301 to -819) of the CYP1A1 gene containing four dioxin response elements ( X R E M ) (P. M . Garrison 1996). The p R L - T K vector contains a thymidine kinase promoter upstream of Rluc. Rluc is the slightly modified c D N A encoding Renilla luciferase from the sea pansy Renilla reniformis (Promega, Madison, Wisconsin). 2.11 C Y P 1 A 1 promoter plasmid cloning 2.11.1 Conventional PCR CYP1A1 promoter Plasmid, p G L 3 B - C T P L 4 / (-2425 to +352) was constructed. 37 The following primers were used in the process of constructing the CYP1A1 promoter sequence: 5'- A C A T G G T A C C C C G A G T G A C C T T G C T T C T C T -3 ' (forward) 5'- A C G A A G A T C T C T G G G T C C T G A A G T C C T G A A -3 ' (reverse). Conventional P C R components in 50 uL of final volume included 0.2 ug of human genomic D N A , 2 m M M g 2 + , 0.1 U / u L of Platinum Taq D N A polymerase, 0.2 m M of dNTP, and 0.5 m M of both forward and reverse primer. The P C R protocol included incubation for 3 min at 94 °C; and 10 cycles of 94 °C for 45 s, 54 °C for 45 s, and 72 °C for 200 s; then, 30 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 200 s followed with an additional 10 min at 72 °C for elongation after the last cycle. 2.11.2 T O P O T A Cloning The P C R product was ligated to pCR®2.1-TOPO vector using T O P O T A Cloning® kit (Invitrogen, Carlsbad, C A ) . The ligation product was transformed into competent cells (Top 10 cells). After being plated onto a petri dish (100 u.g/ml of ampicillin in agar gel), cells were incubated at 3 7 C overnight. 12 clones of bacteria were randomly chosen and incubated in 2 m l L B medium with 100 |J,g/ml of ampicillin, shaking at 250 rpm at 37 °C over night. PureLink™Quick Plasmid Miniprep K i t (Invitrogen, Carlsbad, C A ) was used to purify the plasmid. 2.11.3 Restriction Digestion After purification, plasmids were tested by running in 1% agarose gel. Two of 38 the successful samples were selected for restriction enzyme digestion to double test them. E c o R l (Invitrogen, Carlsbad, C A ) was selected as the restriction enzyme. After confirmation by running the enzyme digestion product in 1% agarose gel, another restriction enzyme digestion was performed for the T O P O T A cloning product and pGL-3 basic vector (4.8 kbp) together, using K p n l (Invitrogen, Carlsbad, C A ) and N c o l (Invitrogen, Carlsbad, C A ) sharing No . 4 buffer from Invitrogen. A 2.8 kbp band and a 4.8 kbp band were cut off and purified with QIAquick®Gel Extraction K i t ( Q I A G E N , Mississauga, ON) . 2.11.4 T4 Ligation and Sequencing Two purified products, CYP1A1 promoter/Kpnl-Ncol and pGL-3 bas i c /Kpn l -Nco l , were ligated by T4 ligase (Invitrogen, Carlsbad, C A ) at 25 °C for 2.5 hours with insert/vector molar ratio around 3:1. The ligation product was transformed into Top 10 cells and after amplification plasmids were extracted using QIAfilter™ Plasmid M a x i kit (Qiagen, Mississauga, ON) . Another restriction enzyme digestion was performed with K p n l and N c o l for the final plasmid. The result was tested on a 1% agarose gel. The D N A Sequencing Laboratory (the University o f British Columbia, Canada) conducted the sequencing for T O P O T A cloning product. 39 2.12 Transfection 2.12.1 Optimization experiment To optimize the transfection experiment with respect to the number o f cells, the transfection incubation time, and the ratio of D N A to Lipofectin® Reagent (Invitrogen, Carlsbad, C A ) experiments were carried out using a 6-well plate. Each well received 0.3 pg/pL of the A h R plasmid, 0.3 pg/pL of the Gudluc 1.1 plasmid, and 0.02 pg/pL of p R L - T K plasmid. In an attempt to obtain 40% confluence after 24 h, 4x 10 5 cells were plated in each well . Similar attempts were made to obtain 50%, 60%, 70%, 80%, and 85% confluence after 24h, with 5*10 5, 6><105, 7*10 5 , 8 x l O 5 and 8.5 x i o 5 cells plated into each well . The volume of Lipofectin was 6 p L and the incubation time was 6h for all the wells in the confluence optimization group. To optimize the ratio of DNA/Lipofect in , 4, 6, 8, 10, 12, and 16 p L of Lipofectin were added with a consistent level of cells at 6x10 5 cells per well and an incubation time o f 6 h. For the incubation time optimization group, 6x10 s cells were plated and 6 p L of Lipofectin was added into each well . The incubation time range was 4, 6, 18, and 24 h. Lipofectin was diluted with serum free media and kept at room temperature for 30 min. D N A was also diluted with serum free media and gently mixed with the Lipofectin dilution. The dilution mixture was kept at room temperature for another 10 min. It was then diluted again with serum free media to attain a final volume of 250 p L per well . The culture media was discarded and the transfection serum free media solution was added to the cells. After adequate transfection time, the transfection media was discarded and the cells were cultured in D M E M with 10% F B S for another 24h. Proteins were harvested using Passive Lysis Buffer (from Dual-Luciferase® 40 Reporter Assay System). After gentle vortexing, the protein samples were stored at - 8 0 C until the time of analysis. 2.12.2Transfection with Gudluc Plasmid Based on the result from optimization experiments the following transfection conditions were used in transfection experiments: 60% confluence by the beginning of transfection, 2 (ig/8 uL of the DNA/reagent ratio, and an incubation time of 6h. The concentration of T C D D was determined according to the literature ( X u et al. 2000). HepG-2 cells were plated in 24-well plates with 1.5 X 10 5 cells each wel l . The following day, cells were transfected with 0.25 u.g of the A h R plasmid, 0.25 ug of the Gudluc 1.1 plasmid, and 0.025 \ig o f p R L - T K plasmid in each well . After incubating for 6 hours, cells were treated with 0, 1, 5, 10, 20, 40 u M of aPPD and aPPT in D M E M with 2% F B S . 20 n M and 2 n M of T C D D were the positive controls for the experimental duplicate. D M S O was used as a negative control. The treatment incubation lasted for 38 h. The cells were lysed as described in section 2.11.1. 2.12.3Transfection with pGL3B-CYPlAl The function o f pGL3B-CYPlAl construct was tested by comparison with the positive control pGL-3 promoter (Promega, Madison, WI) and negative control pGL-3 basic (Promega, Madison, WI) in HepG-2 cells. The amount of plasmids in each well was as follows: 0.3 jj.g pGL-3 basic, 0.3 jxg pGL-3 promoter, 0.3 ug pGL3B-CYP!Al, and 0.03 p,g p R L - T K . Transfection procedure was the same as section 2.11.2. 41 The same amount of pGL3B-CYP!Al construct was co-transfected with 0.3 pg A h R plasmid and 0.03 pg p R L - T K in HepG-2 cells followed by 20 p M aPPD and aPPT treatment for 24h. The positive control was treated with 2 n M T C D D for 24h, while negative control was considered as the addition of the same volume o f D M S O . 2.13 Luciferase assays Luciferase assays were performed using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI) and the E G & G Berthold Microplate Luminometer L B 96V (Berthold Technologies, Bad Wildbad). Luciferase Assay Buffer was added into Luciferase Assay Substrate and inverted for 5 times by hand. 200 u L o f Stop & Glo® substrate was added to Stop & Glo® buffer. The protein samples were thawed from -80 °C and centrifuged at 12,000 rpm for 4min. 20 p L of the supernatant from each sample was added in a 96-well plate for reading. The plate was read using the following conditions: Injection P (Luciferase Assay buffer and substrate) delayed 1.6 s and a measurement interval of 30 s was conducted. Injection M (Stop & Glo® buffer and substrate) delayed 10 s and a measurement interval of 10 s was conducted. For both Injection P and M , the volume was 100 pL. The working temperature was 20 C. 42 C Y P 1 A 1 Promoter Gene Figure 2.1: Structure of the YGL3B-CYP1A1 plasmid. This construct contains a 2425 bp fragment from the upstream region of CYP1A1 gene and the 352 bp down stream sequence including the first exon, and part of the first intron. The plasmid contains a firefly luciferase gene under control of the CYP1A1 promoter sequence. 43 CHAPTER 3 RESULTS 3.1 Study of CYP1 A l induction at the transcriptional and translational level 3.1.1 Determination of the time required for CYP1A1 mRNA to translate protein Before investigating the inductive effects of ginsenosides, it is necessary to determine the rate at which C Y P 1 A l protein is translated. T C D D , a well-known C Y P 1 A l m R N A and protein inducer, was used. The treatment duration for R N A studies was 0, 2, 4, 6, 8, and 12h, while the time for protein studies ranged from 0, 4, 8, 12, 18, to 24h. The C Y P 1 A l m R N A was induced to the highest level (237±31 fold) at 12h within the chosen time course range (Figure 3.1A). Wi th respect to translation, induction was observed from 12h onwards with a 5.32±1.2 fold increase (n=2). From 12h to 24h, C Y P 1 A 1 protein expression was stable at levels 5 to 6-fold greater than the controls (Figure 3.1B). These results were used to determine treatment times for the following experiments. 12h and 24h were chosen as appropriate time points. 44 3.1.2 Cytotoxicity study Every cell line has a unique response to a particular drug. The cytotoxicity experiments involved were performed using a M T T assay. HepG-2 and Caco-2 were the two cell lines used and the cytotoxicity of aPPD and aPPT in these two cell lines was tested in this step. Based on observations that C Y P 1 A l protein was stably expressed 12 h to 24 h after T C D D treatment, and given that the highest level of C Y P 1 A l m R N A was seen at 12 h, cell viability of HepG-2 and Caco-2 cells was measured at 12 h and 24 h after treatment with a range of aPPD and aPPT concentrations. Cytotoxicity results over a range of 0 to 80 u M , shown in Figure 3.2, indicate that aPPT does not have a cytotoxic effect at concentrations o f 40 u M and lower over 12 h and 24 h treatment durations in either cell line (n=3). In contrast, aPPD treatment at 40 u M resulted in death of most cells after a 24 h in both cell lines (n=3). When treated for 12 h with 40 u M aPPD, half o f the Caco-2 cells were dead, but almost no effect was seen on HepG-2 cells (n=3). 3.1.3 Induction study in transcriptional level Real time R T - P C R was used to quantitate C Y P 1 A l m R N A expression in HepG-2 and Caco-2 cells after aPPD and aPPT treatment. The Dose dependent induction of C Y P 1 A l was observed in transcriptional levels following both 12h and 24h treatments of both HepG-2 and Caco-2 cells, with aPPD and aPPT (Figure 3.3). The induction was statistically significant at 5 u M and all concentrations above. 40 45 u M aPPD treatment led to cell death in both Caco-2 and HepG-2 cells at 24h. A t this concentration and at the 24h time point, aPPD caused 32.25 ± 24.65 fold increase in C Y P 1 A l m R N A level in HepG-2 cells (n=2). The reason for the large variation is only a small amount of R N A harvested, which was just enough to perform two experimental repeats. In Caco-2 cells, at 40 u M aPPD, an insufficient amount of R N A was available to make c D N A and as a consequence this treatment data was not collected. However, 20 u M o f aPPD resulted in a clear increase in the expression of C Y P 1 A 1 m R N A (n=3). 3.2 Effects of ginsenoside treatment on protein translation In translation level, Western blotting was used to measure the expression of C Y P 1 A l protein. Treatment with aPPD at 40 n M led to cell death in both cell lines, therefore, insufficient protein was obtained for analysis. For other dosage levels, equal masses of protein were loaded into each lane. We have determined C Y P 1 A l induction at a transcriptional level with both aPPD and aPPT in both cell lines by measuring R N A levels. The induction at the translational level was present and significant in HepG-2 cells treated with 20 u M aPPD for 24h, which caused a 1.78 ± 0.14 (p=0.02) fold change (n=2). In Caco-2 cells, the induction of 1.38 ± 0.2 fold change was not statistically significant (n=2). C Y P 1 A l protein expression was not induced significantly by aPPT in HepG-2 cells at any of the concentrations tested, although expression was decreased by treatment with 46 20 p M of aPPT with a 0.69±0.02 (p=0.04) fold change (n=2). Overall, these results did not show significant translational induction of either aPPD or aPPT with any of the concentrations tested over 24h in HepG-2 and Caco-2 cell lines (Figure 3.4). 3.3 C Y P 1 A 1 metabolic activity P450-Glo™ C Y P 1 A 1 Assay kit was used to evaluate the effect of aPPD and aPPT on C Y P 1 A l metabolic activity. The results described in Figure 3.5 show that aPPD did not increase the C Y P 1 A l protein activity at concentrations below 40 p M , but only at higher concentrations of 80 and 160 p M . aPPT did not increase C Y P 1 A l activity significantly at any of the concentrations tested. T C D D decreased C Y P 1 A 1 activity at 80 and 160 p M . While T C D D is a good inducer of C Y P 1 A 1 in cells, it was not found to be a good activator. In comparison, the positive control for inhibition, a-NF, inhibited C Y P 1 A l activity in a dose dependent fashion. 3.4 Mechanistic studies of C Y P 1 A l 3.4.1 Gudluc 1.1 plasmid, and induction of CYP1A1 by aPPD and aPPT with AhR Figure 3.6 shows the luciferase assay data for aPPD and aPPT binding to A h R . The data of aPPD and aPPT were the average o f the biological duplicate (n=2), while data for the T C D D positive control was the 2 n M concentration. Luciferase signal of samples treated with 20 n M T C D D caused 199.81±54.66 (n=l) fold increase, data not shown here. A h R / p R L - T K ratio was calculated by firefly over Renil la luciferase signal. This ratio in aPPD and aPPT samples was lower than 2.8±0.16 (n=2), which is similar to the negative control D M S O level at 2.1±0.7 (n=2). However, the ratio of positive control T C D D sample was 22.64±7.3 (n=l). From the plot, it was apparent that aPPD and aPPT did not activate the reporter gene expression through A h R . The results suggest therefore that the induction of C Y P 1 A l m R N A by aPPD and aPPT was not regulated through the A h R pathway. 3.4.2 pGL3B-CYP!Al plasmid and induction of CYP1A1 by aPPD and aPPT In pGL3B-CYPlAl plasmid, CYP1A1 promoter sequence has driven the reporter gene expression as shown in Figure 3.7 A . Similar results to those obtained from transfection with Gudluc 1.1 plasmid was obtained in Figure 3.7 B . A h R / p R L - T K ratio was 3.11±0.1 for aPPD, 2.99±0.2 for aPPT, 25.16±8.7 for T C D D and 2.97±0.04 for D M S O (n=2). The results might suggest that the functional sequence was outside of the region spanning from -2425 to +352, which is discussed in Chapter 4. This also proved that aPPD and aPPT induced CYP1A1 gene expression through a mechanism unrelated to A h R pathway and verifies that the Gudluc 1.1 plasmid results were indeed positive evidence of an induction event independent of the A h R pathway. 48 A. Figure 3.1: Determination of the time required for CYP1 A l mRNA to translate protein. The level o f C Y P 1 A 1 m R N A was measured by real time R T - P C R in HepG-2 cells after 10 n M T C D D treatment with time course ranged from 0 to 12 h. (B) C Y P 1 A l protein was examined by western blotting. Vincul in was used as loading control for cytoplasmic fractions. The ratio of C Y P 1 A l over Vincul in intensity was quantitated (n=2). 49 A HepG-2 cells 250 -i 200 4 0 5 10 20 40 80 Dosing Concentration (uM) Figure 3.2: Cytotoxicity study result for aPPD and aPPT in HepG-2 and Caco-2 cell lines. M T T assays were performed in either (A) HepG-2 cells (n=3) or (B) Caco-2 cells (n=3). Concentration o f aPPD and aPPT ranged from 0 to 80 p M . Treatment duration was 12 h and 24 h. The relative difference in absorbance is used as an indicator of the number of cells that are viable in each group. The results o f the mean absorbance observed in each of the treatment groups shows that the relative percentage of surviving cells compared to the untreated control group, which is taken as 100%. 50 A aPPD 60.00 50.00 & 40.00 •S 30.00 I 20.00 PL, io.or o.o$ ~ ~o~ 1 "5 fif "20" 40~ Dosing Concentration ( y M) 5 10 20 40 Dosing Concentration (uM) - HepG-2 24h - « — HepG-2 12h - Caco-2 24h —X— Caco-2 12h Figure 3.3: Induction study of C Y P 1 A 1 by aPPD and aPPT in transcriptional level. Dosing concentration ranged from 0 to 40 u M . Treatment duration was 12 h and 24 h. The result was quantitated by using C T value o f both C Y P 1 A 1 and beta-actin gene of each sample to calculate 2 A - A A C T value. A l l the samples were compared with control, which was treated with same volume of 100% ethanol. Student t-test was used and statistically significant is denoted by * (p<0.05) (n=3). The induction was statistically significant at all concentrations above and including 5 u M . 51 Western blotting image of HepG-2 aPPD 24h O u M l u M 5 u M l O u M M L M 2 0 u M m~* «<•:<» mt **» mm »mi m CYP1A1 v incu l in Effect on CYP1A1 protein egress ion after 24h aPPD/aPPT treatment 5 10 Dosing (11M) - • — Caco-2 aPPD -m— Caco-2 aPPT -*— HepG-2 aPPD -X— HepG-2 aPPT Figure 3.4: The induction study of CYP1A1 after aPPD and aPPT 24h treatment at translational level. The result was quantitated by the intensity ratio of C Y P 1 A l over vinculin, which was a housekeeping protein used as internal control. A l l other sample results were compared to the control, which was treated with same volume of 100% ethanol. Mouse liver microsome ( M L M ) was used as a positive control in western blotting. Results were analyzed using student t-test and statistically significant is denoted by * (p<0.05) (n=2). 52 Effect on CYP1A1 activity after aPPD/aPPT/a -NF /TCDD treatment Figure 3.5: CYP1A1 metabolic activity study after aPPD and aPPT treatment. a-NF was a positive control for inhibition. The luciferase signal of each sample was compared to negative control, which was treated with same volume of D M S O . Results were analyzed using Student t-test and statistically significant is denoted by * (p<0.05) (n=2). 53 Gudluc 1 .1 plasmid and induction o f C Y P 1 A 1 by aPPD and aPPT 35 30 0 1 5 10 20 40 T C D D DMSO Dosing Concentration (uM ) and Positive and Negative Control Figure 3.6: C Y P 1 A l induction mechanism study related to AhR using Gudluc 1.1 plasmid. The result of each sample was determined by calculating the ratio of luciferase signal: firefly luciferase Renil la luciferase Firefly luciferase was produced by the Gudluc 1.1 plasmid, which contains the upstream promoter region (from -1301 to -819) of the CYP1A1 gene, while Renilla luciferase was from p R L - T K , the internal control. HepG-2 cells were co-transfected with Gudluc 1.1, A h R and p R L - T K plasmids (n=2). 54 A pGL3B-CYPlAl construct fuction test 150.0 o ^ i Jj 100.0 i—'—1 PGL-3 promoter pGL3B-CYPlAl PGL-3 basic Plasmid name B p G L 3 B - C Y P l A l plasmid and induction o f C Y P l A l by aPPD and aPPT 40 35 aPPD aPPT TCDD DMSO Dosing Drug Name Figure 3.7: pGL3B-CYPlAl plasmid function test and the use of this construct in C Y P 1 A 1 induction mechanism study (A) CYP1A1 promoter sequence has driven the firefly reporter gene expression in the pGL3B-CYPlAl plasmid (n=2). (B) The responsive element for CYP1A1 induction by aPPD and aPPT was outside of the region from -2425 to +352 in CYP1A1 gene. HepG-2 cells were co-transfected with p G L 3 B - C y P 7 ^ 7 , A h R and p R L - T K plasmids (n=2). 55 CHAPTER 4 DISCUSSION This thesis described a series o f experiments in which HepG-2 and Caco-2 cells were used as models for the study of cytochrome P450. It may be debated as to whether these cell lines are appropriate models for a cytochrome P450 induction study. It is wel l known that results of in vivo animal experiments are unable to predict human C Y P induction because interspecies difference in genomic sequences exist and different C Y P families and subfamilies are also apparent between species (Nelson 2002). Taking this into consideration, in vitro induction studies using human hepatoma cell lines or primary human hepatocytes are preferable and likely to be representative o f human pharmacology. Wilkening et al (2003) compared primary human hepatocytes and HepG-2 cells recently. Their work determined that both provide useful data but are not experimentally equivalent (Wilkening et al. 2003). Human hepatocytes were the preferred model for studying metabolic biotransformation in human liver because they express a number o f important phase II enzymes which were similar to those in human liver samples; the expression level of these enzymes in HepG-2 differed significantly from that seen in primary hepatocytes. However, they found that the up-regulation of specific genes by probe substances was similar in both groups. To this end, they concluded that HepG-2 cells may be useful to study regulation of drug-metabolizing enzymes (Wilkening et 56 al. 2003). Established cell lines have low C Y P expression levels, however, they have an unlimited life span, a more stable phenotype than primary cultures and are readily available (Rodriguez-Antona et al. 2002). HepG-2 cells are the most frequently used and best-characterized human hepatoma cell line. This cell line is still regarded as a good in vitro model for C Y P induction studies, since this it retains a variety o f liver-specific metabolic functions (Allen et al. 2001). Dvorak et al. (2006) provided the most recent evidence o f this, in which the HepG-2 cell line was used to study the effect o f colchicine and nocodazole on CYP1A1 gene and protein expression, as well as protein activity (Dvorak et al. 2006). Caco-2 is a colorectal adenocarcinoma cell line. It exhibits characteristic cytochrome P450 activity as seen in the colonic epithelium(Lampen et al. 1998; Rosenberg & Leff 1993). In 1998, Lampen et al. compared catalytic activities, protein and mRNA-expression of cytochrome P450 isoenzymes in intestinal cell lines. In their study, several P450 isoenzymes were investigated, including C Y P 1 A 1 , C Y P 1 A 2 , CYP2C9/10 , C Y P 2 E 1 and C Y P 3 A . The results showed that among those tested Caco-2 cells were the only cell line which expressed C Y P 1 A 1 at both protein and m R N A level, and was able to produce metabolites similar to those observed in in vivo metabolism studies (Lampen et al. 1998). In summary, we can conclude that HepG-2 and Caco-2 cell lines are representative models for C Y P induction studies. Northern blotting was used to detect C Y P m R N A by Agarwal et al. in 1994 (Agarwal et al. 1994). This method is only semi-quantitative with a non-specific 57 reaction in hybridization. Recently, several PCR-based approaches have been introduced for the quantitation of C Y P m R N A , which include reverse transcription P C R (RT-PCR) (Sumida et al. 2000; Sumida et al. 1999), quantitative R T - P C R (Rodriguez-Antona et al. 2000), and real-time P C R (Bowen et al. 2000; Burczynski et al. 2001). These techniques are extremely sensitive with single-base discrimination capability. However, both R T - P C R and quantitative R T - P C R use agarose gels for detection of P C R amplification at the final phase or end-point of the P C R reaction, which leads to inaccurate quantification of m R N A . In real time P C R , exact doubling of product is accumulating at every cycle, which is known as the exponential phase and provides a distinct advantage over traditional P C R detection. Thus, real time P C R is more quantitative than conventional P C R . Western blotting is still a basic tool for investigating gene regulation at the protein level, however, it is not effective for detecting rapid protein degradation. In this situation, western blotting is not the best technique chosen. In 2000, Lekas et al. determined that C Y P 1 A l m R N A was rapidly degraded in HepG-2 cells, with a half-life of 2.4 ± 0.13 h (Lekas et al. 2000). They also generated a model suggesting that loss of the poly (A) tail is an early step in the degradation of the C Y P 1 A 1 m R N A (Lekas et al. 2000). To determine the time required for C Y P 1 A l m R N A translation to protein, cells should be lysed after a time course, which starts at the time of removal o f inducer. However, considering the rapid degradation of C Y P 1 A 1 m R N A , T C D D , the inducer, was not removed from 58 cells in a time course experiment following progression from m R N A to protein for the purpose o f this thesis research. A s a consequence, the rate of C Y P 1 A 1 m R N A translation was not clearly determined, however, the results obtained in the time-course experiments provided a gross time course for drug treatment in the induction study that followed. The induction study described in this thesis illustrates that the induction function of C Y P 1 A 1 was only observed at the transcriptional level, but was not in translational protein expression. The expression of many genes are known to be regulated after transcription, so it is reasonable to say that an increase in m R N A concentration does not imply an increase in protein expression. A n example of this is provided (Hashimoto et al. 2006). Expression profiles of melanogenesis-related genes and proteins in acquired melanocytic nevus were studied. In that study, Pmel-17/gpl00 protein was seen only in the basal layer. The protein and m R N A profiles were observed to be appreciably different (Hashimoto et al. 2006). Many factors interfere with the process o f R N A degradation and translation to protein. To explain the inconsistency of C Y P 1 A l m R N A and protein expression in this study, the following rationale might be considered: A possibility would be that aPPD and aPPT increases C Y P 1 A 1 protein degradation after induction by magnifying the production of its protease. Induction o f C Y P 1 A 1 is commonly measured through increased Ethoxyresorufin (O) dealkylation (EROD) activity (Chang et al. 2001; Merchant et al. 59 1992; Shimada et al. 2002; Zhi-Hua Chen 2005) In this reaction, the substrate ethoxyresorufin is hydrolyzed to resorufin, a stable and fluorescent compound. Enzyme kinetics are recorded by a fluorimeter. P450 Glo™ C Y P 1 A 1 assay was used in this study, which provided a luminescent method for measuring C Y P 1 A 1 activity. The reaction is performed by incubating human liver microsomes with luminogenic C Y P 1 A 1 substrate (Luciferin 6' chloroethyl ether (Luciferin-CEE)). The amount of luminescent light produced is directly proportional to the activity o f C Y P 1 A 1 . The advantage of the P450-Glo™ C Y P 1 A 1 assay is there are no fluorescent excitation and emission overlaps between analytes, N A D P H and cytochrome P450 substrates. Such an overlap may confound analysis and present misleading or irrelevant data. This method is simple, highly sensitive, and specific, while providing a stable signal with a half-life of greater than 2 hours. Chang et al. (2002) conducted a previous study on the impact o f ginseng on CYP I family activity. They found that ginseng extracts, such as G i l 5 {Panax ginseng extract) and N A G E {Panax Quinquefolius extract) were able to decrease human recombinant C Y P 1 A 1 activities in a dose dependent manner and these inhibitory effects were not identified to be due to any of the seven ginsenosides: R b l , Rb2, Rc , R d , Re, Rf, or R g l , which together account for >90% of the total ginsenoside content in ginseng extract. These inhibitory effects could have been the result of non-ginsenoside compound (Chang et al. 2002). In their experiments, a 7-ethoxyresorufin O-dealkylation assay was used. To date, the results stated in this 60 thesis is the first report about the impact of isolated gisenosides aPPD and aPPT on C Y P 1 A l activity, which was increased at high concentrations of aPPD at 80 and 160 u M , but not lower concentrations including 40 u M . Even though these high concentrations may not be pharmacologically attainable unless the compound is accumulated in the tissue, this result is still interesting and holds a value as mechanism to study C Y P 1 A l activation. T C D D , a potent C Y P 1 A 1 inducer, did not activate C Y P 1 A 1 activity in human liver microsomes. It induced C Y P 1 A 1 m R N A and protein expression indirectly by the A h R (Anderson et al. 2006; Riddick et al. 1994; Shimada et al. 2003), but it is not an activator o f C Y P 1 A l directly. However, aPPD could have acted as an activator of C Y P 1 A 1 , increasing C Y P 1 A 1 activity directly. To discuss the mechanism, some basic pharmacology definition should be noted here: Affinity and intrinsic activity are independent properties o f drugs. Agonists have both affinity, that is, the ability to bind to the receptor, and intrinsic activity, the ability to produce a measurable effect. K d is the equilibrium dissociation constant and is the reciprocal of the affinity. The smaller the K d , the greater the affinity. The ability of a drug to produce a physiological effect is dependent on both receptor occupancy (which is in turn governed by drug concentration and K d value) and the propensity of the drug to activate the receptor. Based on the above theory, i f aPPD binds to a receptor, which triggers C Y P 1 A 1 activity directly, aPPD may have a high K d value, and subsequently a lower affinity, so that higher concentrations of aPPD are needed to activate C Y P 1 A 1 activity. 61 Another possibility is that aPPD may be a substrate of C Y P 1 A 1 or its metabolite a P P D - X may be an allosteric activator of C Y P 1 A 1 . If this is the case, aPPD might compete with Lucifer in-CEE as the substrate of C Y P 1 A 1 in the P450-Glo™ C Y P 1 A l assay. Such a competition would result in a lower luciferase signal caused by luciferin. This reduction would be complemented by the effect o f aPPD-X, which acts as an activator of C Y P 1 A 1 , subsequently increasing the metabolism of Lucifer in-CEE. A s the aPPD concentration increases, more and more a P P D - X is produced; producing an increased C Y P 1 A l activity and resulting in an increase of the metabolism of Lucifer in-CEE resulting in the production of more free luciferin. This might be the reason for lack of induction at low concentrations o f aPPD whereas at higher concentrations the enzyme was induced. The A h R pathway is a well recognized mechanism and regulator o f C Y P 1 A 1 induction at the transcriptional level(Dvorak et al. 2006; Jr. 1999; M a 2001; Seree E 2004). A s part of this thesis, the question of whether aPPD and aPPT are ligands of A h R was answered. Gudluc 1.1 plasmid which contains a portion o f the upstream promoter region (from -1301 to -819) of the mouse CYP1A1 gene contains four dioxin response elements ( X R E M ) (El-Fouly 1994; Fisher 1989; Fisher 1990; P. M . Garrison 1996). Garrison et al. constructed this plasmid in 1996 (P. M . Garrison 1996) and it has since been used in several studies (Abnet et al. 1999; Jeon & Esser 2000; Vrzal et al. 2005). The results of this study demonstrate that both aPPD and aPPT do not activate A h R as indicated by the fact that they did not increase the firefly 62 luciferase signal. This suggests that the induction of C Y P 1 A 1 by aPPD and aPPT is not regulated by the A h R pathway. It is possible that aPPD and aPPT may induce C Y P 1 A 1 by activating other receptors, or binding to the CYP1A1 promoter directly. If this is the case, it is possible that the CYP1A1 promoter sequence in G u d l u c l . l plasmid is too short, resulting in ineffective binding to the promotor. Several different CYP1A1 promoter plasmids have been used to study the A h R pathway and are reported in the literature (Anderson et al. 2006; Shimada et al. 2002; Zhi-Hua Chen 2005). In 2001, Lee and Safe studied the inhibition o f C Y P 1 A l expression by resveratrol in breast cancer cells (Shimada et al. 2002). In their study, the plasmid used had the -1142 to +2434 regulatory region from the human CYP1A1 gene fused to the bacterial CAT reporter gene (Shimada et al. 2002). In 1993, Postlind constructed p L l A 1 N plasmid, which contained a fragment from -1612 to +292 (all o f exon one, a portion of intron one and 1612 bp of 5'-flanking sequences) of the human CYP1A1 gene. Contained within this fragment are three consensus X R E s (Zhi-Hua Chen 2005). In 2004, Galijatovic et al. developed a transgenic mouse line, which carries this p L l A 1 N plasmid, to study the mechanism associated with A h R control of the CYP1A1 gene in vivo (Anderson et al. 2006). Based on the p L I A I N plasmid structure, a longer human CYP1A1 promoter construct, p G L 3 B - l A l (-2425 to +352), was created. A h R , p G L 3 B - / ^ / , and p R L - T K plasmids were co-transfected into HepG-2 cells and the resulting luciferase signal was detected. The result obtained was the same as that observed using Gudluc 1.1 plasmid, which confirmed the 63 conclusion that the induction of C Y P 1 A 1 by aPPD and aPPT was not through A h R pathway. This result also suggested that i f aPPD and aPPT induced the CYP1A1 gene by binding directly to the promoter, the control sequence might be outside the region from -2425 to +352. It is also testimony to the effectiveness of the Gudluc 1.1 plasmid as a good construct for A h R pathway study. However, there are further issues to consider when using the p G L 3 B - L 4 7 plasmid. It is possible that there is a suppressor region within this part of the sequence; as a result, the promoter would not be triggered. This study does not have data to support this hypothesis, but in future studies it would be beneficial to truncate the CYP1A1 promoter construct to investigate the responsive element and the possible existing suppressor. Is C Y P 1 A 1 induction always related to the A h R signaling pathway? Delescluse (2000) asks this question in the paper's title (Delescluse et al. 2000). Several exceptions o f C Y P 1 A 1 induction via the A h R pathway were reported. Stress conditions such as hyperoxia was found to induce C Y P 1 A 1 expression (Hazinski et al. 1995; Okamoto et al. 1993). This demonstrates that C Y P 1 A 1 induction does not always require a ligand. Several non-polycyclic and non-planar compounds have shown they activate CYP1A1, even though they do not compete for the T C D D binding site on A h R (Aix et al. 1994; Daujat et al. 1992; Fontaine et al. 1999; Gradelet et al. 1997; Lee et al. 1996; Lesca et al. 1995). Seree, et al. provided evidence for another new human CYP1A1 regulation pathway involving peroxisome proliferator-activated receptor-a (PPAR-a) and two peroxisome proliferator response 64 element (PPRE) sites which are located within the CYP1A1 promoter (positions -931/-919 and -531/-519) (Seree et al. 2004). Their results indicated that P P A R - a ligands, which are common environmental compounds, induced human C Y P 1 A 1 m R N A expression. Norihito et al. determined a region from -614 to -458 ( X R E M ) in the CYP1A1 promoter, which is important for C Y P 1 A 1 induction. They also found several activators of human pregnane X receptor (hPXR) and human constitutive androstane receptor (hCAR) could dramatically induce CYP1A1 promoter activity through 1A1 X R E M . Their results support a novel role for h P X R and h C A R in the regulation of human CYP1A1 gene (Norihito S 2005). Post-transcriptional stabilization is another possible mechanism of C Y P 1 A 1 induction by aPPD and aPPT. The half-life of C Y P 1 A 1 m R N A may have been prolonged and subsequently increased the gene expression level as measured due to improvement o f m R N A stability, not the increase of transcription. There is still much to be investigated regarding C Y P 1 A l induction mechanism. In conclusion, the results described in this thesis indicate that aPPD and aPPT induce C Y P 1 A 1 m R N A expression significantly, but that the effect did not carry through to protein translation. A t the highest concentrations, 80 and 160 u M , aPPD and aPPT increased C Y P 1 A 1 activity. Finally, we can also conclude that regulation of C Y P 1 A l m R N A expression did not occur via the A h R pathway. The effect o f aPPD and aPPT on cytochrome P450 3A4 expression was initially measured at the transcriptional level as well as C Y P 1 A 1 . In the HepG-2 65 cell line, 12h treatment only slightly induced the expression of C Y P 3 A 4 with 1,5, 10 u M of both aPPD and aPPT and 20uM aPPT, but significantly suppressed C Y P 3 A 4 expression with 20, 40uM aPPD and 40uM aPPT. In the Caco-2 cell line, only 1,5, 10 u M aPPD and l u M aPPT slightly induced C Y P 3 A 4 after 12h treatment. No induction o f C Y P 3 A 4 was seen in other concentrations with both aPPT and aPPD. This result provided further evidences that aPPD and aPPT specifically induced C Y P 1 A 1 m R N A expression. The result in this thesis indicated that commercially available ginseng products are l ikely to be safe and not cause problematic interactions with drugs/environmental chemicals. The amount of circulating ginseng derived compounds are not likely to be as high as 80 and 160 p M , concentrations of aPPD and aPPT which have been shown here to activate C Y P 1 A l protein activity in vitro. 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