University of Wien

Influence of Vitamin D - Metabolites and
 - Analogs on Growth and Apoptosis of C6 -
Rat-Glioma-Cells

Diplomarbeit/ Diploma Thesis

by

Josef Elias

10- 2002

TABLE OF CONTENTS

1. INTRODUCTION ... 1
1.1. General aspects of Vitamin D ... 1
1.1.1. Synthesis and Metabolism ... 1
1.1.2. Biological Role of Vitamin D ... 3
1.2. Mechanisms of Cell Death: Apoptosis versus Necrosis ... 4
1.3. Vitamin D and Apoptosis of Malignant Cells ... 7
1.3.1. Vitamin D as an Anti-cancer Agent ... 7
1.3.2. Anti-tumor Actions of Vitamin D on Glioma ... 8
1.3.3. C6-Rat Glioma Cells: Model to Study Vitamin D Actions ... 9
1.4. Aim of diploma thesis ... 10

2. MATERIALS AND METHODS ... 12
2.1. Used devices and reagents ... 12
2.1.1. Cell culture ... 12
2.1.2. Compounds used in Incubations ... 13
2.1.2.1. Etoposide: solution of 1,2 mg/ml (2 mmol/l) in ethanol (96 vol.%); ... 13
2.1.2.2. Vitamin D-analogues or –metabolites : (were kindly provided by S. Reddy (Brown University, Rhode Island, USA)) ... 14
2.1.3. Materials used in Analytical procedures Materials for DNA-Isolation ... 17
2.1.5. Materials for Capillary electrophoresis ... 17
2.2. Methods ... 18
2.2.1. Cell Culture ... 18
2.2.1.1. Thawing of frozen samples of C6-cells and cultivation ... 18
2.2.1.2. Subcultivation of C6-cells ("splitting") ... 18
2.2.2. Incubations ... 19
2.2.3. Analytical methods ... 19
2.2.3.1. Neutral Red Assay ... 19
2.2.3.2. Trypan-blue-assay and cell-counting ... 21
2.2.3.3. Staining of cell nuclei with Hoechst No. 33258 ... 22
2.2.4. DNA-Isolation ... 23
2.2.5. Capillary gel electrophoresis ... 24
2.2.6. Evaluations, Calculations ... 25

3. RESULTS ... 26
3.1. Effect of 1á,25(OH)2D3 on growth of C6-rat-glioma-cells ... 26
3.1.1. Growth of C6-rat-glioma-cells in serum-containing culture medium ... 26
3.1.2. Growth of C6-rat-glioma-cells in the absence of serum ... 27
3.2. Viability of C6-rat-glioma-cells and induction of apoptosis by 1,25(OH)2D3 and 1,25(OH)2-3-epi-D3 ... 30
3.2.1. Dose-dependent effects on viability ... 30
3.2.2. Time course of apoptosis and dose-dependent effects ... 34
3.3. Influence of various Vitamin D – metabolites and and – analogs on growth and apoptosis of C6-rat-glioma-cells ... 39
3.3.1. Effects of natural metabolites on growth and apoptosis ... 40
3.3.2. Effects of synthetic analogs on growth and apoptosis ... 44
3.4. Detection of DNA-fragmentation in C6-rat-glioma cells by Capillary electrophoresis (CE) ... 49
3.4.1. Cell numbers and yield of DNA ... 50
3.4.2. Separation of DNA by CE ... 51

4. DISCUSSION ... 55
4.1. Anti-cancer effects of 1á,25-(OH)2-D3 on glioma ... 55
4.2. Can 3-epi-vitamin D-analogues offer advantages over 1á,25-(OH)2-D3 in the treatment of gliomas? ... 56
4.2.1. C-3 epimerization is a metabolic pathway of 1á,25-(OH)2-D3 ... 56
4.2.2. Established biological activities of 3á-vitamin D-analogues ... 56
4.3. Vitamin D-metabolites and -analogs tested in this study: actions on growth and apoptosis of C6-glioma-cells ... 57
4.3.1. Relevance of applied methods ... 57
4.3.2. Effects of 1á,25-(OH)2-D3 on cell growth in the presence and in absence of serum ... 58
4.3.3. Comparison: 1á,25-(OH)2-D3 and 1á,25-(OH)2-3-epi-D3 ... 59
4.3.4. Effects of other vitamin D-metabolites ... 60
4.3.5. Vitamin D-analogues : Comparison 3â- and 3á-epimers ... 61
4.4. Availability of vitamin D-metabolites/-analogs at the target site ... 65
4.4.1. Uptake into the brain. ... 65
4.4.2. Availability at the tumor site ... 65
4.5. Conclusions ... 66

5. REFERENCES ... 68

1. INTRODUCTION
1.1. General aspects of Vitamin D
1.1.1. Synthesis and Metabolism
Vitamin D in the human body exists in two forms, derived from two sources. In the form of Vitamin D3, it is generated from 7-dehydrocholesterol in the skin by exposure to ultraviolet light (270-300 nm range), and in the form of Vitamin D2 or Vitamin D3 (Fig. 1), it can be derived from diet.

!! Im PDF-Dokument befinden sich an dieser Stelle Abbildungen !!

Fig. 1. Chemical structures of vitamin D3 and vitamin D2

Fig. 2. Electron transport chain for mitochondrial steroid hydroxylases. Concept for 3-dimensional arrangement of components of mitochondrial cytochrome P-450-containing hydroxylases is shown (Jones et al. (1998)).

Both forms of Vitamin D are precursor of functionally active hormones and undergo the same two-step activation process leading to 1á,25-(OH)2-D2 or 1á^*,25-(OH)2-D3, respectively. These metabolic activations of Vitamin D3 are carried out a by specific P-450-containing enzymes, the vitamin D3-25- hydroxylase (CYP27A) and the 25-hydroxyvitamin D-1á-hydroxylase (CYP27B1). Both hydroxylases are located in the inner mitochondrial membrane of cells (Fig. 2).There is little evidence that the two hormones differ in their mode of action (Jones et al. (1998)).

In contrast to earlier assumptions, which strictly localized 25- hydroxylation in the liver and sequential 1á.hydroxylation in the kidney (Blunt et al. (1968)), many different cell types were shown to be capable of the two-step activation process too. Regulation of both enzymes appears to be tissuespecific: CYP27A is only loosely regulated (Bhattacharyya et DeLuca (1973)) or constitutively expressed (Schussler et al. (2001)). 1á-Hydroxylase in the kidney is tightly regulated by the levels of plasma 1,25-(OH)2D3 and calcium (via the parathyroid hormone (PTH)) (reviewed by Jones et al. (1998)), however constitutively expressed in skin (Schussler et al. (2001)). A third vitamin Drelated mitochondrial cytochrome P-450-containing enzyme is the 25- hydroxyvitamin D-24-hydroxylase (CYP24). This enzyme is strongly inducible by 1á,25-(OH)2-D3 in practically all cell types of the body, prefers 1á,25-(OH)2- D3 as a substrate over 25-OH-D3, and catalyzes several steps of 1á,25-(OH)2- D3 – metabolism, collectively known as the C-24 oxidation pathway (Fig.3., Reddy and Tserng, 1989; Makin et al., 1989).

!! Im PDF-Dokument befinden sich an dieser Stelle Abbildungen !!

Fig. 3. Vitamin D Cascade:Synthesis and metabolism of hormonally active vitamin D.

Besides catalyzing three successive oxidations, two at C-24 and one at C-23, there is strong evidence that CYP24 cleaves the resulting intermediate to give the C-23 alcohol/acid (Beckman et al., 1996; Schuster et al., 2001b). Although the terminal product of this pathway, calcitroic acid, is less active than 1á,25-(OH)2-D3 in vivo, most intermediates still retain substantial biological activity (Schuster et al. 1997, Jones et al. (1998)).

Recently, a novel tissue specific pathway was found, namely the C-3 epimerization pathway, in which 1a,25-(OH)2-3-epi-D3 is formed as a result of the change in the orientation of the hydroxyl group at the C-3 position from ß to a? (see also Fig. 3) (Reddy et al. (1997, 2001), Astecker et al. (2000)). 1a,25-(OH)2-3-epi-D3 also undergoes 24-oxidation, however at a much slower rate than 1a,25-(OH)2-D3 (Astecker et al. 2000, Schuster et al. 2001b). 

1.1.2. Biological Role of Vitamin D
1a,25-(OH)2-D3 is believed to be the major active form of Vitamin D Its mode of action in generating biological responses is analogous to that of the classical steroid hormones like estradiol, testosterone, progesterone, cortisol and aldosterone (Evans (1988), Haussler et al. (1988)).

Genomic mode of action of 1a,25-(OH)2-D3. 1a,25-(OH)2-D3 exerts effects on transcription of so called vitamin D-target genes by means of a nuclear receptor protein, the vitamin D-receptor (VDR). 1a,25-(OH)2-D3 (and its analogues) are lipophilic molecules, that easily pass cellular membranes and enter the nucleus, where they bind with high affinity to the VDR. The hormone-receptor-complex then binds to specific DNA sequences termed response elements (vitamin D responsive elements, VDRE) in the promoter regions of these genes, thereby up- or downregulating them, respectively (reviewed by Jones et al. (1998)). This process includes interaction of the VDR with other nuclear receptors (like the retinoid X receptor, RXR) for the formation of dimeric complexes and contact with cofactors, such as coactivators and corepressors, for modulation of transcriptional activities (Carlberg et al. (2001)). Thus, the presence of the VDR in a cell confers it to become a target cell which is producing the biological response (Stumpf et al. (1979), Reichel et al. (1989a)). 

Actions of 1a,25-(OH)2-D3. 1a,25-(OH)2-D3 regulates the expression of a very wide variety of gene, resulting in pleiotropic activities. Besides its central role in calcium homeostasis and, therefore in bone formation (DeLuca et al. (1990)), 1a,25-(OH)2-D3 is also crucially involved in controlling cellular growth, differentiation and apoptosis in normal as well as in malignant cells (Walters (1992)). Moreover, important immunoregulatory activities have been demonstrated, in particular, inhibition of the expression of Th1-cytokines (interferon ., interleukin 2) and modulation of antigen expressing cells (reviewed by Mathieu and Adorini, 2002). Key functions have been confirmed in the nervous system, including regulation of synthesis of neurotrophic factors and neurotransmitters (reviewed by Garcion et al. 2002) Table 1 displays a selection of genes that have been shown to be responsive to Vitamin D.

[...]

* á steht für alpha (Dies wird im PDF-Dokument richtig dargestellt)