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. [...]
Both forms of Vitamin D are precursor of functionally active hormones
and undergo the same two-step activation process leading to 1α,25-(OH)2-D2 or 1α,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-1α-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 1α−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 (Schüssler et al. (2001)). 1α-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 (Schüssler 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 1α,25-(OH)2-D3 in practically all cell types of the body, prefers 1α,25-(OH)2-
D3 as a substrate over 25-OH-D3, and catalyzes several steps of 1α,25-(OH)2-
D3 – metabolism, collectively known as the C-24 oxidation pathway (Fig.3.,
Reddy and Tserng, 1989; Makin et al., 1989). [...]
Table of Contents
1. INTRODUCTION
1.1. General aspects of Vitamin D
1.1.1. Synthesis and Metabolism
1.1.2. Biological Role of Vitamin D
1.2. Mechanisms of Cell Death: Apoptosis versus Necrosis
1.3. Vitamin D and Apoptosis of Malignant Cells
1.3.1. Vitamin D as an Anti-cancer Agent
1.3.2. Anti-tumor Actions of Vitamin D on Glioma
1.3.3. C6-Rat Glioma Cells: Model to Study Vitamin D Actions
1.4. Aim of diploma thesis
2. MATERIALS AND METHODS
2.1. Used devices and reagents
2.1.1. Cell culture
2.1.2. Compounds used in Incubations
2.1.2.1. Etoposide:
2.1.2.2. Vitamin D-analogues or –metabolites : (were kindly provided by S. Reddy (Brown University, Rhode Island, USA))
2.1.3. Materials used in Analytical procedures
Materials for DNA-Isolation
2.1.5. Materials for Capillary electrophoresis
2.2. Methods
2.2.1. Cell Culture
2.2.1.1. Thawing of frozen samples of C6-cells and cultivation
2.2.1.2. Subcultivation of C6-cells ("splitting")
2.2.2. Incubations
2.2.3. Analytical methods
2.2.3.1. Neutral Red Assay
2.2.3.2. Trypan-blue-assay and cell-counting
2.2.3.3. Staining of cell nuclei with Hoechst No. 33258
2.2.4. DNA-Isolation
2.2.5. Capillary gel electrophoresis
2.2.6. Evaluations, Calculations
3. RESULTS
3.1. Effect of 1α,25(OH)2D3 on growth of C6-rat-glioma-cells
3.1.1. Growth of C6-rat-glioma-cells in serum-containing culture medium
3.1.2. Growth of C6-rat-glioma-cells in the absence of serum
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
3.2.1. Dose-dependent effects on viability
3.2.2. Time course of apoptosis and dose-dependent effects
3.3. Influence of various Vitamin D – metabolites and and – analogs on growth and apoptosis of C6-rat-glioma-cells
3.3.1. Effects of natural metabolites on growth and apoptosis
3.3.2. Effects of synthetic analogs on growth and apoptosis
3.4. Detection of DNA-fragmentation in C6-rat-glioma cells by Capillary electrophoresis (CE)
3.4.1. Cell numbers and yield of DNA
3.4.2. Separation of DNA by CE
4. DISCUSSION
4.1. Anti-cancer effects of 1α,25-(OH)2-D3 on glioma
4.2. Can 3-epi-vitamin D-analogues offer advantages over 1α,25-(OH)2-D3 in the treatment of gliomas?
4.2.1. C-3 epimerization is a metabolic pathway of 1α,25-(OH)2-D3
4.2.2. Established biological activities of 3α-vitamin D-analogues
4.3. Vitamin D-metabolites and -analogs tested in this study: actions on growth and apoptosis of C6-glioma-cells
4.3.1. Relevance of applied methods
4.3.2. Effects of 1α,25-(OH)2-D3 on cell growth in the presence and in absence of serum
4.3.3. Comparison: 1α,25-(OH)2-D3 and 1α,25-(OH)2-3-epi-D3
4.3.4. Effects of other vitamin D-metabolites
4.3.5. Vitamin D-analogues : Comparison 3β- and 3α-epimers
4.4. Availability of vitamin D-metabolites/-analogs at the target site
4.4.1. Uptake into the brain.
4.4.2. Availability at the tumor site
4.5. Conclusions
Objectives and Topics
This thesis investigates the potential of various Vitamin D metabolites and synthetic analogs to inhibit tumor growth and induce apoptosis in C6 rat glioma cells, aiming to identify compounds with an improved therapeutic profile compared to 1α,25-(OH)2D3. The research explores the impact of C-3 stereochemistry on biological activity and tests the feasibility of capillary gel electrophoresis as an advanced method for monitoring apoptosis-related DNA fragmentation.
- Evaluation of Vitamin D-based agents for anti-glioma therapy.
- Comparative analysis of 3β- and 3α-epimers on cell growth and apoptosis.
- Investigation of the apoptotic potential of natural Vitamin D metabolites.
- Assessment of metabolic stability and reduced calcemic side effects of synthetic analogs.
- Validation of capillary gel electrophoresis for quantifying DNA fragmentation.
Excerpt from the Book
1.3.2. Anti-tumor Actions of Vitamin D on Glioma
Malignant gliomas account for approximately 2,5 % of deaths due to cancer. In spite of multimodality therapies, which include neurosurgical resection of the tumor and radiotherapy and/or chemotherapy, the median survival of patients with glioblastoma is less than 20 months following diagnosis (Ushio (1991), Weingart et Brem (1992)).
In recent years several investigations indicated that 1α,25-(OH)2-D3 or its analogues alone or in combination with other therapeutic approaches could be of interest in the treatment of brain glial tumors. In 1994, Naveilhan et al. provided the first evidence for a cytotoxic action of 1α,25-(OH)2-D3 on glioma cells from rat (C6-cells) and man (GHD-cells). Since this toxic effect of 1α,25-(OH)2-D3 was obtained in vitro in malignant glioma cells, but not in primary cultures of normal glial cells, they concluded that this specificity of action could offer a new strategy for the inhibition of glioma growth in vivo. One year later, the group investigated the effects of several vitamin D3-analogues, which had previously been shown to be less calcemic than 1α,25-(OH)2-D3, on C6.9 (rat glioma) cells (Baudet et al. (1996)). The study not only revealed that several of the tested substances were able to induce cell death of C6.9 cells, but also that the toxic effect was accompanied by several of the biochemical features of apoptosis, such as DNA fragmentation and induction of the c-myc protooncogene.
Summary of Chapters
1. INTRODUCTION: Provides an overview of Vitamin D metabolism, its role in apoptosis, and current research regarding its potential as an anti-cancer agent in glioma treatment.
2. MATERIALS AND METHODS: Describes the experimental setup, cell culture protocols for C6-rat-glioma cells, and analytical techniques including Neutral Red assay and Capillary gel electrophoresis.
3. RESULTS: Presents detailed data on the effects of various Vitamin D metabolites and analogs on cell growth, viability, and apoptosis, including DNA fragmentation analysis.
4. DISCUSSION: Interprets the findings, comparing different Vitamin D analogs, exploring the role of C-3 epimerization, and assessing the therapeutic potential and future perspectives for glioma treatment.
Keywords
Vitamin D, Glioma, C6-cells, Apoptosis, Necrosis, Cell viability, 1α,25-(OH)2D3, 3-epi-vitamin D, Capillary electrophoresis, DNA fragmentation, Cancer therapy, Cell culture, Antiproliferative effects, Metabolic stability, Signal transduction.
Frequently Asked Questions
What is the core subject of this research?
The work focuses on the anti-tumor potential of Vitamin D metabolites and synthetic analogs, specifically investigating their ability to inhibit growth and induce apoptosis in malignant C6 rat glioma cells.
What are the primary research themes?
Key themes include the impact of molecular stereochemistry (C-3 position), the metabolic stability of Vitamin D analogs, and the efficacy of these compounds in reducing cancer cell proliferation compared to standard therapies.
What is the main goal of the study?
The primary goal is to evaluate if novel 3α-hydroxyl analogs can provide a therapeutic advantage, such as higher apoptotic capacity or lower calcemic side effects, in the treatment of brain gliomas.
Which scientific methodology is applied?
The study utilizes in vitro cell culture, specifically the Neutral Red assay for cell viability and morphological assessment via Hoechst 33258 staining, alongside capillary gel electrophoresis to detect DNA fragmentation.
What topics are discussed in the main section?
The main section analyzes the cytotoxic response of glioma cells to various Vitamin D derivatives, compares the effects of serum-containing versus serum-free conditions, and validates CE as a tool for apoptosis monitoring.
Which keywords summarize this work?
The work is characterized by terms such as Vitamin D, Glioma, Apoptosis, C6-cells, 1α,25-(OH)2D3 analogs, and DNA fragmentation.
How does the C-3 epimerization affect Vitamin D activity in glioma cells?
The research suggests that C-3 epimerization to the 3α-configuration can significantly alter the biological response, with some analogs showing enhanced apoptotic potential compared to their 3β-counterparts.
What is the significance of the findings regarding synthetic Vitamin D analogs?
The findings indicate that several synthetic analogs, particularly those modified to be more metabolically stable, demonstrate potent anti-tumor effects, suggesting they are promising candidates for future tumor therapy trials.
- Citation du texte
- Josef Elias (Auteur), 2002, Influence of Vitamin D - Metabolites and -Analogs on Growth and Apoptosis of C6-Rat-Glioma-Cells, Munich, GRIN Verlag, https://www.grin.com/document/19715
