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

Inhaltsverzeichnis (Table of Contents)

• 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
    • 2.1.3. Materials used in Analytical procedures..
    • 2.1.4. 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 1a, 25(OH)2D3 on growth of C6-rat-glioma-cells
    • 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
  • 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 1a, 25-(OH)2-D3 on glioma.....
  • 4.2. Can 3-epi-vitamin D-analogues offer advantages over 1a, 25-(OH)2-D3 in the treatment of gliomas? ......
    • 4.2.1. C-3 epimerization is a metabolic pathway of 1a, 25-(OH)2-D3......... 56
    • 4.2.2. Established biological activities of 3a-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 1a,25-(OH)2-D3 on cell growth in the presence and in absence of serum
    • 4.3.3. Comparison: 1a, 25-(OH)2-D3 and 1a, 25-(OH)2-3-epi-D3.
    • 4.3.4. Effects of other vitamin D-metabolites........
    • 4.3.5. Vitamin D-analogues: Comparison 3ẞ- and 3a-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.....
• 5. REFERENCES

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This diploma thesis explores the impact of vitamin D metabolites and analogs on the growth and apoptosis of C6-rat-glioma-cells. The primary objective is to investigate the potential anti-cancer properties of vitamin D and its derivatives against brain tumor cells. The study analyzes the effects of various vitamin D forms, including natural metabolites and synthetic analogs, on cell viability, growth rate, and the induction of apoptosis.

• The role of vitamin D in the regulation of cell growth and apoptosis.
• The anti-cancer effects of vitamin D and its derivatives on glioma cells.
• The comparison of different vitamin D metabolites and analogs in terms of their effectiveness on glioma cells.
• The mechanisms by which vitamin D induces apoptosis in glioma cells.
• The potential therapeutic applications of vitamin D and its derivatives in the treatment of gliomas.

Zusammenfassung der Kapitel (Chapter Summaries)

The introduction provides a comprehensive overview of vitamin D, including its synthesis, metabolism, and biological roles. It discusses the mechanisms of cell death, specifically focusing on apoptosis and necrosis, and delves into the potential of vitamin D as an anti-cancer agent. The chapter also introduces the C6-rat glioma cell line, a model system used in the study to investigate the effects of vitamin D on brain tumor cells.

The materials and methods section describes the devices, reagents, and analytical procedures employed in the study. It outlines the cell culture techniques, the incubation protocols with vitamin D metabolites and analogs, and the various analytical methods used to assess cell viability, apoptosis, and DNA fragmentation.

The results chapter presents the findings of the study, including the effects of 1a, 25(OH)2D3 on the growth of C6-rat-glioma-cells in both serum-containing and serum-free culture medium. It examines the dose-dependent effects of 1a, 25(OH)2D3 and 1a, 25(OH)2-3-epi-D3 on cell viability and the induction of apoptosis. Further, it analyzes the influence of other vitamin D metabolites and analogs on the growth and apoptosis of the glioma cells. The chapter concludes with the detection of DNA fragmentation using capillary electrophoresis.

The discussion section interprets the results of the study, focusing on the anti-cancer effects of 1a, 25-(OH)2-D3 on glioma cells. It evaluates the potential advantages of 3-epi-vitamin D-analogues over 1a, 25-(OH)2-D3 in the treatment of gliomas, considering the metabolic pathways of 1a, 25-(OH)2-D3 and established biological activities of 3a-vitamin D-analogues. It also examines the effects of other vitamin D-metabolites and -analogs on growth and apoptosis of C6-glioma-cells, discussing the relevance of the applied methods, the comparison of 1a, 25-(OH)2-D3 and 1a, 25-(OH)2-3-epi-D3, and the effects of other vitamin D-metabolites. Finally, it considers the availability of vitamin D-metabolites/-analogs at the target site, including uptake into the brain and availability at the tumor site.

Schlüsselwörter (Keywords)

This diploma thesis focuses on the impact of vitamin D metabolites and analogs on the growth and apoptosis of C6-rat-glioma-cells. Key concepts include: vitamin D, glioma, apoptosis, cell growth, anti-cancer effects, 1a, 25(OH)2D3, 1a, 25(OH)2-3-epi-D3, cell viability, DNA fragmentation, capillary electrophoresis, and therapeutic potential.