Apoptotic activity of Vitamin E Phosphate: A novel strategy in the cancer chemotherapy

1. Apoptotic activity of Vitamin E Phosphate: A novel strategy in the cancer chemotherapy Dr. Bashir M. Rezk

3. Biological activity of VE

4. Vitamin E derivatives VE is often supplemented as a chemically stable ester derivative such as • Vitamin E Acetate (VEA) • Vitamin E Succinate (VES) • Vitamin E Phosphate (VEP)

5. Vitamin E derivatives • Unlike the redox-active VE , VES, VEA and VEP are redox-silent • Unlike VEA, VES and VEP have side groups that can be charged at physiological pH • Unlike VEA and VE, VES is a potent selective anticancer agent

6. Structural formula of VE and its derivatives

7. Rank order Assay Reference VEA > VE Rat fetal resorption Harris & Hudwig VEA > VE Rat fetal resorption Leth & Sondergaard VEA > VE Rat fetal resorption Weiser et al. VES > VE Microsomal lipid peroxidation Carini et al. Trolox > VE >>> VEA and VES Lipid peroxidation in cultured endothelial cells Mabile et al. VES > VE  VEA Lipid peroxidation in hepatocytes Fariss et al. Trolox > VE > VEA > VES Microsomal lipid peroxidation Rezk et al. The reported rank order of the biological activity of VE and its ester derivatives

8. Apoptotic activity of VES • VES induced potent morphological differentiation and growth inhibition in B-16 (1982) • VE, VEA, and VEN have no effect • VES inhibited growth and caused morphological changes in B-16, NBP2 and C-6 cells (1983) • VES enhanced the growth inhibitory effects of ADR on a variety of cancer cells (1986) • VES can inhibit the growth of N1E 115 cells (1989) • VES induces growth inhibition and apoptogenic activity on a variety of cell lines without affecting the proliferation of normal cells in vitro and in vivo (1990-2004).

9. Mechanism apoptotic activity of VES • Membrane destabilizing activity • 1- VES has a detergent like activity • 2- Can exist as a deprotonated or protonated • 3- 99% of the total VES is charged at neutral pH • 4- The uncharged will increase to 25% at pH 6.2 • 5- The uncharged VES enter cells by p. diffusion • 6- The cytotoxic effect of VES increased with acidic pH • Dysregulation of signaling pathways

10. Non-apoptogenic (A) and apoptogenic (B) agents

11. Non-apoptogenic (A) and apoptogenic (B) agents • Neither trolox succinate nor phytyl succinate caused apoptosis • VEG has a very poor apoptogenic activity compared to VES, VEM and VEF • VEO has a potent apoptotic activity compared to VES and VEM • Flexibility of the terminal carboxyl containing moiety has a role in the apoptotic activity

12. Non-apoptogenic (A) and apoptogenic (B) agents

13. Vitamin E Phosphate • VEP has been discovered in plant and animal tissues, including human • The concentrations of VEP are in general similar to those of VE • In rat and pig liver VEP levels were higher • In foodstuffs such as chocolate and cheese the concentrations of VEP are 10 to 30 times higher

14. Physiological effect of VEP • Reduced of the intensive rate of metabolism in rabbit muscles • Activation of cAMP phosphodiesterase • Increasing the rate of phenlalanine hydroxylase • Protection against irradiation in rat intestine and cultured mouse skin • Increase in sperm survival • Improvement of acetylcholine-dependent relaxation in the aged aortic strips exposed to oxidative • Protection against LPO in non-heated and heated rat liver microsomes

15. Extraordinary antioxidant activity of VEP Time-course of the effect of VE, Trolox, VEA, VES, & VEP on LPO heated microsomes (A) Induction of hemolysis by VE & VE esters(B)

16. Physiological effect of VEP

17. Goal of the study The present study has been designed to investigate the effect of VEP on the oesteosarcoma cell line, MG-63, using VES as a reference compound

18. Materials and Methods • Cell Culture • Viability assay • Proliferation assay (qualitative and quantitative) • DNA fragmentation assay for apoptosis • Vitamin E determination • Membrane fluidity of human erythrocytes • Erythrocyte Hemolysis

19. Results & Discussion 1 • VEP induced cell death after 6 h incubation • The effect of VEP was superior to that of VES • In a conc. of 80 µM both VEP and VES hardly showed any conversion into VE • In contrast, VEP showed a massive conversion into VE In cultured mouse skin Fig. 1. Cytotoxic effect of VES and VEP on MG-63 cells. The insert shows the formation of VE from VES and VEP

20. Results & Discussion 2 • The antiproliferative effect of VEP is more potent than that of VES Fig. 2. Qualitative & Quantative antiproliferative effect of VE, VES and VEP on MG-63 cells

21. Results & Discussion 3 • VEP has a more potent apoptogenic activity (64 ± 11% ) than VES (46 ± 9%) • VEP induced a more DNA fragmentation and nucleus condensation Fig. 3. Apoptotic effect of VE, VES, and VEP on MG-63 cells

22. Induction of apoptosis Low concentrations (5-46 µM) had no effect on DNA laddering, whereas high concentrations (46-184) induced significant DNA degradation Munteanu et al. (Azzi group) BBRC (2004)

23. Induction of apoptosis VEP, 55.3% di-VEP, 30.6% VE, 5%

25. Results & Discussion 4 • Neither trolox succinate nor phytyl succinate caused apoptosis • Methylation of the free succinyl carboxyl group (giving VE methyl succinate) completely abolished the apoptogenic activity of VES • VEG has a very poor apoptogenic activity compared to that of VES, VEM and VEF • Apoptogenic potency also differs between vitamin E oxalate (VEO), vitamin E malonate (VEM) and VES. • The apoptogenic activity correlates with the flexibility of the terminal carboxyl containing moiety • Fewer than four carbon atoms in the terminal dicarboxylic moiety were necessary for the potent apoptogenic activity

26. Results & Discussion 6 • Four carbon atoms or more in the terminal carboxyl moiety are implicated in the flexibility of the ester derivatives • More flexibility, less apoptotic activity Vitamie E pimelate

27. Structural requirements • Domain I is considered as functional domain • Domain II is regarded as signalling domain • Domain III is an orientation domain Fig. 4. Structures of VES and VEP showing major domains

28. Results & Discussion 7 • Both VES and VEP decreased membrane fluidity of erythrocytes • VE has no significant effect Fig. 6. Effect of VE, VES, and VEP on membrane fluidity of erythrocytes

29. Results & Discussion 8 • The cytotoxicity of VEP on erythrocytes is higher at acidic pH Fig. 6. Induction of hemolysis of erythrocytes by VEP

30. Membrane destabilizing activity of VES or VEP • 1- VES has a detergent like activity • 2- Can exist as a deprotonated or protonated • 3- 99% of the total VES is charged at neutral pH • 4- The uncharged will increase to 25% at pH 6.2 • 5- The uncharged VES enter cells by p. diffusion • 6- The cytotoxic effect of VES increased with acidic pH • Possibly protonation of the phosphate group is involved in the cytotoxic effect of VEP

31. Conclusion • VEP induces cell death, prevents cell proliferation and provokes apoptosis in MG-63 • This is probably due to a membrane destabilising activity by acting as a detergent • The apoptogenic activity requires the combination of the three structural domains, i.e. functional, signalling and orientation domains

32. Plans of the Pharmacology Unit • The ICGEB project on vitamin E succinate • The PhD program on vitamin E succinate • The PhD program on vitamin E phosphate • Collaboration with a Japanese group on vitamin E oxalate Dr. Bashir M. Rezk, Dr. Hafez F. Hafez, Prof. dr. Mohamed M. El-Khayal and Prof. dr. Mahmoud M. El-Merzabani

33. Thank you