1. Iv. Javakhishvili Tbilisi State University Faculty of Exact and Natural Sciences Department of Biology Division of Cellular and Molecular Biology Prostate Cancer Epithelial Cells and The Changes That Take Place During Their Malignant Transformation PhD Student – Liana Ramishvili Scientific supervisor - Prof. N. Kotrikadze

2. Normal Epithelial Cells of Prostate Partial Activity of Krebs Cycle Low level of Respiration and Terminal Oxidation Energetically Inefficient Can only produce small amount of Electorns Low Levels of Reactive oxygen Species (ROS) Mitochondria

3. Incresed production of Reactive Oxygen Species (ROS) Intensification of Freeradical Processes Mitochondrial Defects Krebs Cycle Functions Properly Malignant transformation of Epithelial Cells of Prostate Increased electons Flow to the Electon transport Chain Increased rate of Mitochondrial DNA Mutations

4. To study the metabolic changes that take place in prostate epithelial cells during their malignant transformation. Tasks: To study of prostate tumor tissue by fluorescence spectroscopy. To study the mitochondrial defects (respiratory chain enzymes and gluthatione dependent system) in epithelial cells of prostate tumor. The Goal of the Work

5. Object of investigation : Tumour tissue of patients with prostate tumours: - Prostate benign hyperplasia; - Prostate benign hyperplasia with PING(3-4) regions; - Prostate Cancer. Method of Investigation : Laser induced Fluorescence Spectroscopic Methods

6. Histo-morphological pictures of Prostate Tumours. A. Controle group B Benign Hyperplasia C. Benign Hyperplasia with PIN regions D. Prostate adenocarcinoma AB C D

7. Laser Canceroscope Consists of 4 blocks : Lights Source block; Sample block Registration block Data collecting and processing block. Excitation was carried out by N 2 laser : = 337nm wavelength. Recording of Spectra was carried out in the 300- 500 nm wavelength region. Light source Sample Data collection and processing N 2 Laser registration

8. The Study of Prostate Tumour tissue by Laser induced Fluorescence

9. Tumour tissue fluorescence spectrum of men with prostate benign hyperplasia λ (nm) 440-460nm (I=0,48) 390-400nm (I=0,38) Fluorescence intensity (I)

10. Tumour tissue fluorescence spectrum of men with prostate benign hyperplasia with PING (3-4) regions λ (nm) 440-460nm (I=0,9) 400-410nm (I-=0,65) Fluorescence intensity (I)

11. 460-470nm (I=0,8) 400-410nm (I=0,45) Tumour tissue fluorescence spectrum of men with prostate adenocarcinoma λ (nm) Fluorescence intensity ( I )

12. 460-470nm (I=0,8) 400-410nm (I=0,45) λ (nm) Fluorescence intensity (I) (I) λ (nm) 440-460nm (I=0,9) 400-410nm (I-=0,65) Fluorescence intensity (I) λ (nm) 440-460nm (I=0,48) 390-400nm (I=0,38) Fluorescence intensity (I) Benign tumour Benign tumour with PING (3-4) regions Prostate Cancer

13. The Changes of NADH Fluorescennce peaks intensities tumor tissue of prostate (440-460 nm) 1- Prostate Benign Hyperplasia; 2- Prostate Benign Hyperplasia with PING 3-4 regions; 3- Prostate Cancer.

14. Conclusion Sharply Increased intensity of the Nicotinamide Coenzymes peak (440-460 nm) in benign prostate tumor with PING 3-4 regions and in prostate adenocarcinoma compared with benign tumor tissue spectra, reflects the type of metabolism that is typical to prostate malignant tumor cells.

15. The Study of Mitochondrial respiratory chain enzymes (complex II and Complex IV )

16. The Activity of Succinatedehydrogenase 1- Prostate Benign Hyperplasia; 2- Prostate Benign Hyperplasia with PING 3-4 regions; 3- Prostate Cancer. The sharp increase in SD activity presumably indicates on the enhanced electrons flow in respiratory chain of mitochondria.

17. 1- Prostate Benign Hyperplasia; 2- Prostate Benign Hyperplasia with PING 3-4 regions; 3- Prostate Cancer. The Activity of Cytochromeoxidase The insignificant changes in COX activity presumably indicates on the low level of terminal oxidation.

18. Thus, - Sharp increase of the activity of SDH (complex II); - Insignificant changes of COX (complex IV) activity ; These changes Presumably indicates to activation of Krebs cycle in mitochondria and increase of electrons flow in respiration chain on the one hand, and to impairment of the terminal oxidation of oxygen, on the other.

19. m-aconitase oxidates NADH- Dehydrogenase (I complex) GSH Citrate Isocitrate Enhanced Electron transfer O2-O2- H2O2H2O2 O2O2 Reduced Electron transfer 2H´+ 1/2O 2 H20H20 Isocitrate Dehydrogenase NADPH NADP + Succinate Dehydrogenase (SDH) (II complex) Ubiquinone/Cytochromeb (III complex) Cytochrome Oxidase (COX) (IV complex) GSSG H 2 O + O 2 Krebs Cycle activation Enhanced production activates Glutathione reductase Glutathione peroxidase Epithelial Cells of Prostate malignant Tissue (PIN G 3-4,, Cap) General scheme of energy metabolism Possible alterations in mitochondria of epithelial cells of prostate malignant tissue (BHP with PIN G 3-4, regions, CaP).

20. Glutathione-dependent Enzymes : Glutathione peroxidase (GSH-Px); Glutathione reductase (GR); Reduced Glutathione (GSH).

21. The Activity of Glutathione Peroxidase 1 23 1- Prostate Benign Hyperplasia; 2- Prostate Benign Hyperplasia with PING 3-4 regions; 3- Prostate Cancer.

22. The Activity of Glutathione Reductase 1- Prostate Benign Hyperplasia; 2- Prostate Benign Hyperplasia with PING 3-4 regions; 3- Prostate Cancer.

23. The Amount of Reduced Glutathione 1- Prostate Benign Hyperplasia; 2- Prostate Benign Hyperplasia with PING 3-4 regions; 3- Prostate Cancer.

24. Thus, sharp activation of mitochondrial antioxidant system, (GSH-Px, GR) revealed in BHP with PING(3-4) regions and malignant tumor epithelial cells, indicates to intensification of defensive abilities of tumor cells. (to withstand switching of the mitochondrial way of apoptosis, induced by free radicals).

25.  Thus, stimulation of the activity of SDH and retention of COX activity in epithelial cells of prostate malignant tissue may be responsible for sharp activation of isocitrate dehydrogenase and correspondingly, for significant accumulation of NADP(H).  Laser Induced Fluorescence spectra have shown the incresead intensity of NADP(H) peak in case of malignant tumor tissue that corresponds with investigations in Mitochondria of tumor epithelial cells.  Accumulation of NADP(H). may stipulate a sharp activation of the glutathione-depended system, which was proved by our investigations. Activation of the GSH-dependent system (GSH-Px, GR) presumably would be responsible for resistance of cancer cells against the oxidative stress.  Changes in the activity of enzymes of the II and IV complexes of mitochondrial respiration chain and antioxidant system, in case of prostate malignant trabsformation, are reflection of specific metabolic changes in mitochondria.  All the Above mentioned indicates to resistance of prostate malignant cells and correspondingly, to intensification of proliferation processes. Conclusions:

26. m-aconitase oxidates NADH- Dehydrogenase (I complex) GSH Citrate Isocitrate Enhanced Electron transfer O2-O2- H2O2H2O2 O2O2 Reduced Electron transfer 2H´+ 1/2O 2 H20H20 Isocitrate Dehydrogenase NADPH NADP + Succinate Dehydrogenase (SDH) (II complex) Ubiquinone/Cytochromeb (III complex) Cytochrome Oxidase (COX) (IV complex) GSSG H 2 O + O 2 Krebs Cycle activation Enhanced production activates Glutathione reductase Glutathione peroxidase Epithelial Cells of Prostate malignant Tissue (PIN G 3-4,, Cap) General scheme of energy metabolism Possible alterations in mitochondria of epithelial cells of prostate malignant tissue (BHP with PIN G 3-4, regions, CaP).

27. Thank you for attention

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