PhD Student – Liana Ramishvili Scientific supervisor - Prof . N. Kotrikadze

Iv. Javakhishvili Tbilisi State UniversityFaculty of Exact and Natural Sciences

Department of BiologyDivision 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

NormalEpithelial Cells of Prostate

Partial Activity of Krebs Cycle

Low level of Respiration and

Terminal Oxidation

Energetically InefficientCan only produce small

amount of Electorns

Low Levels of Reactive oxygen Species (ROS)

Mitochondria

Mitochondria

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

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

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 FluorescenceSpectroscopic Methods

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

A B

C D

TeTr i si naT l i s wyar o

F L1

N2-l az er i

L2

Ar+ - l az er i

YAG: Nd3+ - l az er i

CCD speqt r omet er i

FM

2

3

4

Ch

PM

IBM PC: monacemTa

Segr oveba da damuSaveba

ni muSebi s sadgami

Laser Canceroscope

Consists of 4 blocks :

•Lights Source block;

•Sample block

•Registration block

•Data collecting and processing block.

Excitation was carried out by N2 laser:

=337nm wavelength.

Recording of Spectra was carried out in the 300- 500 nm wavelength region.

Light source

Sample

Data collection and

processing

N2 Laser

registration

The Study of Prostate Tumour tissue by Laser induced Fluorescence

Tumour tissue fluorescence spectrum of men with prostate benign hyperplasia

λ (nm)

440-460nm (I=0,48)

390-400nm (I=0,38)

Flu

ores

c en

ce in

ten

sity

(I

)

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)

Flu

ores

cen

ce in

ten

sit y

(I)

460-470nm (I=0,8)

400-410nm (I=0,45)

Tumour tissue fluorescence spectrum of men with prostate

adenocarcinoma

λ (nm)

Flu

ores

cen

ce in

ten

sit y

(I

)

460-470nm (I=0,8)

400-410nm (I=0,45)

λ (nm)

Flu

ores

cen

ce in

ten

sit y

(I)

(I)

λ (nm)

440-460nm (I=0,48)

390-400nm (I=0,38)

Flu

ores

c en

ce in

ten

sity

(I) Benign tumour

Benign tumour with

PING(3-4) regions

Prostate Cancer

0

0.2

0.4

0.6

0.8

1

1 2 3

The Changes of NADH Fluorescennce peaks intensities tumor tissue of prostate (440-460 nm)

1- Prostate Benign Hyperplasia;

2- Prostate Benign Hyperplasia with PING3-4 regions;

3- Prostate Cancer.

Conclusion

• Sharply Increased intensity of the Nicotinamide Coenzymes peak

(440-460 nm) in benign prostate tumor with PING3-4 regions and in

prostate adenocarcinoma compared with benign tumor tissue spectra,

reflects the type of metabolism that is typical to prostate malignant tumor

cells.

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

The Activity of Succinatedehydrogenase

0

5

10

15

20

25

1 2 3

1- Prostate Benign Hyperplasia;

2- Prostate Benign Hyperplasia with PING3-4 regions;

3- Prostate Cancer.

The sharp increase in SD activity presumably indicates on the enhanced electrons flow in respiratory chain of mitochondria.

0

20

40

60

80

100

120

140

160

1 2 3

1- Prostate Benign Hyperplasia;

2- Prostate Benign Hyperplasia with PING3-4 regions;

3- Prostate Cancer.

The Activity of Cytochromeoxidase

The insignificant changes in COX activity presumably indicates on the low level of terminal oxidation.

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.

m-aconitase

oxidates

NADH-Dehydrogenase

(I complex)

GSH

Citrate

Isocitrate

Enhanced Electron transfer

O2-

H2O2O2

Reduced Electron transfer

2H´+ 1/2O2

H20

Isocitrate DehydrogenaseNADPHNADP+

Succinate Dehydrogenase(SDH)

(II complex)

Ubiquinone/Cytochromeb(III complex)

Cytochrome Oxidase(COX)

(IV complex)

GSSGH2O + O2

Krebs Cycle

activation

Enhanced production

activates

Glutathione reductase

Glutathione peroxidase

Epithelial Cells of Prostate malignant Tissue (PIN G3-4,, Cap)

General scheme of energy metabolism Possible alterations in mitochondria of epithelial cells of

prostate malignant tissue (BHP with PIN G3-4,regions, CaP).

Glutathione-dependent Enzymes : Glutathione peroxidase (GSH-Px);

Glutathione reductase (GR);

Reduced Glutathione (GSH).

The Activity of Glutathione Peroxidase

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1 2 3

1 2 3

1- Prostate Benign Hyperplasia;

2- Prostate Benign Hyperplasia with PING3-4 regions;

3- Prostate Cancer.

The Activity of Glutathione Reductase

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035

1 2 3

1- Prostate Benign Hyperplasia;

2- Prostate Benign Hyperplasia with PING3-4 regions;

3- Prostate Cancer.

The Amount of Reduced Glutathione

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 2 3

1- Prostate Benign Hyperplasia;

2- Prostate Benign Hyperplasia with PING3-4 regions;

3- Prostate Cancer.

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).

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:

m-aconitase

oxidates

NADH-Dehydrogenase

(I complex)

GSH

Citrate

Isocitrate

Enhanced Electron transfer

O2-

H2O2O2

Reduced Electron transfer

2H´+ 1/2O2

H20

Isocitrate DehydrogenaseNADPHNADP+

Succinate Dehydrogenase(SDH)

(II complex)

Ubiquinone/Cytochromeb(III complex)

Cytochrome Oxidase(COX)

(IV complex)

GSSGH2O + O2

Krebs Cycle

activation

Enhanced production

activates

Glutathione reductase

Glutathione peroxidase

Epithelial Cells of Prostate malignant Tissue (PIN G3-4,, Cap)

General scheme of energy metabolism Possible alterations in mitochondria of epithelial cells of

prostate malignant tissue (BHP with PIN G3-4,regions, CaP).

Thank you for attention

winamdebare jirkvlis lokalizacia da zonaluri anatomia CZ – centraluri zona; PZ – periferiuli zona; TZ – gardamavali

zona

prostatis keTilTvisebiani simsivne

viTardeba prostatis kranialur

nawilSi ZiriTadad

periureTraluri jirkvlebidan

PIN ubnebiani keTilTvisebiani da avTvisebiani simsivneebi

(ukana kaudalur nawilSi)

mTavari samozne jirkvlovani epiTeliumis sekretoruli ujredebia

prostatis epiTeliumSi arCeven bazalur, sekretorul da parakrinul-endokrinul ujredebs

prostatis keTilTvisebiani hiperplazia prostatis adenokarcinoma