APOPTOSIS AND ITS RELATION TO CANCER

Engin ULUKAYA (MD, PhD)

Uludağ University, Department of Biochemistry, 16059 Bursa / TURKEY

Talk about....

1. APOPTOSIS

2. DEREGULATION OF APOPTOSIS IN MALIGNANCIES

3. POTENTIAL ROLE OF APOPTOSIS IN CANCER TREATMENT

APOPTOSIS

Cells are born, live for a given period of time and then die

Bowen, 1998

--- Physiological cell death--- Cell suicide--- Cell deletion--- Programmed cell death

APOPTOSIS

Cells are born, live for a given period

of time and then die Bowen, 1998

WHERE can APOPTOSIS be WHERE can APOPTOSIS be ENCOUNTERED ?ENCOUNTERED ?

... Growth of Embrio

... Tissue Homeostasis

... Immunology

... Chronic viral diseases

... Neurodegenerative diseases

... Reperfusion injury

... Insuline-dependent Diabetes

... Atheroschlerosis

... Miyokard Infarction

... AIDS

... Development and Treatment of Malignancies

0

2000

4000

6000

8000

10000

12000

1984-1985

1986-1987

1988-1989

1990-1991

1992-1993

1994-1995

1996-1997

1998-1999

Years

Num

ber o

f Apo

ptos

is-R

elev

ant

Pub

licat

ions

GENERAL FEATURES OF APOPTOSIS

... Occupation of death receptors

... Dimerization of Bcl-2 family members

... Release of cytochrome c

... Activation of caspases

... Activation of DNase

1) A number of activities take place

2) Translocation of phosphatidylserine

3) ATP-dependency

4) Internucleosomal DNA fragmentation (ladder pattern)

5) No apoptosis at +4 oC

6) No inflammation

CASPASES

Caspase-1 (ICE)

Caspase-2 (ICH-1, Nedd-2)

Caspase-3 (CPP32, Apopain, Yama)

Caspase-4 (ICH-2, TX, ICEreıı)

Caspase-5 (ICErelııı, TY)

Caspase-6 (Mch2)

Caspase-7 (ICE-LAP3, Mch3, CMH-1)

Caspase-8 (FLICE, Mch5, MACH)

Caspace-9 (Mch6, ICE-LAP6)

Caspase-10 (Mch4)

SUBSTRATES for CASPASES

... PARP

... DNA-PK

... pRb

... Lamins

... NuMA

... Fodrin

... -Aktin

... Mdm2

... Cyclin A2

... Presenilin

... Others

THE APOPTOTIC PATHWAY

Triggers Modulators Effectors Substrates DEATH

. FADD

. TRADD

. FLIP

. Bcl-2 family

. Cytochrome c

. p53

. Mdm2

. Caspases . Many cellular proteins. DNA

. Growth factor Deprivation. Hypoxia. Loss of adhesion. Death receptors . Radiation . Chemotherapy

From the archive of Dr Ulukaya

Apoptotic Cells (Electron Microscopy Image)

From the archive of Dr Ulukaya

Apoptotic and Necrotic Cells (Fluorescence Microscopy Images)A

pop

tosi

sN

ecro

sis

DEREGULATION OF APOPTOSIS IN

MALIGNANCIES

Transfection studies in rat fibroblasts

Ras

Apoptosis

Tumor growth

c-myc

Apoptosis

Tumor growth

CASPASES CAN BE INHIBITED BY VIRUSES

... CrmA

... Baculovirüs p35

... Ebstein Barr Virüs BHRFI proteini

... Ebstein Barr Virüs LMP-1 proteini

3

APOPTOSIS-RELATED CELLULAR PROTEINS INVOLVE IN THE

PROGRESSION OF MALIGNANCIES

... p53

... pRb

... Fas

... Mdm2

... c-myc

... c-Jun

... Bcl-2 family

4

Bcl-2 FAMILY

- Bcl-2- Bcl-XL

- Mcl-1*******************- p35 (Baculovirüs)-BHRF1 (Ebstein-Barr Virüsü)- LMW5 HL (“African Swine Fever Virus”)- p19 (E1B) (Adenovirüs)

- Bax- Bcl-XS

- Bak- Bad***************-????

Anti-apoptotic Pro-apoptotic

Bcl-XL

Bad

Bcl-XL

BaxBcl-2Bax

Bax Bax

Bcl-2Bad

CELL SURVIVAL

CELL DEATH

. Increased Bcl-2 –--------------------------------- Poor prognosis . Increased FasL –--------------------- Decreased CTL number

. FasL induction (with Doxorubicin)----------------Determines chemosensitivity

. Overexpression of Bax---------------- Improve the efficacy of chemotherapy

. p53 antibodies ------------------- Resistance to chemotherapy with cisplatin + 5-Fluorouracil

Various Expression Levels of Apoptosis-Related Proteins Determine Patient-Specific Malignancy ?

5

• "Right now we lump patients together and treat them with the same drugs and then deal with their variable response to treatment. We're essentially treating different diseases with the same medicine.”

• Richard Klausner, 1997

Is Cancer Puzzling ?

Question 1 ...

Does Apoptosis Held a Key Position in the Treatment of

Cancer ?

Question 2 ...

POTENTIAL ROLE OF APOPTOSIS IN CANCER TREATMENT

Things to do ....

Determination of Both Apoptosis Itself and Apoptosis-Related Proteins

(1)

. p53 gene status--------------- Modulates the chemosensitivity

. p53 level –---------- Predictor for the response to chemo- or radiotherapy (Advanced Head and Neck Carcinomas, Epithelial Ovarian Ca)

. Mutant p53 --------- Overall shortened survival (Breast Ca)

. Ratio of Bcl-2/Bax -----------------------–--- Prognostic factor (Hematologic Malignancies, Colon Ca)

. Bcl-2 alone –-------- Prognostic factor (Advanced Over Ca)

Assaying of Cytotoxic (Maybe Apoptosis-Inducing) Potential of Chemotherapeutic

Agents on Individual Cancer Tissue Specimens Removed from Cancer Patients

Things to do ....

(2)

In Other Words...

• Designation of Patient-Specific Chemotherapy

SOME METHODS FOR THE CHEMOSENSITIVITY TESTING

1... Thymidine Incorporation Assay

2... Tissue Explant Assay

3... MTT assay

4... Fluorescence Assay

5... DISC Assay

6... The ISCO* ATP-Tumor Chemosensitivity Assay (ATP-TCA)

*ISCO, International Society of Chemosensitivity Testing in Oncology

ATP-Tumour ChemosensitivityAssay

Tumour1mm3 Fragments

Overnight enzymedissociation

Wash cells, count andestimate viability

Plate at 20,000cell/well

Incubate for 5-7 days,extract ATP and readin a luminometer

Kindly supplied from Dr Cree

... A working tumor chemosensitivity assay (TCA) could be of immense benefit to the pharmaceutical industry, oncologists and their patients (Cree and Kurbacher, 1997)

... ATP-TCA can be used to select patients who might benefit from specific chemotherapeutic agents alone or in combination (Cree et al, 1999)

In the literature (1)....

In the literature (2)....

Chemotherapy guided by the ATP-TCA

... Retrospective clinical correlation in breast carcinoma (Cree et al, 1996): 97% assay evaluability, 76% accuracy, 27% imrovement in clinical response rate

... A greater benefit with regard to both ORR and PFS in platinum refractory patients (Kurbacher et al, 1998): Overall survival, 97 weeks / 69 weeks; Response rate, 64% / 37%

TWO GREAT BENEFITS

• Exclusion of chemotherapeutic agents which are not likely to be effective, thereby avoidance of their potential toxicity

• Selection of chemotherapeutic agents with the greatest likelihood of clinical effectiveness for improved response rates and prolonged survival

SUMMARY

• It is considered that defective apoptosis is a feature of malignant development

• Induction of apoptosis in malignancies is to be aimed

• Detection of apoptosis-related proteins may be of importance in the prediction for response to chemo- or radiotherapy as well as for survival

• Chemosensitivity testing, thereby individualised chemotherapy, seems to be promising in the succesful treatment of malignancies on the basis of patient-specificity