APOPTOSIS AND ITS RELATION TO CANCER

Engin ULUKAYA (MD, PhD)

Uluda 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, for for Cells are born, live live a given period a given period die of time and then Bowen, of time and then die 1998
Bowen, 1998

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

WHERE can APOPTOSIS be 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

Number of Apoptosis-Relevant Publications

12000 10000 8000 6000 4000 2000 0 1984- 1986- 1988- 1990- 1992- 1994- 1996- 19981985 1987 1989 1991 1993 1995 1997 1999 Years

GENERAL FEATURES OF APOPTOSIS

1) A number of activities take place
... Occupation of death receptors

... Dimerization of Bcl-2 family members

... Release of cytochrome c ... Activation of caspases ... Activation of DNase

2) Translocation of phosphatidylserine
3) ATP-dependency 4) Internucleosomal DNA fragmentation (ladder pattern) 5) No apoptosis at +4 oC

6) No inflammation

Calbiochem, Inc

CELL SURFACE DEATH RECEPTORS

CASPASES
Caspase-1 (ICE) Caspase-2 (ICH-1, Nedd-2) Caspase-3 (CPP32, Apopain, Yama) Caspase-4 (ICH-2, TX, ICEre) Caspase-5 (ICErel, 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

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

. FADD . TRADD . FLIP . Bcl-2 family . Cytochrome c . p53 . Mdm2

. Caspases

. Many cellular proteins . DNA

AN APOPTOTIC CELL IN CULTURE

Collins JA, et al. 1997

From the archive of Dr Ulukaya

DEREGULATION OF APOPTOSIS IN MALIGNANCIES

Transfection studies in rat fibroblasts

Apoptosis Ras Tumor growth

Apoptosis c-myc Tumor growth

Igney and Krammer 1999

3
CASPASES CAN BE INHIBITED BY VIRUSES

... CrmA ... Baculovirs p35 ... Ebstein Barr Virs BHRFI proteini ... Ebstein Barr Virs LMP-1 proteini

4
APOPTOSIS-RELATED CELLULAR PROTEINS INVOLVE IN THE PROGRESSION OF MALIGNANCIES
... p53 ... pRb ... Fas ... Mdm2 ... c-myc ... c-Jun ... Bcl-2 family

Bcl-2 FAMILY
Anti-apoptotic
- Bcl-2 - Bcl-XL - Mcl-1 ******************* - p35 (Baculovirs) -BHRF1 (Ebstein-Barr Virs) - LMW5 HL (African Swine Fever Virus) - p19 (E1B) (Adenovirs)

Pro-apoptotic
- Bax - Bcl-XS - Bak - Bad *************** -????

Bad Bcl-XL CELL SURVIVAL

Bad Bcl-2

Bcl-XL Bax

Bcl-2 Bax

Bax

Bax

CELL DEATH

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

. 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

 "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

OncoTech, Inc

Question 1 ...

Is Cancer Puzzling ?

Question 2 ...
Does Apoptosis Held a Key Position in the Treatment of Cancer ?

POTENTIAL ROLE OF APOPTOSIS IN CANCER TREATMENT

Things to do ....
(1)
Determination of the ApoptosisRelated Proteins

. 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)

Things to do ....
(2)
Measurement of the Cytotoxic (ApoptosisInducing) Effects of Chemotherapeutic Agents on Individual Cancer Tissue Specimens Removed from Cancer Patients

In Other Words...

Designation of Patient-Specific Chemotherapy

METHODS FOR THE CHEMOSENSITIVITY TESTING

1... Clonogenic assay 2... Thymidine Incorporation Assay 3... Tissue Explant Assay 4... MTT assay 5... Fluorescence Assay 6... DISC Assay 7... The ISCO* ATP-Tumor Chemosensitivity Assay (ATP-TCA)
*ISCO, International Society of Chemosensitivity Testing in Oncology

From the archive of Dr Ulukaya

ATP-Tumour Chemosensitivity Assay

Tumour 1mm3 Fragments Overnight enzyme dissociation

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

Plate at 20,000 cell/well

Wash cells, count and estimate viability

Kindly supplied from Dr I Cree

In the literature (1)....

... 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 (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 of patients response to chemo- or radiotherapy as well as of survival rates Chemosensitivity testing, thereby individualised chemotherapy on the basis of patient-specificity, seems to be promising in the succesful treatment of malignancies. This testing, thereby, may revolutionize the way we use anticancer drugs in near future