apoptosis and its relation to cancer engin ulukaya (md, phd) uludağ university, department of...
TRANSCRIPT
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
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
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2000
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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
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ant
Pub
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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 and Necrotic Cells (Fluorescence Microscopy Images)A
pop
tosi
sN
ecro
sis
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
. 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
. 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)
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