Transcript - PMAS-Arid Agriculture University Rawalpindi

Introduction to Cancer
Apoptosis, a Developmental and Defense Mechanism
The Structure and Function of IAP Proteins
Survivin, a Crucial IAP Target in Cancer Therapy
Conclusion
Normally cells utilize different death mechanisms,
e.g.:

Apoptosis,
Autophagy,
Necrosis
to maintain homeostasis and physiological integrity of the organism,
Cancer is typically a consequence of

Imbalance between cell death and proliferation in a way favorable to
cell proliferation and survival
APOPTOSIS
A pathway of cell death induced by a tightly regulated
suicidal program, in which the cells destined to die activate
enzymes that degrade cells own nuclear DNA and nuclear,
cytoplasmic proteins.
4
Why should a cell commit suicide?
1. Programmed cell death is as needed for proper normal
development as mitosis is.
Examples:
o The resorption of the tadpole tail in frog .
o The formation of the fingers and toes of the fetus
requires the removal, by apoptosis.
o The sloughing off of the endometrium at the start of
menstruation.
o The formation of the proper connections (synapses)
between neurons in the brain.
2. Programmed cell death is needed to destroy cells that
represent a threat to the integrity of the organism.
• Examples:
o Cells infected with viruses
o Cells of the immune system
o Cells with DNA damage
Intrinsic pathway (damage):
Mitochondria
BAX
BAK
BOK
BCL-Xs
BAD
BID
B IK
BIM
NIP3
BNIP3
Cytochrome c release
Pro-caspase 9 cleavage
Pro-execution caspase (3) cleavage
BCL-2
BCL-XL
BCL-W
MCL1
BFL1
DIVA
NR-13
Several
viral
proteins
Caspase (3) cleavage of cellular proteins,
nuclease activation,
etc.
Death
Oxidative damage from free radicals, Radiation, Virus infection, Nutrient
deprivation, Pro-apoptotic Factors
Damage to the mitochondrial membrane increasing permeability
Entry of Cytochrome C into the cytoplasm
Cytochrome C binds to Apaf-1 forming an apoptosome
Apoptosome activates procaspase-9 to caspase-9
Caspase-9 cleaves and activates caspase-3 and caspase-7.
This executioner caspases activate a cascade of proteolytic activity that leads to:
Chromatin condensation, DNA fragmentation, Protein cleavage, Membrane
permeability
Proapoptotic and anti apoptotic proteins containing
BHdomains
Balance shift from critical level in favor of proapoptotic
proteins
PROTEINS BIND TO MITOCHONDRIAL MEMBRANE AND
DECREASE INTEGRITY
MEMBRANE DEPOLARIZATION AND RELEASE OF
MITOCHONDRAIL CONTENTS INTO CYTOPLASM

The inhibitor apoptotic proteins family contain one to three
baculovirus IAP repeat or BIR domains

cellular IAPs also possess new gene (RING) domain at their Ctermini, and some also contain a caspase recruitment domain or
CARD

X-IAPS attach the caspases and inhibit their function
(controversial)

142 amino acid residues

smallest IAP, with the unique characteristic of having a single
BIR domain

Survivin levels and localization can be regulated by changes
in transcription, physical association with chaperones, altering
proteosomal degradation, and by other post-translational
mechanisms such as phosphorylation and acetylation of key
amino acid residues.

Expressed from the fetal stage instead of adulthood

Plays role in normal cells (t-cells,hoemopoetic cells, vascular
endothelial cells and Hepatic cells etc)

Most of the cancers have been found Survivin positive

Nodal proteins(various cellular actions)

Mitotic regulator for many divisions

Chromosomal passenger complex and localize mitotic apparatus

INCEP+BOREALIN +Survivin=localization of
enzymes=alignment+cell formation in mitosis

DNA damage-induced activation of the checkpoint kinase 2
(CHK2) results in rapid release of Survivin from the
mitochondria and consequently inhibition of cell death

DNA damage stimuli also stabilize p53, which in turn can
repress the transcription of Survivin and help balance the
degree to which activation of CHK2 promotes Survivin
release and activation of caspases

Survivin suppresses radiation-induced cytotoxicity

Survivin mRNA expression and radio sensitivity are inversely
related

inverse relationship between Survivin expression and Radio
sensitivity observed in colorectal cancer, glioblastoma, and
melanoma, pancreatic cancer

Survivin inhibition= enhanced radiation and chemotherapy

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expression of Survivin is substantially different in tumor
cells compared to normal cells
Survivin has emerged as a potential early predictor of
malignancies.
Survivin’s over-expression at the mRNA and proteins
levels appears to correspond with higher malignant
grades and reduced survival rates in different cancers
Studies suggest that the survival rate for Survivin
negative patients is much more than Survivin negative
patients
nuclear
Survivin
localization correlated
with
significantly lower survival rate than that of patients
with low nuclear Survivin levels(acts as a biomarker)
Survivin is not an enzyme nor it is a cell surface protein( so
targeting it can be difficult studies are still being conducted)
 Properties of drugs used:
1.
function at the transcription level and inhibit the transcription of
Survivin
2. inhibit Survivin at post-translational level include
3. vaccines that are based on cytotoxic activities of CD8+ T
lymphocytes against specific Survivin epitopes
 gene therapy methods including transfecting with dominant
negative mutants

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Antisense oligonucleotides (AO) are short, single stranded
RNA or DNA sequences that are complimentary to a specific
RNA

It acts by hybridization to the target mRNA strand to suppress
the expression of the particular gene

targeting Survivin triggered apoptosis in human melanoma
cell lines hence leading to increased apoptosis

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Ribozymes are small RNA molecules that cleave target RNA by
their endonucleolytic activity
transfection of human melanoma cell lines with ribozymes led
to reduced expression of Survivin protein
SiRNAs are often more effective than other antisense
approaches, rendering them better candidates for
clinical treatments Small interfering RNAs (siRNAs) are short,
double-stranded RNAs that inhibit gene expression. triggered
apoptosis
because lower concentrations of them is needed for each
treatment resulting in less side effects compared to other
techniques

Delayed mitosis, misaligned chromosomes

decreased Survivin expression, enhanced cell death, reduced
cell proliferation and inhibited tumor Growth

sensitivity of cancer cell lines to multiple treatments such as
vincristine
Therapies:


HSP90inhibitors:interaction of Survivin with the
molecular chaperone Hsp90 stabilizes the Survivin
protein Shepherdin is a cell-permeable antagonist of the
Hsp90-Survivin complex and acts through counteracting
the binding of these two proteins
Cyclin-Dependent Kinase (CDK) Inhibitors:CDK
inhibitors can counteract mitotic phosphorylation of
Survivin on Thr34 and thereby lead to the destruction of
Survivin and its function.it leads to increased apoptotic
activities through different pathways.

Promoter Inhibitors: imidazolium-based compound
is the most important in this group of low molecular
weight antagonists.

YM155 specifically inhibits Survivin gene
expression by suppressing promoter activity and
subsequent gene expression in cervical, colon, lung,
and other cancer cells.

other low molecular weight antagonist of Survivin is
terameprocol

The recognition of specific tumor-associated antigens by T
lymphocytes forms the basis for cancer immunotherapy.

These vaccines are able to induce tumor suppression in
multiple cancers(first tested on animals)
1.
Dominant negative mutants:

one of the earliest strategies used to inhibit Survivin activity

studies reported that transfection with dominant-negative
mutants of Survivin led to suppression of growth and
increased apoptosis in gastric cancer cell lines and some other
types of cancers in animal models. also increased sensitivity to
other treatments.

“genome editing” techniques, which are based on site-specificdirected nucleases, has raised enhanced feasibility of targeting the
main causes of many diseases. The potential for therapeutic
application of Zinc-finger nuclease (ZFNs),

transcription activator-like effector nucleases (TALENs) or clustered
regulatory interspaced short

palindromic repeat (CRISPR/Cas-based) RNA-guided DNA
endonucleases
has enabled a paradigm-shift compared to
conventional gene therapy methods

Domains of the tools:
1.
a designable and sequence-specific DNA-binding domain
2.
a nuclease domain, which modifies the specific sequence of
interest by leaving double-strand breaks behind
The differential expression of Survivin in malignant versus normal cells
is a strong rationale for development of Survivin-based cancer
therapeutics.
Tumor-specific promoters like that of Survivin, may allow future studies
to potentially direct the expression of other relevant apoptosis-inducing
genes or apoptosis-inhibiting mutants in tumors.
Approaches support the idea that the Survivin gene and promoter might
have continued utility for developing novel agents to target and attack
tumors of various types
REVIEW :Survivin as a Preferential Target for Cancer Therapy
Mahsa Mobahat , Aru Narendran and Karl Riabowol
Int. J. Mol. Sci. 2014, 15, 2494-2516;
doi:10.3390/ijms15022494