Download Apoptotic cell death signaling in the Human Colon Cancer Cell line

Shubhranshu Debnath
Apoptotic cell death signaling in the Human Colon Cancer Cell line
(HCT-116)
The development of an organism depends upon three basic fundamental cellular processes:
proliferation, differentiation and death. Each of these processes is of equal importance in
shaping an adult individual. Tumor is defined by uncontrolled proliferation and avoidance
of cell death. Hence, most anti-cancer treatments aim to eradicate tumor cells through
activation of various cell death processes, including apoptosis. Unfortunately, development
of resistance to chemotherapeutic drugs during the course of treatment is a substantial
problem in the clinics today. Therefore, a more precise understanding of cell death
mechanisms would facilitate development of novel methods for treatment.
Programmed cell death, or apoptosis, regulates cell numbers and forms complex organ
structures by cell removal. Apoptosis also occurs when a cell is damaged beyond repair.
During apoptosis several protein breaker enzymes (proteases) are activated, particularly
from the cystine aspartate protease family known as caspases. In apoptosis, caspase-8 and 9 serve as the most upstream activating caspases, respectively in the apoptotic cascade. The
activation of caspase-8 as a result of Death Induced Signalling Complex (DISC) formation
is a well studied event. If sufficient amounts of active caspase-8 are generated at the DISC
to directly process effector caspases then the cells are classified as type I apoptotic cells,
while in type II cells, the amount of caspase-8 processed in the DISC is insufficient.
Therefore, apoptosis in type II cells is dependent mitochondria, which is also known as the
power house of the cell, provides energy to the cell and permiabilization of the outer
mitochondrial membrane further damages the cell’s energy production mechanism and thus,
intensify the apoptotic process.
Our aims for the present project study are to map upstream regulatory events in the
apoptotic cell death cascade described and by this means build a more detailed model of
apoptotic mechanisms as a response to DNA damage by drug Flurouracil (5-FU) in Human
colon carcinoma cells (HCT116). Previous studies show that 5-FU creates cell stress
provoked by DNA damage and follow type I cell death. Our results reveal that apoptosis
indeed is a death receptor mediated process in this experimental model, by using the gene
silencing technique (short interfering RNA- “siRNA”), targeting the DISC regulatory
protein Flip (FLICE (Caspase-8) like inhibitor protein) and by creating a stable cell
expressing a dominant negative FADD (FAS- associated death domain) protein. Further
analyses defined DR5 (Death receptor 5) as the most important death receptor for the
signalling cascade studied. Two interesting findings then contributed to following
investigations. Firstly, apart from clear plasma membrane localization, DR5 also showed a
cytoplasmic punctuate expression pattern. Secondly, upon 5-FU treatment, HCT116 cells
are positive for several specific autophagy markers (autophagy- self eating; another cell
death mechanism). The present study contains several interesting data which eventually will
provide tools to further analyse upstream signalling events in 5-FU induced cell death.
More detailed analyses are, however, required to completely map the DR5 mediated
apoptotic process in HCT116 cells.
Supervisor: Prof. (Dr.) Boris Zhivotovsky
Degree project 60 credits in Protein Science – September 2009 to June 2010
Department of Chemistry, Lund University
Institution: Division of Toxicology, Institute of Environmental Medicine, Karolinska Institute
Shubhranshu Debnath
The role of Autophagy for DNA damage-induced Apoptotic signaling
in the Human Colon Carcinoma Cell line (HCT-116)
The best-characterized apoptotic pathways are the receptor-mediated (extrinsic) and
cellular stress-induced (intrinsic) pathways. In which caspase-8 and -9 serve as the
most upstream activating caspases, respectively. The activation of caspase-8 as a
result of DISC formation is well studied and represents the commencement of the
extrinsic apoptotic pathway. In type I cells, sufficient amount of active caspase-8 is
generated at the DISC to directly process effector caspases. In type II cells, the
amount of caspase-8 processed in the DISC is insufficient. Therefore, apoptosis in
type II cells is dependent on the cleavage of the pro-apoptotic Bcl-2 family protein
Bid. Conversion of Bid to tBid then leads to oligomerization and activation of Bax/
Bak, succeeded by the opening of specific pores in the outer mitochondrial membrane
(OMM) and resulting in the release of cytochrome c and other proteins from the
mitochondrial inter membrane space to the cytoplasm.
Our aims for the present project are to define upstream regulatory elements in the
apoptotic cascade described and by this mean build a more detailed model of
apoptotic mechanisms as a response to 5-FU induced DNA-damage in HCT116 cells.
Previous findings state that although 5-FU creates cell stress provoked by DNA
lesions, it seems that an extrinsic type of apoptosis is activated.
Our results revels that apoptosis indeed is death receptor mediated in this
experimental model by using siRNA targeting the DISC regulatory protein Flip and
by creating a stable cell expressing a dominant negative FADD protein, Further
analyses defined DR5 as the most important death receptor for the signalling cascade
examined. Two interesting findings then contributed to following investigations.
Firstly, apart from clear plasma membrane localization, DR5 also showed a
cytoplasmic punctuate expression pattern in immunostainings. Secondly, upon 5-FU
treatment, HCT116 cells are positive for several specific autophagy markers. The
present investigation contains several interesting data which eventually will provide
tools to further analyse upstream signalling events in 5-FU induced cell death. More
detailed analyses are, however, required to completely map the DR5 mediated
apoptotic process in HCT116 cells.
Supervisor: Prof. (Dr.) Boris Zhivotovsky
Institution: Division of Toxicology, Institute of Environmental Medicine, Karolinska Institute,
Degree project 60 credits in Protein Science – September 2009 to June 2010
Department of Chemistry, Lund University