الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Most of the world’s countries reported that in 2020, BC ranked first for incidence and
mortality among women. Many different types of cancer seem to be successfully under
control with radiation therapy, but the radio-resistance that tumors develop totally
compromise its efficacy. Necroptosis, an alternate process of programmed cell death that
overcomes apoptosis resistance, has challenged the conventional knowledge and apoptosis
is the only type of programmed cell death. Our purpose was to investigate the
interrelationship between apoptotic and necroptotic death pathways and their influence on
the response of breast cancer cells to radiotherapy in vitro. Human BC cell lines MCF-7
and MDA-231 were treated with different combinations of radiation doses and MLKL
siRNA and followed-up up to 48 h. Investigation of cell survival was dose using MTT and
Annexin V-FITC/PI. Expression of genes related to survival, apoptosis and necroptosis
was quantified using real-time PCR and protein expression of necroptotic markers both
total and activated was done using western blot.
Our results showed regarding to survival signals, cIAP1 and 2 have demonstrated
considerable dose- and time-dependent downregulation. Both cell lines demonstrated an
increase in TRAF2 expression 24 hours after irradiation; however, the expression level
dropped dramatically when the radiation dose was increased to 8 Gy and follow up time
for 48 hours. TRAF2 expression was also correlated to cIAP1 and cIAP2 expression in
both cell lines. Upon treatment with MLKL siRNA, elevated cell survival and
overexpression of cIAP1 and cIAP2 and TRAF2 expression was observed indicating that
the cells demonstrated radio-resistance and evaded cell death by inhibiting necroptosis.
Regarding apoptosis modulators, although caspase-8 and TRADD showed an initial
early dose-dependent increase in MCF-7 cells that did not last with follow-up up to 48 h.
Upon MLKL knockdown, caspase and TRADD levels also increased early but dropped
later. Irradiated MDA-231 cells, on the other hand, they levels showed a late elevation 48 h
post irradiation. In absence of MLKL expression, caspase 8 and TRADD did not show a
Summary, Conclusion & Recommendations
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significant change 24 h post irradiation but after 48 h their levels were significantly
elevated. This indicates that the apoptotic pathway is activated in MCF-7 earlier and more
significantly compared to MDA-231 cells.
RIPK1, a marker in the crossroads between apoptosis and necroptosis showed a
significant time and dose dependent increase upon exposure to ionizing radiation in both
cell lines. MLKL knockdown lead to a significant initial elevation in RIPK1 expression in
MDA-231 cells that declined dramatically after 48 h which was consistent with the
radioresistant features of MDA-231. On the other hand, RIPK1 expression was
significantly increased in MCF-7 cells at both time intervals in a dose-dependent manner
suggesting the activation of apoptotic signals to compensate the silencing of necroptotic
modulator.
RIPK3 and MLKL, the specific necroptotic markers’ gene expression was
significantly increased, particularly at higher radiation doses in both cell types and the
expression increased over time. In both cell lines, their expression was negatively
correlated with survival at both time points. MLKL knockdown significantly reduced
RIPK3 and MLKL expression compared to groups that were not treated with siRNA which
indicates an escape strategy from necroptosis. This strong correlation with cell survival
emphasizes the crucial role of necroptosis in both cell lines regardless of the activation of
caspase 8-dependent apoptosis.