الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Summary and conclusio
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Cancer is among the frequent causes of human death in the
world. It has been proven that overexpression, mutations and
constitutive activation of several tyrosine kinase receptors
contribute to tumorigenesis. Sunitinib, a small molecule tyrosine
kinase inhibitor, has been approved by the FDA to be used in the
treatment of renal cell carcinoma, imatinib-resistant GIST and
pancreatic neuroendocrine tumor primarily through its inhibitory
actions on various tyrosine kinase receptors as VEGFR, c-kit and
PDGFR.
Over the last few years, increasing studies reported rapid
development of sunitinib-resistant cells. It has been shown that
pro-survival mechanisms have been activated in sunitinib-resistant
cancer cells. Autophagy is a double-edged weapon; on one side, it
is regarded as programmed cell death type II through which
several chemotherapeutic agents mediate its cytotoxicity. On the
other side, cancer cells may induce autophagy as a cytoprotective
mechanism. In this regards, relevant studies reported conflicting
data regarding the stimulatory versus inhibitory effect of sunitinib
on autophagy.
To our knowledge, the current study is the first to explain the
differential modulatory effect of sunitinib on human colorectal
cancer (HCT116), cervical cancer (Hela), osteosarcoma (U2OS)
Summary and conclusi-237-
and embryonic renal (293T) cells. The “pro-survival” versus “prodeath”
role of autophagy modulated by sunitinib has been
investigated. The effect of sunitinib on autophagy regulators (Bcl-
2 anti-apoptotic family and mTOR pathway) has been explored.
The following parameters were investigated:
! Cytotoxic activity of sunitinib (40 mg/kg) on murine EAC
solid tumor model and its effect on autophagy.
! Cytotoxic activity of sunitinib on human colorectal cancer
(HCT116), cervical cancer (Hela), osteosarcoma (U2OS)
and embryonic kidney (293T) cells.
! Effect of varying concentrations of sunitinib on autophagy
assessed by:
o Examining the presence of cytoplasmic
autophagosomes and autophagolysosomes in
untreated and treated cells.
o Checking the abundance of GFP-LC3 puncta in
untreated and treated cells.
Summary and conclusion-238-
o Checking the protein level of p62/SQTSM1 in
untreated and treated HCT116, Hela, U2OS and 293T
cells.
! Effect of “autophagy modulation” on sunitinib cytotoxicity
using transduced 293T cells stably expressing shscrambled,
shAtg5 and shAtg7.
! Effect of sunitinib on autophagy upstream regulators; Bcl-2
anti-apoptotic family and mammalian target of rapamycin
(mTOR) as assessed by:
o Checking the protein levels of Mcl-1 and Bcl-2 in
untreated and treated HCT116, Hela, U2OS and 293T
cells.
o Checking p-p70S6k and total p70S6k protein levels
in untreated and treated HCT116, Hela, U2OS and
293T cells.
! Role of Mcl-1 and/or mTOR in attenuating sunitinib
cytotoxicity as assessed by:
o Investigating the effect of Mcl-1 deletion on sunitinib
cytotoxicity.
Summary and conclusions239-
o Exploring whether outer membrane and/or matrix
localized Mcl-1 mediates compromised sunitinib
cytotoxicity.
o Exploring the effect of Mcl-1/mTOR
pharmacological modulators as sorafenib and
rapamycin when combined with sunitinib.
! Molecular insights by which sunitinib modulates Mcl-1
protein levels/mTOR activity by:
o Checking the effect of sunitinib on Mcl-1
transcription.
o Checking the effect of sunitinib on Mcl-1 translation.
o Checking the effect of sunitinib on Mcl-1 stabiliy.
o Checking the effect of sunitinib on upstream
regulators of Mcl-1 stability/mTOR activity.
The findings of the present investigation can be summarized as
follows:
! Unlike lower concentration range of sunitinib, higher ones
switches on autophagic machinery as indicated by increased
Summary and conclusions
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cytoplasmic autophagic vesicles, GFP-LC3puncta and
degraded SQTSM1/p62 protein levels.
! Autophagic cell death is required for sunitinib cytotoxicity.
! Similarily, unlike higher concentration range, clinically
relevant concentrations of sunitinib increase Mcl-1 protein
levels and mTOR activity human colorectal cancer
(HCT116), cervical cancer (Hela), osteosarcoma (U2OS)
and embryonic kidney (293T) cells.
! Genetic and/or pharmacological inhibition of Mcl-1/mTOR
augments sunitinib cytotoxicity.
! Outer membrane and not matrix localized Mcl-1 mediates
attenuated sunitinib chemotherpeutic activity.
! Sunitinib does not alter Mcl-1 transcription or translation.
! Suntinib reduces Mcl-1 ubiquitination presumably through
ERK phosphorylation.
! PI3K/AKT inhibition and AMPK activation reverse
sunitinib-induced mTOR activation.
In conclusion, the present study is the first which showed that the
differential effects of sunitinib on autophagy is “concentration
range“ dependent. The present study also identified a novel effect
Summary 241-
of sunitinib where clinically relevant concentrations of sunitinib
increased Mcl-1 protein levels and activated mTOR; both of
which contributed to its compromised cytotoxicity. This could be
one of the mechanisms explaining acquired resistance to sunitinib.
Investigating the relevance of the findings of the present study in
sunitinib-resistant tumors warrants further exploration. The
preliminary findings of this study merit exploration of sunitinib
and Mcl-1/mTOR pharmacological inhibitors incorporation in a
novel combination in prospective controlled clinical trials in
sunitinib-resistant cases.