الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Breast cancer ‘BC’ ranks as the fifth cause of death from cancer overall and known as the second leading cause of cancer death in after lung cancer. It is a heterogeneous disease that can be classified using a variety of clinical and pathological features. TNBCs are sub-type of breast cancer which lack ER, PR and HER-2/neu receptors. TNBCs constitute 10%–20% of all breast cancers; more frequently affect younger patients. They are generally larger in size, are of higher grade, have lymph node involvement at diagnosis, and are biologically more aggressive. TNBCs are further classified into 6 subtypes. Thus, the molecular heterogeneity of TNBC confounds the clinical approach to TNBC treatment, consequently, it is considered as one of the most difficult subtypes of breast cancer to treat due to a lack of a targeted therapy. Several studies have pointed out to the possibilities for benefits from targeting different signaling pathways including Notch pathway. Notch signaling is the most widely used intercellular communicating pathway that is involved in carcinogenesis through regulating many cellular responses, survival, proliferation, migration, differentiation, and apoptosis. Thus, deregulation of Notch pathway may lead to critical tumor hallmarks, including oncogene expression, angiogenesis, stem cell maintenance, deregulated cell cycle progression, and anti-apoptotic mechanisms. Furthermore, Notch signaling found to be oncogenic or tumor suppressor in different types of cancers. Generally, Notch plays a key role in the pathogenesis of BC and TNBC in specific; it has a role in tumor initiation, proliferation, angiogenesis, and metastasis as well as tumor suppressor function. Hence, Notch signaling may represent a potentially promising therapeutic target for patients with TNBC. On the other hand, Autophagy process, the evolutionarily conserved catabolic process of self-digestion, plays a vital role in the mechanism of keeping cellular homeostasis and survival. It acts as a strategy for survival under stress conditions. It plays an active role in programmed cell death when over activated by starvation, ROS, hypoxia, drug stimuli, and endoplasmic reticulum stress via complex signal transduction pathways, excess autophagy results in cell death. In cancer, Autophagy has both positive and negative functions, depending on the type of cancer and the context, autophagy fulfills a dual role; having both tumor promoting and suppressing properties. The function of autophagy in BC still unclear; it can be reduced at various developmental and metastatic phases. It can also be a primary cell death pathway in some tumors in apoptosis deficiency. Therefore, all these led to controversy over whether or how autophagy manipulation should be attempted in cancer therapy.
Summary 83
There is minimal data on the effects of the Notch pathway on autophagy in cancer. However;
increasing evidence suggests that Notch signaling pathway affects autophagosomal trafficking, and
its inhibition induces autophagy in some types of cancer as well as during differentiation of stem
cells. Besides, some studies showed that inhibition of autophagy in combination with Notch
inhibitor can give better clinical outcome than using notch inhibitor alone.
Although there is no approved targeted therapy available for TNBCs, molecular-profiling
efforts have revealed promising molecular targets, with several candidate compounds entered
clinical trials for patients with TNBC. Metal-based antitumor drugs play a relevant role in
chemotherapy; there has been a rapid expansion in research and development of novel metal-based
anticancer drugs to improve clinical effectiveness, to reduce general toxicity and to broaden the
spectrum of activity. Among non-Pt compounds, copper complexes are potentially attractive as
anticancer agents. Special emphasis has been focused on the identification of structure-activity
relationships for the different classes of copper (I, II) complexes. Recently, special attention has
been paid on the copper (I)-nicotinate complex (CNC), which shows antioxidant activity, antiinflammatory
and anti-tumor activities.
The present study is an attempt to shed more lights on the potential effect of CNC as a
targeted therapy for Notch1 signaling pathway in autophagy modulated TNBC cell lines. This
might explain more about the possible cross talk between Notch1 signaling pathway and
autophagy. To approach the objective of the current study, two different subtypes of TNBC cell
lines were utilized; the acantholytic squamous basal-like HCC1806 cell line and the mesenchymal
stem-like MDA MB 231 cell line. According to treatment, both TNBC cell lines were divided
into:
I. Control Groups:
1. Untreated control cultured for 24 hrs.
2. Untreated control cultured for 48 hrs.
II. CNC-treated Groups:
1. CNC50 –treated group: treated with IC50 of CNC.
2. CNC10 –treated group: treated with 10% of IC50 of CNC.
III. Doxo-treated Group: treated with IC50 of doxorubicin.
IV. CNC-Doxo treated Groups:
1. CNC50-Doxo: treated with combination of IC50 of CNC and IC50 of doxorubicin.
2. CNC10-Doxo: treated with combination of 10% of IC50 of CNC and IC50 of
doxorubicin.
Summary 84
V. Torin1-treated Group: treated with 10% of IC50 of Tor; the autophagy inducer.
VI. Chloroquine-treated Group: treated with 10% of IC50 of CQ; the autophagy inhibitor.
VII. Tor-CNC-treated Groups:
1. Tor-CNC50–treated group: cells were pre-treated with10% of IC50 of Tor for 24hrs then
treated with IC50 of CNC.
2. Tor-CNC10–treated group: cells were pre-treated with10% of IC50 of Tor for 24hrs then
treated with 10% of IC50 of CNC.
VIII. Tor-Doxo-treated Group: cells were pre-treated with10% of IC50 of Tor for 24hrs then
with IC50 of Doxo.
IX. CQ-CNC-treated Groups:
1. CQ-CNC50–treated group: cells were pre-treated with10% of IC50 of CQ for 24hrs then
with IC50 of CNC.
2. CQ-CNC10–treated group: cells were pre-treated with10% of IC50 of CQ for 24hrs then
treated with 10% of IC50 of CNC.
X. CQ-Doxo-treated Group: cells were pre-treated with10% of IC50 of CQ for 24hrs then with
IC50 of Doxo.
XI. Tor-CNC-Doxo treated Groups:
1. Tor-CNC50-Doxo: cells were pre-treated with10% of IC50 of Tor for 24hrs then treated
with combination of IC50 of CNC and IC50 of doxorubicin.
2. Tor-CNC10-Doxo: cells were pre-treated with10% of IC50 of Tor for 24hrs then treated
with combination of 10% of IC50 of CNC and IC50 of doxorubicin.
XII. CQ-CNC-Doxo treated Groups:
1. CQ-CNC50-Doxo: cells were pre-treated with10% of IC50 of CQ for 24hrs then treated
with combination of IC50 of CNC and IC50 of doxorubicin.
2. CQ-CNC10-Doxo: cells were pre-treated with10% of IC50 of CQ for 24hrs then treated
with combination of 10% of IC50 of CNC and IC50 of doxorubicin.
In the present study, the half maximal inhibitory concentration for each treatment was
assessed and represented by (IC50) value by MTT assay. Biochemically, gene expressions of
Notch signaling pathway including Notch 1, Jagged 1 and Hes 1 were assessed by RT-PCR using
specific primers and β-actin as a reference. Also, Light chain (LC3) protein level was assessed
using flow cytometry as marker for autophagy. Cycle analysis was also carried using flow
cytometer. Morphological changes were also observed using inverted microscope. TEM was used
in specific groups including untreated, CQ-treated, Tor-treated, CNC50-treated, CNC10-treated and
Doxo-treated cells. Inverted microscope showed morphological alterations, shrinking and
Summary 85
detachment of most of cells after the treatment of studied drugs, while TEM showed the presence
of autophagosomes in cells both treated and untreated cell lines.
The two cells lines responded differently to the applied treatment; and this might be due to the
biological and heredity heterogeneity of TNBC subtypes. Generally, the present study showed that
treating HCC 1806 cell line with CNC; CNC50, resulted in down regulation of Notch1 signaling
genes with increased the level of apoptosis and autophagy. While, in MDA MB 231 cell line,
CNC10 led to up regulation of Notch1 signaling genes with increasing the levels of both autophagy
and apoptosis. CNC10 produced similar effects as that of Doxo in the two cell lines.
The combination of CNC and Doxo led to synergistic effect in both cell lines. In HCC 1806,
co-treatment of Doxo and CNC50; CNC50-Doxo, displayed a pro-apoptotic and anti-proliferative
effect associated with a minor elevation in autophagy after the up regulation effect appeared in
Notch1signlaing genes. Regarding MDA MB 231, CNC50-Doxo was found to up regulate the
relative gene expression of Notch1 and JAG1 genes as well as, down regulated Hes1 relative gene,
revealing its pro-apoptotic and anti-proliferative effects.
Induction of autophagy with Tor resulted in a significant down regulation in Notch1 signaling
genes in HCC1806 cell line besides an increase in cellular proliferation. Likewise, inhibition of
autophagy by CQ produced significantly pronounced down regulation in the expression of the
Notch1 signaling genes. Besides, the increase in number of proliferating cells appeared in the cell
cycle. However, in MDA MB 231 cell line, treatment of cells with either Tor or CQ led to up
regulation of Notch1 signaling genes. While, Tor appeared as a proliferative agent after inducing
autophagy, CQ gave pro-apoptotic effects after inhibiting autophagy.
Furthermore, in Tor-induced autophagy in HCC1806 cells, treating cells with CNC promoted
an anti-autophagic, pro-apoptotic and anti-proliferative effect after increasing the relative gene
expression of Notch1 signaling genes. Though, addition of Doxo to Tor-pretreated cells of HCC
1806 gave almost the same effect of CNC. Likewise, pro-apoptotic, anti-proliferative and antiautophagic
effects of CNC appeared in Tor pre-treated cells of MDA MB 231 cell line. With regard
to Notch1 signaling pathway in Tor pre-treated MDA MB 231 cells, CNC50 produced a significant
increase in the expression of the Notch1 signaling genes, while CNC10 resulted in down regulation
of Notch1 and Hes1 genes with slight increase in the expression of JAG1 gene. Also, treatment of
Tor-pretreated MDA MB 231 cells with Doxo revealed its pro-apoptotic and anti-proliferative
abilities as in CNC10.
Summary 86
In the same line, in CQ-inhibited autophagy of HCC1806 cells CNC increased accumulated
apoptotic cells in a concentration dependant manner exhibiting pro-apoptotic, anti-proliferative
effects with pro-autophagic activity in addition to a significant up-regulation in the relative gene
expression of Notch1 signaling genes. Similarly, treatment with Doxo elevated the accumulation of
apoptotic cells, reflecting it pro-apoptotic activity beside its anti-proliferative and pro-autophagic
effects with up-regulation of Hes1 gene expression. Meantime, in CQ-pre-treated cells of MDA
MB 231 cells, CNC50 resulted in significant up-regulation in the expression of the Notch1 and
JAG1 genes, with significant down regulation of Hes1 gene, accompanied by anti-autophagic and
proliferative effects. While CNC10 led to significant down regulation of the gene expression of
Notch1 and Hes1 genes with anti-proliferative and pro-autophagic effects. Moreover, treatment
with Doxo revealed its anti-proliferative and pro-autophagic effects with elevated level of
expression of Notch1 and JAG1 genes.
Compared to Tor-treated HCC 1806 cells, addition of CNC50-Doxo led to significant increase in
the expression of Notch1 signaling genes. These were associated with pro-apoptotic and antiproliferative
effects as well as slight elevation in autophagy. However, treatment with CNC10-Doxo
exhibited pro-apoptotic, anti-proliferative and anti-autophagic effects with significant downregulation
in Hes1 gene expression. On the other hand, treating Tor-pre-treated MDA MB 231 cells
with the combination of CNC and Doxo; CNC50-Doxo and CNC10-Doxo, increased the level of
apoptosis and anti-proliferation effects associated with up regulation of Notch1 signaling genes.
Treatment with CNC50-Doxo was found to have anti-autophagic effect but almost no change with
CNC10-Doxo.
Treating CQ-pre-treated cells of HCC 1806 with CNC50-Doxo gave significant elevation of
Notch1, JAG1 and Hes1 genes while CNC10-Doxo significantly elevated JAG1 gene expression
only. Also, CNC-Doxo caused elevation of both levels of autophagy and apoptosis. Whilst, cotreatment
with CNC-Doxo in MDA MB 231-CQ-autophagy inhibited cells exhibited proautophagic
effects with variable changes in cell cycle distribution and obvious changes in the gene
expression of Notch1 signaling pathway.
The resultant conclusion from this study is TNBC subtypes are different in their genetic
makeup and this can influence the choice of the right treatment. Moreover, Notch signaling
pathway is considered to be a convenient target for chemotherapeutic drugs in different subtypes of
TNBC. Besides, CNC exerted antitumor activities in TNBC. It can induce apoptosis and inhibit
proliferation through provoking Notch1 signaling genes. CNC can also be given in combination
with Doxorubicin to ameliorate its cytotoxic effects and reduces its side effects.
Summary 87
The present study also proposed the use of Notch1 targeted therapy with inhibiting autophagy
process in basal –like TNBC, but with its inducing in mesenchymal stem like TNBC. However the
Autophagy-Notch1 crosstalk in different subtypes of TNBC is complicated and requires further
investigations to be clarified and utilized in treating TNBC.