الفهرس 
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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 subtype
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 pathways including Autophagy.
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.
Although there is no approved targeted therapy available for TNBCs, molecularprofiling
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-Platinum compounds, copper complexes are potentially attractive as
anticancer agents. Special emphasis has been focused on the identification of structureactivity
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, anti-inflammatory and anti-tumor activities.
The present study is an attempt to shed more lights on the potential effect of CNC as
a targeted therapy in autophagy modulated TNBC cell lines.
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:
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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 CNC.
III. Doxo-treated Group: treated with IC50 of doxorubicin.
IV. CNC-Doxo treated Groups:
1. CNC50-Doxo: treated with combination of IC50 CNC and IC50 of Doxorubicin.
2. CNC10-Doxo: treated with combination of 10% of IC50 of CNC and IC50 of
Doxorubicin.
V. Torinl-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 with 10% 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 with 10% 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 of IC50 of CQ for 24hrs then with
IC50 of CNC.
2. CQ-CNC10 treated group: cells were pre-treated with 10% of IC IC50 of CQ for
24hrs then treated with of IC50 of CNC.
X. CQ-Doxo-treated Group: cells were pre-treated with of IC50 of CQ for 24hrs then
with of Doxo.
XI. Tor-CNC-Doxo treated Groups:
1. Tor-CNC50-Doxo: cells were pre-treated 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 Of IC50 of Tor for 24hrs then treated with
combination IC50 of CNC and IC50 of Doxorubicin.
XII. CQ-CNC-Doxo treated Groups:
1. CQ-CNC50-Doxo: cells were pre-treated 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 of IC50 of CQ for 24hrs then treated with
a combination of 10% of IC50 of CNC and IC50 of doxorubicin.
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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 different stages of autophagy including Beclin1 representing nucleation,
ATG16L1 representing elongation, and RAB24 representing maturation were assessed by
RT-PCR using specific primers and ß-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, Tortreated,
CNC50 –treated, CNC 10-treated and Doxo-treated cells. Inverted microscope
showed morphological alterations, shrinking and 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 cell lines responded differently to the applied treatments; and this might be
due to the biological and heredity heterogeneity of TNBC subtypes. Generally, the present
study showed that treatment HCC 1806 cell line with CNC; CNC50, resulted in
upregulation in relative gene expressions of Beclin1 and ATG16L1, with increasing the
level of apoptosis and autophagy. In contrast, treating HCC1806 TNBC with the lower
concentration CNC10 led to significant down-regulation in the relative expressions of
Beclin, ATG16L1, and RAB24 genes reflecting anti autophagic effect along with
proliferative effect. While, in MDA MB 231 cell line CNC; CNC50, resulted in a
significant down regulation in relative gene expression of Beclin1, and up regulation in the
relative expression of RAB24, ATG16L1 genes to exert pro autopahagic anti-proliferative
effects but still couldn‘t drive cells into apoptosis. Treating HCC1806 TNBC cells with
Doxo resulted in an up-regulation of the relative expression of Beclin1 and ATG16L1
genes associated with proliferative activities reflecting the chemoresistance of BL TNBC
cell line.
As for MSL TNBC cell line treatment with CNC10 produced similar effects as that
caused Doxo Pro autophagic pro apoptotic effect also upregulation in relative gene
expression of Beclin1 and down regulation in relative genes expression of ATG16L1 and
RAB24.
In attempt to attain better response of Doxo, co-treatment with CNC as a possible
anti autophagic agent was considered in the present study to appraise the possibility of
sensitizing TNBC to treatment of Doxo. In HCC 1806, co-treatment of Doxo and CNC,
displayed a pro-apoptotic and anti-proliferative effect associated with a minor decrease in
autophagy after the non-significant down regulation of relative expressions of both Beclin1
and ATG16L1 genes. Similarly MDA MB 231, CNC-Doxo with both concentration caused
down regulation of Beclin1 signaling gene and up regulation of ATG16L1 and RAB24
genes revealing anti autophagic, pro-apoptotic and anti-proliferative effects. This may gave
a privilege of CNC-Doxo treatment over treatment with Doxo solely and hence, reducing
the negative side effects of Doxo treatment in both cell lines.
On the other hand, Induction of autophagy with Tor resulted in a significant up
regulation in Autophagy genes in both cell lines exerting cyto-protective effect in HCCI
1806 cell line and cyto-toxic effect in MDA MB 231 cell line. In contrast, inhibition of
autophagy by CQ produced down regulation in the relative gene expression of Autophagy
genes in both cell lines to be significantly pronounced in MDA MB 231 cell line, also a
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pro-apoptotic and anti-proliferative effects after inhibiting autophagy was noticed in MDA
MB 231 cell line. In contranst, the increase in number of proliferating cells appeared in the
cell cycle in HCC 1806 TNBC cell line after autophagy inhibition.
Furthermore, in Tor-induced autophagy in HCC1806 cells, treating cells with CNC
promoted an anti-autophagic, pro-apoptotic and anti-proliferative effect after
downregulating the relative expression of Beclin1, ATG16L1, and RAB24 genes to be
more significant in the lower concentration of CNC10. Though, addition of Doxo to Torpretreated
cells of HCC1806 gave similar effect to the one exerted by CNC10.
Likewise, pro-apoptotic, anti-proliferative and anti-autophagic effects of CNC10
appeared in Tor pre-treated cells of MDA MB 231 cell line along with significant
downregulating the relative expression of Beclin1, ATG16L1, and RAB24 genes. With regard
to the exact genes in Tor pre-treated MDA MB 231 cells, CNC50 produced general
upregulation in their relative expression. Also, treatment of Tor-pretreated MDA MB 231 cells
with Doxo was synergistic revealing their pro autophagic, pro-apoptotic and anti-proliferative
abilities as in CNC50. As a result of these findings, inducing the process of autophagy can be
used to achieve good clinical outcome in both BL and MSL subtypes of TNBC.
In the same line, in CQ-inhibited autophagy of HCC1806 cells CNC increased
accumulated apoptotic cells in a concentration dependent manner exhibiting pro-apoptotic,
anti-proliferative effects with pro-autophagic activity reflected by the significant
upregulation of Beclin1 and ATG16L1 relative gene expressions as essential autophagy
genes. Similarly, treatment with Doxo elevated the accumulation of apoptotic cells,
reflecting its pro-apoptotic and anti-proliferative activity but also its anti-autophagic
activity caused by general down regulation in relative expression of Beclin1, ATG16L1,
and RAB24, along with atophagosome accumulation.
from the previously discussed observations; Tor-CNC50 was found to give more
apoptotic effect claiming that induction of autophagy in MSL subtype of TNBC would
give convenient cell fate, unlike BL subtype which responded better to autophagy
inhibition using CQ-CNC50.
In the present study an attempt to explore the influence of autophagy modulation on
the potential cytotoxic role of autophagy, CNC-Doxo co-treatment was carried out. Thus,
Tor-activated autophagy HCC 1806 cells were co-treated with CNC50-Doxo which led to a
significant up regulation in Beclin relative gene expression. These were associated with
pro-apoptotic and anti-proliferative effects as well as slight elevation in LC3 positive cells.
However, treatment of autophagy activated HCC1806 with CNC10-Doxo exhibited proapoptotic,
anti-proliferative and anti-autophagic effects. The increase in apoptotic cell
death could be referred to the significant down regulation in autophagy related genes
Beclin1 and ATG16L1 with the reduced autophagic outpot (LC3).
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 antiproliferation
effect. However, with regard to autophagy the co-treatment with different CNC
concentrations gave different effects, as CNC50-Doxo was found to have anti-autophagic
effect due to significant down regulation of both autophagy related genes Beclin1 and
ATG16L1. In contrast, treating cells with CNC10-Doxo resulted in significant upregulation
of both genes along with slight increase in LC3. Accordingly, it could be suggested that
CNC-Doxo co-treatment in Tor-induced autophagy can be a promising treatment strategy for
both BL and MSL TNBC considering CNC concentration for each subtype.
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In an attempt to establish a more effecint therapeutic strategy, co-treatment of CNC
and Doxo into CQ-inhibited autophagy cells was carried out. Thus, on one hand, treating
CQ-inhibited cells of HCC 1806 with CNC50-Doxo caused down regulation of autophagy
related genes relative expressions; Beclin and ATG16L1 along with autophagosome
accumulation. Also, co treatment of CNC-Doxo of CQ-inhibited autophagy cells led to
shift into apoptosis. Regardless of the mechanism (s) involved in fate of cells CQpretreated,
it could be suggested that autophagy inhibition is a convenient strategy to attain
worthy clinical outcomes on co-treating BL TNBC with CNC-Doxo.
On the other hand, co-treatment of MDA MB 231-CQ-autophagy inhibited cells with
CNC-Doxo exhibited pro-autophagic effects with presented by up-regulation in Beclin1,
ATG16L1, and RAB24 relative genes expression. These were associated with proliferative
activity observed by decrease in apoptotic cells and increase proliferative cellular
population. Thus, co-treatment with CNC-Doxo regimen overcomes the autophagyinhibitory
effect of CQ. Under these conditions, autophagy may exhibit its cyto-protective
and chemo-resistance activities (172). This, in turn, may lead to the suggestion that
autophagy inhibition is not the strategy of choice in treating this subtype of TNBC. This
suggestion is supported by the obtaining the same observations on treating CQ-autophagy
inhibited cells of MSL TNBC with Doxo.
Collectively, biological and heredity heterogeneity of TNBC may have a crucial role
in drug sensitivity and chemo resistance. This could be the corner stone for the observed
different responds of autophagy related genes upon treatment with CNC or Doxo or their
combinations.In the present study, the ability of CNC to induce apoptosis and to inhibit
cellular proliferation may support more CNC as a promising targeted anticancer agent for
treatment of both BL and MSL subtypes of TNBC considering its utilized concentration.
Also, the ability of CNC to potentiate the cytotoxicity of chemotherapy in both
subtypes of TNBC was revieled. This, in turn may reduce the side effects of Doxo through
decreasing of its utilized therapeutic dose and attain promising clinical outcomes.
Furthermore, several questions concerning autophagy modulation in TNBC upon
efficacy of the chemotherapeutic regimen and cell fate were raised. Thus, although many
studies favored autophagy inhibition based protocols in treating TNBC, in the view of the
results of the present study, it could be suggested that autopagy induction either with Doxo,
CNC, or specially their combination can be considered as a promising line of treatment
either for BL or MSL TNBC.