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 female 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 including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem–like (MSL), and a luminal androgen receptor (LAR) subtype. Thus, the molecular heterogeneity of TNBC confounds the clinical approach to TNBC treatment, thus, it is 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 several signals including Wnt/β-Catenin signaling pathways. It 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, activation of Wnt/β-Catenin pathway may lead to critical tumor hallmarks, including oncogene expression, angiogenesis, stem cell maintenance, deregulated cell cycle progression, and antiapoptotic mechanisms. Generally, activation of Wnt 1 plays a key role in the pathogenesis of TNBC cancer; it has a role in tumor initiation, proliferation, angiogenesis, and metastasis as well as tumor suppressor function. On the other hand, autophagy, an evolutionarily conserved catabolic process of self-digestion, plays an important role in the mechanism of keeping cellular homeostasis and survival. In cancer, Autophagy has both positive and negative roles and this has 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, 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
Summary 83.
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 role of CNC as a
targeted therapy for Wnt/β-Catenin signaling pathway in autophagy modulated TNBC cell lines.
This might explain more about the possible cross talk between Wnt/β-Catenin 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 stemlike
MDA MB 231 cell line. According to treatment, both TNBC cell lines were divided into:
group I: untreated TNBC cell line will serve as control group.
group II: CNC-treated Sub-Groups:
1. CNC Treated TNBC cell line.
2. CNC Treatment in up-regulated Autophagy TNBC cell line.
3. CNC Treatment in down-regulated Autophagy TNBC cell line.
group III: Doxorubicin (Doxo) treated Sub-Group:
1. Doxo Treated TNBC cell line.
2. Doxo Treatment in up-regulated Autophagy TNBC cell line.
3. Doxo Treatment in down-regulated Autophagy TNBC cell line.
group IV: CNC-Doxo treated Sub-Groups
1. CNC and Doxo Treated TNBC cell line.
2. Treatment with CNC and Doxo in up-regulated Autophagy.
3. Treatment with CNC and Doxo in down-regulated Autophagy.
Autophagy was up-regulated by torin-1 “Tor” treatment, while down-regulation was
carried out by chloroquin “CQ” treatment. Therefore, two additional groups of TNBC cell line
were considered; group V: torin-1 treated group and group VI: chloroquin treated group.
In present study, the half maximal inhibitory concentration for each treatment was
assessed and represented by (IC50) value by MTT assay. Biochemically, gene expressions of
Wnt/β-Catenin signaling pathway including Wnt 1, β-Catenin 1 and Cyclin D 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
Summary 84.
flow cytometer. Morphological changes were also observed using microscope. EM was used in
specific groups including untreated cell,CNC50, CNC10, DOXO, Tor, and CQ
The treating of both cell lines HCC1806 and MDA MB 231 with CNC10 resulted in down
regulation in the gene expressions of Wnt1, β-Cat and Cyclin D1. These were associated with weak
pro-autophagic, pro-apoptotic and proliferative activities. While with CNC50 a significant down
regulation in β-Cat gene expression was observed. This was associated with pro-autophagic activity
and pro-apoptotic or anti-proliferative.
When treated the two cell lines HCC1806 and MDA MB 231 with IC50 of DOXO which
resulted in a significant down regulation in gene expressions of Wnt1, β-Cat and Cyclin D1 expect
for the later in HCC1806 where significant up-regulation in CyclinD1 expression was noticed
associated with pro-autophagic activity. Interestingly, whereas Doxo exhibited a proliferative effect
in TNBC HCC 1806, a pro-apoptotic effect was noticed in TNBC MDA MB 231.
In HCC1806 cell, treatment with combination of DOXO-CNC10 resulted in a significant upregulation
in β-Cat gene expression and down regulation in Wnt1 and Cyclin D1gene expressions
with reduction in autophagic machinery and associated with the pro-apoptotic and anti-proliferative
activities. Upon treatment of HCC1806 with the combination of DOXO-CNC50, a significant upregulation
in Wnt1 gene expression associated with elevation of apoptosis ,anti-proliferative and
insignificant change in autophagy.
Treating TNBC MDA MB 231 cells with DOXO-CNC50 caused a significant up-regulation
Wnt1, as in HCC 1806 cells, and Cyclin D1 gene expressions. These associated with pro-apoptotic,
pro-autophagic and anti-proliferative activities. While treating cell with DOXO-CNC10 resulted a
significant up regulation with the increase in the autophagy, apoptosis and proliferation.
Treating HCC 1806 cells with TOR resulted in a significant up-regulation in β-Cat and
CyclinD1 gene expressions with enhanced cellular proliferation, autophagy and anti-apototic
effects. Whereas, treatment with CQ resulted a significant down-regulation in the expression of
Wnt1, β-Cat and Cyclin D1 genes in HCC1806 cells. These results associated with an increased
number of anti-apoptotic, anti-autophagic and proliferative effects.
While, treating MDA MB 231 cells with Tor caused a significant up-regulation in Wnt1 and
Cyclin D1 gene expression with pro-apoptotic and anti-proliferative effects. Treating MDA-MB-
231 cells with CQ revealed a significant up-regulation in Wnt1 gene expression associated with an
inhibition of autophagy and promotion of apoptosis.
Summary 85.
Treatment of Tor-pretreated HCC 1806, with CNC50 resulted in insignificant changes in
Wnt, β-Cat and CyclinD1 gene expressions associated with the pro-apoptotic and anti-autophagic
effects.
Interestingly, treating Tor-pretreated HCC 1806 cells with CNC10 caused a significant upregulation
in Wnt1 gene expression and down-regulation in β-Cat and Cyclin D1 gene expressions.
Also, treating Tor-pretreated HCC 180 cells with CNC10 exhibited anti-autophagic, pro-apoptotic
and anti-proliferation effects.
However, with regard to Tor-pretreated MDA MB 231, the response to treatment was
different that observed in HCC 1806 cells. Upon treatment with CNC; CNC50 or CNC10: a
significant up-regulation in Wnt1and Cyclin D1 gene expressions was noticed. Also, CNC
treatment showed anti-autophagic, pro-apoptotic and anti-proliferation effects.
On the other hand, with regard to CQ-pretreated HCC 1806 cells, treatment with CNC50
resulted in a significant up-regulation in β-Cat gene expression. These was associated with proautophagic
and pro-apoptotic effects. . Meanwhile, CQ-pretreated HCC 1806 cells, treatment with
CNC10 caused significant down-regulation Wnt1 and β-Cat gene expressions. And associated with
pro-autophagic, pro-apoptosis and proliferation.
In CQ-pretreated MDA MB 231 cells, treatment with CNC50 significantly up-regulated
Wnt1, β-Cat and Cyclin D1 gene expressions with enhancement of proliferation and decrease in cell
death pathways, apoptosis and autophagy. While treatment with CNC10 resulted a significant up
regulation in the Wnt1 associated with pro-autophagic , pro-apoptosis and proliferation.
Meanwhile, addition of Doxo to Tor-pretreated HCC 1806 cells exhibited a pro-apoptotic,
slight pro-autophagic and anti-proliferation activities. These observations were associated with a
significant down-regulation in Wnt1 gene expression a significant up-regulation in Cyclin D1 gene
expression. With regard to modulation of autophagy with CQ, an autophagy inhibitor, treatment of
CQ-pre-treated HCC 1806 cells with Doxo resulted in an a significant down-regulation in wnt1 and
up regulation β -cat1 gene expression. Also, it demonstrated promotion of autophagy, apoptosis and
anti-proliferative effects. Similarly, also, treatment of Tor-pretreated MDA MB 231 with Doxo
revealed its role in promoting cell death by autophagy and apoptosis.However, such treatment
demonstrated a significant up-regulation in Wnt1 and down-regulation in β-Cat gene expressions.
Summary 86.
In HCC 1806 cells Co-treatment of Tor-treated with CNC-Doxo; CNC50-Doxo or CNC10-
Doxo, promote apoptosis and anti-proliferation effects. Whereas, co-treatment with CNC50-Doxo
showed pro-autophagic effect, co-treatment with CNC10-Doxo exhibited anti-autophagic
activity.The later also down-regulated significantly gene expression of Wnt1 signaling genes; Wnt1,
β-Cat1 and Cyclin D1.while treatment with CNC50-Doxo exhibited a significant up-regulation in
gene expressions of Wnt1 and Cyclin D1 with significant down-regulation in β-Cat gene
expression.
With regard to Co-treatment of CQ-treated HCC 1806 cells with CNC-Doxo; CNC50-Doxo
or CNC10-Doxo, promote apoptosis, autophagy and anti-proliferation activities accompanied by
down regulation in Wnt1 gene expression.
In MDA MB 231 pre-treated cells with Tor, treatment with either CNC50-Doxo or CNC10-
Doxo promotes apoptosis and anti-proliferation of cells. Moreover, treatment of with CNC50-Doxo
up-regulated gene expressions of WNt1, β-Cat and Cyclin D1. While the co-treatment with CNC10-
Doxo where generally it elevated the expression of Wnt1, β-Cat and Cyclin D1.
CQ pre-treated in MDA MB 231 cells, with combination of CNC50-Doxo led to significant
down-regulation of Wnt1 gene expression with significant up-regulation β-Cat and Cyclin D1 gene
expression with pro-autphagic and other changes in cell cycle distributions. However, in CQ pretreated
MDA MB 231and co-treated with CNC10-Doxo exhibited also a pro-autophagic effect with
significant up-regulation in Wnt1 and β-Cat gene expressions.
Collectively, Wnt1 signaling genes in either basal like subtype or mesenchymal stem-like
cells have responded similarly upon treatment with CNC or Doxo and consequently, it may
represent a convenient target for chemotherapy in TNBC. The correlations among its Wnt1
signaling genes in MSL sub-type may provide it as an aim for targeted therapy in this sub-type of
TNBC. The ability of CNC to induce apoptosis and decrease the oncogenic activity of Wnt1 may
support CNC as a promising anticancer agent for treatment of TNBC considering its concentration.
Interestingly, the co-treatment of CNC and Doxo can serve as a potential cytotoxic regimen which
may reduce the side effects of Doxo besides, the synergistic effect of both CNC and Doxo.
Furthermore, the data of the present study may fulfill that inhibiting autophagy process in BL
subtype, but its induction in MSL subtype might ameliorate the clinical outcome. However, the
autophagy-Wnt1 crosstalk in different TNBC subtypes may be complicated and require further
investigations to address the relation between these two crucial processes and their impact on the
treatment of TNBC considering its sub-types.