Influence of Copper (I) Nicotinate Complex on the Notch1 Signaling Pathways in Triple Negative Breast Cancer Cell Lines

doi:10.21203/rs.3.rs-2451740/v1

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Influence of Copper (I) Nicotinate Complex on the Notch1 Signaling Pathways in Triple Negative Breast Cancer Cell Lines

https://doi.org/10.21203/rs.3.rs-2451740/v1

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TNBC is a subtype of breast cancer which is characterized by its aggressiveness, poor survival and short overall survival. Notch signaling pathway has received great attention as one of the most important potential targets for developing a novel therapeutic strategy.

Purpose, the present study is an attempt to assess the promising chemotherapeutic activities of copper (I) nicotinate (CNC) through its impact on the expression of downstream genes of Notch1 signaling pathway and the cell fate of TNBC. The co-treatment of TNBC cells with doxorubicin (Doxo) and CNC was also investigated.

Methods, to approach the objective of the present study, triple negative breast cancer HCC1806 and MDA MB 231 cell line were utilized. Cell viability assay, MTT assay, was used to determine IC50 values of CNC and Doxo. After treatment, cell cycle distribution and indirect antibody labeling of microtubule-associated protein light chain 3 (LC3) were determined by flow cytometry. Real time PCR was used to detect the changes in gene expressions that are involved Notch1 signaling pathway. Moreover, autophagosomes were monitored and imaged by TEM.

Results, treatment of TNBC cells with CNC modulated Notch1 signaling pathways in different manners with respect to the type of cells and the applied dose of CNC.

Conclusion, the observed pro-apoptotic of CNC in addition to its pro- or anti- autophagic activities may reflect the possible anti-cancer activities of CNC in both types of TNBC. However, cell type and CNC dose should be considered.

Triple negative breast cancer

Copper (I) nicotinate complex

Doxorubicin

Apoptosis

Autophagy

Notch1 signaling pathway

Based on specific gene expression patterns, breast cancers are categorized into five intrinsic or molecular subtypes. Among the intrinsic subtypes, basal-like triple-negative breast cancer (TNBC) accounts for 12-20% of breast cancers (1). In Egypt, according to (NCI-CU) cancer 12-years registry; TNBC constitutes about 13.5% of breast cancer (2). TNBC has drawn specific attention due to the lack of expression of all three receptors, ER, PR, and Her2. Thus, it cannot be treated using anti-estrogen hormonal therapies or trastuzumab (3). Thus, developing a more effective therapeutic method for patients with TNBC is necessary.

However, inappropriate activation of signal transduction, and indeed the inability to respond to negative signals, is a hallmark of cancer. Deeper insights into the pertinent and targetable signaling pathways in TNBC may offer new avenues for therapeutic intervention. Notch signaling is aberrantly activated in breast cancer, with increased Notch intracellular domain accumulation and target gene expression detected in a range of breast cancer cell lines and primary samples (4-6). Over- expressions of Notch receptors and ligands have been reported in breast tumours, and is correlated with poorer patient prognosis (7). Aberrant Notch signaling has also been extensively linked to the triple negative breast cancer (TNBC) subtype; Notch receptor overexpression is correlated with the aggressive, metastatic and therapy resistance phenotype characteristic of TNBC (8,9).

On the other hand, since the discovery of cisplatin, a great deal of research has been conducted on the therapeutic applications of metal-based complexes. Despite the challenges emanating from the clinical use of platinum compounds, there is a growing demand for metal-based compounds in cancer therapy. In this regard, copper complexes were found to have a less toxic effect on normal cells and could stimulate apoptotic cell death (10,11). Thus, many of copper complexes have shown anticancer activity due to their ability to produce reactive oxygen species (12). Generally, it should be noted that trafficking of copper (I) complexes in cells is preferable than copper (II) compound (13). Evidentially, copper (I) nicotinate complex (CNC) displayed several anti-tumor activities in TNBC including pro-apoptosis, anti-autophagy as well as anti-angiogenesis (11). Accordingly, CNC was suggested as a promising anti-cancer agent in treatment of TNBC despite of further studies were recommended. Therefore, and based on the previously mentioned observations concerning CNC, the present study was undertaken to reveal the possible utilization of Notch1 signaling pathways as a molecular target in the therapy of TNBC with CNC.

Preparation of Copper (I)-Nicotinate Complex

The [Cu (I)-(nicotinic acid)2] +Cl- complex‘CNC’ was synthesized as described by Gohar and Dratoviscky in 1975 (14) through the reaction of ethanolic solution of CuCl2-2H2O with nicotinic acid in the presence of L+- ascorbic acid. The obtained pure bright red needle crystals were examined by an infrared spectrum to confirm the chemical structure of Cu (I)-(nicotinic acid)2Cl-2H2O (15).

Cell Culture

TNBC cell lines, HCC1806 (ATCC®CRL-2335™) and MDA-MB-231 (ATCC®HTB-26™), were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells of both cell lines were maintained at 37 °C, 5% CO2 in 10% DMEM (Lanza Bioproduct, Belgium) supplemented with 10% fetal bovine serum (Sera Lab, UK).

Cell Proliferation Analysis

Cell viability and proliferation of both cell lines HCC 1806 and MDA-MB231 was measured by MTT Cell Proliferation Assay, according to the manufacturer’s protocol (ATCC). Cells were cultured into 96-well tissue culture plates and treated with different concentrations of CNC and Doxo, optical density was measured at 490 nm by Plate reader (BioTek Company). The half maximal inhibitory concentration is represented by (IC50) values.

Design of Experiment

Both cell lines were treated with either CNC50, or CNC10, or Doxo, or CNC50- Doxo, or CNC10- Doxo and incubated for 24 hours. Where CNC50 and CNC10 are the IC50 and 10% of IC50 of CNC, respectively.

Cell Morphology

Alterations in cell morphology were examined under an inverted microscope with magnification of 200X.

Cell Cycle Analysis

Cells of HCC 1806 and MDA MB 231 were collected and trypsinized after treatment according to the previously mentioned experimental design. After fixation with ice-cold 70 % ethanol, cells were centrifuged for 5 min at 600×g, washed and re-suspended with PBS (16). Afterwards cells were stained with 100μg/ml PI (Immunostep S.L, Spain) and examination of cell cycle distribution was carried out using FACS Calibur (Becton Dickinson, CA, USA).

Indirect Antibody Labeling of LC3-II

Cells were collected, re-suspended in 1xPBS, fixed in 4% formaldehyde for 10minutes. Afterwards,cells are permeabilized by addition of ice-cold 100 % methanol to pre-chilled cells, while gently vortexing to give final concentration of 90% methanol then incubated for 30mins. Immune-staining by re-suspension of cells in 100μL of primary antibody is done then samples are analyzed by a flowcytometer (17).

Monitoring autophagy by electron microscopy

Specimen of cells is fixed, sectioned and stained (18) to be prepared for imaging of autophagosomes by JEM-1400 plus Electron Microscope.

Real-Time PCR Analysis of Notch1, Jagged1 and Hes1 genes

Real-time PCR was performed by iCycler-iQ Optical (Bio-Rad). Total RNA is extracted by TRIzol method according to the mRNA extraction protocol made by abcam®. Reverse-transcriptase enzyme was synthesized to convert the extracted RNA into cDNA using the protocol provided by HISenScript™ RH [-] cDNA Synthesis Kit (iNtRON, Korea). The analysis was performed using the following primers;

βactin-F:5’-TGGCACCACACCTTCTACAATGAGC-3’,R:5’- GCACAGCTTCTCCTTAATGTCACGC-3’

Notch1-F:5’-CCGCCTTTGTGCTTCTGTT-3’,R:5’-TCCTCCTCTTCCTCGCTGTT-3’,

Jagged1-F:5’-GATCCTGTCCATGCAGAACG-3’,R:5’-GGATCTGATACTCAAAGTGG-3’,

Hes1-F:5’-GACAGCATCTGAGCACAGAAATG-3’,R:5’-GTCATGGCATTGATCTGGGTCAT-3’.

PCR amplification was performed using SYBR Green with low ROXTOPreal™ qPCR 2XpreMIX (Enzynomics, Korea). The relative mRNA expression levels were calculated using the comparative (Ct) method (2^-ΔCTequation) where, ΔCT equals (CT of gene of interest - CT of housekeeping gene). Data were expressed as relative mRNA expression/million cells.

Statistical analysis

Statistical analysis was carried out with SPSS 22.0 for windows. For comparison of the quantitative variables, Mann Whitney’s test was used to compare between means of different studied groups. Biochemical Correlation among studied parameters was done using Spearman’s correlation coefficient. Generally, (p) values were considered statistically significant at level ≤ 0.05.

IC₅₀ values of CNCN and DOXO

IC₅₀ values for CNC and Doxo in HCC 1806 are; 100.0 μg/ml and 061.5 μg/ml respectively. while, IC₅₀ values for CNC and Doxo in MDA MB 231 cells are 02.50 mg/ml and 00.15 mg/ml respectively.

Cell Morphology

Visualization of morphological alterations by inverted microscope showed the shrinking and detachment of most of cells after treatment of drugs in accordance to the previously stated protocol, Fig.1.

Cell Cycle kinetics

Effect of treatment of CNC/ Doxo

Treatment of HCC1806 cells with CNC50 resulted in accumulation of cells in sub G1 phase with slight decrease in cells at G0/1 phase. However, treatment with CNC10 decreased the accumulated cells in G0/1 and increased cells at S phases. In same manner, treating of HCC 1806 cells with Doxo caused an increase in cells at S and G2/M phases and decrease in cells in subG1 and G0/1 phases, (Figure. 2) (Figure.4).

On the other hand, treatment of MDA MB 231 cell line with CNC50 caused an increase in cells arrested at G0/1 phase with a decrease in cells at G2/M phase. Also, treatment with CNC10 showed an increase in apoptotic cells at sub G1 phase. Similarly, treating MDA MB 231 cells with Doxo resulted in a slight increase in apoptotic cells at sub G1 phase, (Figure.2) (Figure.4).

Effect of co-treatment of CNC and Doxo

In HCC1806 cells, compared to Doxo-treated cells, treatment with both CNC50-Doxo and CNC10-Doxo gave rise to an increase in the apoptotic cells accumulated at sub G1 phase with decrease in proliferating cells at S phase, (Figures 3&.4). Similarly, treating cells of MDA MB 231 cell line with either CNC50-Doxo or CNC10-Doxo led to an increase in apoptotic cells at sub G1 phase, and decrease in cells at S phase, (Figures 3&.4).

Indirect Antibody Labeling of LC3-II

Effect of treatment of CNC/Doxo

Treatment of HCC 1806 with either CNC50 or CNC10 slightly increase the number of LC3 positive cells, (Figures 5&.7). Also, treatment with Doxo resulted in an increase in the number of LC3 positive cells, ((Figures 5&.7). In MDA MB 231 cell line, treatment with CNC; CNC50 or CNC10, increased the number of LC3 positive cells in a dose dependent manner, (Figures 5&.7). Meanwhile, treatment with Doxo led to greater increase in the number of LC3 positive cells, (Figures 5&.7).

Effect of CNC and Doxo co-treatment

Compared to Doxo-treated cells of HCC 1806, treating cells with a combination of CNC50-Doxo gave rise to minute elevation of the LC3 positive cells whereas; the combination of CNC10-Doxo decreased the number of LC3 positive cells, (Figures 6&.7). With regard to MDA MB 231 cell line, reduced number of LC3 positive cells was observed upon treatment with a combination of CNC50-Doxo, but no change was noticed with CNC10-Doxo treatment, (Figures 6&.7).

Electron Microscopy Monitoring of Autophagy

Transmission electron microscopy (TEM) was used for detection and identification of autophagosomes in selected control groups used in this study; untreated cells, CNC50, CNC10 and Doxo, (Figure.8).

RT-PCR Analysis of Notch1, JAG1 and Hes1 gene expressions

Effect of CNC/Doxo treatment

Treatment of HCC 1806 TNBC cell line with either CNC50 or CNC10 resulted in a down regulation in the gene expression of Notch1, JAG1 and Hes1genes. Sharp and significant down-regulation in the gene expression of Notch1 and JAG1 genes were observed in HCC1806 TNBC when treated with CNC50 but was significant for Notch1, JAG1 and Hes1 genes upon treatment with CNC10 (p≤0.05), (Table. 1). Also, the gene expression of Hes1 gene was significantly down regulated by the effect of CNC10 when compared to that of CNC50 (p ≤0.05), (Table. 1). In contrast to HCC 1806 cell line, treatment of MDA MB 231 cell line with CNC50 or CNC10 resulted in up regulation in the gene expression of Notch1, JAG1 and Hes1genes. Significant up regulation was observed with the gene expression of JAG1 and Hes1genes (p≤0.05), (Table. 1). Also, the gene expression of JAG1 gene was significantly up regulated by the effect of CNC50 when compared to that of CNC10 (p ≤0.05), (Table. 1). However, Treatment of both cell lines; HCC 1806 and MDA MB 231, with Doxo induced the same effects as CNC in the corresponding cell line. Where, in HCC 1806 cell line Doxo caused a significant down regulation in the gene expression of Notch1and JAG1 genes (p≤0.05), (Table. 1). Meanwhile, in MDA MB 231 it led to a significant up regulation in the gene expression of Notch1, JAG1 and Hes1genes (p≤0.05), (Table. 1).

Table (1): Effect of CNC/Doxo treatment on the relative gene expressions of Notch1, JAG1 and Hes1 genes in TNBC cell lines, HCC 1806 and MDA MB 231

	Notch1	JAG1	Hes1
HCC1806 cell line
Untreated48hrs
X̅ ± S.E.	220 ± 48	32.8 ± 4.0	2186 ± 806
CNC50
X̅ ± S.E.	8.97 ± 1.58 ^a	6.35 ± 0.89 ^a	631 ± 82
CNC10
X̅ ± S.E.	5.77 ± 0.78 ^a	4.9 ± 1.4 ^a	26.3 ± 11.4 ^a,b
Doxo
X̅ ± S.E.	1.2 ± 0.15 ^a	5.2 ± 1.6 ^a	1170 ± 287
MDA MB 231 cell line
Untreated 48hrs
X̅ ± S.E.	140 ± 1.96	25.0 ± 3.1	835 ± 350
CNC50
X̅ ± S.E.	34830 ± 7355 ^a	499366 ± 208594 ^a	1793791 ± 1089429 ^a
CNC10
X̅ ± S.E.	195 ± 26.3 ^b	97 ± 12.8 ^a,b	27656 ± 6149 ^a
Doxo
X̅ ± S.E.	4163 ± 923 ^a	81833 ± 34152 ^a	18177149 ± 4064394 ^a

Relative gene expression was measured per million of cells.

a: Statistically significant when compared to untreated control group

b: Statistically significant when compared to CNC50-treated group

(p) Values were considered significant at level ≤ 0.05

Effect of CNC and Doxo co-treatment

Compared to Doxo-treated cells, treatment of HCC 1806 cell line with a combination of CNC50-Doxo or CNC10-Doxo resulted in a significant up regulation in the gene expression of Notch1 and JAG1 genes (p≤0.05), (Table. 2). Also, a significant up regulation in the gene expression of Hes1 gene was observed upon treatment with CNC50-Doxo (p≤0.05), (Table. 2). However, down regulation in the gene expression of Hes1 gene with CNC10-Doxo (p≤0.05), (Table. 2). On the other hand, treating cells of MDA MB 231 with CNC50-Doxo caused a significant up regulation in the gene expression of JAG1gene with significant down regulation of Hes1 gene (p≤0.05), (Table. 2). While, CNC10-Doxo led to a significant down regulation in the gene expression of Notch1 and Hes1 genes (p≤0.05), (Table. 2).

Table (2): Effect of CNC-Doxo treatment on the relative expression of Notch1, JAG1 and Hes1 genes in TNBC cell lines; HCC1806 and MDA MB 231.

	Notch1	JAG1	Hes1
HCC1806 cell line
Doxo
X̅ ± S.E.	1.2 ± 0.15	5.2 ± 1.6	1170 ± 287
CNC50-Doxo
X̅ ± S.E.	2267 ± 585 ^a	1168 ± 173 ^a	101509 ± 31723 ^a
CNC10-Doxo
X̅ ± S.E.	96.5 ± 24.4 ^a,b	28.9 ± 7.17 ^a,b	661 ± 67 ^b
MDA MB 231 cell line
Doxo
X̅ ± S.E.	4163 ± 923	81833 ± 34152	18177149 ± 4064394
CNC50-Doxo
X̅ ± S.E.	13911 ± 5048	668966 ± 99711 ^a	500252 ± 114009 ^a
CNC10-Doxo
X̅ ± S.E.	392 ± 89 ^a	662 ± 265	10996 ± 4969 ^a,b

Relative gene expression was measured per million of cells.

a: Statistically significant when compared to Doxo-treated control group

b: Statistically significant when compared to CNC50-Doxo-treated group

(p) Values were considered significant at level ≤ 0.05

Two different subtypes of TNBC were utilized in the present study; the acantholytic squamous (19) basal-like HCC 1806 cell line, and the mesenchymal stem-like MDA MB 231 cell line (20). Presently, cell cycle analysis of untreated cells of both cell lines revealed that the percentage of proliferating cells, in HCC1806 cell line was obviously less than that in MDA MB 231 cell line. Also, the basal level of autophagy in the two cell lines was different, where positive cells for LC3 were 53% and 81.9% in HCC 1806 and MDA MB 231 cell lines, respectively. Additionally, electron micrograph of the two cell lines showed that autophagosomes in MDA MB 231 were more obvious than in HCC 1806 cell lines. In consistence with previous report (11), the IC50 values for CNC in both cell lines showed that HCC 1806 was more sensitive. All of these observations may contribute to the differences in the malignant behavior and chemosensitivity in both cell lines. Volk-Draper, et al., demonstrated the dramatic differences in sensitivity to chemotherapeutic drugs where HCC1806 cells were more sensitive (20), while MDA MB 231 cells are highly resistant to multiple cytotoxic agents and possess stem cell phenotypes (21). Thus, expectedly the two cell lines will respond differently to the applied treatment.

Effect of treatment CNC or Doxo

Cell cycle analysis of HCC1806 TNBC treated with CNC50 revealed an increase in cell population arrested in Sub G1 revealing its pro-apoptotic effect. Meanwhile, treating HCC1806 TNBC with CNC10 resulted in increased the cell cycle arrest at S phase manifesting its proliferative effect. Unlike HCC 1806 TNBC cell line, treating MDA MB 231 TNBC cell line with CNC50 resulted in arrested of cells at G0/G1 with decreased cell population at G2/M phase, showing its proliferative effect. Treatment with CNC10 displayed an increased cell at sub G1 phase reflecting its pro-apoptotic activity.

Furthermore, sharp and significant down-regulation in the gene expression of Notch1 and JAG1 genes were observed in HCC1806 TNBC when treated with CNC50 but was significant for Notch1, JAG1 and Hes1 genes upon treatment with CNC10. This was associated with increase in LC3 positive cells denoting elevation in autophagy. These observations are in consistence with the previous reports which claimed that; inhibition of Notch expression in different types of cancers resulted in growth arrest (22) and induction of autophagy (23,24) and during stem cell differentiation (25). Evidently, inhibition of Notch has most frequently slow cancer cell proliferation and trigger apoptosis in cancer cells (26). These observations might result from the ability of CNC to produce ROS (27) in concentration dependent manner. ROS have been reported to be involved in the regulation of Notch1 signaling pathway (28) and promote autophagy and apoptosis (29). Thus, it could be suggested that the final fate of cells will mainly be CNC concentration dependent. The existence of autophagy and apoptosis together in the same cell is not surprising (30), as long as programs of cell death can involve autophagy working alongside apoptosis (31). Autophagy may occur before, but independently from, apoptosis (30), it facilitates apoptotic activation (32), also can be directly stimulated by apoptosis (33). Thus, as previously suggested (11), CNC can be used as an anticancer agent in HCC1806 cell line targeting Notch1 signaling pathway but its concentration should be considered.

The previous suggestion may be more supported by the observation that CNC almost induced similar effects as that observed upon treating HCC 1806 cells with Doxo especially with CNC10. Thus, treatment with Doxo led to down regulation of Notch1 signaling genes; this down regulation was significant with Notch1 and JAG1 genes. Regarding its effect on autophagy process, Doxo resulted in an increase in LC3 positive cells to 78.8% and presence of autophagosomes. These observations are in accordance with previous studies suggested the pro-autophagic effect of Doxo through induction of cytoprotective autophagic flux which diminishes the cytotoxic effects of Doxo (34,35). Thus, may explain the observed proliferative activities of either Doxo or CNC10 through increasing cell cycle arrest at S phase and decreasing in G0/1 cell arrest. This is in accordance with the observed significant negative correlation between proliferation and relative gene expressions of Notch1 and JAG1 in HCC1806 cell line.

Treating MDA MB 231 TNBC cell line with CNC50 resulted in a sharp and significant up regulation in the expression of the Notch1 signaling genes associated with an increase in the number of LC3 positive cells and the presence of autophagosomes. In consistence with cell cycle analysis, Notch hyper-activity favors cell survival and inhibition of apoptosis and may be essential for cell division (34,35). Meanwhile and with regard to CNC10 treatment, it resulted in up regulation of Notch1 signaling genes with pro-autophagic and pro-apoptotic activities. Recently, Tamagnone, et al., 2018, have reported that Notch can also act as a tumor suppressor in certain contexts (36). Moreover, several reports have pointed out to the possibility of Notch1 signaling may have a potent tumor suppressor role of in both hematological malignancies and solid tumors and its activation may inhibit cellular growth (37-40). Generally, Notch activation is known to promote cancer development, whereas it can also play a tumor suppressor role. Biologically, this is not surprising, in view of the opposite role that Notch signaling can exert either a pro- or anti-apoptotic function through multiple mechanisms that are highly cell- and context dependent (41). Therefore, it could be suggested that the increase in apoptosis in MDA MB 231 cell line upon CNC10 treatment may attributed to provoking the tumor suppressing effect of Notch1 signaling. As explained before, CNC-anticancer activity might be attributed to ROS production (27) which has been involved in the regulation of Notch1 signaling pathway (28), induction of autophagy and apoptosis through ROS/JNK signaling pathways (29,42,43). These observations are may provide more evidence for CNC as a promising chemotherapeutic agent in treating MSL subtype of TNBC.

As noticed in HCC 1806 cells, treating MDA MB 231 cells with Doxo has induced almost similar effects as that produced after treatment with CNC10; revealing the pro-apoptotic and pro-autophagic activities of Doxo. This pro-apoptotic effect of Doxo (44) was previously reported in MDA MB 231 cell line. Moreover, Doxo led to sharp and significant increase in the expression of Notch1 signaling genes, and according to previous studies, Doxo can strongly increase the expression of multiple Notch pathway components in cancer cells (45). Huang, et al., 2018, have pointed out to the Doxo-driven apoptosis appeared due to the induced Hes1 gene expression (45). Taken all together may confirm the pro-apoptotic role of Doxo in MDA MB 231 that, in part, may due to its upregulating effect on Notch signaling pathway. Also, considering these effects induced by Doxo with that produced by CNC may favor the suggestion that CNC may be a promising anti-cancer therapeutic agent for both types of TNBC; BL and MSL subtypes. However, CNC concentration should be taken into consideration.

Effect of CNC and Doxo co-treatment

It should be recognized that Doxo, as an anthracycline, has a high efficacy in treating TNBC but it can result in poor outcomes due to chemoresistance induction (46). Therefore, complexation of Doxo with copper has been reported to be a potential anti-breast cancer agent, having a higher efficacy than the parent drug and could potentially induce apoptosis through increased ROS production (20).Presently, co-treatment of Doxo and CNC was used to appraise the possibility of sensitizing TNBC to treatment of Doxo. CNC50–Doxo resulted in significant up regulation of Notch1 signaling genes in HCC 1806 cells. Also, it displayed a pro-apoptotic and anti-proliferative effect associated with a minor elevation in autophagy. Additionally, CNC10–Doxo also gave the same effect on the cell cycle distribution after up regulating of Notch1 and JAG1 and down regulation in Hes1 gene expression with a reduction in autophagy. This difference in the response of autophagy after both treatments may be attributed to the positive correlation found between Hes1 gene expression and autophagy in HCC 1806. Accordingly, combined treatment of CNC and Doxo may sensitize the cells to Doxo leading to obvious synergistic antitumor effect that appeared in inhibition of cell viability, promotion of apoptosis. This is in consistence with a previous report stated that Notch inhibitors; such as CNC in the present case, may synergize the effect of DNA damaging agents like Doxo in a variety of breast cancer cell lines (47).

With regard to MDA MB 231 cell line, treatment with CNC50–Doxo up regulated the relative gene expression of Notch1 and JAG1 genes as well as, down regulated Hes1 relative gene expression, however, CNC10–Doxo caused down regulation of Notch1 signaling genes. Like HCC 1806, and as expected, combination of CNC with the chemotherapeutic drug, Doxo, showed pro-apoptotic and anti-proliferative effects. Besides, CNC50-Doxo reduced the number of LC3 positive cells but, almost no change with CNC10-Doxo. Thus, co-treatment of CNC and Doxo may have a substantial improvement in clinical outcome exhibiting an effective strategy for TNBC treatment, as well as, modulation of Notch pathway could give synergistic effect and, therefore, might reduce Doxo-induced side effects and resistance in breast cancer treatment (33).

In conclusion, co-treatment of CNC with Doxo can be valid as a potential strategy in treating both TNBC subtypes; BL and MSL. Briefly, the produced cytotoxic effects could be imputed to the formation of copper-Doxo complex and its production of ROS. In addition, CNC may facilitate the binding of Doxo to DNA and exert therapeutic effects (48) besides, the regulation of Notch1 signaling pathway by ROS production (28). All together may explain, in part, the enhanced cytotoxic effects of the co-treatment of CNC and Doxo. Notably, this complex formation could be through a dose dependent manner.

Statements & Declarations
Ethics approval and consent to participate: Not applicable
Consent for publication: Not applicable

Author Contributions: Conceptualization, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B, M.A.A (Morsy A. Abu-Youssef), M.A.Y ,L.A.S and S.A.A; formal analysis, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A,A; investigation, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A.A; project administration, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A.A; software, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A.A; validation, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A.A; visualization, M.A.A (Mohamed A. Abdel-Mohsen); writing—original draft, A.M.B; writing—review and editing, M.A.A, (Mohamed A. Abdel-Mohsen) A.M.B and S.A.A; All authors have read and agreed to the published version of the manuscript.

Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data Availability Statement: Data are contained within the article.

Competing interests: The authors declare no conflict of interest.

Acknowledgements: Not applicable

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Influence of Copper (I) Nicotinate Complex on the Notch1 Signaling Pathways in Triple Negative Breast Cancer Cell Lines

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