Impact of copper (I) Nicotinate complex on Notch signaling pathway in autophagy-modulated triple-negative breast cancer cell lines

doi:10.21203/rs.3.rs-5263120/v1

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype and is distinguished by a short overall survival, chemoresistance, recurrence, and a poor prognosis. Despite the increase in the disease's incidence globally, the development of numerous targeted medications and innovative combination therapies has improved the overall prognosis for individuals with breast cancer. However, this generally encouraging image is tarnished by the development of resistance or the reduced effectiveness of pharmaceutical combinations, even if the basic processes are not understood. For instance, autophagy, a catabolic process intended to recycle damaged cellular components and provide energy, is quickly activated by cancer cells to evade most therapies. Consistent with this theory, there is a rising need for metal substances such as copper complex, which may promote cell death and have a less harmful effect on normal cells. On the other hand, deregulated Notch signaling within the breast tumor and its microenvironment is linked to poor clinical outcomes in the treatment of resistant breast cancer.

Objectives: In autophagy-modulated TNBC cell lines, the current study aimed to clarify the possible significance of copper (I) nicotinate complex (CNC) as a targeted therapy for the Notch signaling pathway and evaluated any potential communication between the autophagic process and the Notch signaling pathway.

Method: Two distinct TNBC cell lines, HCC1806 and MDA-MB231 cells, were used. To assess the autophagy process, Torin1 was used to stimulate autophagy, while chloroquine was used to inhibit it. The MTT assay was used to determine the cytotoxicity of CNC and the reference treatment, doxorubicin. The indirect antibody labeling of microtubule-associated protein light chain 3 (LC3) was measured using flow cytometry. The Notch signaling pathway-related gene expressions were determined using real-time PCR technique.

Results and conclusion: Treatment of TNBC cells with the CNC-modulated Notch signaling pathway significantly differed depending on the type of cell line and CNC concentration used. In conclusion, given CNCs’ concentration and use with Doxo, it may be a viable targeted anticancer treatment for TNBC due to its ability to inhibit Notch signaling in autophagy-modulated TNBC.

Triple negative breast cancer

Drug resistance

Autophagy

Notch signaling pathway

Copper (I) nicotinate complex

Triple-negative breast cancer (TNBC) differentiates itself by the lack of expression of the molecular targets, estrogen, progesterone, and HER2 receptors, and accounts for roughly 10-15% of all diagnosed breast cancers (1). It is distinguished by its high grade, poor prognosis, high recurrence, elevated metastatic rates, high resistance to hormonal and most targeted therapies, and high mortality rate (2), making it the most severe of all breast cancer subtypes (3). Cytotoxic chemotherapy remains the primary treatment for TNBC (2). The most effective therapeutic active substance for TNBC treatment now on the market is doxorubicin (Doxo). Doxo is a nonselective chemotherapeutic drug that belongs to the anthracycline chemotherapeutic class (4) and produces necrosis and apoptosis in tumor cells, healthy brain, liver, kidney, and heart tissue (5). So, metal-based drugs emerged as potential anticancer agents to avoid chemo-resistance and toxicity (6) as copper complexes that showed a relevant role in chemotherapy (7). Copper (I)-nicotinate complex (CNC) draws great attention due to its wide spectrum as an antimicrobial, antiviral, anti-inflammatory, and antitumor agents (9). In previous research, the pro-apoptotic and antitumor effects of the copper complexes revealed an interaction between autophagy and apoptosis (10,11). Recently, CNC showed anticancer activity in different types of TNBC (12).

Autophagy is an essential and conserved lysosomal degradation process that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles to maintain cytoplasmic homeostasis (13). This process occurs at a basal rate in most cells and is considered a cellular strategy for survival under stress conditions (14). It plays an active role in programmed cell death when over-activated by starvation, reactive oxygen species (ROS), hypoxia, drug stimuli, and endoplasmic reticulum stress via complex signal transduction pathways, so excess autophagy results in cell death (15). Depending on the type of cancer and the context, autophagy fulfills a dual role, having both tumor-promoting and tumor-suppressing properties (16). The evolutionarily conserved role of autophagy in promoting cell survival during metabolic stress has stimulated research to determine whether it promotes chemoresistance (16). Its pharmacological modulators have been extensively used in basic research and preclinical studies. Chloroquine (CQ) is a well-known late-phase autophagy inhibitor (17-19) and a weak base easily transported throughout the human body when administered. It can permeate through the plasma membrane, causing a rise in pH and the inactivation of lysosomal hydrolysates and increasing autolysosome formation by blocking autophagy. This activity enhances the lethal effect of radiation and chemotherapy in cancer cells (20). Furthermore, Torin1 is a highly effective and selective ATP-competitive Mammalian target of rapamycin “mTOR” inhibitor, mTORC1 and mTORC2, and reduces cell growth and proliferation, on the other hand, induces autophagy (21).

Furthermore, Notch signaling is a commonly used intercellular communication pathway and is implicated in a wide range of biological activities and some clinical disorders (22). Deregulation of the Notch signaling may lead to critical tumor hallmarks, including oncogene expression (23,24), angiogenesis (25), stem cell maintenance (26), deregulated cell cycle progression (27), and anti-apoptotic mechanisms (28). In mammals, Notch receptors (Notch1-4) are expressed on cell membranes and cleaved by ADAM17 or ADAM10 proteases and a presenilin-dependent Ɣ-secretase complex (32). The Notch Intracellular Domain (NICD) then translocates into the nucleus to interact with the ubiquitous transcription factor CBF-1/suppressor of hairless/Lag1 (CSL) (32), resulting in gene transcriptional activation of Hes and Hey target genes. There is growing evidence that Notch signals are oncogenic in breast and other cancers (33) by collaborating with other oncogenic signaling pathways (34). So, it plays a vital role in tumor initiation, proliferation, angiogenesis, and metastasis as well as tumor suppressor function (34). However, Notch expression has been linked specifically to TNBC, as evidenced by high JAG1 and Notch1 expressions. As a result, Notch signaling may represent a promising therapeutic target for patients with TNBC (35).

Nevertheless, there is minimal data on the effects of the Notch pathway on autophagy in cancer (36), suggesting that the Notch signaling pathway affects autophagosome trafficking (37), and its inhibition induces autophagy as well as during differentiation of stem cells (38). Moreover, it has been shown that inhibition of autophagy caused elevation of the levels of Notch1, its activated cleaved form, NICD, and the protein levels of its target gene, Hes1 (39). Furthermore, pharmacological blockade of Notch induced protective autophagy, resulting in chemoresistance (40). Accordingly, in the present study, CNC may be suggested as a promising targeted anti-cancer agent for TNBC. Also, it may be employed to reveal the possible crosstalk of Notch-Autophagy signaling pathways as a novel molecular target in the TNBC treatment.

Preparation of copper (I)-nicotinate complex

The [Cu (I)-(nicotinic acid)2] +Cl- complex ‘CNC’ was prepared through the reaction of ethanolic solution of CuCl₂-2H₂O with nicotinic acid in the presence of L+- ascorbic acid (41). The produced bright red needle crystals were examined by aninfrared spectrum to confirm the chemical structure of Cu (I)-(nicotinic acid)2Cl-2H₂O (42).

Cell culture

TNBC cell lines, HCC1806 (ATCC®CRL-2335™) and MDA-MB231 (ATCC®HTB-26™) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines maintained at 37°C, 5% CO2 in 10% Dulbecco's Modified Eagle Medium (DMEM) (Lanza Bioproduct, Belgium) supplemented with 10% fetal bovine serum (Sera Lab, UK).

Cytotoxicity assay

Cytotoxic effects of CNC, Doxo, Tor1, and CQ were assessed in MDA-MB231 and HCC1806 cells using an MTT assay. 10,000 cells per well were seeded in 96-well plates and incubated at 37°C with 5% CO₂ until 80% confluence. Different drug concentrations were added to the wells and completed to the final volume of 200μl with DMEM, followed by 48-hour incubation. Afterward, 10μl of MTT solution (5 mg/mL) was added to cells and incubated for another 4 hours at 37°C. Control wells contained 50µl of cells and 50µl of MTT solution. After incubation and removing the media, 100µl of dimethyl sulfoxide (DMSO) was added to cells. Optical density was measured at 590nm using an ELISA reader (BioBioTek Company, UA). Living cell counts were recorded with trypan blue (1:1 ratio), allowing for the calculation of relative viability (% control) using the formula: (experimental absorbance - background absorbance) / (absorbance of untreated controls - background absorbance) × 100%, with the negative control set at 100%. IC₅₀ values were derived from relative viability measurements. The experiment was performed in triplicate.

Design of Experiment

The HCC1806 and MDA-MB231 cell lines were treated with either the IC₅₀ of CQ to induce autophagy or TOR to inhibit it. After 48 hours, and according to the experimental design, cells received either 50% or 10% of the IC₅₀ of CNC alone or in combination with the IC₅₀ of DOXO. Table.1

Autophagy Assessment

Autophagy was assessed using indirect LC3-II antibody Alexa Fluor® 488 (ab185036) using flow cytometry. Cells were collected, re-suspended in 1x PBS, and fixed in 4% formaldehyde for 10 minutes. They were then permeabilized with ice-cold 100% methanol, achieving a final concentration of 90% methanol, and incubated for 30 minutes. Following this, the cells were re-suspended in 100 μL of primary antibody for analysis using flow cytometry (43). The experiment was performed in triplicate

Notch signaling pathway assessment

Total RNA was extracted from harvested cells from each group according to the manufacturer's instructions of RNA-spin™ Total RNA Extraction Kit (iNtRON, Korea, ca.no 17211). Purified RNA was used to synthesize cDNA using Reverse-transcriptase enzyme according to the protocol provided by HiSenScript™ RH(-) RT PreMix Kit (iNtRON, Korea. ca. no 25087). Synthesized cDNA was used to detect the expression of targeted genes of the Notch signaling pathway via quantitative Real-time polymerase chain reaction (qRT-PCR). RT was performed by thermal cycler at 25^oC for 5 minutes, 45^oC for 1hr, and then 85^oC for 10 minutes before proceeding to qRT-PCR. PCR amplification was performed using SYBR Green with low ROXTOPreal™ qPCR 2XpreMIX (Enzynomics, Korea) on 7500 applied-Biosystem with the following conditions: denaturation at 95^oC for 10 minutes, followed by 45 cycles at 95^oC for 15 secs, 55oC for 15 secs, and at 72^oC for 30secs (Fluorescence detection point). The relative gene expression levels were calculated using the comparative method (2-ΔCTequation) where ΔCT equals (CT of the gene of interest - CT of housekeeping gene). The experiment was performed in six replicas.

Statistical analysis

Statistical analysis was performed using SPSS 22.0 for descriptive data analysis. Mann-Whitney U test was employed to compare means across different groups, while Spearman’s correlation coefficient assessed biochemical correlations among parameters. P-values ≤ 0.05 were deemed statistically significant.

Characterization of CNC complex

The IR spectra of The Cu(Nic-H)2Cl.2H2O exhibit two bands around 1700 and 1640 cm-1 associated with the v(C=O) and v(COO-), respectively. Thus the v(C==O) band in free Nic-H is a little shifted to lower frequencies in this complex. The other v(COO) band suggests the presence of a zwitter ion form of Nic-H. This complex, therefore, contain a neutral nicotinic acid molecule as well as a zwitterion molecule. The two water molecules are most likely hydrogen bonded to these two different Nic-H molecules. The existence of O-H. . .O and N-H.. .O bonds are indicated by the absorption bands in the region 3500 & 2000 cm-1 as shown in Fig.1.

This result suggests that the complex contains two types of nicotinic acid molecules; a neutral molecule acting as a bidentate ligand, coordinating through its heterocyclic nitrogen and the carboxy group giving rise to chelate ring with copper(I) atom, and two zwitterions acting as monodentate ligands. Fig.1.

Cytotoxicity results

The cytotoxicity percentages (%) of CNC, DOXO, Tor and CQ were evaluated against HCC1806 and MDA-MB231 cells showed significant changes in both cell lines (p<0.05). CNC concentrations were 0.1 and 2.55µM, DOXO were 0.062 and 0.15µM, Tor was 0.01 and 0.074µM and CQ were 0.012 and 1.0µM against HCC1806 and MDA-MB231 cells, respectively. Table.2 & Fig.2.a & Fig.2.b

Cell Morphology

Visualization of morphological alterations by inverted microscope showed the shrinking and detachment of most of the cells after treatment of drugs by the previously stated protocol. Fig.3.a & 3.b

Autophagy assessment

Treatment of the HCC1806 cells with Tor significantly increased the LC3-positive cell numbers, while CQ treatment decreased it. Whereas the number of LC3 positive cells in MDA-MB231 cells didn’t change after treatment of cells with Tor while CQ treatment led to a significant reduction in its number. Fig.4.a

In Tor-pretreated HCC1806 cells, treatment with CNC50 showed a very minute decrease in the number of LC3 positive cells, while CNC10 reduced this number markedly. However, treatment with Doxo gave a slight increase in the number of LC3 positive cells in Tor-pretreated HCC1806 cells. Referring to Tor-pretreated MDA-MB231 cells, treatment with CNC; CNC50 or CNC10, reduced the number of LC3 positive cells in a dose-dependent manner. In contrast, treatment with Doxo increased the number of LC3-positive cells. Fig.4.b

In CQ-pretreated HCC1806 cells, treatment with CNC50 or CNC10 increased the number of LC3-positive cells. Also, treatment with Doxo increased the number of LC3 positive cells in CQ-pretreated cells of the HCC1806 cell line. Concerning MDA-MB231, the level of LC3 positive cells was reduced after treatment of CQ-pretreated cells with CNC50. However, CNC10 increased it. Moreover, treatment of CQ-pretreated MDA-MB231 cells with Doxo led to an increase in LC3-positive cells. Fig.4.c

Co-treatment of CNC50 and Doxo with Tor-pretreated cells of the HCC1806 cell line slightly increased the number of LC3-positive cells, whereas CNC10-Doxo caused a reduction in the number of LC3-positive cells. A marked decrease in the number of LC3-positive cells was noticed after co-treating Tor-pretreated MDA-MB231 cells with CNC50 and Doxo. Fig.4.d, while CNC10-Doxo gave no change in the number of LC3-positive cells. A sharp increase in LC3 positive cell numbers appeared after treating CQ-pretreated HCC1806 cells with CNC-Doxo or CNC10-Doxo. Similarly, in CQ-pretreated MDA-MB231 cells, co-treatment of CNC and Doxo, CNC50-Doxo, or CNC10-Doxo significantly increased in LC3 positive cell numbers.Fig.4.e

Notch signaling pathway assessment

In the HCC1806 cell line, treatment with either Tor or CQ resulted in a significant downregulation in the gene expression of Notch1, JAG1, and Hes1 genes (p≤0.05). The down-regulating effect of CQ was more pronounced than that produced by Tor (p≤0.05). However, in the MDA-MB231 cell line, a significant upregulation in the gene expression of the Notch1 gene (p≤0.05) was observed upon treatment with Tor. In addition, significant upregulation in the gene expression of Notch1 and Hes1 genes (p≤0.05) after treating cells with CQ. Moreover, CQ treatment caused a significant downregulation in the gene expression of the Notch1 gene (p≤0.05) besides a significant upregulation in the gene expression of the Hes1 gene when compared to Tor–treated cells. Table.3 & Fig.5

In Tor-treated HCC1806 cells, treatment with CNC, CNC50, or CNC10 significantly upregulated Notch1 and Hes1 gene expression (p≤0.05), with the effect being particularly strong for CNC50 (p≤0.05). JAG1 expression was significantly increased with CNC50 but not with CNC10 (p≤0.05). Additionally, Doxo treatment of Tor-treated HCC1806 cells upregulated JAG1 and Hes1 expression, showing a significant effect only for Hes1 (p≤0.05), while Notch1 expression remained unchanged. Moreover, treating Tor-treated cells of the MDA-MB231 cell line CNC50 significantly upregulated the gene expression of Notch1, JAG1, and Hes1 genes (p≤0.05). Inversely, CNC10 caused downregulation in the gene expression of the previously stated genes. Regarding Doxo treatment, treating Tor-treated cells of the MDA-MB231 cell line with Doxo showed a significant upregulation of JAG1 gene expression (p≤0.05). Table.4 & Fig.6

In CQ-treated HCC1806 cells, treatment with CNC; CNC50, or CNC10, significantly upregulated the gene expression of Notch1, JAG1, and Hes1 genes (p≤0.05). CNC10 caused a more pronounced significant upregulation of JAG1 gene expression than that caused by CNC50 (p≤0.05). On the other hand, treatment of CQ-treated HCC1806 cells with Doxo led to significant downregulation of Notch1 gene expression associated with significant upregulation of Hes1 gene expression (p≤0.05). Table (5) For MDA-MB231, treatment of CQ-treated cells with CNC50 revealed a significant upregulation in the gene expression of Notch1 and JAG1 genes associated with a significant downregulation in the gene expression of Hes1 gene (p≤0.05). However, treating CQ-treated cells of MDA-MB231 with CNC10 resulted in a significant downregulation in Notch1 and Hes1 gene expression (p≤0.05). Treatment with CNC10 caused a significantly more pronounced downregulation in Hes1 gene expression than that caused by CNC50. Similar to the treatment with CNC50, treatment of CQ-treated cells of MDA-MB231 with Doxo showed a significant upregulation in the gene expression of Notch1 and JAG1 genes with a significant down-regulation of Hes1 gene expression (p≤0.05). Table.5 & Fig.7

In Tor-treated HCC1806 cells, co-treatment of CNC50 and Doxo, CNC50-Doxo, significantly upregulated the relative gene expression of Notch1, JAG1, and Hes1 genes (p≤0.05). However, co-treatment of CNC10 and Doxo, CNC10-Doxo, significantly downregulated the gene expression of the Hes1 gene (p≤0.05). Table (6) Similarly, a significant upregulation in the gene expression of the previously stated genes was noted upon co-treatment of CNC50 and Doxo in Tor-treated cells of the MDA-MB231 cell line (p≤0.05). While co-treatment of CNC10 and Doxo, CNC10-Doxo, significantly upregulated the gene expression of Notch1 and JAG1 genes only (p≤0.05). CNC-Doxo's regulatory effect on the expression of Notch1 and JAG1 genes was dose-dependent. Table.6 & Fig.8

In CQ-treated HCC1806 cells, co-treatment of CNC50 and Doxo, CNC50-Doxo, significantly upregulated the gene expression of Notch1, JAG1, and Hes1 genes (p≤0.05). While, CNC10-Doxo, significantly upregulated the gene expression of the JAG1 gene only (p≤0.05). Upregulation of JAG1 gene expression in CQ-pretreated cells of the HCC1806 cell line after co-treatment of CNC and Doxo was in a CNC dose-dependent manner. Interestingly, co-treatment of CNC50 and Doxo in CQ-treated cells of the MDA-MB231 cell line induced a significant upregulation of JAG1 gene expression (p≤0.05). Meanwhile, CNC10-Doxo led to obvious changes in the gene expression of Notch1 signaling genes (p≤0.05). Table.7 & Fig.9

The study examined TNBC cell lines HCC1806 (basal-like) and MDA-MB231 (mesenchymal stem-like) to investigate Notch-autophagy interactions. In Tor-induced autophagy-HCC1806 cells, CNC demonstrated an anti-autophagic effect by enhancing the expression of Notch signaling genes. This suggests that Notch's reported tumor suppressor role may be activated in the autophagy-induced HCC1806 cells following CNC treatment. These observations support the idea that autophagy down-regulated by CNC may surpass the autophagy-promoting effect of Tor. However, adding Doxo to Tor-pretreated HCC1806 cells resulted in pro-autophagic effects. Interestingly, there is a tendency to believe that Doxo-induced autophagic and apoptotic cell deaths have different mechanisms (48) however, it may involve both processes. In Tor-treated cells, treatment with Doxo showed a significant over-expression in the Hes1 gene only. In addition, as previously explained, Doxo-induced over-expression of Hes1 can further enhance its role in promoting apoptosis (49). Thus, the induction of autophagy and its interactions with apoptosis, as well as the implication of Hes1 gene overexpression could have profound effects on the clinical outcome of the tumor cell.

In the HCC1806 cell line treated with Tor and in MDA-MB231 cells, the anti-autophagic effect of CNC appears to be concentration-dependent, linked to the generation of ROS. CNC50 significantly increased the expression of Notch signaling genes, while CNC10 downregulated Notch1 and Hes1 genes with a modest increase in JAG1 expression. Notch signaling plays a dual role in cancer cells, promoting proliferation or apoptosis through various mechanisms. Furthermore, CNC may enhance the tumor suppressor function of Notch in Tor-induced autophagic cells, (46) potentially leading to cell death.

Furthermore, treatment of Tor-pretreated MDA-MB231 cells with Doxo revealed its ability to promote cell death pathways, autophagy, and apoptosis (50,51) as CNC10 through the increasing LC3 positive cells. Besides, Doxo increased the expression of multiple Notch pathway components in cancer cells and the Hes1 over-expression that drives cells to apoptosis (52). As a result of these findings, inducing the process of autophagy can be used to achieve good clinical outcomes in the MSL subtype of TNBC.

In CQ-inhibited autophagy of HCC1806 cells, CNC demonstrated pro-autophagic activity in a concentration-dependent manner and significantly upregulated Notch signaling gene expression. Additionally, treatment with CNC was associated with increased ROS production (53), similar to CQ (54). The findings suggest that inhibiting autophagy in the BL subtype of TNBC may lead to better clinical outcomes.

In the same line, treating CQ-inhibited autophagy cells of the HCC1806 cell line with Doxo elevated the level of LC3-positive cells, reflecting its pro-autophagic effects. This role could be seen due to its capability of ROS production in addition to the upregulation of Hes1 gene expression linked to Doxo-driven apoptosis (55). Also, the involvement of other mechanisms leading to an increase in the programmed death machinery after activation of the Notch signaling pathway should not be ignored (56,57). These findings suggest that inhibiting autophagy may lead to positive clinical outcomes in basal-like TNBC.

Meantime, in CQ-treated cells of MDA-MB231 cells, CNC50 resulted in significant up-regulation in the expression of the Notch1 and JAG1 genes, with significant downregulation of the Hes1 gene, accompanied by anti-autophagic effect. On the other hand, CNC10 led to significant downregulation of the gene expression of Notch1 and Hes1 genes with pro-autophagic effect. Regarding Notch signaling genes, it was stated that JAG1 regulated both pro- and anti-apoptosis gene expressions, depending on cell type (58). However, Hes1 levels are not always coupled with the activated Notch signaling pathway; it can also be enhanced via the hedgehog signaling pathway and other distinct pathways (59) that may explain the different patterns of expression of Notch1 signaling genes after treatment. Therefore because of the oncogenic/tumor suppressor roles of Notch signaling (60), the cytotoxic effect of Notch downregulation is well known. The implications of its upregulation still require further clarification. Moreover, treatment with Doxo revealed its pro-autophagic effect with elevated levels of expression of Notch1 and JAG1 genes. As previously illustrated, Doxo can increase the expression of multiple Notch pathway components in cancer cells (49) to reveal its anti-proliferative (61) and pro-autophagic properties (50).

Based on earlier observations, Tor-CNC50 suggests that inducing autophagy in a mesenchymal-like subtype of TNBC may lead to a favorable cell fate, unlike in the basal subtype. Thus, further research is recommended to explore the effects of autophagy modulation as a potential treatment for various TNBC subtypes.

In an attempt to explore the influence of autophagy modulation on the potential cytotoxic role of the CNC-Doxo combination, Tor-pretreated cells or CQ-pretreated were further co-treated with a combination of CNC and Doxo. Compared to Tor-treated HCC1806 cells, the addition of CNC50-Doxo led to a significant increase in the expression of Notch1 signaling genes. These were associated with a slight elevation in autophagy. One part may include mechanisms that involve the production of large amounts of ROS because of CNC50-Doxo treatment. ROS, in turn, may affect various signaling pathways including a stress-activated protein kinase (SAPK); JNK, which regulates many cellular events, including apoptosis and autophagy in TNBC (48,56). The other part may involve the Notch1 signaling pathway in which regulation of apoptosis in different cancer cell lines through several different mechanisms including inhibition of JNK function (62) and other different pathways were observed. However, treatment with CNC10-Doxo exhibited anti-autophagic effects. The significant down-regulation in Hes1 gene expression with the reduced autophagy, as long as, inhibiting Notch signaling in cancer cells induces apoptosis (63). On the other hand, treating Tor-pre-treated MDA-MB231 cells with the combination of CNC and Doxo; CNC50-Doxo, and CNC10-Doxo led to the upregulation of Notch signaling genes. However, treatment with CNC50-Doxo was found to have an anti-autophagic effect but almost no change with CNC10-Doxo. Thus, apoptosis may be induced with CNC-Doxo combined treatment due to the previously illustrated mechanisms in Tor-treated HCC1806 cells.

In an endeavor to establish a more efficient therapeutic intervention co-treatment of CNC and Doxo into CQ-pretreated cells was carried out. Thus, on the one hand, treating CQ-pre-treated cells of HCC1806 with CNC50-Doxo gave a significant elevation of Notch1, JAG1, and Hes1 genes while CNC10-Doxo significantly elevated JAG1 gene expression only. Also, CNC-Doxo caused an elevation of the level of autophagy. Regardless of the mechanisms involved in the fate of those CQ-pretreated cells, it could be suggested that autophagy inhibition is a convenient way to improve clinical outcomes and chemotherapeutic response in HCC1806 as an example of BL-TNBC. On the other hand, co-treatment with CNC-Doxo exhibited pro-autophagic effects that might be due to the obvious changes that came out in the gene expression of the Notch signaling pathway after treatment of MDA-MB231-CQ-autophagy inhibited cells with CNC-Doxo. These results might be due to the previously proposed mechanisms. Notably, in light of the present observations, it may seem that a convenient clinical outcome may be attainable in MSL TNBC upon treatment with CNC50-Doxo conditioned with the promotion of autophagy.

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.M; 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.M; project administration, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A.M; software, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A.M; validation, M.A.A (Mohamed A. Abdel-Mohsen), A.M.B and S.A.M; 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.M; 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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Table.1 Experiment design

Groups	Time of treatment administration
Groups	First 48hrs	Second 48hrs
Un-Treated cells	--
Tor	Tor	--
CQ	CQ	--
Tor-CNC₅₀	Tor	CNC₅₀
Tor-CNC₁₀	Tor	CNC₁₀
Tor-Doxo	Tor	Doxo
CQ-CNC₅₀	CQ	CNC₅₀
CQ-CNC₁₀	CQ	CNC₁₀
CQ-Doxo	CQ	Doxo
Tor-CNC₅₀-Doxo	Tor	CNC₅₀-Doxo
Tor-CNC₁₀-Doxo	Tor	CNC₁₀-Doxo
CQ-CNC₅₀-Doxo	CQ	CNC₅₀-Doxo
CQ-CNC₁₀-Doxo	CQ	CNC₁₀-Doxo

CQ; Choloroquine, Tor; Torin, CNC; Copper incotinate (I) complex, Doxo; Doxorubicin, CNC50; 50% of IC₅₀ of CNC, CNC10; 10% of IC₅₀ of CNC

Table.2 The cytotoxicity percentage (% control) of of IC₅₀ values of CNC, Doxo, Tor and CQ on HCC1806 and MDA-MB231 cells

Cell line	Cytotoxicity (% Control)
	Control	CNC	DOXO	Tor	CQ
HCC1806	99.4±4.6	49.2±1.5	52.8±1.6	48.6±1.4	44.9±5.4
MDA-MB2321	101±5.2	50.0±4.1	54.0±2.0	56.2±5.5	51.2±8.6

CQ; Choloroquine, Tor; Torin, CNC; Copper incotinate (I) complex, Doxo; Doxorubicin

Table. 3 Statistical analysis of autophagy modulation effect on Notch signaling pathway in TNBC cell lines, HCC1806 and MDA-MB231 cells

	Notch1	JAG1	Hes1
HCC1806 cell line
Un-Treated cells
X̅± S.E.	52.3±3.5	13.13±0.89	2731±424
Tor
X̅± S.E.	23.8±3.9^*	4.81±1.03^*	753±59^*
CQ
X̅± S.E.	6.01±0.40^*,^#	0.52±0.11^*,^#	399±101^*,^#
MDA-MB231 cell line
Un-Treated cells
X̅± S.E.	37.1±14.4	5.6±1.5	741±239
Tor
X̅± S.E.	1108±421^*	7.8±0.4	20541±12924
CQ
X̅± S.E.	92±5.3^*,^#	24.3±8.3	190116±79014^*,^#

CQ; Choloroquine, Tor; Torin, CNC; Copper incotinate (I) complex, Doxo; Doxorubicin

Relative gene expression was measured per million of cells.

* Statistically significant when compared to untreated control groups

^# Statistically significant when compared to Tor-treated group

(p) Values were considered significant at level ≤ 0.05

Table. 4 Statistical analysis of CNC or Doxo treatment effects on Notch signaling pathway in Tor-induced Autophagy TNBC cell lines; HCC1806 and MDA-MB231 cells

	Notch1	JAG1	Hes1
HCC1806 cell line
Tor
X̅ ± S.E.	23.8±3.9	4.81±1.03	753±59
Tor-CNC50
X̅± S.E.	2010±596^*	181±24.8^*	30591±6600^*
Tor-CNC10
X̅± S.E.	101±24.8^*,^#	7.69±1.42^#	1442±188^*,^#
Tor-Doxo
X̅± S.E.	23.2±3.6	20.8±6.8	5062±1149^*
MDA-MB231 cell line
Tor
X̅ ± S.E.	1108±421	7.8±0.4	20541±12924
Tor-CNC50
X̅± S.E.	4044±890^*	213328±1325239^*	755796±216582^*
Tor-CNC10
X̅± S.E.	451±92	14.04±3.5	3481±641
Tor-Doxo
X̅± S.E.	573±115	4285±1658^*	37476±13329

CQ; Choloroquine, Tor; Torin, CNC; Copper incotinate (I) complex, Doxo; Doxorubicin, CNC50; 50% of IC₅₀ of CNC, CNC10; 10% of IC₅₀ of CNC

Relative gene expression was measured per million of cells.

* Statistically significant when compared to untreated control groups

^# Statistically significant when compared to Tor-treated group

(p) Values were considered significant at level ≤ 0.05

Table. 5 Statistical analysis of CNC or Doxo treatment on Notch signaling genes in CQ-inhibited Autophagy TNBC cell lines; HCC1806 and MDA-MB231 cells

	Notch1	JAG1	Hes1
HCC1806 cell line
CQ
X̅ ± S.E.	6.01±0.40	0.52±0.11	399±101
CQ -CNC50
X̅± S.E.	57.8±10.14^*	7.7±2.98^*	1508±114^*
CQ -CNC10
X̅± S.E.	38.1±11.9^*	23.5±6.3^*,^#	3411±712^*
CQ –Doxo
X̅± S.E.	2.3±0.4^*,^#^,^Ѱ	1.2±0.4^#,Ѱ	1751±238^*^,Ѱ
MDA-MB231 cell line
CQ
X̅ ± S.E.	92±5.3	24.3±8.3	190116±79014
CQ -CNC50
X̅± S.E.	2871±36.04^*	237±36.9^*	51582±2017^*
CQ -CNC10
X̅± S.E.	79±6^*	27.5±9.2	132±28^*,^#
CQ –Doxo
X̅± S.E.	1640±578^*,^Ѱ	527±67^*,^#^,^Ѱ	627±135^*,^#,Ѱ

CQ; Choloroquine, Tor; Torin, CNC; Copper incotinate (I) complex, Doxo; Doxorubicin, CNC50; 50% of IC₅₀ of CNC, CNC10; 10% of IC₅₀ of CNC

Relative gene expression was measured per million of cells.

* Statistically significant when compared to CQ-treated group.

# Statistically significant when compared to CQ-CNC₅₀-treated group.

Ѱ Statistically significant when compared to CQ-CNC₁₀-treated group.

(p) Values were considered significant at level ≤ 0.05

Table. 6 Statistical analysis of CNC and Doxo combination effect on the Notch signaling pathway in Tor-induced Autophagy TNBC cell lines; HCC1806 and MDA-MB231 cells

	Notch1	JAG1	Hes1
HCC1806 cell line
Tor
X̅± S.E.	23.8±3.9	4.81±1.03	753±59
Tor-CNC50-Doxo
X̅± S.E.	96.5±24.4^*	28.9±7.2^a	60659±6844^*
Tor-CNC₁₀-Doxo
X̅± S.E.	19.1±3.4^#	5.6±1.92^#	270±69.9^*,^#
MDA-MB231 cell line
Tor
X̅± S.E.	1108±421	7.8±0.4	20541±12924
Tor-CNC50-Doxo
X̅± S.E.	444772±11566^*	51017±14158^*	244798±82260^*
Tor-CNC10-Doxo
X̅± S.E.	7392±2309^*,^#	473±102^*,^#	38611±8967

CQ; Choloroquine, Tor; Torin, CNC; Copper incotinate (I) complex, Doxo; Doxorubicin, CNC50; 50% of IC₅₀ of CNC, CNC10; 10% of IC₅₀ of CNC

Relative gene expression was measured per million of cells.

* Statistically significant when compared to Tor-treated group.

# Statistically significant when compared to Tor-CNC₅₀-Doxo-treated group.

(p) Values were considered significant at level ≤ 0.05

Table. 7 Statistical analysis of CNC and Doxo combination on Notch signaling pathway in CQ-inhibited Autophagy TNBC cell lines; HCC1806 and MDA-MB231 cells

	Notch1	JAG1	Hes1
HCC1806 cell line
CQ
X̅± S.E.	6.01±0.40	0.52±0.11	399±101
CQ -CNC50-Doxo
X̅± S.E.	1202±230^*	638±126^*	481646±119856^*
CQ -CNC10-Doxo
X̅± S.E.	5.79±1.4	1.87±0.4^*,^#	951±218^#
MDA-MB231 cell line
CQ
X̅± S.E.	92±5.3	24.3±8.3	190116±79014
CQ -CNC50-Doxo
X̅± S.E.	131±58.4	1345±772^*	60219±5511
CQ -CNC10-Doxo
X̅± S.E.	385±222	74.8±18.4^#	4407±1265^#

Relative gene expression was measured per million of cells.

* Statistically significant when compared to CQ-treated group.

# Statistically significant when compared to CQ-CNC₅₀-Doxo-treated group.

(p) Values were considered significant at level ≤ 0.05

Impact of copper (I) Nicotinate complex on Notch signaling pathway in autophagy-modulated triple-negative breast cancer cell lines

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Declarations

References

Tables

Status:

Version 1