Influence of copper(I) nicotinate complex on the Notch1 signaling pathway in triple negative breast cancer cell lines.

Triple negative breast cancer (TNBC) is a subtype of breast cancer which is characterized by its aggressiveness, poor and short overall survival. In this concept, there is a growing demand for metal-based compounds in TNBC therapy as copper complex that have a less toxic effect on normal cells and could stimulate apoptotic cell death. Additionally, Notch1 signaling pathway has received great attention as one of the most important potential targets for developing a novel therapeutic strategy. 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. To approach the objective of the present study, TNBC cell lines; HCC1806 and MDAMB231, were utilized. MTT assay was used to determine the IC50 values of CNC and Doxo. After treatment, microtubule-associated protein light chain3 (LC3) were determined by flow cytometry. Additionally, qRT-PCR technique was used to detect the changes in genes levels that are involved Notch1 signaling pathway. Moreover, autophagosomes were monitored and imaged by Transmission electron microscopy. Treatment of TNBC cells with CNC modulated Notch1 signaling pathway in different manners with respect to the type of cells and the applied dose of CNC. The observed effects of CNC may reflect the possible anti-cancer activities of CNC in both types of TNBC. However, cell type and CNC dose should be considered.

IL-6 Impairs the Activity of Vitamin D3 in the Regulation of Epithelial-Mesenchymal Transition in Triple Negative Breast Cancer.

Objective: The present study aimed to investigate the possible role of IL-6 and 1α,25-dihydroxyvitamin D3 (1,25D) signaling in epithelial-mesenchymal transition (EMT) and stemness in triple-negative breast cancer (TNBC) cell line. Methods: TNBC cell line, HCC 1806, was treated with IL-6 and 1,25D for three and six days. Also, the role of vitamin D receptor (VDR) was studied by transfection of TNBC cell line with VDR gene and transfection efficiency was assessed using Human VDR enzyme-linked immunosorbent assay (ELISA). Changes in E-cadherin gene expression were analyzed by quantitative real-time PCR (qRT-PCR). Also, changes in CD44+ cells were analyzed by flow cytometry. Finally, morphological changes were investigated by light microscopy after 6 days. Results: Treatment of HCC1806 cells with IL-6 has no significant effect either on E-cadherin gene expression or CD44+ cells, (p > 0.05). However, E-cadherin gene expression was significantly up-regulated after treatment with 1,25D for 6 days, (p < 0.05). Also, CD44+ cells were significantly reduced after treatment with 1,25D either for 3 or 6 days, (p < 0.05). Transfection of TNBC cell line with VDR gene significantly up-regulated VDR protein expression, (p < 0.05). In addition, overexpression of VDR in TNBC cells and treatment with 1,25D significantly up-regulated E-cadherin gene expression, (p < 0.05) and reduced CD44+ cells, (p < 0.05). Moreover, transfection with VDR and treatment with a combination of 1,25D and IL-6 significantly down-regulated E-cadherin gene expression and increased CD44+ cells compared with transfected cells with VDR treated with 1,25D alone, (p < 0.05). No significant morphological changes were observed in treated cells, 6 days post-treatment. Conclusion: The presence of IL-6 in the breast tumor microenvironment may impair the activity of vitamin D3 signaling, limiting its anti-tumor effects in TNBC.

Effect of Polycyclic Aromatic Hydrocarbons Exposure on Sperm DNA in Idiopathic Male Infertility.

BACKGROUND: Biological mechanisms contribute to the relationship between polycyclic aromatic hydrocarbon (PAH) exposure and infertility in males by altering semen quality. OBJECTIVES: The aim of the present study was to evaluate the impact of PAHs on male infertility using the sperm chromatin dispersion test (Halo sperm assay). METHODS: Sixty-six (66) infertile males under 45 years of age were examined for the determination of urinary metabolite and oxidative stress by measuring lipid peroxidation and antioxidant activity of glutathione and glutathione-s-transferase, as well as hormonal activity of follicle stimulating hormone (FSH), testosterone and prolactin and semen quality. RESULTS: There was an increased level of urinary metabolite of 1-hydroxy pyrene, 1-hydroxy naphthalene and 2-hydroxy naphthalene in the urine of the infertile group. In addition, elevated concentrations of malondialdehyde coincided with a decreased level of antioxidants, leading to oxidative stress in the infertile group. Semen samples showed 30% sperm deoxyribonucleic acid (DNA) fragmentation. CONCLUSIONS: The data provide strong evidence of a statistical threshold for semen samples containing 30% sperm DNA fragmentation resulting in a reduced level of pregnancy success. PARTICIPANT CONSENT: Obtained. ETHICS APPROVAL: Study approval was given by the ethics committee of Alexandria University (United States Department of Health and Human Services, institutional review board registration (IRB), IORG0008812 Medical Research Institute, expires 4/8/2019, OMB No: 0990-0279). COMPETING INTERESTS: The authors declare no competing financial interests.

Influence of copper (I) nicotinate complex and autophagy modulation on doxorubicin-induced cytotoxicity in HCC1806 breast cancer cells.

PURPOSE: Doxorubicin is regarded as the most therapeutic active agent available for triple-negative breast cancer (TNBC) treatment. However, the development of drug resistance and toxicity limits its effectiveness. Thus, developing novel strategies for TNBC treatment remains a significant challenge and doxorubicin-based combinations either by metal complexes (Copper I nicotinate complex) or with autophagy modulators could provide novel strategies and alternative strategies contributed to cancer cell death pathways, autophagy and apoptosis. MATERIALS AND METHODS: The viability of HCC1806 TNBC cells and IC50 values of Doxorubicin (DOX), Torin-1 (TOR), Chloroquine (CQ) and Copper (I) nicotinate complex (CNC) were assessed by MTT assay. ELISA was used for detecting microtubule-associated protein 1 light chain 3 (LC3) level. Real time PCR was used to determine (NBR1) gene expression. Cell cycle analysis and quantitative detection of acid vesicular organelles (AVOs) was performed by flow cytometry. TOR and CQ were used as autophagy modulators for induction and suppression of autophagy, respectively. RESULTS: The half-maximal inhibition effect of TOR combination with DOX was revealed to the induction of autophagic cell death and apoptotic cell death. On the other hand, combination of CQ with DOX increased the growth inhibitory effect, induced accumulation of AVOs and suppressed apoptotic cell death. However, combination of CNC with DOX inhibited autophagy and induced cell cycle arrest. CONCLUSION: Doxorubicin drug based combinations either with TOR, CQ or CNC could positively affect DOX effectiveness and reduce DOX doses applied on HCC1806 cells through modulation of autophagy.

Oncostatic treatment effect of triple negative breast cancer cell line with copper (I)-nicotinate complex.

The treatment of triple-negative breast cancer (TNBC) remains a major challenge. The present study aimed to throw more light on the role of copper (I)-nicotinate complex (CNC) as an antitumor as well as a proapoptotic agent. In this study, the HCC-1806 cell line was used as a model for TNBC. Cell cycle, apoptosis assay, and programmed cell death protein-1 were investigated by flowcytometry. Besides, the comet assay was performed using a fluorescence microscope. The enzyme-linked immunosorbent assay technique was used for the detection of phospho-Chk1 at ser 317 and caspase-3. Moreover, the gene expression of survivin was identified by real-time polymerase chain reaction. Finally, superoxide dismutase (SOD) was calorimetrically assayed. The viability of HCC-1806 cells treated with CNC was decreased in a dose-dependent manner. The tendency for apoptotic machinery was observed through the increase in the sub G0 peak, the percentage of early and late apoptotic phases, and the elevation in caspase-3 levels associated with a downregulation of the survivin gene expression. The antioxidant property of the complex, reflected by elevated SOD activity, may contribute to mediate the cell death pathways. Low concentrations of CNC were found to favor the apoptotis-mediated mechanism. However, one cannot neglect the abundance of cell necrosis-mediated death of cells via CNC, especially at higher concentrations.

Antitumor Activity of Copper (I)-Nicotinate Complex and Autophagy Modulation in HCC1806 Breast Cancer Cells.

BACKGROUND: Squamous cell cancer is a heterogeneous aggressive disease, therefore, its treatment is challenging. Increased attention has been paid to metal complexes as anticancer drugs. However, new insights towards autophagy have been recognized due to its role in tumor cell death or survival. OBJECTIVE: To clarify the antitumor activity of copper (I) nicotinate complex (CNC) as new therapeutic agent and understand the role of autophagy modulation as a prospective target for the advancement of efficient therapeutic agent for treatment. METHOD: Viability of MDA-MB-231 and HCC1806 cells and IC50 values of CNC for both cell lines were assessed by MTT assay. Also, the viability and IC50 values of Torin1 and Chloroquine (CQ) were assessed only in HCC1806 cells by MTT assay. The level of microtubule-associated protein 1 light chain 3 (LC3) was assessed by ELISA. Real time PCR was used to detect the changes in NBR1 gene expression. Cell cycle distribution and quantitative detection of acid vesicular organelles (AVOs) were determined by flow cytometry. Fluorescence microscope was used for qualitative detection of AVOs. Modulation of autophagy was carried out by Torin1 as inducer and CQ as inhibitor. RESULTS: CNC restrained the growth, in a dose-dependent manner, and induced cell death in human HCC1806 cell line. In addition, the CNC treated cells displayed inhibition of autophagy, as indicated by reduction of AVOs, decrease in LC3 protein level and up regulation of NBR1 gene expression. CONCLUSION: CNC, as an autophagy inhibitor and pro-apoptotic agent, could be a promising anti-cancer agent either alone or in combination with other therapeutic drugs.

Impact of Cellular Genetic Make-up on Colorectal Cancer Cell Lines Response to Ellagic Acid: Implications of Small Interfering RNA.

BACKGROUND: K-Ras activation is an early event in colorectal carcinogenesis and associated mutations have been reported in about 40% of colorectal cancer patients. These mutations have always been responsible for enhancing malignancy and silencing them is associated with attenuation of tumorigenicity. Among downstream effectors are the RAF/MEK/ERK and the PI3K/Akt signaling pathways. PI3K/Akt signaling leads to reduction of apoptosis, stimulated cell growth and enhanced proliferation. Ellagic acid (EA), a naturally occurring antioxidant, has recently emerged as a promising anti-cancer agent. PURPOSE: To evaluate the impact of cellular genetic make- up of two colon cancer cell lines with different genetic backgrounds, HCT-116 (K-Ras-/p53+) and Caco-2 (K-Ras+/ p53-), on response to potential anti-tumour effects of EA. In addition, the influence of K-Ras silencing in HCT- 116 cells was investigated. MATERIALS AND METHODS: Cellular proliferation, morphology and cell cycle analysis were carried out in addition to Western blotting for detecting total Akt and p-Akt (at Thr308 and Ser473) in the presence and absence of different concentrations of EA. Cell proliferation was also assessed in cells transfected with different concentrations of K-Ras siRNA or incubated with ellagic acid following transfection. RESULTS: The results of the present study revealed that EA exerts anti-proliferative and dose-dependent pro-apoptotic effects. Cytostatic and cytotoxic effects were also observed. p-Akt (at Thr308 and Ser473) was downregulated. Moreover, EA treatment was found to (i) reduce K-Ras protein expression; (ii) in cells transfected with siRNA and co-treated with EA, pronounced anti-proliferative effects as well as depletion of p-Akt (at Thr308) were detected. CONCLUSIONS: Cellular genetic makeup (K-Ras-/p53-) was not likely to impose limitations on targeting EA in treatment of colon cancer. EA had a multi-disciplinary pro-apoptotic anti-proliferative approach, having inhibited Akt phosphorylation, induced cell cycle arrest and showed an anti-proliferative potential in HCT-116 cells (expressing mutant K-Ras).

Biological monitoring of occupational exposure to electrophilic compound during rubber industry.

Electrophilic compounds are widely used in industry. Rubber processing is the famous example of sites where workers are exposed to electrophilic compounds. Besides their immediate effect on different body systems, electrophilic compounds include most mutagenic and carcinogenic substances. The present study was carried out to elucidate the possibility of using non selective assays in the biological monitoring of occupational exposure to electrophilic compounds. The study included a total number of 40 workers exposed to chemicals during rubber industry (n=25 exposed) and comparison group (n=15). The biochemical tests carried out on each subject included: (1) Estimation of thioether levels in urine as a biological indices of electrophilic compounds. (2) Non selective assays as urinary RNase and alpha esterase, glutathione-S-transferase and aryl sulphatase. Their determination is recommended in the biological monitoring of workers exposed to such agents beside the medical surveillance of workers exposed to such chemicals. The study finding showed significant increase in the levels of thioether and all the non-selective assays among exposed workers rather than the comparison group.