الفهرس 
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Abstract
Female breast cancer is one of the most common malignancies. It has now surpassed lung cancer for the first time as the most common cancer diagnosed globally the fifth leading cause of cancer mortality worldwide.
Conventional treatments, most notably chemotherapeutic drugs, especially combination chemotherapy, have become the standard of care for advanced and invasive breast cancer and certain subtypes like triple-negative breast cancer. But unfortunately, these conventional therapies are limited due to drug resistance and low solubility. In addition, these therapies are systemic treatments that lack selectivity for target tumor cells. They spread all over the body and reach normal cells and cancer cells, causing severe toxic effects and low therapeutic index. As a result, developing a unique therapeutic approach for advanced breast cancer that overcomes drug resistance and includes selective delivery of cytotoxic agents to the tumor mass is important for improving the therapeutic index (efficacy/toxicity ratio) and reducing toxic side effects.
Nano therapies combination avoid all limitation of traditional breast cancer treatments. The permeability and accumulation of chemotherapeutical nanoparticles passively target tumor tissue, resulting in low systemic toxicity and increased drug concentration inside tumors compared to standard chemotherapy treatment. To overcome multidrug resistance, nanoparticles provide the ability to co-deliver multiple drugs, allowing for more accurate control of each drug’s ratio to special target.
CuONPs is the simplest copper compounds. It also has a number of potential physical properties, such as small size, a high surface-to-volume ratio, dissolution, functional groups, and aggregation behavior. These properties make it more toxic and effective as a nano-carrier and anticancer drug. CuONPs have shown excellent anticancer activities in different types of cancer as monotherapy due to their ability to passively target cancer cells. Its killing power against cancer cells can be increased while its toxicity against normal cells is decreased by binding it with one of the natural antioxidant products such as Q. However, quercetin is regarded as a powerful antioxidant and a number of studies have demonstrated that it has anti-carcinogenic properties.
The combination of CuONPs with Q has been utilized as novel promising therapeutic strategy for breast cancer treatment. In our separate experiments to evaluate the cytotoxic effect of CuONPs and Q in combination, we used three combination doses of CuONPs with Q. The three combination doses of CuONPs with Q showed a significant more potent synergistic growth inhibitory effect than the individual doses. High doses of Q are used in breast cancer prevention and treatment due its low circulating concentrations, aqueous solubility, stability, bioavailability, and target specificity in the body. As a result, there is an urgent need to improve its limitations by conjugation with CuONPs coated with chitosan.
The present study is aimed at developing novel Q conjugated copper oxide nanoparticles (CuO-ChNPs-Q) as an anti-breast cancer agent and assessing its anti-tumor effect in vitro and in vivo. Initially, we evaluated the cytotoxic effect of CuONPs, Q and CuO-ChNPs-Q on cell viability, potent anti-proliferative action of the CuO-ChNPs-Q against HePG-2, MCF-7 and CaCo-2 human cell lines as well as against mammary carcinoma induced by DMBA confirming synergistic effect. Even CuO-ChNPs-Q has less toxic effect against WI38 normal lung cells than doxorubicin as standard anticancer treatment, this reflects that CuO-ChNPs-Q safety on normal cells.
In our in vivo study, although, the percentage of breast tumor apoptotic cells of both CuONPs and Q was approximately the same, the percentage of breast tumor apoptotic cells of DMBA-induced rats treated with CuO-ChNPs-Q was significantly higher. Our data showed that apoptotic effect of CuO-ChNPS-Q is the strongest as the minimized tumor mass via apoptosis activation among other compounds, whether CuONPs or Q.
We evaluated the effect of CuONPs, Q and CuO-ChNPs-Q on the alteration of the cell cycle phases. Our results showed that all treated group specially the DMBA induced rats treated with CuO-ChNPS-Q had triggered the cell cycle arrest at G0/G1 along with minimized mitosis (G2/M) with activating apoptosis, leading to a small mammary carcinoma mass in these rats.
Our data confirms a significant increase in p53 marker (%) in CuONPs and Q treated groups, with a markedly elevation in the CuO-ChNPs-Q treated group compared with the DMBA group. An activation in p53 marker in CuO-ChNPs-Q group which inhibits the breast cancer from proliferation, activating apoptosis and by triggering cell cycle arrest at G0/G1 phase that leads to obvious reduction in mammary tumor mass.
In our study, the caspase-dependent mechanism based on the intrinsic pathway was detected when significant elevation in the cytochrome c levels were detected in both CuONPs and Q treated groups with a markedly elevation in the CuO-ChNPs-Q treated group compared with the DMBA group. That results established the apoptotic pathway improvement in the CuO-ChNPs-Q treated rats as the level of cytochrome c was highly increased, leading to production of caspase-9 and finally cleavage of caspase-3.
We have further evaluated some of the key molecular markers that play a critical role in apoptosis to estimate the involvement of these markers in cell death induced by our CuO-ChNPs-Q treated groups. The expression levels of apoptotic markers caspase 3 was significantly increased in our CuO-ChNPs-Q treated groups with the significant elevation in the apoptotic pathway that is detected in our study.
The anti-proliferative effect as a result of apoptosis enhancement that has been detected using our CuO-ChNPs-Q treatment was also found to be modulated by a reduction in the PCNA percentage that led to a low proliferation rate of the mammary carcinoma cells, and this was observed by the tiny tumor mass of the CuO-ChNPs-Q treated rats. Also, the safety of the CuO-ChNPs-Q on the liver and kidney of the treated rats revealed its low toxicity compared with the CuONPs high toxicity due to the presence of Q. Therefore, the CuO-ChNPs-Q might be a promising chemotherapeutic agent for the treatment of breast cancer with minimal toxicity to vital organs.
Overall, our data revealed that the anti-proliferative effect of CONPs-Q against DMBA induced mammary carcinoma is via the p53 activation pathway that arrests the cell cycle at G0/G1 with the release and elevating secretion of cytochrome c, which activates caspase-3 splitting, leading to apoptotic cancer cell death along with PCNA down-regulation with minimal side effects.