الفهرس 
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Abstract
Despite advances in early detection, breast cancer remains the second most frequent cause of cancer mortality in female. It is the most common invasive and metastatic malignancy of breast cancer that confounds treatment. Novel agents or treatments are urgently needed to improve the outcome for the large number of patients who relapse after receiving the currently available breast cancer therapies, since the existing therapies are no longer effective in these patients.
In Egypt, breast cancer is estimated to be the most common cancer among females accounting for 38.8% of all female cancer. Breast cancer is also the leading cause of cancer mortality in females accounting for 13.7% of female cancer-related mortality globally compared to 29.1% in Egypt.
Chemotherapeutic agents represent the most common treatment option for breast cancer.However, their deleterious side effects and the increase in multidrug resistance had augmented the need for developing new strategies based on a deeper understanding to the molecular mechanisms underlining the cancer development.The uses of phytochemicals, which are natural dietary constituents of fruits and vegetables, have currently been explored to target the molecular subtypes of breast cancer.Phytochemicals are generally non-toxic and display a remarkable spectrum of biological activities, including anti-proliferative, anti-allergic, anti-inflammatory, antioxidant, and modulation of enzymatic activities.
In Vitro Studies
In the present study, our data showed the role of the natural product pomegranate extracts (PE) and/or temsirolimus individually and combined in vivo, on albino mice and in vitro, on Estrogen receptor positive T-47D human breast cancer cell line. Cell viability assay (MTT assay) was performed on temsirolimus treated T-47D cells after 72 h to evaluate its IC50.Our data showed the morphological changes of T-47D cell line in the tested cell line treated with different concentrations of temsirolimus.Additionally, our data showedapoptotic induction by measuring the released histones using ELISA technique after treatment with 1/2 IC50 concentration of temsirolimus for 72 h and 1/2 IC50 concentration of pomegranate extract for 48 h combined for 24 h in T-47D breast cancer cell line. 1/2 IC50 of temsirolimus and 1/2 IC50 of pomegranate extract and/or combined effect on caspase-3 activity was investigated calorimetrically in the tested T-47D cell line.Furthermore, our data showed the protein expression levels of mTOR and cyclin D1 by western blot analysis.Also, our data showedapoptosis and the cell cycle analysis using flow cytometry after the treatment of T-47D with 1/2 IC50 concentration of temsirolimus for 72 h and 1/2 IC50 concentration of pomegranate extract for 48 h and/or combined.Finally, immunocytochemical assay was used to visualize the nuclear and cytoplasmic localization of oncogenic β-catenin.
Our results indicated that temsirolimus exerts potent anticancer effect in T-47D breast cancer cell line evidenced by significant decrease in cell viability and proliferation of the cells after 72 h treatment, in a concentration dependent manner.Moreover, dramatic morphological changes which include rounding up, chromatin and cytoplasmic condensation, formation of apoptotic bodies and a significant reduction in cell population was also observed. Histone release and caspase-3 activity were also induced by 1/2
IC50concentration of temsirolimus for 72 h and 1/2 IC50 concentration of pomegranate extract for 48 h and/or combined for 24 h in T-47D cells, suggesting apoptosis as a mechanism of cell death.To further investigate the effect of treatments on oncogenesity and cell cycleour data showedthe effect of temsirolimus, pomegranate extract and/or combined treatment on the expression of mTOR and cyclin D1. Indeed, the results showed a significant decrease of both signal transduction in case of combined treatment more than each treatment PE and temsirolimus individually. In addition, combined treatment significantly downregulated the expression of mTOR and major cell cycle regulator; cyclin D1, suggesting a possible cross talk between them.Also, the effect of 1/2 IC50 concentration of temsirolimus for 72 h and 1/2 IC50 concentration of pomegranate extract for 48 h and/or combined for 24 h treatment on oncogenic β-catenin was further confirmed by immunocytochemical analysis of nuclear and cytoplasmic localization of β-catenin compared to untreated cells.
In Vivo Studies
Also, our data showed the pomegranate extracts, temsirolimus individually and/or combined on mice with MNU induced mammary tumor. Mice were divided into five groups:
group I: Twenty female healthy mice as control.
group II: Sixtyfemale mice were given MNU I.P. for induction of ductal carcinoma (induced) as a negative control.
After the ductal carcinoma has appeared after three months, the induced group was divided into the three following groups for treatment three times a week for one month.
(a) Twentyfemale mice were treated with pomegranate extract, through orogastric gavage.
(b) Twenty female mice were treated with temsirolimus by I.P. injections.
(c) Twenty female mice were treated with a combined temsirolimus and pomegranate extract.
The results demonstrate that treatment with the combination therapy significantly reduced growth of tumors, compared to controls. In addition, serum blood biochemical parameters (ALT, AST, urea, creatinine, Albumin, and total protein) were performed on blood serum samples collected from all groups.Moreover, histopathological studies showed a decrease in β-catenin expression and cytoplasmic localization of β-catenin in treated groups compared to induce untreated animals.
Collectively, in vivo data was consistent with in vitro data and both confirming the more potent anticancer effect for combined treatment of PE and temsirolimus over their individual treatment.