الفهرس 
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Abstract
Breast cancer is the most diagnosed cancer in women. The existence of hormonal therapies with marked efficacy, lead to better prognosis and survival. For over three decades, tamoxifen has represented an efficient standard therapy of ER+ breast cancer. However, the development of resistance remains a major problem. Several signaling pathways were found to be related to the development of tamoxifen resistance. They form a complicated interconnected network of cross talks,regulating their expression. In this study, we focused on some of the signaling molecules that contributed to tamoxifen resistance such as; cyclin D1, p53, HER2, IGF1, NFκB, β-catenin and VEGF. This study is an approach to finding new promising and effective combinations with tamoxifen. Two natural products; diosmin a flavonoid compound and berberine an isoquinoline alkaloid were evaluated as antitumor agents either alone or when combined with tamoxifen on breast cancer cell lines. In this study, the parent ER+ MCF7 and the tamoxifen resistant but estrogen sensitive MCF7/LCC2 cell lines were used. Cell viability and cytotoxicity assays using MTT reagent, gene expression analysis using qRT-PCR and protein expression analysis using ELISA and colorimetric assays were performed.
For the evaluation of the effect of these drugs on the cellular proliferation, protein expression of the key cell cycle regulator cyclin D1 was assessed. cyclin D1 was found to bind and activate ER in a ligand independent manner and prevents its inhibition by tamoxifen, thus, contributing to tamoxifen resistance. In the present study, the combination therapy of both diosmin and Berberine each with 4-OHT showed a significant decrease in the level of cyclin D1 protein expression as compared to the upregulation produced by 4-OHT treatment alone, in MCF7 and MCF7/LCC2 cell lines. Moreover, for better understanding of the effect on proliferation and its balance with apoptosis, caspase 3 activity and p53 gene expression were measured. The combination of berberine with 4-OHT gave a highly significant increase in caspase 3 activity in both cell lines, while, the combination of diosmin with 4-OHT showed only a significant increase with MCF7 cells. Loss of p53 expression or function during tumorigenesis results in downregulation of ER and therefore, lack of tamoxifen response. This study showed that the two combination regimens showed a significant upregulation in p53 mRNA expression in the resistant cell line, however, this upregulation was significantly higher with the combination of berberine. Therefore, the antiproliferative and apoptotics effect mediated by diosmin and berberine in combination with 4-OHT involved cyclin D1, caspase 3 and p53 regulation.
ER represents a very important transcription factor associated with breast cancer initiation and progression. One of the mechanisms underlying resistance is the ability of the cancer cells to activate ER in a ligand independent manner. In these pathways, ER is phosphorylated by membrane RTKs like the growth factor receptors; HER2 and IGF1-R, followed by the activation of transcription and enhanced downstream signaling pathways leading to cellular proliferation, differentiation and survival and most importantly, leading to tamoxifen resistance. In this study, the combination of both diosmin and berberine each with 4-OHT showed a downregulation of HER2 on the gene and protein expression levels when compared to treatment with 4-OHT alone. However, on the gene expression level, the effect of the berebrine combination was superior in MCF7 cells, whereas, in MCF7/LCC2 cells, diosmin combination was more effective. Similarly, the two combinations produced a downregulation of IGF1 on the gene and protein expression
levels as compared to the effect produced by 4-OHT treatment alone, in the resistant cell line.
One of the key regulatory molecules that shares a common mechanistic link in hormonal resistance is NFκB. NFκB activation is linked to loss of ER expression, likewise, ER was found to cause inhibition of NFκB activation. In fact, this inhibitory cross-talk revealed that an increase in both NF-κB DNA-binding activity and expression of NFκB target genes is associated with a shift from estrogen dependence to estrogen independence in breast cancer. NFκB was found to be regulated by HER2 and IGF1. In breast cancer, tamoxifen might cause reactivation of NF-κB, potentially rerouting a proliferative signal to breast cancer cells and contributing to hormone resistance. In this study, both diosmin and berberine and their combinations with 4-OHT showed a downregulation of the mRNA expression and protein phosphorylation of NFκB in both cell lines when compared to the upregulation caused by 4-OHT treatment alone.
Amongst the important signaling pathways that plays an important role in cell migration, invasion, metastasis, adhesion and survival is the Wnt/β-catenin pathway. Wnt/β-catenin signaling pathway was found to be upregulated in acquired tamoxifen resistance and its activation inhibits the effects of tamoxifen. Moreover, its inhibition re-sensitizes the resistant cells to tamoxifen. In the presentstudy, the two combination regimens produced a significant downregulation of β-catenin mRNA expression in both cell lines. However, the two combinations did not show any difference neither in the total β-catenin protein nor in the normalized ratio of the active dephosphorylated β-catenin to the total β-catenin protein expression among all treatment groups.
One of the signaling moleculesidentified to be related to tamoxifen resistance is VEGF. VEGF is a potent inducer of angiogenesis that is significantly increased in tumors. The combination of tamoxifen and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER+ tumors. In this study, the two combination regimens showed a significant downregulation of VEGF, as compared to the 4-OHT groups, in both cell lines. Also, on the gene expression level, the combination of diosmin downregulated VEGF, when comapred to the 4-OHT group, in MCF7 cells. In contrast, the combination of berberine showed a significant upregulation of VEGF, in both cell lines.
In summary, there were no significant differences between the effects of the two combination regimens of berberine and diosmin on the protein expression of the measured parameters in both cell lines. However, on the gene expression level, berberine combination therapy showed superior benefits in the regulation of HER2 in MCF7 cells and p53 in MCF7/LCC2 cell line. On the other hand, the combination therapy of diosmin was superior in the regulation of VEGF in MCF7 cells and HER2 in MCF7/LCC2 cell line.