الفهرس 
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Abstract
Over the last few decades, there have been advances in breast cancer management leading to earlier detection of the disease and the development of more effective treatments, improving the outcomes for women living with the disease and resulting in declines in breast cancer deaths.
Breast cancer is no longer seen as a single disease but rather a disease comprised of distinct biological subtypes with diverse natural history, presenting a varied spectrum of clinical, pathologic and molecular features with different prognostic and therapeutic implications (Nguyen et al., 2008).
1. Objectives of the present study: were to investigate the protein expression of cyclin D1 and E in correlation with the expression of human epidermal growth factor-receptor 2 (Her-2) and the endocrine receptors in human breast cancer samples and to study the effect of inhibition of Estrogen receptor (ER) by Tamoxifen (Tam) on the apoptosis and expression of cell cycle signaling proteins in the ER-responsive breast cancer cell line MCF-7 and the ER-negative MDAMB-231 ”as a negative control”.
2. Methods used in the present study:
1. Immunohistochemical detection of Her-2, cyclin D1 and cyclin E in 15 breast cancer samples obtained from the Medical Research Institute, Alexandria-Egypt.
2. In vitro study: the effect of 17β-estradiol (E2) and Tamoxifen (Tam) on the cell proliferation, apoptosis as well as on the expression of cell cycle proteins were studied in a human ER+ve breast cancer cell line ”MCF-7” and an ER-ve breast cancer cell line ”MDAMB-231”.
3. The results obtained could be summarized as follows:
3.1 Immunohistochemical detection of Her-2: The overexpression of Her-2 was obtained in 20% of the breast carcinoma (3/15 of the cases); one sample was 3+ (6.66%) and two samples were 2+ (13.33%). There was no correlation between the level of Her-2 expression and the histopathological findings or with the ER and progesterone receptor (PR) levels.
3.2 Immunohistochemical detection of cyclin D1: Eight samples showed high expression of cyclin D1 (57% of tumors) and were assessed as positively stained and six samples showed low expression of cyclin D1 and were considered as negatively stained (43%).
Overexpression of cyclin D1 has been associated with ER positivity in 75% of the positively stained tumor cells, with the highest level of cyclin D1 in the case with double strongly positive ER/PR expression (3+/3+). It was also observed that 4/14 cases showed simultaneously low cyclin D1 levels and low (+1) level of ER expression.
Summary and Conclusion
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3.3 Immunohistochemical detection of cyclin E: Overall cyclin E overexpression was evident in 8/15 Patients (53.3%). Seven tumors showed low expression of cyclin E (46.6%).
Overexpression of cyclin E has been associated with ER positivity in 7/8 patients (87.5 %). Whereas, 71.4% of the samples (5/7 patients) with low cyclin E were ER negative, indicating a strong association between Cyclin E overexpression and ER positivity.
It was also demonstrated that cyclin E deregulation occurred through altered localization of the protein showing cytoplasmic expression. This was noted in 20% of the cases.
Despite of the few number of samples in this current study, it can be concluded that high cyclin D1 or high cyclin E levels can be strongly linked with the ER status in breast carcinoma.
3.4 E2 and cell cycle proteins:
To study the effect of E2 on the cell cycle proteins, the MCF-7 and MDAMB-231 breast cell lines were treated with 10nM E2 for 24 h.
Data from the western blot analysis showed that the treatment of MCF-7 cells with E2 led to a pronounced overexpression of the Cyclin dependent kinases (Cdk-4 & Cdk-6), epidermal growth factor receptor (EGFR), phosphorylated extracellular-signal-regulated kinases (pERk) and a slight activation of of pCdk-1T161, cyclins A and B, ER, insulin-like growth factor -1 receptor (IGF-1R) and pIGF-1R. An overexpression of cyclins D1 and E was also observed, this may indicate that cyclin D1-Cdk-4 complex is essential for E2 action.
However, neither of these alterations was observed in the MDAMB-231 breast cell line.
3.5 Effect of E2 on the cell proliferation of the breast cancer cell lines:
E2 induced an increase in the cell growth rate of MCF-7 cells after 48 h but did not affect the MDAMB-231 cell line.
These results illustrated that E2 stimulated the cell proliferation and activated the key cell cycle proteins in human breast cancer cells in an ER dependent manner.
3.6 Antiestrogen treatment with Tamoxifen decreased the expression of cell cycle proteins and induces apoptosis in MCF-7 cell line:
In the present study, the effect of Tam treatment was examined in the two breast cancer cell lines, MCF-7 and its negative control MDAMB-231, by treating the cells with 1 μM Tam for 24 h.
In the MCF-7 cell line, Tam induced a pronounced decrease in the expression of cyclin E, a slight decrease in cyclin B and Cdk6 and a pronounced induction of p21 and Her-4. A slight increase in the expression of ER was also observed. Also, Tam downregulated the expression of EGFR, Erk, pErk and pAkt. Tam did not have an effect on the cell cycle proteins in the ER-negative MDAMB-231 cell line.
Summary and Conclusion
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MCF-7 cells underwent massive apoptosis after 24 h exposure to Tam. While the control MCF-7 cells showed only 1.4% of apoptotic cells after 24 h of treatment. The percentage of apoptotic cells in Tam-treated MCF-7 cells was 15%. In contrast, the ER-negative cell line MDAMB-231 showed no difference in apoptosis between Tam-treated or control cells (1.3% and 1.6%, respectively).
Therefore, this indicates that the changes observed in the levels of cell cycle proteins are due to the inhibition of the ER by Tam.
Recommendation
Further studies addressed to elucidate the function of Her-receptor family as well as that of other molecular markers may help to identify patients most likely to benefit from adjuvant treatment, this could contribute to design efficient therapeutic strategies which may lead to therapy optimisation in the future.
Also, an understanding of how estrogens and antiestrogens influence the cell cycle.