Estrogen-reactive Protein And Breast Cancer Research

Breast cancer is one of the leading cause of cancer deaths in women. Although the earlier detection and more effective treatments have attained remarkable successes in past years, the curative effect is still not satisfied. It is very important to detect the tumor markers for earlier diagnosis and monitoring response to therapy. We have been focusing on the specific protein regulated by estrogen, which synthesized in target organs for estradiol.Our hypothesis is that this kind of protein would be changed accompanying the estradiol induced cancerization. To establish a correlation between the protein and breast cancer would be a way to find novel marker of breast cancer, and which may be available in the cancer diagnosis and treatment.In our previous studies, a 250kD protein regulated by estradiol was identified from fluid of rat uterus, and nominated estrogen-responsive protein y (ERPy). The preliminary study showed that some similar antigen epitopes were recognized in tumor and normal breast tissue by anti-ERPy antibody.Objectives of the present study are: to characterize the ERPy further, to investigate the correlation between ERPy and breast cancer, to elucidate the correlation of estrogen-responsive protein and breast cancer by examining the MUC1 expression in cultured human breast cancer cell lines after administrating estradiol or tamoxifen; Moreover, to examine MUC1 and E-cadherin in the cultured cell for ascertaining possible association between the two proteins in tumor.The techniques were used in the present study including two-dimensional gel electrophoresis, Mass spectrometry, Bio-information, immuno-chemistry, cell culture, Laser confocal microscope and Flow cytometry, etc. The results are as follows:1, Two protein bands at 140kD and 160kD were specifically recognized by rabbit anti-250kD ERPy polyclonal antibody from breast cancer tissue extract by using Western blot analysis. Furthermore, the result showed that quantity and quality of the recognized protein bands varied from patient to patient. The difference may reflect the different stage in tumor, which is significant in evaluating prognosis for patient. 2, One protein band about 50kD was specifically recognized by rabbit anti-250kD ERPy polyclonal antibody from MCF7 cell extract by Western blot analysis. The 2DE combined with Western blotting were performed. The results also showed that two protein spots at about 50kD, pl of two spots were 6.0 and 6.5 separately, were recognized by the antibody. The two spots were digested in gel by trypsin and the mass of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Peptide mass fingerprinting combined with Internet searching of protein database revealed that both two spots are identical to enolase.3, The enolase expression were further identified by commercial anti-enolase antibody. The 2DE showed that enolase was increased in cell extract of MCF7 after administrating E₂ at 10^-8 mol/l for 48 h. The enolase were also examined in tissues of breast cancer and near cancer from 14 patients' specimen. The results revealed that expression of enolase was higher in tissue of tumor than that in normal. It suggested that perhaps enolase is one of the changed enzymes which accompanying the estradiol induced cancerization. Therefore, enolase may be used as a marker in breast cancer diagnosis.4, The correlation of estrogen-responsive protein and breast cancer was investigated by examining the effect of E₂ or TAM on MUC1 expression in cultured cells. First of all, MCF7 and MDA-MB-231 cells were inoculated and grown well in the plate. The different concentration of E₂ or TAM were administrated, and the optimal concentration were selected according to MTT assay.5, The localization of MUCI in MCF7 and MDA-MB-231 cells was observed by Confocal analysis. The results indicated that the location of MUC1 in both cells were not changed after drug treatment. It suggested that the location of MUC1 is not fit for evaluating the effect of TAM treatment, since the MUC1 location would not reflect the progress of tumor.6, Flow cytometry analysis ascertained that the MUC1 is one of estrogen-responsive proteins, and the effect of E₂ or TAM on MUC1 expression by ER approach. The result suggested that when treating ER(+) patient, the change of MUC1 expression would reflect the inhibit effect of TAM on tumor cells and MUC1 was suitable as prognostic marker. However, when treating ER(-) patient, it is not suitable.7, The localization of MUC1 and E-cadherin was observed by Confocal analysis in MCF7 cells after treating with E₂. The results indicated that the location of both MUC1 and E-cadherin were not valuable marker for prognosis. The Flow cytometry analysis result also showed that E-cadherin did not change with MUC1. It means that E-cadherin did not cooperate with MUC1 directly.The results of the study provide significant information about the correlation of estrogen-responsive protein and estradiol in breast cancer. This information could be used for developing novel diagnostic and therapeutic strategies, and that will be useful for future research on evaluating the effect of estrogen receptor regulating therapy.