Breast cancer is the most common cancer among women in the world. One of the approaches for diagnosis of breast cancer is detection of its tumor-associated markers. Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Also, human epidermal growth factor receptor-2 (HER2/erbB-2) belongs to the one of four members of tyrosin kinase type 1 family in which overexpression of HER2 is associated with malignancy in breast cancer. This study was aimed to bioinformatics analysis and designing a recombinant chimeric protein containing MUC1 and HER2 antigens to express in prokaryotic host (Escherichia coli) as breast cancer diagnosis tools. The immunogenic sequences of MUC1 and HER2 were extracted and fused together by a linker. The chimeric construct was analyzed by bioinformatics softwares. The optimization and purification, evaluation of the expression of chimeric protein was performed using Western blotting, ELISA, and immunohistochemistry. The results showed that the chimeric construct was stable and immunogenic domains were exposed. The pET-28a vector containing chimeric gene had high level of protein expression. The recombinant chimeric protein was confirmed using Western blotting, and it was investigated using ELISA and IHC. Then, the MUC1 and HER2 combined peptides can be used as coating antigens in ELISA for detection of antibodies against MUC1 or HER2 in human serum.
Objective(s) Breast tumors show heterogeneity containing cancer stem cells as a small subpopulation of a tumor mass. CD44 as a cancer stem cells antigen is abnormally expressed by carcinomas of epithelial origin. Also, overexpression of CD44 variable isoforms (CD44v) is associated with malignancy in breast cancer. In the present research, our objective was to evaluate the immunogenicity of prepared nanoparticles containing a novel recombinant CD44v (rCD44v) protein in the mouse model. Materials and methods CD44 gene was expressed in E. coli BL21 DE3 using the pET28a-CD44 vector. The expressed rCD44v protein was purified, encapsulated into the chitosan nanoparticles, and administered to BALB/c mice. ELISA was used to evaluate the immunoglobulin levels of immunized animals. For challenge experiment, 2 × 106 4T1-CD44 tumor cells were injected subcutaneously in mice, and tumor size, necrosis, and metastases were measured. Finally, cell proliferation assay, cytokines assay, and neutralization assay of the mouse anti-rCD44v on the human breast cancer cell line were examined. Results The measured size of chitosan-rCD44v nanoparticles was 146.5 nm. Recombinant CD44v encapsulated by chitosan nanoparticles increases immunological responses via the adjuvant nature of chitosan nanoparticles. In the immunized mice, IgG and IgA titers were significantly increased. Tumor growth in injection and nano-injection test groups compared with the mice control groups displayed a significant reduction (P < 0.05). A high amount of splenocytes secreting IFNγ and IL-17 was seen in immunized mice with rCD44v (P < 0.05). Furthermore, a smaller size of lung metastases compared to the control mice groups was detected. Conclusion The encapsulated rCD44v within the chitosan nanoparticles induced a significant immune response in mice and can establish significant protection against breast cancer. Therefore, it can be considered a vaccine candidate for breast cancer therapeutic modalities.
Background: CD44, as a tumor-associated marker, can be used to detect stem cells in breast cancer. While CD44 is expressed in normal epithelial cells, carcinoma cells overexpress CD44.Aims: In the current study, we designed a recombinant protein that included the variable component of the CD44 (CD44v) extracellular domain to apply in clinical diagnosis of breast cancer.Methods: A total of 100 CD44v amino-acid residues were determined, and the structure was examined using bioinformatics tools. The construct was inserted into the PET28a vector and transformed in E. coli BL21(DE3). A nearly 12 kDa fusion protein was obtained by Ni-NTA affinity metal chromatography. Recombinant CD44v was examined by Western blotting, ELISA, and immunohistochemistry (IHC) assays. Results:The findings revealed that the structure of rCD44v was stable, and its antigenic domain was exposed. The recombinant CD44v was confirmed by western blotting, and the presence of antibodies against recombinant CD44v protein in the patient's serum was detected by the ELISA. Our data demonstrated a link between CD44v serum levels and the prevalence of breast cancer. Conclusion:Assessments of antiCD44v antibodies with rCD44v could be a useful tool for identifying breast cancer in its early stages, which can lead to better outcomes.
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