Urolithin B protects PC12 cells against glutamate-induced toxicity.

BACKGROUND: The involvement of malfunctioning glutamate systems in various central nervous system (CNS) disorders is widely acknowledged. Urolithin B, known for its neuroprotective and antioxidant properties, has shown potential as a therapeutic agent for these disorders. However, little is known about its protective effects against glutamate-induced toxicity in PC12 cells. Therefore, in this study, for the first time we aimed to investigate the ability of Urolithin B to reduce the cytotoxic effects of glutamate on PC12 cells. METHODS: Different non-toxic concentrations of urolithin B were applied to PC12 cells for 24 h before exposure to glutamate (10 mM). The cells were then analyzed for cell viability, intracellular reactive oxygen species (ROS), cell cycle arrest, apoptosis, and the expression of Bax and Bcl-2 genes. RESULTS: The results of MTT assay showed that glutamate at a concentration of 10 mM and urolithin B at a concentration of 114 µM can reduce PC12 cell viability by 50%. However, urolithin B at non-toxic concentrations of 4 and 8 µM significantly reduced glutamate-induced cytotoxicity (p < 0.01). Interestingly, treatment with glutamate significantly enhanced the intracellular ROS levels and apoptosis rate in PC12 cells, while pre-treatment with non-toxic concentrations of urolithin B significantly reduced these cytotoxic effects. The results also showed that pre-treatment with urolithin B can decrease the Bax (p < 0.05) and increase the Bcl-2 (p < 0.01) gene expression, which was dysregulated by glutamate. CONCLUSIONS: Taken together, urolithin B may play a protective role through reducing oxidative stress and apoptosis against glutamate-induced toxicity in PC12 cells, which merits further investigations.

Preparation of chitosan nanoparticle containing recombinant CD44v antigen and evaluation of its immunization capacity against breast cancer in BALB/c mice.

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.

In silico designing and expression of novel recombinant construct containing the variable part of CD44 extracellular domain for prediagnostic breast cancer.

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.

Immunogenicity of chimeric MUC1-HER2 vaccine against breast cancer in mice.

OBJECTIVES: Breast cancer is one of the most common cancers in the world and is on the increase. MUC1 and HER2 as tumor-associated antigens (TAAs) are abnormally expressed to some extent in 75-80% of breast cancers. In our present research, a novel chimeric MUC1-HER2 (HM) protein was designed and used to study whether an immune response can be generated against these TAAs. In vitro analysis of the HER2-MUC1 construct confirmed the co-expression of MUC1 and HER2. MATERIALS AND METHODS: BALB/c mice were immunized with this novel chimeric protein. The humoral immune response was assessed by enzyme-linked immunosorbent assay (ELISA). Then, BALB/c mice were injected subcutaneously 2×105 4T1-MUC1-HER2 tumor cells. Subsequently, tumor size and tumor necrosis measurements, MTT, cytokines assay and survival test were performed. RESULTS: The results implied a critical role of HER2 and MUC1 antibodies in vaccination against breast cancer. This engineered protein can be a good vaccine to stop breast cancer. CONCLUSION: The results implied a critical role of HER2 and MUC1 antibodies in vaccination against breast cancer. This engineered protein can be a good vaccine to stop breast cancer.

Designing a recombinant chimeric construct contain MUC1 and HER2 extracellular domain for prediagnostic breast cancer.

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.