Hesperidin Plays Neuroprotective Effects Against Quinolinic Acid in Human SH-SY5Y Cells: Focusing on ROS Levels and Cell Cycle Arrest

Background and objectives: In some neurodegenerative diseases, an aberrant accumulation of quinolinic acid is frequently associated with the loss of nerve cells and a condition known as neuritis. This is typically caused by an excessive production of free radicals. Studies have shown that hesperidin has potent antioxidant effects, but nothing is known about how it protects against the neurotoxicity induced by quinolinic acid. This study aimed to evaluate the protective effect of hesperidin against quinolinic acid-induced neurotoxicity in the SH-SY5Y neuroblastoma cell line. Methods: The MTT test was used to determine cell viability and protective dosage of hesperidin. Flow cytometry using propidium iodide (PI) staining was used to determine the cell cycle of SH-SY5Y cells after exposure to quinolinic acid in combination with hesperidin. Reactive oxygen species (ROS) levels within cells were also measured using 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) in the mentioned groups. Results: Our results demonstrated that hesperidin had a protective effect against quinolinic acid-induced toxicity at nontoxic concentrations (p<0.001). Moreover, the percentage of apoptotic cells in the sub-G1 phase increased significantly (p<0.001). Hesperidin pretreatment significantly decreased sub-G1 arrest that was induced by quinolinic acid (p<0.001). Hesperidin significantly decreased ROS levels generated by quinolinic acid (p<0.001). Conclusion: The current study showed that hesperidin exerts its effect through antioxidant activity and can be considered a promising neuroprotectant agent against quinolinic acid-induced neurotoxicity in neurodegenerative disorders; however, more research is necessary in this area for the treatment

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

Abstract 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

<em>In silico</em> analysis of chimeric subunit vaccine containing HER-2-MUC1 against breast cancer

224-233Breast cancer is a leading cause of cancer-related deaths in women worldwide. Although tumorectomy, radiotherapy, chemotherapy and hormone replacement therapy have been used for the treatment of breast cancer, there is no effective therapy for patients with invasive and metastatic breast cancer. Targeting tumors using cancer vaccine therapeutics has several advantages including the induction of long-term immunity, prime boost strategies for additional treatments and reduced side effects compared to conventional chemotherapeutics. However, one problem in targeting tumor antigens directly is that it can lead to antigen loss or immune editing. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu- (480–620) and seven tandem repeats of MUC1 (VNTR) that represents a three-dimensional epitope. The construct was analyzed by bioinformatics softwares. Linear and discontinuous B-cell epitopes, MHC class I and II binding peptides of chimeric protein were predicted. Results suggest that the construct can be an appropriate vaccine candidate against breast cancer

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

Objective(s): 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