Tuning spacer length improves the functionality of the nanobody-based VEGFR2 CAR T cell.

BACKGROUND: The chimeric antigen receptor-expressing T (CAR-T) cells for cancer immunotherapy have obtained considerable clinical importance. CAR T cells need an optimized intracellular signaling domain to get appropriately activated and also for the proper antigen recognition, the length and composition of the extracellular spacer are critical factors. RESULTS: We constructed two third-generation nanobody-based VEGFR2-CARs containing either IgG1 hinge-CH2-CH3 region or hinge-only as long or short extracellular spacers, respectively. Both CARs also contained intracellular activating domains of CD28, OX40, and CD3ζ. The T cells from healthy individuals were transduced efficiently with the two CARs, and showed increased secretion of IL-2 and IFN-γ cytokines, and also CD69 and CD25 activation markers along with cytolytic activity after encountering VEGFR2+ cells. The VEGFR2-CAR T cells harboring the long spacer showed higher cytokine release and CD69 and CD25 expression in addition to a more efficient cytolytic effect on VEGFR2+ target cells. CONCLUSIONS: The results demonstrated that the third-generation anti-VEGFR2 nanobody-based CAR T cell with a long spacer had a superior function and potentially could be a better candidate for solid tumor treatment.

"Combination treatments of imatinib with astaxanthin and crocin efficiently ameliorate antioxidant status, inflammation and cell death progression in imatinib-resistant chronic myeloid leukemia cells".

BACKGROUND: Imatinib resistance remains a major obstacle in the treatment of chronic myelogenous leukemia (CML). Crocin (CRC) and astaxanthin (ATX) are phytochemicals with anti-cancer properties. AIMS: This study aimed to explore the effects of combination treatment of Imatinib with CRC and ATX on Imatinib-resistant K562 (IR-K562) cells. METHODS AND RESULTS: After the establishment of IR-K562 cells, growth inhibitory activity was determined by the MTT assay. To test the regeneration potential, a colony formation assay was performed. Cell cycle analyses were examined by flow cytometry. Cell injury was evaluated by lactate dehydrogenase (LDH) leakage. Real-time PCR was applied to assess the expression of IL6, TNF-α, STAT3, BAD, CASP3, TP53, and Bcl-2 genes. Caspase-3 activity was determined by a colorimetric assay. Antioxidant activity was measured using a diphenylpicrylhydrazyl (DPPH) assay. After 48 h of treatment, ATX (IC50 = 30µM) and CRC (IC50 = 190µM) significantly inhibited cell proliferation and colony formation ability, induced G1 cell cycle arrest and cell injury, upregulated the expression of apoptosis-associated genes, and downregulated the expression of anti-apoptotic and inflammatory genes. The combination of IM with ATX and/or CRC synergistically reduced cell viability (combination index [CI] < 1). CONCLUSION: Our data suggest that IM shows better therapeutic efficacy at lower doses when combined with ATX and/or CRC.

Glucose increases proliferation and chemoresistance in chronic myeloid leukemia via decreasing antioxidant Properties of ω-3 polyunsaturated fatty acids in the presence of Iron.

BACKGROUND: There is a strong association between hyperglycemia, oxidative stress, inflammation and the onset and progression of diabetes which causes a higher risk of cancer. This study investigated, the effect of concomitant use of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) with iron supplements in hyper-glucose conditions on the K-562 cell line. METHODS: The effects of iron, ω-3 PUFAs, and a combination of both on K-562 cells were investigated under normal and high glucose conditions. The impact of these treatments was evaluated using multiple methodologies, including the MTT assay for cell viability, quantification of oxidative stress markers [total antioxidant capacity (TAC) and malondialdehyde (MDA)], and analysis of the cell cycle. Furthermore, the expression levels of TNFα and p53 mRNA were measured using Real-time PCR. RESULTS: The co-treatment of ω-3 PUFAs and iron in the presence of high glucose had notable effects, as evidenced by an increase in cell survival, resistance to imatinib chemotherapy, TNFαmRNA expression levels, MDA levels, and percentage of cells in the G2/S phase. Additionally, there was a decrease in the mRNA expression of p53 and TAC levels compared to treatment in the normal-glucose condition. CONCLUSION: Hyperglycemic conditions in conjunction with the combined treatment of theω-3 PUFAs and iron, led to reduced anticancer capacity, chemosensitivity, anti-inflammatory and antioxidant properties of the K-562 cells. These effects were found to be mediated by oxidative stress.

Astaxanthin decreases the growth-inhibitory dose of cytarabine and inflammatory response in the acute lymphoblastic leukemia cell line NALM-6.

BACKGROUND: In spite of the great progress in acute lymphoblastic leukemia (ALL) treatment, a large number of patients still suffer from chemotherapy drug toxicity. As a routine medication for ALL treatment, cytarabine (Ara-C) has many side effects on the patients. Astaxanthin (ASX), on the other hand, is a carotenoid with antioxidant, anti-inflammatory and anti-cancer properties. PURPOSE: The present study investigated the effects of ASX in combination with Ara-C on cell proliferation, apoptosis induction, and cell cycle arrest in NALM-6 cell line. METHODS: NALM6 cells were treated with different concentrations of ASX, Ara-C, and their co-treatment. Cytotoxic effects were evaluated using MTT assay. After treating the cells with the IC50 dose of ASX, Ara-C and their co-treatment, we studied apoptosis induction, cell cycle arrest, and expression of apoptotic, anti-apoptotic, and inflammatory genes. RESULT: MTT assay demonstrated that co-treatment of cytarabine and ASX had greater cytotoxicity effects compared with the IC50 dose of Ara-C alone. After 48 h of treatment of NALM-6 cells with the combination dose, expression levels of apoptotic genes (P53, caspase-8, 3), the anti-apoptotic gene (Bcl-xL) and inflammatory genes (IL-6, TNF-α) changed significantly compared to the untreated group (p < 0.05). CONCLUSIONS: Co-treatment of ASX and Ara-C has synergism effects on apoptosis pathways, cell proliferation inhibition, and decreased inflammation.

Orbital Lateral Cantal Distance Can Predict the Cephalometric Characteristic of Mandible in Iranian Population.

ABSTRACT: Orbitofacial anthropometrics have become an important tool used in reconstructive surgery. The authors attempt to evaluate the relation between orbital lateral canthal distance and the cephalometric characteristic of mandible in Iranian population.In a cross-sectional study, anthropometric parameters of face in 200 subjects (100 males and 100 females) with mean age of 34.39 ±â€Š18.83 were evaluated by three-dimensional computed tomography imaging.In this study, there was not a significant difference in the age of sex groups (P = 0.183). Also, there was no significant difference in the left and right mandible angle in different sex groups (P = 0.25, P  =  0.124, respectively). There were significant differences in the anterior mandible distance, inferior mandible angle distance (P = 0.0001) and lateral cantus distance of sex groups (P = 0.0001). There was a significant correlation between lateral contuse distance and left mandible angle (r = 0.226, P = 0.001), right mandible angle (r = 0.283, P = 0.00), mandible angle (r = -0.266, P = 0.00), anterior mandible angle distance (r = 0.655, P = 0.00), and inferior mandible angle distance (r = 0.582, P = 0.00).Here, we conclude that orbital lateral canthal distance can predict the cephalometric characteristic of mandible in Iranian population.

The anti-inflammatory effect of omega-3 polyunsaturated fatty acids dramatically decreases by iron in the hippocampus of diabetic rats.

AIMS: Receptor for advanced glycation end products (RAGE) production is induced by diabetes. Microglial cells are activated by RAGE and produce inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and oxidative stress markers. Persistent production of TNF-α can provide a link between diabetes and Alzheimer's disease (AD). The purpose of this study was to investigate the effect of concomitant use of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) with iron supplements on microglial cell activation and inflammatory conditions in the hippocampus of type 2 diabetic rats. MAIN METHODS: Diabetic and normal Wistar rats were divided into six groups. Oxidative stress markers (total oxidant status (TOS), total antioxidant capacity (TAC), and malondialdehyde (MDA)), mRNA expression and protein levels of RAGE and TNF-α were evaluated in the hippocampus of the controls and supplemented with ferrous sulfate and ω-3 PUFAs alone and together rats. Also, the entry of microglia cells into the hippocampus was evaluated by immunohistochemistry technique. KEY FINDINGS: Levels of the microglial activation (2.4 fold, p < 0.0001), MDA (84%, p < 0.0001) and oxidative stress index (OSI) (11%, p = 0.0094), mRNA expression and protein contents of RAGE (1.83 fold and 82% respectively) and TNF-α (2.25 fold and 86% respectively) were strongly influenced by negative effect of iron compared to the group receiving only ω-3 PUFAs which was dramatically improved by vitamin E. SIGNIFICANCE: These observations indicated that the co-supplementation of ferrous sulfate with ω-3 PUFAs decreases the anti-inflammatory ability of ω-3 PUFAs in the hippocampus of diabetic rats via RAGE/TNF-α-induced oxidative stress pathway up-regulation.

miR-155 effectively induces apoptosis in K562 Philadelphia positive cell line through upregulation of p27kip1.

Introduction: Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia chromosome translocation, at (9; 22), which results in BCR-ABL fusion tyrosine kinase oncoprotein. This fusion induces down-regulation of miR-155. Upregulation of miR-155 can influence cell fate via the effect on p27kip1 and apoptosis. The aim of this study was to induce apoptosis in K562 CML cell line by overexpression of miR-155. Methods: The K562 cell line was transfected with pLenti-III-pre mir155-GFP constructs through electroporation. Then, overexpression of miR-155 as well as the expression level of p27kip1 and c-Myc was analyzed by quantitative PCR (qPCR). The level of p27 (Kip1) protein expression was measured by Western blot and the Annexin V method was carried out to investigate apoptosis. Results: Flow cytometric analysis results of K562 cells transfected with pLenti-III-pre mir155-GFP construct showed a significant increase in cell apoptosis. Gene expression and protein level of p27kip1 were upregulated. However, there was no change in c-Myc expression profile. Conclusion: miR-155 could be a promising approach to aid in the treatment of CML. However, further studies are required in this respect.

Tumor suppressive function of microRNA-192 in acute lymphoblastic leukemia.

Non-coding RNAs play a critical role in gene regulation in cancer cells. Reduced expression of microRNA-192 (miR-192) has been detected in many cancers. In this study, we investigated the role of miR-192 in cell proliferation and cell cycle control in NALM-6 cell line, a model of acute lymphoblastic leukemia (ALL). Cell cycle analysis by DNA content using propidium iodide staining and cell apoptosis analysis using Annexin V assay were carried out. Cell proliferation changes were monitored using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, the relative changes in P53, BAX, CASP3, and BCL-2 gene expression were determined by quantitative reverse transcription PCR. Overexpression of miR-192 resulted in cell proliferation arrest in ALL cells. After 72 and 96 hours of transduction, apoptosis was significantly increased in the cells transduced with miR-192-overexpressing virus compared with control cells. The expression of P53, BAX, and CASP3 increased after 48 hours of transduction in miR-192-overexpressing cells, but no change was observed in BCL-2 expression. The G0/S and G1/S ratio changed to 7.5 and 4.5, respectively, in the cells overexpressing miR-192 compared with controls. The results of our study suggest, for the first time, tumor suppressive effects of miR-192 in ALL cells.