Evaluation of the effects of simulated hypoxia by CoCl2 on radioresistance and change of hypoxia-inducible factors in human glioblastoma U87 tumor cell line.

PURPOSE: Glioblastoma (GBM) is considered the most common and lethal type of brain tumor with a poor prognosis. GBM treatment has challenges due to its aggressive nature, which often causes treatment failure and recurrence. Hypoxia is one of the characteristics of glioblastoma tumors that contribute to radioresistance and malignant phenotypes of GBM. In this study, we aimed to determine the effects of hypoxia on the radiosensitivity of U87 GBM cells by the hypoxia-mimicking model. METHODS: Following the treatment of cells with different concentrations of CoCl2, an MTT assay was used to evaluate the cytotoxicity of CoCl2. To understand the effects of Ionizing radiation on CoCl2-treated groups, cells were exposed to irradiation after pretreating with 100 µM CoCl2, and a clonogenic survival assay was performed to determine the radiosensitivity of U87 cells. Also, the intracellular Reactive oxygen level was measured by 2',7'-dichlorofluorescein diacetate (DCFDA) probe staining. Additionally, the expression of hypoxia-associated genes, including HIF-1α, HIF-2α, and their target genes (GLUT-1), was monitored by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Our study revealed that the cell viability of CoCl2-treated cells was decreased in a concentration-dependent manner. Also, CoCl2 did not cause any cytotoxicity on U87 cells at a concentration of 100 µM after treatment for 24 h. Colony formation assay showed that CoCl2 pretreatment induced radioresistance of tumor cells compared to non-treated cells. Also, CoCl2 can protect cells against irradiation by the clearance of ROS. Moreover, Real-time results showed that the mRNA expression of HIF-1α and GLUT-1 were significantly upregulated following hypoxia induction and/or irradiation condition. However, the level of HIF-2α mRNA did not change significantly in hypoxia or irradiation alone conditions, but it increased significantly only in hypoxia + irradiation conditions. CONCLUSION: Taken together, our results indicated that simulating hypoxia by CoCl2 can effectively increase hypoxia-associated genes, specially HIF-1α and GLUT-1, but did not affect HIF-2α gene expression. Also, it can increase the clearance of ROS, respectively, and it leads to inducing radioresistance of U87 cells.

Association of cytotoxic T-lymphocyte-associated protein 4 polymorphisms with recurrent pregnancy loss: A case-control study.

Background: A large proportion of cases of recurrent pregnancy loss (RPL) are associated with immunological factors. Objective: This study investigated the association between single nucleotide polymorphisms of cytotoxic T-lymphocyte-associated protein (CTLA)-4 gene in women with a history of RPL compared to healthy women. Materials and Methods: A case-control study was performed on 2 groups consisting of 120 healthy women with no history of abortion and at least one delivery (control) and 120 women with a history of 2 or more primary RPLs (case). In addition, 5 mL of peripheral blood sample was taken from all subjects. The frequencies of CTLA-4 rs3087243 and rs231775 polymorphisms were assayed by restriction fragment length polymorphism polymerase chain reaction and rs5742909 using the high-resolution melting real-time polymerase chain reaction method. Results: The mean age of the women in the control and RPL groups were 30.03 ± 4.23 (range 21-37), and 28.64 ± 3.61 yr (range 20-35), respectively. Pregnancy loss numbers ranged between 2-6 in women with a history of RPL, and between 1 and 4 in the successful pregnancy group. Statistical analysis showed a significant difference between the genotypes of GG and AG in the 2 groups in rs3087243 polymorphism (OR 1.00 for GG genotype and OR 2.87 for AG genotype, p = 0.0043). No significant difference was observed in the genotype frequencies of rs231775 and rs5742909 polymorphisms, of the 2 groups (p = 0.37, and p = 0.095), respectively. Conclusion: Our findings indicated that CTLA-4 polymorphism, rs3087243, might be associated with a risk of RPL in Iranian women.

Triptorelin Peptide Conjugated Alginate Coated Gold Nanoparticles as A New Contrast Media for Targeted Computed Tomography Imaging of Cancer Cells.

OBJECTIVE: Increasing research has been focused on the development of various nanocomplexes as targeted contrast media in diagnostic modalities, mainly in computed tomography (CT) scan imaging. Herein, we report a new method that uses Triptorelin [a luteinizing hormone-releasing hormone (LHRH) agonist]-targeted gold nanoparticles (AuNPs) via alginate for early detection of cancer by molecular CT imaging. MATERIALS AND METHODS: In the experimental study, the formed multifunctional AuNPs coated with alginate conjugated with Triptorelin peptide (Triptorelin-Alginate-AuNPs) were synthesized and characterized via different techniques, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and fourier transform infrared (FTIR) spectroscopy. The MTT assay was applied to calculate the toxicity of the NPs. RESULTS: The results indicated that the formed Triptorelin-Alginate-AuNPs with an Au core size of ~18 nm are noncytotoxic at 127-, 254-, 381- and 508-mM concentrations and revealed significant improvement in the attenuation of X-rays intensity and contrast to noise ratio (CNR), compared with non-targeted cells at the highest energies (90, 120, 140 kVp). At 90 kVp, compared to non-targeted cells, targeted cells (Triptorelin-Alginate-AuNPs) enable 1.58, 1.69, 3.7 and 3.43 times greater contrast at a concentration of 127 mM, 254 mM, 381 mM, and 508 mM, respectively. CONCLUSION: These results suggest that the developed Triptorelin-Alginate-AuNPs may be considered an effective contrast agent for molecular CT imaging of gonadotropin-releasing hormone (GnRH) receptor-expressing cancer cells.

Alteration in Expression of Trim29, TRIM37, TRIM44, and ß-Catenin Genes After Irradiation in Human Cells with Different Radiosensitivity.

Introduction: Radiotherapy is a crucial component of treatment for ∼70% of all cancer patients. The identification of effective biomarkers of radiosensitivity (RS) is a fundamental goal of radiobiology. The authors hypothesize that the RS of human normal and tumoral cells is correlated by the level of expression of TRIM29, TRIM37, TRIM44, and ß-catenin genes. Materials and Methods: Clonogenic assay was performed and RS of four cell lines was determined by survival fraction at 2 Gy. To determine the level of gene expression 6 and 24 h after irradiation, RNA was extracted from each cell line, and expression of the above-mentioned genes in cell lines with different RS was determined by real-time polymerase chain reaction (PCR). Results: The clonogenic assay showed that human dermal fibroblasts (fibroblast) and HT-29 (colorectal) cells are radioresistant, while human foreskin fibroblasts (fibroblast) and QU-DB (lung) cells are radiosensitive. Analysis of the real-time PCR data, 6 h after irradiation, showed that the increase and decrease of the expression of TRIM29 and TRIM37 genes were directly correlated with the RS of normal and tumor cells. At 24 h postirradiation, a considerable difference was only observed in the expression of the ß-catenin gene. Conclusion: This study showed that the TRIM29 and TRIM37 genes are involved in the cell response to radiation and proposed that these genes may be biomarkers for predicting RS in normal and tumoral cell lines.

Association between rs1049174 NKG2D gene polymorphism and idiopathic recurrent spontaneous abortion in Iranian women: a case-control study.

Natural killer group 2, member D (NKG2D) is one of the best known activating receptors of NK cells, which recognises its ligand on altered or stressed cells and activates NK cells to kill them. In this study, the single nucleotide polymorphism of the NKG2D gene for rs1049174 mutation was compared in 140 women with recurrent spontaneous abortion (RSA) and 175 control women with at least one successful pregnancy and without any known pregnancy loss. The findings just revealed that GG genotype and G allele were significantly higher in the case group compared with the control group (p < .001). Our results regarding decreased risk of RSA in C allele (OR = 0.438; 95%CI = 0.310-0.619; p < .001), and GC genotype (OR = 0.492; 95%CI = 0.214-0.574; p < .001) compared with G allele and GG genotype respectively. This study demonstrated a significant association between NKG2D gene polymorphism (rs1049174 G/C) and the risk of RSA in Iranian women.Impact statementWhat is already known on this subject? According to previous investigations, maternal immune responses may affect the foetus, causing recurrent spontaneous abortion (RSA). The main cause of RSA has not yet been detected in nearly 50% of the cases.What do the results of this study add? The results showed that the frequency of G allele and C allele were significantly different in the case group and control group.What are the implications of these findings for clinical practice and/or further research? The results suggest a protective function of C allele because it significantly decreased the risk of RSA compared to G allele. It improves inhibition of NK cells and probably participates in maintaining pregnancy in fertile controls; whereas, G allele is related to a slight inhibition of NK cells, probably leading to increase effectiveness of NK activation and undesirable inflammation, which consequently causes foetal rejection.