Annexin-A5 Overexpression Increases Sensitivity of MCF-7 and MCF-7/ADR Cells to Epirubicin.

Background: Multi-drug resistance is an important challenge in the chemotherapy of cancer. The role of annexin A5 (ANXA5) in the biology of cancer has been the focus of many studies. Breast Cancer (BC) is frequent cancer in women with high morbidity and mortality rate. The present study aimed to investigate the effects of ANXA5 overexpression on the anti-tumor activity of Epirubicin (EPI) in MCF-7 and MCF-7/ADR cells. Methods: MCF-7 and MCF-7/ADR cells were transfected with the pAdenoVator-CMV-ANXA5-IRES-GFP plasmid or mock plasmid. The overexpression of ANXA5 was evaluated using qPCR. The effects of ANXA5 overexpression and EPI on the cell viability of MCF-7 and MCF-7/ADR cells were measured using an MTT assay. Cell apoptosis was measured by annexin V/7-AAD flow cytometry assay. Results: Following the overexpression of ANXA5, the viability of MCF-7 and MCF-7/ADR was significantly decreased. Furthermore, the overexpression of ANXA5 in MCF-7 cells increased the cytotoxic effects of EPI in all doses and reduced the IC50 of EPI from 17.69 µM to 4.07 µM. Similarly, the overexpression of ANXA5 in MCF7-ADR cells reduced the IC50 of EPI from 27.3 µM to 6.69 µM. ANXA5 overexpression alone or combined with EPI treatment increased the apoptosis of MCF7 and MCF7-ADR cells. Conclusion: The results of the present study demonstrate that ANXA5 overexpression increases the sensitivity of MCF-7 and MCF-7/ADR to EPI, suggesting a possible beneficial role of ANXA5 in the therapy of BC.

Protective effects of Panax Ginseng against 131I-induced genotoxicity in patients with differentiated thyroid cancer.

BACKGROUND: Radioiodine (131I) therapy (RAIT) is associated with oxidative stress (OS)-induced DNA damage in patients with differentiated thyroid cancer (DTC). The goal of this study was to evaluate the possible ameliorating effects of Panax Ginseng (PG) on RAIT-induced genotoxicity in patients with DTC. MATERIALS AND METHODS: Forty DTC patients who had received 131I (100 to 175 mCi) were enrolled in this study. The patients were randomly classified (n = 10) into control, placebo, PG1 groups (receiving 500 mg/day of PG for 2 days before RAIT), and PG2 group (receiving 500 mg/day of PG for 2 days before to 1 day after RAIT). Blood samples were collected before and 2 days after RAIT. Lymphocyte micronuclei (MN) frequency was measured using the MN assay. Serum total antioxidant capacity (TAC) and ischemia-modified albumin (IMA) were measured using colorimetric assays. Serum albumin, blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using commercial kits. RESULTS: The mean of baseline MN frequency was the same in the four groups. RAIT increased the MN frequencies to at least three times the baseline values in the control (39 ± 5) and placebo groups (38 ± 6) (P < 0.001). PG caused a significant decrease in the MN frequencies in the treated groups compared to the control and placebo groups (P < 0.001). RAIT and PG administration had no significant effects on the serum IMA, TAC, and markers of liver and kidney toxicity. CONCLUSION: PG could be considered a useful remedy for the protection against RAIT-induced chromosomal damage in DCT patients.

The effect of Annexin A5 overexpression on invasiveness and expression of the genes involved in epithelial-mesenchymal transition of HCT 116 cell line.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in colorectal cancer (CRC) metastasis. In the present study, we evaluated the effects of annexin A5 (ANXA5) overexpression on invasiveness as well as the expression of genes involved in EMT of HCT 116 cell line. PCMV6-AC-IRES-GFP plasmid harboring ANXA5 cDNA was constructed. HCT 116 cell line was transfected with recombinant plasmids using Lipofectamine 3000. Fluorescent microscopy was used to determine the efficiency of plasmid transfection. Cell viability was determined using the MTT assay. HCT 116 cell migration was evaluated using wound healing assay and transwell migration assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of genes involved in EMT. The results of RT-qPCR showed overexpression of ANXA5 compared to the control group. ANXA5 overexpression had no significant effects on cell viability but significantly decreased the rate of wound closure in the wound healing assay as well as the number of migrated cells in transwell assay. Furthermore, ANXA5 overexpression decreased the expression of N-cadherin, Snail, Slug, MMP-2, and MMP-9 while the expression of E-cadherin increased following ANXA5 overexpression. However, VEGF expression did not significantly change after ANXA5 overexpression. Results of the present study suggest that ANXA5 overexpression might have inhibitory effects on the metastasis of CRC through modulating the expression of EMT- related genes.

Diagnostic values of ischemia modified albumin in diabetes-related complications: a narrative review.

Purpose: Oxidative stress (OS) plays a critical role in the onset and progression of macro and microvascular complications of diabetes mellitus (DM). Ischemia-modified albumin (IMA) is a novel and simple test for evaluating OS. In the present study, we reviewed the available information on the alteration of circulating IMA in DM and its possible prognostic and diagnostic value in DM-related complications. Methods: Relevant studies regarding IMA alteration in DM published until May 30, 2022 were extracted from Google Scholar, PubMed, Scopus, and Science Direct databases. The following key words were used: IMA, DM, diabetes complications, retinopathy, nephropathy, diabetic foot, and vascular complications. Results: This review revealed increased circulating IMA levels in the patients with type 1, type 2, and gestational DM. Furthermore, IMA showed a close relationship with the severity of DM complications including hyperglycemia, dyslipidemia, diabetic retinopathy, diabetic nephropathy, peripheral arterial disease, and diabetic foot ulcer. However, lack of assay standardization and low specificity are major obstacles to the use of IMA as a promising biomarker. Conclusion: IMA levels are associated with DM complications and can be applied as a practical test for evaluating the risk and predicting the severity of DM complications.

Targeted Overexpression of NDRG2 using Survivin Promoter Reduces Viability and Invasiveness of A549 Cell Line.

Background: Anti-tumor effects of N-myc Downstream Regulated Gene2 (NDRG2) have been demonstrated in many tumors. In the present study, NDRG2 was specifically overexpressed in lung cancer cell line using Survivin Promoter (Sur-P). Then, the effects of NDRG2 overexpression on viability, apoptosis, migration, and invasion of A549 cells were evaluated. Methods: Recombinant pAdenoVator-Sur-P-NDRG2-IRES-GFP plasmid harboring NDRG2 gene under transcriptional control of Sur-P and mock plasmid were constructed. A549 lung tumor cells and LX-2 cells (non-tumor cell line) were transfected with pAdenoVator-Sur-P-NDRG2-IRES-GFP, pAdenoVator-CMV-NDRG2-IRES-GFP, or mock plasmids. Tumor specificity of Sur-P was evaluated using fluorescent microscopy for GFP expression. The effects of NDRG2 overexpression on cell viability, apoptosis, and migration of A549 cells were measured using MTT, annexinV/7-AAD flow cytometry, and transwell migration assay, respectively. NDRG2 and matrix metalloproteinase-2 (MMP-2) expression were measured using real time-PCR. Results: pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection resulted in a huge GFP expression in A549 cells, but not in LX-2 cells. The results of real time-PCR analysis also showed that pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection led to an abundant NDRG2 expression in A549 cells. NDRG2 overexpression decreased A549 cell viability through increasing cell apoptosis. In addition, migration, invasion, and MMP-2 expression decreased following NDRG2 overexpression in A549 cells. Conclusion: The findings indicate that the targeted overexpression of NDRG2 using Sur-P can reduce the viability and invasiveness of A549 cells, suggesting possible benefits of this approach in lung cancer therapy.

Association of serum kynurenine/tryptophan ratio with poor glycemic control in patients with type2 diabetes.

PURPOSE: The role of indoleamine 2,3-dioxygenase (IDO) has been shown in insulin resistance and metabolic syndrome. The present study aimed to measure serum IDO activity in patients with type 2 diabetes (T2DM) and to determine its association with glycemic control, oxidative stress, and insulin resistance. METHODS: Seventy-four patients with T2DM and 74 healthy subjects were selected to participate in this study. Fasting serum biochemical parameters including fasting blood sugar (FBS), HbA1c, insulin, uric acid, albumin, tryptophan, kynurenine, and total antioxidant capacity (TAC) were measured. HOMA-IR, QUICKI, and HOMA-B were calculated using serum FBS and insulin values. IDO activity was estimated using kynurenine/tryptophan ratio (KTR). Data were analyzed using SPSS software (Version 15) and p < 0.05 was considered as a significant difference. RESULTS: The findings showed higher levels of FBS, HbA1c, HOMA-IR, and KTR in the patients compared to the controls. TAC and HOMA-B were significantly lowered in the T2DM patients compared to controls. KTR was significantly correlated with the level of HbA1c, and T2DM patients with poor glycemic control (HbA1c ≤ 8) had significantly higher level of KTR. HOMA-B was significantly correlated with serum tryptophan and inversely correlated with HbA1c. CONCLUSION: Serum KTR is increased in T2DM patients with poor glycemic control. Potential clinical implications and possible pathogenic roles of IDO in T2DM development should be further elucidated.

Adrenomedullin increases cAMP accumulation and BDNF expression in rat DRG and spinal motor neurons.

OBJECTIVES: Adrenomedullin (AM) has high expression in the spinal cord. In this study, we investigated the expression of AM and its receptor components, including calcitonin receptor-like receptor (CLR) and receptor activity modifying proteins (RAMPs) in dorsal root ganglion (DRG) and spinal motor (SM) neurons. Furthermore, the effects of AM on cAMP/cAMP response element-binding protein (CREB), AKT/glycogen synthase kinase-3 beta (GSK-3ß) signaling pathways, and expressions of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were evaluated. MATERIALS AND METHODS: Rat embryonic DRG and SM neurons were isolated, purified, and cultured. Real-time PCR was used to assess expressions of AM, CLR, and RAMPs. cAMP levels, p-CREB, BDNF, and NT-3 were determined using an enzyme-linked immunosorbent assay. p-AKT and p-GSK-3ß levels were determined by western blotting. Real-time PCR showed expressions of AM, CLR, RAMP2, and RAMP3 in both DRG and SM neurons. RESULTS: AM increased cAMP accumulation and p-CREB levels in DRG and SM neurons. AM increased p-AKT and p-GSK-3ß in DRG, but not SM neurons. AM significantly increased BDNF expression in both DRG and SM neurons. There was also an increase in NT-3 level in both DRG and SM neurons, which is statistically significant in SM neurons. CONCLUSION: These results showed both DRG and SM neurons are targets of AM actions in the spinal cord. An increase in BDNF expression by AM in both DRG and SM neurons suggests the possible beneficial role of AM in protecting, survival, and regeneration of sensory and motor neurons.

Diverse Effect of Vitamin C and N-Acetylcysteine on Aluminum-Induced Eryptosis.

PURPOSE: The role of oxidative stress in Aluminum (Al)-induced apoptotic effects has been investigated and suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and phosphatidylserine externalization (PSE) at the surface of the erythrocyte cell membrane. Eryptosis is stimulated by an increase in cytosolic Ca2+ concentration and reactive oxygen species (ROS). This ex vivo study was conducted to evaluate the effect of well-known antioxidants including vitamin C (vit C) and N-acetylcysteine (NAC), against Al-induced hemolysis and eryptosis. METHODS: Isolated erythrocytes from the healthy volunteers were partitioned into various groups (6 replicates/group) and treated by various concentrations of Al (3-100 µM) in the presence and absence of vit C (0.6 mM) and NAC (1 mM). After 24 hours of treatment, hemolysis was determined from hemoglobin levels in the supernatant. Flowcytometric methods were applied to measure PSE, cell shrinkage, Ca2+ content, and ROS abundance using annexin V-binding, forward scatter, Fluo3-fluorescence, and DCFDA dependent fluorescence, respectively. Reduced glutathione (GSH) was measured by the ELISA method. RESULTS: The results showed that a 24 hours' exposure of the erythrocytes to Al (10-100 µM) significantly increased hemolysis in a dose and Ca2+dependent manner. Al also dramatically decreased forward scatter. The percentage of PSE cells, Fluo3-fluorescence, and DCFDA fluorescence were increased by Al. Furthermore, cotreatment with NAC inhibited the effect of Al on hemolysis, eryptosis, and ROS production. Vit C decreased Al-induced ROS production. However, increased Al-induced eryptosis. There were no significant changes in glutathione after the ALCL3 treatment. CONCLUSIONS: Al-induced eryptosis and hemolysis through triggering oxidative stress, while NAC could diverse this effect. In contrast, vit C might intensify Al-induced eryptosis at particular doses through a less known mechanism.

Adrenomedullin protects rat dorsal root ganglion neurons against doxorubicin-induced toxicity by ameliorating oxidative stress.

OBJECTIVES: Despite effective anticancer effects, the use of doxorubicin (DOX) is hindered due to its cardio and neurotoxicity. The neuroprotective effect of adrenomedullin (AM) was shown in several studies. The present study aimed to evaluate the possible protective effects of AM against DOX-induced toxicity in dorsal root ganglia (DRGs) neurons. MATERIALS AND METHODS: Rat embryonic DRG neurons were isolated and cultured. The effect of various concentrations of DOX (0.0 to 100 µM) in the absence or presence of AM (3.125 -100 nM) on cell death, apoptosis, oxidative stress, expression of tumor necrosis-α (TNF-α), interleukin1- ß (IL-1ß), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP) 3 and 13, and SRY-related protein 9 (SOX9) were examined. RESULTS: Based on MTT assay data, DOX decreased the viability of DRG neurons in a dose and time-dependent manner (IC50=6.88 µm) while dose-dependently, AM protected DRG neurons against DOX-induced cell death. Furthermore, results of annexin V apoptosis assay revealed the protective effects of AM (25 nm) against DOX (6.88 µM)-induced apoptosis and necrosis of DRG neurons. Also, AM significantly ameliorated DOX-induced oxidative stress in DRG neurons. Real-time PCR results showed a significant increase in the expression of TNF-α, IL-1ß, iNOS, MMP 3, and MMP 13, and a decrease in the expression of SOX9 following treatment with DOX. Treatment with AM (25 nM) significantly reversed the effects of DOX on the above-mentioned genes expression. CONCLUSION: Our findings suggest that AM can be considered a novel ameliorating drug against DOX-induced neurotoxicity.

Combination of NDRG2 overexpression, X-ray radiation and docetaxel enhances apoptosis and inhibits invasiveness properties of LNCaP cells.

OBJECTIVE: N-myc downstream regulated gene 2 (NDRG2) is identified as a promising candidate tumor suppressor in several human malignancies including prostate cancer (PCa). Here, we investigated the effect of combined NDRG2 overexpression, x-ray radiation (RTX), and docetaxel (DTX) against viability and invasiveness properties of LNCaP cells. MATERIAL AND METHODS: A plasmid harboring NDRG2 gene under transcriptional control of prostate-specific enhancing sequence regulatory element was constructed to overexpress NDRG2 in PCa cell lines. The effects of NDRG2 overexpression in combination with RTX and DTX on viability, proliferation, and apoptosis of LNCaP cells were evaluated using MTT, colony formation, and annexin V flowcytometirc assays. Migration and invasion of NDRG2-overexpressed cells as well as expression of matrix metalloproteinses-2 (MMP2) and -9 (MMP9) were also assessed using transwell chamber assay and real-time PCR. RESULTS: The results of fluorescence microscopy and real-time PCR showed a high and specific overexpression of NDRG2 in LNCaP cells. Overexpression of NDRG2 significantly reduced cell viability and increased apoptosis of LNCaP cell. Migration, invasion, as well as the expression of MMP2 and MMP9, was decreased following NDRG2 overexpression. Combination of NDRG2 overexpression with RTX and DTX decreased the viability, invasion, and migration of LNCaP cells synergistically. CONCLUSION: These results indicate that a combination of NDRG2 overexpression with chemotherapy and radiotherapy can be considered for effective treatment of PCa.

CRISPR/Cas9-mediated knockout of MLL5 enhances apoptotic effect of cisplatin in HeLa cells in vitro.

Mixed lineage leukemia 5 (MLL5) transactivates the expression of E6 and E7 oncogenes in cervical cancer cells. In this study, we utilized CRISPR/Cas9 system with the aim to target HPV-E6 and MLL5 to enhance apoptosis efficiency in HPV-18 positive HeLa cells and to improve chemotherapeutic efficacy of Cisplatin as the most common anticancer drug, used for cervical cancer. sgRNAs against MLL5 and E6 were designed and cloned into PX458 plasmid vector. Real-time PCR was used to determine knockout expression of MLL5 and E6 following, transfection with cloned plasmids. Cell viability and apoptosis were evaluated, using Dimethyl-thiazolyl diphenyl tetrazolium bromide (MTT) assay and Annexin V flow cytometry. Cellular p 53 level was measured, using enzyme linked immune sorbent assay (ELISA). Real-time PCR indicated the downregulation of E6 and MLL5 in the transfected cells. A significant increase in the accumulation of P53 was observed due to targeting MLL5 and E6 genes. MTT and apoptosis assays showed a significant decrease in cell viability and enhanced apoptosis rate of transfected cells. Combination therapy showed that targeting E6 and MLL5 enhanced apoptotic effect of Cisplatin in MLL5 knockout cells in a synergistic manner. The results suggest that CRISPR/Cas9 targeting of E6 and MLL5 genes can increase apoptotic effects of Cisplatin and can be considered as a potential combination therapy for the treatment of HPV- related cervical cancer.

The Hydroalcoholic Extract of Saffron Protects PC12 Cells against Aluminum-Induced Cell Death and Oxidative Stress in Vitro.

BACKGROUND: Aluminum (Al) exposure is among the environmental risk factors that may involve in the pathogenesis of neurodegenerative diseases. Oxidative stress has a critical role in the Al-induced toxicity. Saffron is a plant with potent radical scavenging and anti-oxidative properties. This investigation was designed to evaluate the possible protective effects of saffron extract (SE) on aluminum maltolate (Almal)-induced oxidative stress and apoptosis in PC12 cell line. METHODS: In this in vitro study, PC12 cells were divided into four groups including control, Almal (500 µM), Almal+SE (50 µg/ml), and Almal+SE (100 µg/ml). After 48 hours of treatment with Almal in the absence and presence of SE, cell viability and apoptosis were determined using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay and Annexin V flow cytometry, respectively. Catalase activity was determined as an index of oxidative stress. Statistical analyses were performed using one-way ANOVA (SPSS version 16.0). P<0.05 was accepted as a statistically significant difference between groups. RESULTS: Almal decreased the PC12 cells viability dose-dependently (IC50=500µM). Co-treatment of 50 and 100 µg/ml of SE with 500 µM of Al increased cell viability to 79% (P=0.04) and 86% (P=0.02) of the control group, respectively. Al also increased PC12 cells apoptosis and catalase activity to 37 and 2.7 folds of those of the control group (P<0.001 and =0.001respectively). 100 µg/ml of SE blunted the effects of Al on the increased cell apoptosis (P=0.02) and changes in the catalase activity (P=0.003). CONCLUSION: SE has protective effects against Al-induced apoptosis and oxidative stress and may possess therapeutic values in the treatment of Al-neurotoxicity.

The effects of iron overload, insulin resistance and oxidative stress on metabolic disorders in patients with ß- thalassemia major.

BACKGROUND: Serum lipids and glycemic dysregulation are the known characteristics of ß- thalassemia major (ß-TM). Here, we evaluated the association of these disorders with insulin resistance (IR), oxidative stress and serum ferritin values in patients with ß-TM. METHODS: This case-control study was performed in thalassemia unite of Darab Hospital (Darab, Fars province, Iran) from December 2016 to December 2017. Forty-eight patients with ß-TM and 33 healthy individuals were enrolled. Serum fasting blood sugar (FBS), insulin, total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), ischemia modified albumin (IMA), and ferritin were measured. The values of HOMA-IR, LDL: TG ratio, atherogenic index (AI), atherogenic index of plasma (AIP), and coronary risk index (CRI) were calculated. RESULTS: The level of serum ferritin, IMA, FBS, TG, AIP, LDL: TG ratio, and the prevalence of IR (HOMA-IR < 3.8) were significantly higher while TC, LDL-C, HDL-C, and AI were significantly lower in the patients compared to the control group. In patient with ß-TM, serum ferritin revealed to have a positive association with serum insulin, HOMA-IR, AI, and CRI levels while serum IMA showed positive association with TG and AIP and inverse association with hypocholesterolemia. HOMA-IR had positive correlation with HDL levels. CONCLUSIONS: Oxidative stress and iron overload are predictors of serum glycemic and lipid dysregulation, suggesting possible beneficial effect of antioxidants and efficient iron chelating therapy in reducing the risk of metabolic disorders in ß- thalassemia.

The BDNF Protein and its Cognate mRNAs in the Rat Spinal Cord during Amylin-induced Reversal of Morphine Tolerance.

The pancreatic peptide, Amylin (AMY), reportedly affects nociception in rodents. Here, we investigated the potential effect of AMY on the tolerance to morphine and on the expression of BDNF at both levels of protein and RNA in the lumbar spinal cord of morphine tolerant rats. Animals in both groups of control and test received a single daily dose of intrathecal (i.t.) morphine for 10 days. Rats in the test group received AMY (1, 10 and 60 pmoles) in addition to morphine from days 6 to10. Morphine tolerance was established at day 5. AMY alone showed enduring antinociceptive effects for 10 days. Real-Time PCR, western blotting and ELISA were used respectively to assess levels of BDNF transcripts and their encoded proteins. Rats tolerant to i.t. morphine showed increased expression of exons I, IV, and IX of the BDNF gene, and had elevated levels of pro-BDNF and BDNF protein in their lumbar spinal cord. AMY, when co-administered with morphine from days 6 to 10, reversed morphine tolerance and adversely affected the morphine-induced expression of the BDNF gene at both levels of protein and mRNAs containing exons I, IV and IX. AMY alone increased levels of exons I and IV transcripts. Levels of pro-BDNF and BDNF proteins remained unchanged in the lumbar spinal cord of rats treated by AMY alone. These results suggest that i.t. AMY not only abolished morphine tolerance, but also reduced the morphine induced increase in the expression of both BDNF transcripts and protein in the lumbar spinal cord.

NDRG2 Regulates the Expression of Genes Involved in Epithelial Mesenchymal Transition of Prostate Cancer Cells.

BACKGROUND: Metastasis is the main cause of prostate cancer (PCa) death. The inhibitory effect of N-myc downstream-regulated gene 2 (NDRG2) on the invasiveness properties of PCa cells has been demonstrated previously. However, its underlying mechanisms have not yet been investigated. The present study aimed to investigate the effects of NDRG2 overexpression on the expression of genes involved in epithelial-mesenchymal transition (EMT) including E-cadherin (E-CAD), α- and ß-catenins, Slug and Snail, transforming growth factor (TGF)-α and -ß, and vascular endothelial growth factor (VEGF). METHODS: In the present in vitro study, LNCaP cells were divided into three groups, namely NDRG2 group (transfected with PSES-pAdenoVator-PSA-NDRG2-IRES-GFP plasmid), mock group (transfected with mock plasmid), and control group (without transfection). The effect of NDRG2 overexpression on the migration and invasion of LNCaP cells were investigated using the transwell assay. Real-time PCR was used for the evaluation of gene expression. For the statistical analyses, one-way ANOVA, student t test or Mann-Whitney U test were applied using the SPSS software (version 15.0). P values <0.05 were considered statistically significant. RESULTS: The results demonstrated that the overexpression of NDRG2 reduced the invasion and migration of LNCaP cells compared to the control and mock groups (P<0.001). A decreased expression of TGF-ß (P=0.002), VEGF (P=0.014), Slug (P=0.005), and Snail (P=0.012); and an increased expression of E-CAD (P=0.009) were observed following NDRG2 overexpression in LNCaP cells. CONCLUSION: The results of the present study suggest that NDRG2 inhibits the invasiveness properties of LNCaP cells probably through changes in the expression of genes involved in EMT.

Enhancing Stability of Destabilized Green Fluorescent Protein Using Chimeric mRNA Containing Human Beta-Globin 5' and 3' Untranslated Regions.

BACKGROUND: In spite of recent progress in mRNA technologies and their potential applications for treatment of human diseases, problems such as the transient nature of mRNA limit the stability of gene up-regulation and, thus, potentially reduce mRNA efficiency for gene therapy. Using human ß-globin 5' and 3' untranslated regions (UTRs), this study aimed to develop the different chimeric constructs of mRNAs to increase the stability of destabilized green fluorescent protein (EGFPd2) in HEK 293 cells. METHODS: Purified human ß-globin (HBG) 5'-3'UTRs, and the coding sequence of destabilized green fluorescent protein (EGFPd2) were amplified separately and ligated to each other using SOEing PCR method in a different format. As controls, the original construct of EGFPd2 under the control of T7 promoter was used. Following in vitro transcription, HEK 293 cells were then transfected with several constructs and incubated at 37°C in a CO2 incubator. They were monitored under a fluorescence microscope every four hours for the first 24 hr, then every 12 hr afterwards. The resulting fluorescence was measured as a surrogate for translation efficiency and duration. RESULTS: By monitoring the HEK cells over 48 hr, cells transfected with mRNA with various HBG UTRs showed significantly different fluorescence intensity and stability in comparison with the pEGFPd2 prototype (control transcript) overtime. Overall, the images show that replacement of the 3' UTR end of the prototype vector pGFPd2 with the 3' end of ß-globin mRNA increases the half-life of the chimeric mRNA for more than 32 hr. CONCLUSION: This result indicates that ß-globin 3' UTR would definitely increase the half-life of mRNA and may help to decrease the mRNA therapeutic dosage in the treatment of diseases associated with mRNA therapy.

Erythrocyte glucose-6-phosphate dehydrogenase activity and risk of gestational diabetes.

PURPOSE: Glucose-6-phosphate dehydrogenase (G6PD) is the regulating enzyme in the pentose phosphate pathway. A link between the activity of G6PD and diabetes mellitus has previously been reported. The association of G6PD activity with the pathogenesis of gestational diabetes mellitus (GDM) has not yet been investigated. The aim of the present study was to investigate the association of erythrocyte G6PD activity with major characteristics of GDM. METHODS: This case-control study was conducted at Hafez Hospital, Shiraz University of Medical Sciences, Shiraz, Iran from March to November 2017. Eighty-four age-matched pregnant women including GDM (n = 33), impaired glucose tolerance (IGT; n = 7), and normal glucose tolerance (NGT; n = 44) subjects were enrolled in this study. The levels of erythrocyte G6PD activity, fasting plasma glucose (FPG), insulin, malondialdehyde (MDA), and ferric reducing power (FRAP) of serum were measured. The level of homeostasis model for the assessment of insulin resistance (HOMA-IR) was calculated. The data were analyzed using SPSS software. P < 0.05 was considered statistically significant. RESULTS: The values of FPG, insulin, HOMA-IR, G6PD activity, and FRAP were significantly higher in GDM patients compared to NGT subjects. G6PD activity was correlated with FPG ((r = 0.224; P = 0.041). Binary logistic regression analysis revealed independent association of body mass index >25.88 [OR = 3.23, 95% CI 1.071-9.75, P = 0.037], HOMA- IR >2.33 [OR = 7.15, 95% CI 2.26-22.56, P < 0.001], and G6PD activity>21.17 U/g Hb [OR = 4.63, 95% CI 1.49-14.38, P = 0.008] with an increased risk of GDM. No significant change was observed among serum MDA levels in the three groups. CONCLUSION: The findings demonstrate that increased G6PD activity is positively associated with the risk of GDM.

Sodium valproate ameliorates aluminum-induced oxidative stress and apoptosis of PC12 cells.

OBJECTIVES: According to recent studies, valproate shows some protection against oxidative stress (OS) induced by neurotoxins. Current investigation tried to determine the possible ameliorating effects of sodium valproate (SV) against aluminum (Al)-induced cell death, apoptosis, mitochondrial membrane potential (MMP), and OS in PC12 cells. MATERIALS AND METHODS: In this in vitro study, PC12 cells were treated with different concentrations of aluminum maltolate (Almal) with and without SV (50-400 µM). Cell viability was assessed by MTT assay. To measure quantitatively the effects of SV on Al-induced apoptosis and reactive oxygen species (ROS), flowcytometry using 7AAD/annexin-V and 2', 7'-dichlorofluorescein diacetate staining were employed, respectively. MMP was monitored using the retention of rhodamine 123. Catalase (CAT) activity was assayed by the rate of decomposition of hydrogen peroxide. RESULTS: Exposure of PC12 cells for 48 hr to Almal (125-2000 µM) significantly reduced cell viability (IC50=1090 µM), increased ROS generation and apoptosis, and reduced MMP and CAT activity. SV reduced the Almal-induced cell death and apoptosis. Furthermore, the effects of Almal on ROS generation, catalase activity, and MMP reduction were significantly diminished by SV. CONCLUSION: Data from this study suggest that SV can inhibit Al-induced cell death and apoptosis of PC12 cells via ameliorating OS.

Effects of Two-by-Two Combination Therapy with Valproic Acid, Lithium Chloride, and Celecoxib on the Angiogenesis of the Chicken Chorioallantoic Membrane.

BACKGROUND: The synergistic effects of valproic acid (VPA), lithium (Li), and celecoxib (CX) have been shown in combination therapy against the proliferation and metastasis of numerous cancers. Angiogenesis plays a critical role in the pathogenesis of tumor growth and metastasis. The aim of the present study was to evaluate the antiangiogenic effects of VPA, lithium chloride (LiCl), and CX, alone or in 2-by-2 combinations, using the chicken chorioallantoic membrane (CAM) assay. METHODS: Fertilized chicken eggs were randomly divided into 10 groups: control, VPA (1.8 and 3.6 µmol/CAM), Li (0.15 and 0.60 µmol/CAM), CX (0.02 and 0.08 µmol/CAM), VPA+Li, VPA+CX, and CX+Li (n=10 per group). A window was made on the eggshells and the CAMs were exposed to a filter disk containing VPA, LiCl, and CX, alone or in 2-by-2 combinations. The control CAMs were treated with distilled water (vehicle). Three days after the treatment, the number of vessel branch points was counted in each CAM. The data were analyzed using SPSS, version 15.One-way ANOVA, followed by the Tukey tests, was used to compare the groups. A P<0.05 was considered a statistically significant difference between the groups. RESULTS: According to the results, all the tested drugs decreased the number of the vessel branch points in a dose-dependent manner compared to the control group (P<0.001). In addition, combinations of the drugs were more effective in decreasing angiogenesis than the use of each drug alone. CONCLUSION: These findings suggest that 2-by-2 combinations of VPA, CX, and LiCl can be considered an effective antiangiogenesis therapeutic modality.

The Synergistic Effects of Celecoxib and Sodium Valproate on Apoptosis and Invasiveness Behavior of Papillary Thyroid Cancer Cell Line In-vitro.

Metastasis to lymph nodes and distant organs is the main challenge in the treatment of papillary thyroid cancer. In the current investigation, we aimed to evaluate the synergistic effects of celecoxib (CX) and sodium valproate (VPA) against cell survival, invasiveness properties, and expression of metalloproteinase-2 and -9 (MMP-2 and MMP-9) in papillary thyroid cancer cell line, B-CPAP cells. The effect of CX and VPA on B-CPAP cells viability and apoptosis were investigated using MTT assay and annexin V/7-AAD flowcytometry, respectively. The effects of the drugs on invasiveness properties of B-CPAP cells and expression of MMP-2 and MMP-9 were evaluated using transwell assay and real time PCR, respectively. MTT assay showed that CX and VPA decreased viability of B-CPAP cells dose dependently (IC50 32.4µM and 6.8 mM, respectively). Combination of CX (5 µM) and VPA (2.5 and 5 mM) increased apoptosis, and reduced cell migration and invasion of B-CPAP cell, synergistically. Real time PCR results showed that both CX (5 µM) and VPA (2.5 and 5 mM) reduced MMP-2 expression (P < 0.05) but had no significant effects on the expression of MMP-9. Our findings suggest that CX and VPA synergistically increase apoptosis and suppress migration and invasion of B-CPAP cells through inhibition of MMP-2 expression.