Eslicarbazepine induces apoptosis and cell cycle arrest in C6 glioma cells in vitro and suppresses tumor growth in an intracranial rat model.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant brain tumor, with a poor prognosis and life expectancy of 14-16 months after diagnosis. The standard treatment for GBM consists of surgery, radiotherapy, and chemotherapy with temozolomide. Most patients become resistant to treatment after some time, and the tumor recurs. Therefore, there is a need for new drugs to manage GBM. Eslicarbazepine (ESL) is a well-known antiepileptic drug belonging to the dibenzazepine group with anticancer potentials. In this study, for the first time, we evaluated the potential effects of ESL on C6 cell growth, both in vitro and in vivo, and examined its molecular effects. METHODS: To determine the effect of ESL on the c6 cell line, cell viability, proliferation, and migration were evaluated by MTT assay, colony formation, and wound healing assay. Also, apoptosis and cell cycle were examined by flow cytometry, qRT-PCR, and western blotting. In addition, an intracranial model in Wistar rats was used to investigate the effect of ESL in vivo, and the tumor size was measured using both Caliper and MRI. RESULTS: The obtained results are extremely consistent and highly encouraging. C6 cell viability, proliferation, and migration were significantly suppressed in ESL-treated C6 cells (p < 0.001), as determined by cell-based assays. ESL treatment led to significant enhancement of apoptosis (p < 0.01), as determined by flow cytometry, and upregulation of genes involved in cell apoptosis, such as the Bax/Bcl2 ratio at RNA (p < 0.05) and protein levels (5.37-fold). Flow cytometric analysis of ESL-treated cells revealed G2/M phase cell cycle arrest. ESL-treated cells demonstrated 2.49-fold upregulation of p21 alongside, 0.22-fold downregulation of cyclin B1, and 0.34-fold downregulation of cyclin-dependent kinase-1 at the protein level. Administration of ESL (30 mg/kg) to male rats bearing C6 intracranial tumors also suppressed the tumor volume and weight (p < 0.01). CONCLUSIONS: Based on these novel findings, ESL has the potential for further experimental and clinical studies in glioblastoma.

CLEC19A overexpression inhibits tumor cell proliferation/migration and promotes apoptosis concomitant suppression of PI3K/AKT/NF-κB signaling pathway in glioblastoma multiforme.

BACKGROUND: GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. METHODS: To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. RESULTS: Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. CONCLUSIONS: To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

Efficacy of topical curcumin on mild to moderate carpal tunnel syndrome: a randomized double-blind, placebo-controlled clinical trial.

OBJECTIVES: Recently, there has been a renewed interest in traditional medicine for carpal tunnel syndrome (CTS). Curcumin has been reported as an agent with antioxidant, anti-inflammatory, analgesic, and neuroprotective attributes. This study is one of the first investigations to assess the effect of curcumin gel on CTS. METHODS: This study is a prospective, 8-week, randomized, placebo-controlled, parallel-group clinical trial. A total of 70 patients with CTS were analyzed. The intervention group (n = 35) received a topical curcumin gel and a night wrist splint and the control group (n = 35) received a placebo gel and a night wrist splint for 8 weeks. The primary outcome was the assessment of the symptom severity scale (SSS) and functional status scale (FSS) of the participants using the Boston Carpal Tunnel Questionnaire (BCTQ) after 8 weeks. In addition, all participants were evaluated by electrodiagnostic (EDX) test at baseline and after 8 weeks. RESULTS: The mean scores of SSS demonstrated a significant decrease in the curcumin group compared to the placebo group; P-value= 0.021. The mean change score of SSS after the intervention was 12.45 ± 8.18 in curcumin and 3.28 ± 7.06 in the placebo group; P-value = 0.0001 and the mean change score of FSS were 6.24 ± 4.91 and 2.31 ± 4.95 in curcumin and placebo groups, respectively; P-value = 0.002. However, the EDX study showed no significant changes in both groups. CONCLUSIONS: It seems that curcumin gel could be effective in the improvement of the symptom severity and daily activity of patients with CTS.

ANXA1 with Anti-Inflammatory Properties Might Contribute to Parkinsonism.

We here describe the identification of a novel variant in the anti-inflammatory Annexin A1 protein likely to be the cause of disease in two siblings with autosomal recessive parkinsonism. The disease-segregating variant was ascertained through a combination of homozygosity mapping and whole genome sequencing and was shown to impair phagocytosis in zebrafish mutant embryos. The highly conserved variant, absent in healthy individuals and public SNP databases, affected a functional domain of the protein with neuroprotective properties. This study supports the hypothesis that damaged microglia might lead to impairments in the clearance of accumulated and aggregated proteins resulting in parkinsonism. ANN NEUROL 2021;90:319-323.

The implication of single-nucleotide polymorphisms in ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is an undesirable complication in the course of ovarian stimulation. This kind of stimulation is aimed at acquiring a sufficient number of high-quality oocytes in in vitro fertilization (IVF). Whereas the predisposition to OHSS could be impacted by genetic polymorphisms in susceptible genes, the present study has been jointly conducted with an Iranian cohort to scrutinize its relevant implication. Genomic DNA was extracted from blood samples of patients with a normal ovarian response (NOR) or with OHSS. Samples were analyzed to detect polymorphisms MTHFR rs1801131, MTHFR rs1801133, AMHR2 rs2002555, LHCGR rs2293275, PGR rs10895068, and SERPINE1 rs1799889. Variations of MTHFR, AMHR2, LHCGR, and PGR genes were significantly associated with the developing OHSS. After correction for multiple analysis, this difference was not evident for PGR genotypes. The polymorphic alleles of MTHFR (rs1801131 C-allele and rs1801133 T-allele), AMHR2 (rs2002555 G-allele), and LHCGR (rs2293275 G-allele) were significantly more prevalent among patients with OHSS compared to those in the NOR group. In contrast, the minor allele of PGR single-nucleotide polymorphism (SNP) (rs10895068, A-allele) was more prominent among patients with a NOR than those with OHSS. No significant difference was observed in genotypes or alleles of SERPINE1 rs1799889. The observations indicated that the minor alleles of MTHFR, AMHR2, and LHCGR genes could be considered an independent risk factor in susceptibility to OHSS. Nevertheless, polymorphic allele in the PGR rs10895068 SNP contributes to preventing OHSS occurrence. Therefore, it can be argued that these genes have a significant impact on OHSS.

Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis.

BACKGROUND: Hepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells. METHODS: IC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated. RESULTS: N-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 µg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 µg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h. CONCLUSION: N-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.

CCAT2 knockdown inhibits cell growth, and migration and promotes apoptosis through regulating the hsa-mir-145-5p/AKT3/mTOR axis in tamoxifen-resistant MCF7 cells.

AIMS: Tamoxifen (TAM) selectively modulates estrogen receptors and is widely used in breast cancer treatment. However, resistance to this drug appears in 40 % of estrogen receptor-positive breast cancer patients due to deregulated non-coding RNAs. This study sought to identify a long non-coding-RNA/miRNA/mRNA axis that is involved in the development of resistance to TAM- in MCF7 cells (MCF7-R). MAIN METHODS: Study genes were selected using RNA-seq. The expression of genes was assessed using TCGA cohort analyses and RT-qPCR. To identify potential resistant pathways in MCF7-R, the DAVID and DIANA-miRPath were carried out. The prediction software (RNAhybrid, TargetScan, and LncTar), and RT-qPCR were used to determine the relationship between genes. Next, the MCF7-R was established and RT-qPCR, cell cycle, apoptosis, and wound healing assays were carried out to verify MCF7-R and identify the effects of CCAT2 overexpression and knockdown on the cells. KEY FINDINGS: Based on bioinformatics analyses, CCAT2, AKT3, and mTOR were up-regulated in breast cancer cell lines, tissues, and TAM-resistant cells, while hsa-miR-145-5p was down-regulated. According to DAVID and DIANA-miRPath, PI3K/AKT/mTOR was a pathway involved in MCF7-R. According to the prediction software, and RT-qPCR results, CCAT2/hsa-miR-145-5p and hsa-miR-145-5p/AKT3 had a negative correlation. CCAT2 knockdown could prevent cell growth, and migration, and promote apoptosis in MCF7-R, while CCAT2 overexpression induced the opposite effects. RT-qPCR revealed that the expression of BAX and Bcl-2 genes were regulated in favor of apoptosis, upon CCAT2 knockdown. SIGNIFICANCE: CCAT2 regulates cell cycle, migration, and apoptosis in MCF7-R via the hsa-miR-145-5p/AKT3/mTOR axis. Therefore, CCAT2 may be a target to enhance the sensitivity of resistant MCF7 cells to TAM.

Strategies to overcome the side effects of chimeric antigen receptor T cell therapy.

Chimeric antigen receptor (CAR) therapy is a method directing T lymphocytes against antigens on the surface of tumors, increasing target cell elimination. Genetic engineering enhances the capability of immune cells to detect new antigens expressed on cell surfaces. CAR T cell therapy is a significant breakthrough for treating human malignancies; however, different side effects (e.g., cytokine release syndrome) restrict its application. Improving design and using various combined receptors enhance the performance of these cells. This review discusses limitations and risk factors associated with CAR T cell therapy. We also review some alternative approaches for developing the next generation of CAR T cells.

A Comparative Study of HOTAIR Expression in Breast Cancer Patient Tissues and Cell Lines.

OBJECTIVE: Recent data suggest that increased levels of the HOTAIR long non-coding RNA (lncRNA) are involved in the development of various types of malignancy, including breast cancer. The aim of present study was to investigate HOTAIR lncRNA expression profile in breast cancer (BC) patients and cell lines. MATERIALS AND METHODS: In this experimental study, expression level of HOTAIR lncRNA was evaluated in BC and normal tissues of 15 patients as well as MDA-MB-231, MCF-7 and MCF-10A cell lines, using quantitative reversetranscription polymerase chain reaction (qRT-PCR). HOTAIR lncRNA expression levels were estimated using 2-ΔΔCt method. Further, receiver operating characteristic (ROC) curve analysis was done to evaluate the selected lncRNA diagnostic potential. The Cox's proportional hazards regression model was performed to evaluate the predictive value of this lncRNA level in BC patients. RESULTS: The results of present study demonstrated no significant difference in the expression of HOTAIR lncRNA in MCF7 and MDA-MB-231 cancer cell lines compared to MCF-10A as normal cell line (P>0.05). However, we observed a significantly increase in the expression of HOTAIR in BC patients compared to normal tissues (P<0.001). Significant associations were found between gene expression and tumour size and margin. We found 91.1% sensitivity and 95.7% specificity of circulating HOTAIR with an area under the ROC curve of 0.969. The Kaplan-Meier analysis indicated significant correlation between HOTAIR expression and overall survival. CONCLUSION: This study demonstrated that expression of HOTAIR is increased in BC and might be associated with its progression. According to these findings, HOTAIR expression could be proposed as biomarkers for BC early diagnosis and prognosis.

Evaluation of Neuroprtective Effects of L-Carnitine and Fat Emulsion in the CVA Patients: A Prospective, Randomized, Double Blind, Clinical Trial.

Cerebral infarction presents with neurological deficits caused by the death of neurons in a focal area of the brain. S100B is a biomarker that increases in brain damage. Neuroprotectives can reduce the brain sequels after neurological insult. The purpose of this study was to evaluate the neuroprotective effects of L-carnitine and Fat emulsion (Lipofundin®) alone and in combination in patients with ischemic stroke. In a prospective, RCT, and double-blind study 100 patients with MCA ischemic cerebrovascular accident who were admitted in the first 24 h of injury entered the study. The patients were randomly assigned into four groups of L-carnitine, fat emulsion, L-carnitine plus fat emulsion and control. Fat emulsion 10%, 500 mL, was infused over 6 to 12 h and 1 gr of L-carnitine (10 mL of solution) was administered orally to patients in addition to common therapies, according to the American Heart Association and American Stroke Association (AHA/ASA) guidelines. The patients in the control group received only the usual treatment according to stroke guidelines. Blood samples before the intervention, then after 24 h, 48 h, and 7 days later were taken and immunoenzymatic colorimetric method was used for quantitative determination of S100B concentration in the patients' serum. In the within group analysis, all of our treatment interventions (except control group) have decreased S100B levels statistically significant (P < 0.05). Moreover, changes in observed levels of S100B before and after intervention were different between the groups and the observed differences were statistically significant (P = 0.01). In the GEE model, it was found that S100B levels in the L-carnitine plus fat emulsion group decreased more than the control group and this decline has been statistically significant [P = 0.02, 20.47 (CI 95%: 6.25-34.41)], but in comparison of L-carnitine and fat emulsion group with control group, did not reached statistical significance (P > 0.05). Based on the results obtained from this study, it seems that L-carnitine with fat emulsion could lead to neuroprotective effects with a significant reduction in the S100B biomarker.

Novel Mutations in TACSTD2 Gene in Families with Gelatinous Drop-like Corneal Dystrophy (GDLD).

In the current study, we conducted a mutation screening of tumor-associated calcium signal transducer 2 (TACSTD2) gene in six consanguineous Iranian families with gelatinous drop-like corneal dystrophy (GDLD), in order to find the causative mutations. Detailed eye examination was performed by ophthalmologist to confirm GDLD in patients. To detect the possible mutations, direct Sanger sequencing was performed for the only exon of TACSTD2 gene, and its boundary regions in all patients. In the patients with GDLD, the corneal surface showed lesions with different shapes from mild to severe forms depending on the progress of the disease. The patients showed grayish corneal deposits as a typical mulberry form, corneal dystrophy along with corneal lipid deposition, and vascularization. Targeted Sanger sequencing in TACSTD2 gene revealed the causative mutations in this gene in all studied families. Our study expanded the mutational spectrum of TACSTD2 which along with the related symptoms could help with the diagnosis, and management of the disease.

SNAP-25 gene variations and attention-deficit hyperactivity disorder in Iranian population.

OBJECTIVES: Attention deficit hyperactivity disorder (ADHD) is a common psychiatric condition of childhood characterized by persistent symptoms of hyperactivity, inattention, and impulsivity. The objective of this study was to investigate the association of synaptosomal-associated protein of 25 kDa (SNAP-25) gene variants with ADHD. METHODS: A case-control study with a total of 150 children with ADHD (mean age 9.61; range 6-16; gender ratio 105m/45f) and 150 normal children (mean age 10.02; range 6-16; gender ratio 98m/52f) was conducted. Genomic DNA was extracted from peripheral blood of all samples and SNPs rs78428954 and rs3746544 located in SNAP-25 gene were genotyped. RESULTS: Our analysis indicated that there is no significant association between none of studied variants in SNAP-25 and ADHD. DISCUSSION: To our knowledge, it is the first report of SNAP-25 genotyping in Iranian patients with ADHD. Further investigations with larger populations are needed in order to clarify the exact role of SNAP-25 variations in susceptibility to ADHD.