Effect of folate-targeted Erlotinib loaded human serum albumin nanoparticles on tumor size and survival rate in a rat model of glioblastoma.

The aim of this study was to prepare folate-targeted Erlotinib loaded human serum albumin nanoparticles (FA-ERL-HSA NPs) and investigate in vitro cytotoxic and apoptotic effects using cell lines (U87MG and C6 cells) and an in vivo rat bearing C6 glioma model. The mean size of the FA-ERL-HSA NPs prepared using a desolvation method was 135 nm. In vitro MTT assays demonstrated that FA-ERL-HSA NPs had an IC50 value of 52.18 µg/mL and 17.53 µg/mL compared to free ERL which had an IC50 value of 119.8 µg/mL and 103.2 µg/mL for U87MG and C6 cells for 72 h, respectively. Flow cytometry results showed the apoptosis rate with FA-ERL-HSA NPs (100 µg/mL, 72 h) was higher compared to free ERL for both U87MG and C6 cells. Experiments using a rat glioblastoma model via TUNEL assay indicated that the apoptosis index of FA-ERL-HSA NPs was 48 % compared to 21 % for free ERL and the tumor size effectively decreased after a daily injection of 220 µg (2.5 mg/kg) from 87.45 mm3 (19th day) to 1.28 mm3 (60th day). The median survival rate of the rats increased after treatment to >100 days which was greater than controls.

Microfluidics for detection of exosomes and microRNAs in cancer: State of the art.

Exosomes are small extracellular vesicles with sizes ranging from 30-150 nanometers that contain proteins, lipids, mRNAs, microRNAs, and double-stranded DNA derived from the cells of origin. Exosomes can be taken up by target cells, acting as a means of cell-to-cell communication. The discovery of these vesicles in body fluids and their participation in cell communication has led to major breakthroughs in diagnosis, prognosis, and treatment of several conditions (e.g., cancer). However, conventional isolation and evaluation of exosomes and their microRNA content suffers from high cost, lengthy processes, difficult standardization, low purity, and poor yield. The emergence of microfluidics devices with increased efficiency in sieving, trapping, and immunological separation of small volumes could provide improved detection and monitoring of exosomes involved in cancer. Microfluidics techniques hold promise for advances in development of diagnostic and prognostic devices. This review covers ongoing research on microfluidics devices for detection of microRNAs and exosomes as biomarkers and their translation to point-of-care and clinical applications.

Effect of Paclitaxel/etoposide co-loaded polymeric nanoparticles on tumor size and survival rate in a rat model of glioblastoma.

The aim of this work is to co-load paclitaxel (PTX) and etoposide (ETP) in methoxy poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (mPEG-PLGA NPs) to overcome pharmacokinetics and physiological limitations and enhance therapeutic efficacy for treating intracranial glioblastoma. Both drugs were loaded into mPEG-PLGA NPs by a nano-precipitation method. The resultant NPs demonstrated an enhanced cytotoxic effect indicated by lower IC50 values and augmented cell apoptosis to U87 and C6 glioma cell lines compared to both free drugs. Additionally, blood compatibility assays showed that the PTX/ETP co-loaded mPEG-PLGA NPs did not induce blood hemolysis, blood clotting, or platelet aggregation. In vivo anti-glioma efficacy evaluation in rats bearingintracranialC6glioma revealed a superior anti-glioma activity for the treatment with PTX/ETP co-loaded mPEG-PLGA NPs compared to other formulations, particularly a significantly longer median survival, 76 days compared to 36 days for free PTX and 37 days for free ETP treatment, respectively, and higher tumor regression, proved by magnetic resonance imaging (MRI).

Effects of multiple injections on the efficacy and cytotoxicity of folate-targeted magnetite nanoparticles as theranostic agents for MRI detection and magnetic hyperthermia therapy of tumor cells.

Folate-targeted iron oxide nanoparticles (FA@Fe3O4 NPs) were prepared by a one-pot hydrothermal method and then used as cancer theranostic agents by combining magnetic resonance imaging (MRI) and magnetic hyperthermia therapy (MHT). Crystal structure, morphology, magnetic properties, surface functional group, and heating efficacy of the synthesized nanoparticles were characterized by XRD, TEM, VSM, FTIR, and hyperthermia analyses. The results indicated that the crystal structure, magnetic properties, and heating efficacy of the magnetite nanoparticles were improved by hydrothermal treatment. Toxicity of the prepared NPs was assessed in vitro and in vivo on the mammary cells and BALB/c mice, respectively. The results of the in vitro toxicity analysis showed that the FA@Fe3O4 NPs are relatively safe even at high concentrations of the NPs up to 1000 µg mL-1. Also, the targetability of the FA@Fe3O4 NPs for the detection of folate over-expressed cancer cells was evaluated in an animal model of breast tumor using MRI analysis. It was observed that T2-weighted magnetic resonance signal intensity was decreased with the three-time injection of the FA@Fe3O4 NPs with 24 h interval at a safe dose (50 mg kg-1), indicating the accumulation and retention of the NPs within the tumor tissues. Moreover, the therapeutic efficacy of the MHT using the FA@Fe3O4 NPs was evaluated in vivo in breast tumor-bearing mice. Hyperthermia treatment was carried out under a safe alternating magnetic field permissible for magnetic hyperthermia treatment (f = 150 kHz, H = 12.5 mT). The therapeutic effects of the MHT were evaluated by monitoring the tumor volume during the treatment period. The results showed that the mice in the control group experienced an almost 3.5-fold increase in the tumor volume during 15 days, while, the mice in the MHT group had a mild increase in the tumor volume (1.8-fold) within the same period (P < 0.05). These outcomes give promise that FA@Fe3O4 NPs can be used as theranostic agents for the MRI and MHT applications.

Brain Derived Neurotrophic Factor Modification of Epileptiform Burst Discharges in a Temporal Lobe Epilepsy Model.

INTRODUCTION: Transforming Growth Factor-Beta 1 (TGF-ß1) is a pleiotropic cytokine with potent anti-inflammatory property, which has been considered as an essential risk factor in the inflammatory process of Ischemic Stroke (IS), by involving in the pathophysiological progression of hypertension, atherosclerosis, and lipid metabolisms. -509C/T TGF-ß1 gene polymorphism has been found to be associated with the risk of IS. The aim of this meta-analysis was to provide a relatively comprehensive account of the relation between -509C/T gene polymorphisms of TGF-ß1 and susceptibility to IS. METHODS: Male Wistar rats were divided into sham (receiving phosphate buffered saline within dorsal hippocampus), pilocarpine (epileptic model of TLE), single injection BDNF (epileptic rats which received single high dose of BDBF within dorsal hippocampus), and multiple injections BDNF (epileptic rats which received BDNF in days 10, 11, 12, and 13 after induction of TLE) groups. Their electrocorticogram was recorded and amplitude, frequency, and duration of spikes were evaluated. RESULTS: Amplitude and frequency of epileptiform burst discharges were significantly decreased in animals treated with BDNF compared to pilocarpine group. CONCLUSION: Our findings suggested that BDNF may modulate the epileptic activity in the animal model of TLE. In addition, it may have therapeutic effect for epilepsy. More studies are necessary to clarify the exact mechanisms of BDNF effects.

cAMP-Epac Pathway Stimulation Modulate Connexin-43 and MicroRNA-21 Expression in Glioma Cells.

INTRODUCTION: Malignant astrocytic gliomas are the most common and lethal brain malignancies due to their refractory to the current therapies. Nowadays, molecular targeted therapy has attracted great attention in treatment of glioma. Connexin 43 (Cx43) and micro ribonucleic acid-21(miR-21) are among molecules that are involved in glioma development and progression. These molecules showed potential to be as target molecules with regard to glioma. Some studies have reported that cyclic adenosine monophosphate (cAMP) signaling could be effective on Cx43 and miR-21 in tissues other than in brain. We investigate possible relationship between ß-adrenergic receptor and its newly described downstream, exchange protein directly activated by cAMP (Epac) signaling pathway and expression of Cx43 and miR-21 in low (1321N1) and high grade (U87MG) glioma cell lines. METHODS: We treated cells with ß-adrenergic agonist and Epac activator with and without adenyl cyclase inhibitor. Cx43 and miR-21 expression were measured with real-time PCR. RESULTS: Our data showed that in 1321N1 cells, ß-adrenergic-Epac pathway stimulation up and down-regulated Cx43 and miR-21 expression respectively. Whereas, in U87MG cells these interventions had no effect on Cx43 and miR-21 expression. DISCUSSION: These findings demonstrate that low grade astrocytoma cells have better response to our pharmacological interventions.

Fluoxetin upregulates connexin 43 expression in astrocyte.

INTRODUCTION: Recent studies have shown that astrocytes play major roles in normal and disease condition of the central nervous system including multiple sclerosis (MS). Molecular target therapy studies in MS have revealed that connexin-43 (Cx43) and Aquaporin-4 (AQP4) contents of astrocytes undergo expression alteration. Fluoxetine had some effects in MS patients unrelated to its known antidepressant effects. Some of fluoxetine effects were attributed to its capability of cAMP signaling pathway stimulation. This study aimed to investigate possible acute effects of fluoxetine on Cx43 and AQP4 expression in astrocyte. METHODS: Astrocytoma cells were treated for 24 hours with fluoxetine (10 and 20 µg/ml) with or without adenyl cyclase (AC) and protein kinase A (PKA) inhibition. Cx43 expression at both mRNA and protein levels and AQP4 expression at mRNA level were evaluated. RESULTS: Acquired results showed that fluoxetine with and without AC and PKA inhibition resulted in Cx43 up-regulation both in mRNA and protein levels, whereas AQP4 expression have not changed. DISCUSSION: In conclusion, data showed that fluoxetine alone and in the absence of serotonin acutely up-regulated Cx43 expression in astrocytes that can be assumed in molecular target therapy of MS patients. It seems that cAMP involvement in fluoxetine effects need more researches.

BDNF modifies hippocampal KCC2 and NKCC1 expression in a temporal lobe epilepsy model.

Excitatory GABA actions, induced by altered expression of chloride transporters (KCC2/NKCC1), can contribute to seizure generation in temporal lobe epilepsy. In the present study, we evaluated whether BDNF administration can affect KCC2/NKCC1 expression, ictogenesis and behavioral alterations in this paradigm. Status epilepticus was induced in male rats with pilocarpine, followed by a treatment of either a single high dose or multiple injections of BDNF during the latent phase of temporal lobe epilepsy. Chloride transporters expression, spontaneous recurrent seizures, and hyperexcitability post-seizural behaviors were evaluated after treatment. NKCC1 protein expression was markedly upregulated, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Application of BDNF (both single high dose and multiple injections) increased KCC2 expression in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyperexcitability behaviors were ameliorated for a short period of time. To prevent GABA-A mediated depolarization and design appropriate treatment strategies for temporal lobe epilepsy, chloride transporters can be considered as a target. Future studies are warranted to investigate any possible therapeutic effects of BDNF via altering chloride transporters expression.

Selective ß2 adrenergic agonist increases Cx43 and miR-451 expression via cAMP-Epac.

It has been demonstrated that connexin 43 (Cx43) and microRNAs have significant roles in glioma. Cyclic adenosine monophosphate (cAMP) is suggested to be a regulator of connexins and microRNAs. However, it remains elusive whether cAMP and exchange protein directly activated by cAMP (Epac2), have a regulatory effect on Cx43 and microRNA-451 (miR-451) in astrocytoma cells. We treated 1321N1 astrocytoma cells with a selective ß2 adrenergic agonist and a selective Epac activator with and without adenyl cyclase and protein kinase A inhibition. Cx43 and miR-451 expression were measured. Next, we evaluated the effect of miR-451 overexpression on Cx43 expression. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated that cAMP-Epac2 increased Cx43 and miR-451 expression. However, the alteration of miR-451 expression required a higher dose of drugs. Overexpression of miR-451 had no significant effect on Cx43 expression. The MTT assay showed that cAMP-Epac stimulation and miR-451 overexpression had a synergic inhibitory effect on cell proliferation. These findings may expand our understanding of the molecular biology of glioma and provide new potential therapeutic targets.

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture.

INTRODUCTION: A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues. METHODS: The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers (Oct4, GFRα-1, PLZF, Mvh(VASA), Itgα6, and Itgß1), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance. RESULTS: The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) (P≤0.001). The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82%) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells. CONCLUSION: Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro.

Bumetanide reduces seizure frequency in patients with temporal lobe epilepsy.

Alterations in the balance of K-Na-2Cl cotransporter (NKCC1) and Na-Cl cotransporter (KCC2) activity may cause depolarizing effect of γ-aminobutyric Acid (GABA), and contribute to epileptogenesis in human temporal lobe epilepsy. NKCC1 facilitates accumulation of chloride inside neurons and favors depolarizing responses to GABA. In the current pilot study we provide the first documented look at efficacy of bumetanide, a specific NKCC1 antagonist, on reduction of seizure frequency in adult patients with temporal lobe epilepsy. According to our results, seizure frequency was reduced considerably in these patients. Furthermore, epileptiform discharges decreased in two of our patients. If the efficacy of bumetanide is proven in large scale studies, it can be used as a supplemental therapy in temporal lobe epilepsy.