PEGylated nanohydrogels delivering anti-MicroRNA-21 suppress ovarian tumor-associated angiogenesis in matrigel and chicken chorioallantoic membrane models.

Introduction: Recently, MicroRNAs have gained increasing popularity as a novel nucleic acid-mediated medicine to regulate cancer-related protein expression. MicroRNA-21 (miR-21) is known as an oncogenic microRNA which is overexpressed in almost all cancers, including ovarian carcinoma that causes cisplatin (cis-Pt) resistance and vascular endothelial growth factor (VEGF) upregulation. So, miRNA-based therapy can be regarded as knocking down miR-21 expression, inducing tumor cell apoptosis, and suppressing tumor-associated angiogenesis. Methods: PEG5k-carboxymethylated polyethyleneimine nanohydrogels (PEG5k-CMPEI) were loaded with AntagomiR-21 (As-21) at different ratios of nitrogen to phosphorus (N/P). Particle size and ζ potential were determined for the As-21 loaded nanohydrogels. In the cellular experiments, miR-21 expression, cytotoxicity, and cis-Pt sensitivity were studied on A2780 ovarian cancer cell lines. Finally, tumor cell apoptosis and tumor cell-associated angiogenesis were explored in vitro and in vivo. Results: The nanohydrogels, featuring homogeneous size distribution and redox-responsiveness, were steadily loaded by As-21 at the optimum N/P ratio of 5 without any aggregation as determined by transmission electron microscopy (TEM). As-21-loaded nanohydrogels caused sequence-specific suppression of miR-21 expression and provoked apoptosis through ROS generation and caspase 3 activation. Cisplatin cytotoxicity was remarkably enhanced in A2780R as compared to A2780S following co-incubation with As-21-loaded nanohydrogels. Interestingly, the condition of the medium derived from As-21 nanohydrogel-treated A2780R cells inhibited VEGF suppression in human umbilical vein endothelial cells (HUVECs) and the formation of tubes in Matrigel. Moreover, the condition medium caused angiogenesis inhibition in the chicken chorioallantoic membrane (CAM) model. Conclusion: These results suggest that nanohydrogel-based delivery of As-21 can be a promising neoadjuvant therapy for treating resistant tumors via apoptosis induction and angiogenesis suppression.

Evaluating the Immunogenicity of recombinant VP1 protein from the foot-and-mouth disease virus encapsulated in nanoliposome in guinea pig animal model.

Foot-and-mouth disease (FMD) is considered as contagious in livestock, which is caused by the Picornavridae virus family known as FMD virus (FMDV). In the present study, the VP1 gene from FMDV (O strain) was expressed and purified. In addition, nanoliposomes were utilized as an adjuvant. After formulating the nanoliposomes with DMPC, DMPG, and cholesterol, the recombinant VP1 protein was encapsulated in the nanoliposomes. Further, the intended nanoliposomes in the sizes of 400 and 200 nm, nanoliposome-inactivated FMDV, Freund's adjuvant-inactivated FMDV, and Freund's adjuvant-recombinant VP1 mixtures, and PBS buffer (negative control) were injected to six groups of five guinea pigs. Furthermore, the guinea pig serums were analyzed through using ELISA and serum neutralization tests after four boosting vaccinations. Based on the results, the immunogenicity of the 200 nm-nanoliposomes encapsulating recombinant VP1 protein was more than that of 400 nm-ones so that 200 nm-nanoliposomes could trigger the immune response against FMDV in guinea pigs.

CRISPRi-mediated knock-down of PRDM1/BLIMP1 programs central memory differentiation in ex vivo-expanded human T cells.

Introduction: B lymphocyte-induced maturation protein 1 (BLIMP1) encoded by the positive regulatory domain 1 gene (PRDM1), is a key regulator in T cell differentiation in mouse models. BLIMP1-deficiency results in a lower effector phenotype and a higher memory phenotype. Methods: In this study, we aimed to determine the role of transcription factor BLIMP1 in human T cell differentiation. Specifically, we investigated the role of BLIMP1 in memory differentiation and exhaustion of human T cells. We used CRISPR interference (CRISPRi) to knock-down BLIMP1 and investigated the differential expressions of T cell memory and exhaustion markers in BLIMP1-deficient T cells in comparison with BLIMP1-sufficient ex vivo expanded human T cells. Results: BLIMP1-deficiency caused an increase in central memory (CM) T cells and a decrease in effector memory (EM) T cells. There was a decrease in the amount of TIM3 exhaustion marker expression in BLIMP1-deficient T cells; however, there was an increase in PD1 exhaustion marker expression in BLIMP1-deficient T cells compared with BLIMP1-sufficient T cells. Conclusion: Our study provides the first functional evidence of the impact of BLIMP1 on the regulation of human T cell memory and exhaustion phenotype. These findings suggest that BLIMP1 may be a promising target to improve the immune response in adoptive T cell therapy settings.

Circulating status of microRNAs 660-5p and 210-3p in breast cancer patients.

BACKGROUND: MicroRNAs (miRs), which are stable in the blood, comprise small non-coding RNAs that regulate gene expression. They have important roles in almost all biological pathways, especially in cancer-relevant processes, and have an abnormal expression in breast cancer. In recent studies, the aberrant expression level of various microRNAs has been demonstrated in human cancer. In the present study, the status of serum microRNA-210-3p and microRNA-660-5p expression levels in breast cancer patients was determined compared to healthy controls. METHODS: Serum samples were collected from 40 newly diagnosed breast cancer patients and 40 healthy controls. A real-time quantitative polymerase chain reaction was utilized to detect the expression levels of these microRNAs. Data analysis was conducted with p < 0.05 being considered statistically significant. RESULTS: The data obtained showed that serum levels of miR-660-5p and miR-210-3p were significantly up-regulated in breast cancer patients compared to healthy controls (p < 0.001 and p = 0.001, respectively). In addition, significant up-regulation was observed in the early stage (in situ, stage I and II) of breast cancer patients (n = 25) compared to healthy (n = 40) controls (p < 0.001 and p < 0.05, respectively). Receiver-operating characteristic curve analysis indicated that the serum miR-660-5p and miR-210-3p levels have reasonable sensitivity (79% and 68%) and specificity (61% and 51%) for the detection of breast cancer patients (area under the receiver-operating curve = 0.774 and 0.716, respectively). CONCLUSIONS: Although the results show a reasonable diagnostic accuracy of these microRNAs for detection of breast cancer in this small and preliminary study, further large-scale studies are essential to confirm the presented results.

Redox-sensitive, PEG-shielded carboxymethyl PEI nanogels silencing MicroRNA-21, sensitizes resistant ovarian cancer cells to cisplatin.

A series of branched polyethylenimine (PEI) modifications including PEGylation (PEG2k-PEI) for steric shielding, redox-sensitive crosslinking for synthesis PEG2k-PEI-ss nanogels and subsequent carboxymethylation (PEG2k-CMPEI-ss) for modulation of the polymer pka have been introduced for cellular delivery of Anti-miR-21. The synthesis was characterized using 1H NMR, FTIR, TNBS, potentiometric titration, particle size and ζ potential. Loading of Anti-miR-21 at various N/P ratios was investigated by gel retardation, ethidium bromide dye exclusion, heparin sulfate competition and DNase I digestion experiments. The miR-21 silencing was measured by stem-loop RT PCR in A2780 ovarian cancer cell lines whether it is sensitive to resistant to cisplatin. It has been shown that PEG2k-CMPEI-ss was well suited for delivery of Anti-miR-21 in terms of nucleic acid loading, preservation against extracellular matrix and nucleases and sequence-specific silencing of miRNA-21 in vitro. Moreover, it has been demonstrated that pre-treating cells with Anti-miR-21 loaded nanogels can sensitize them to cis-Pt even at non-toxic concentraions. The results indicate that PEG2k-CMPEI-ss mediated microRNA delivery can be considered as a novel strategy for ovarian cancer therapy.

Coenzyme Q0 immobilized on Magnetic Nanoparticle: Synthesis and Antitumoral Effect on Saos, MCF7 and Hela Cell Lines.

Many attempts in medical community focused on the preparation of anticancer agents. Various Coenzyme Q such as CoQ0 analogs have been reported as anti-inflammatory, anticancer, and antioxidant substances. In this study a novel derivatives of Coenzyme Q as an anticancer agent have been introduced. The prepared magnetic nanoparticle, containing CoQ0 were prepared using common chemical methods and also characterized by means of nuclear magnetic resonance (NMR), fourier transform infrared (FT-IR), thermal gravimetric analysis (TGA), and differential scanning calorimetric (DSC). To evaluate the antiproliferative effects of the nanoparticle, the prepared compound was treated with cell lines such as Hela, MCF-7 and Saos. Moreover, the outcomes were compared with normal fibroblast cell line. These assessments were performed by means of MTT assay. Investigation on the capability of this prepared nanoparticle showed some reliable results including cytotoxicities against MCF7, Saos and Hela cancer cell lines which were illustrated by displaying the morphology of the treated cells using AO/EB dual staining fluorescent technique. Employing simple method for preparation as well as the promising cytotoxic results makes it as a promising candidate for further bioexperiments.

Progresses in microRNA Delivery Using Synthetic Nanovectors in Cancer Therapy.

MicroRNAs are small noncoding RNAs with key roles in gene expression. It has been revealed that aberrant expression of microRNAs is related to gene expression abnormality, and they have the potential to be used as anti-cancer drugs. However, the delivery of microRNAs is limited due to barriers, such as low uptake and insufficient endosomal release, intracellular nucleases degradation, phagocytic elimination, and renal filtration. To overcome these issues, novel delivery systems are developed for improving the efficiency of microRNAs therapy ranging from viral to synthetic; some are further developed with targeted ligands for active targeting purposes. Such delivery systems provide efficient cellular uptake and endosomal release as well as low cytotoxicity and minimum unwanted host immune response. Nevertheless, more complementary studies are warranted before being applied in human studies. This review deals with recent updates on the challenges and achievements of the various nanotechnology-based gene delivery vehicles with a special emphasis on the miRNA delivery in cancer therapy. In addition, we attempted to categorize the designed delivery systems based on miRNA therapeutic molecule. The related cellular signaling pathways and pharmacological action against cancer promotion have also been highlighted.

Cytotoxic effect of methanolic extract, alkaloid and terpenoid fractions of Stachys pilifera against HT-29 cell line.

Stachys pilifera (S. pilifera) Benth (Lamiaceae) is used in traditional medicine to treat a variety of diseases. Despite some reports on the antitumor effects of some species of this genus, anticancer activity of S. pilifera has not been yet reported. Here, we examined the cytotoxic effect and cell death mechanisms of methanolic extract of S. pilifera and its alkaloid and terpenoid fractions on the HT-29 colorectal cell line. HT-29 cells were cultivated and then incubated in the methanolic extract of S. pilifera and its fractions at various concentrations for 24 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Morphology of cells was evaluated by contrast microscopy. Furthermore, effects of the tested extract and fractions were tested on some regulators of cell death and proliferation such as caspase-8, caspase-9, nuclear factor-κB (NF-κB), and nitric oxide (NO). Cisplatin was used as positive control. The estimated IC50 values of the methanolic extract, alkaloid and terpenoid fractions, and cisplatin against HT29 cell after 24 h were determined to be 612, 48.12, 46.44, and 4.02 µg/mL, respectively. Morphological changes such as plasma membrane blebbing, cell size reduction, and apoptotic bodies were observed in cells faced with the extract and fractions. S. pilifera extract and its fractions induced apoptosis through inhibition of NF-κB, NO, and activation of caspase-8 and caspase-9. Data showed considerable cytotoxic and antiproliferative effects of S. plifera on colorectal cell line through induction of apoptosis. These findings provide a basis for the therapeutic potential of S. pilfera in the treatment of colon cancer.

Structural and functional consequences of EF-hand I recovery in mnemiopsin 2.

Mnemiopsin 2 from Mnemiopsis leidyi is a calcium-regulated photoprotein which has luminescence properties in the presence of calcium and coelenterazine. All calcium-regulated photoproteins contain EF-hand loops consisting of 12 individual residues in which the 6th position is occupied by Gly. However, the 6th residue in mneniopsin 2 is Glu rather than Gly. Here, we investigated the structural and functional consequences of substitution of Glu by Gly (E50G variant) using site-directed mutagenesis and spectroscopic procedures. It was revealed that the luminescence activity of the variant was about 17 times greater than that of wild-type (WT) photoprotein. In comparison with WT protein, our variant showed higher optimum temperature and calcium sensitivity as well as slower rate of luminescence decay. Homology modeling and sequence analysis with other known photoproteins showed that EF-hand I loop can affect the luminescence activity of E50G variant. Structural studies using circular dichroism and fluorescence spectroscopy revealed that mutation leads to the reduction in secondary structural content and local structural alterations. Finally, it can be concluded that the activity of E50G variant increases as a result of more flexibility that brought about by Gly essential for adopting the correct conformation for functional activity.

Hepatoprotective effect of Stachys pilifera ethanol extract in carbon tetrachloride-induce hepatotoxicity in rats.

CONTEXT: Stachys pilifera Benth (Lamiaceae) has long been used to treat infectious diseases, respiratory and rheumatoid disorders in Iranian folk medicine. Antitumor and antioxidant activity of the plant have been reported. OBJECTIVE: The study was designed to assess the hepatoprotective activity of ethanol extract of Stachys pilifera in carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. MATERIALS AND METHODS: The rats were randomly divided into six equal groups (n = 7). Group I was treated with normal saline; Group II received CCl4 (1 mL/kg. i.p., twice a week) for 60 consecutive days; Groups III, IV and V were given CCl4 plus Stachys pilifera (100, 200 and 400 mg/kg/d,p.o.); Group VI received the extract (400 mg/kg/d, p.o.). Histopathological analysis and measurement of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), malondialdehyde (MDA), total protein (TP) and albumin (ALB) were performed. RESULTS: CCl4 caused a significant increase in the serum levels of AST, ALT, ALP and MDA as well as decreased ALB, and TP serum levels (p < 0.001). The extract (200 and 400 mg/kg/d) significantly normalized the CCl4-elevated levels of ALT, AST, ALP and MDA (p < 0.001). The extract (200 and 400 mg/kg/d) also increased the serum levels of TP compared to CCl4 group (p< 0.01). The extract (200 and 400 mg/kg/d) also decreased the histological injuries (inflammation and fatty degeneration) by CCl4. DISCUSSION: The results revealed that the Stachys pilifera extract could provide considerable protection against CCl4 hepatotoxicity in rats that may be related to its antioxidant properties.

Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit.

Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The Km and Vmax values of the enzyme for casein were obtained to be 1.29mg/ml and 0.035µmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses.

miR-21, An Oncogenic Target miRNA for Cancer Therapy: Molecular Mechanisms and Recent Advancements in Chemo and Radio-resistance.

In the past decade, miRNAs have been extensively attracted the scientist's attentions as tumor suppressors or oncogenes that have been implicated in tumor progression, metastasis and intrinsic resistance to various cancer therapies. microRNA-21 (miR-21) demonstrates a potential oncogenic function and targets tumor inhibitor proteins in almost all types of cancer. miR-21 overexpression has been studied in terms of cell proliferation, migration, invasion, metastasis, and apoptosis regulation. Inhibition of miRNA expression using antisense technology by various nanovectors of different sizes, shapes and compositions has been evolved progressively to overcome the barriers confronted by miRNA delivery. Application of miR-21 antisense oligonucleotides for treating cancerous cells has become a promising achievement for cancer therapy. Moreover, miR-21 can mediate resistance to radiation and chemotherapy. The expanding role of miR-21 functions in human cancers with an emphasis on its regulatory targets and mechanisms, miR-21 related achievements against cancer promotion have been discussed.

Development of a highly-potent anti-angiogenic VEGF8-109 heterodimer by directed blocking of its VEGFR-2 binding site.

Angiogenesis is a hallmark of various pathological conditions and is controlled by a variety of angiogenic factors. Blockade of vascular endothelial growth factor (VEGF) as the most pivotal stimulator of angiogenesis offers a promising therapeutic approach for some diseases, typically cancer. In the present study, a heterodimeric antagonistic VEGF was precisely designed based on structural information of recently-crystallized VEGF/VEGF receptor-2 (VEGFR-2/fetal liver kinase 1/kinase domain region) complex. Directed blocking of kinase domain region occurs via substitution of a VEGF receptor binding site by two peptide segments in one pole, whereas the binding domain of the other pole of VEGF was intact. Candidate peptides for substitution were selected considering to some sequence and structural criteria. A reliable model of modified VEGF was built, refined using molecular dynamics simulation and docked with VEGFR-2. Docking analysis revealed that binding affinity of mutant VEGF was notably diminished, corroborating our design. Heterodimeric VEGF was expressed, refolded and highly purified by two-step affinity chromatography. Dimerization of this antagonist was confirmed using some analytical techniques. Spectroscopic studies assured us to obtain the heterodimeric form of VEGF. Some angiogenic in vitro assays such endothelial cell proliferation and tube formation indicated that this antagonist is not only strongly capable of inhibiting angiogenesis (half maximal inhibitory concentration of 33 and 24 ng · mL(-1) , respectively), but also showed the highest inhibitory effect compared to all other heterodimeric VEGF variants. The high anti-angiogenic potency of this VEGF antagonist may allow its future use as an anti-tumor agent.

Cloning, sequencing, expression and structural investigation of mnemiopsin from Mnemiopsis leidyi: an attempt toward understanding Ca2+-regulated photoproteins.

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl(2). The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.

A unique EF-hand motif in mnemiopsin photoprotein from Mnemiopsis leidyi: implication for its low calcium sensitivity.

Up to now, all reported Ca(2+)-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca(2+) sensitivity was lower than aequorin. Results of pK(a) calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.