Down-regulation of miR-135b in colon adenocarcinoma induced by a TGF-β receptor i kinase inhibitor (SD-208)

Objective(s): Transforming growth factor-β (TGF-β) is involved in colorectal cancer (CRC). The SD-208 acts as an anti-cancer agent in different malignancies via TGF-β signaling. This work aims to show the effect of manipulation of TGF-β signaling on some miRNAs implicated in CRC. Materials and Methods: We investigated the effects of SD-208 on SW-48, a colon adenocarcinoma cell line. The cell line was treated with 0.5, 1 and 2 μM concentrations of SD-208. Then, the xenograft model of colon cancer was established by subcutaneous inoculation of SW-48 cell line into the nude mice. The animals were treated with SD-208 for three weeks. A quantitative real-time PCR was carried out for expression level analysis of selected oncogenic (miR-21, 31, 20a and 135b) and suppressormiRNAs (let7-g, miR-133b, 145 and 200c). Data were analyzed using the 2-��CT method through student�s t-test via the GraphPad Prism software. Results: Our results revealed that SD-208 could significantly down-regulate the expression of one key onco-miRNA, miR-135b, in either SW-48 colon cells (P=0.006) or tumors orthotopically implanted in nude mice (P=0.018). Our in silico study also predicted that SD-208 could modulate the expression of potential downstream tumor suppressor targets of the miR135b. Conclusion: Our data provide novel evidence that anticancer effects of SD-208 (and likely other TGF-β inhibitors) may be owing to their ability to regulate miRNAs expression. © 2015, Mashhad University of Medical Sciences. All rights reserved

Evaluation of SD-208, a TGF-β-RI kinase inhibitor, as an anticancer agent in retinoblastoma

Retinoblastoma is the most common intraocular tumor in children resulting from genetic alterations and transformation of mature retinal cells. The objective of this study was to investigate the effects of SD-208, TGF-β-RI kinase inhibitor, on the expression of some miRNAs including a miR-17/92 cluster in retinoblastoma cells. Prior to initiate this work, the cell proliferation was studied by Methyl Thiazolyl Tetrazolium (MTT) and bromo-2�-deoxyuridine (BrdU) assays. Then, the expression patterns of four miRNAs (18a, 20a, 22, and 34a) were investigated in the treated SD-208 (0.0, 1, 2 and 3 μM) and untreated Y-79 cells. A remarkable inhibition of the cell proliferation was found in Y-79 cells treated with SD-208 versus untreated cells. Also, the expression changes were observed in miRNAs 18a, 20a, 22 and 34a in response to SD-208 treatment (P<0.05). The findings of the present study suggest that the anti-cancer effect of SD-208 may be exerted due to the regulation of specific miRNAs, at least in this particular retinoblastoma cell line. To the best of the researchers� knowledge, this is the first report demonstrating that the SD-208 could alter the expression of tumor suppressive miRNAs as well as oncomiRs in vitro. In conclusion, the present data suggest that SD-208 could be an alternative agent in retinoblastoma treatment. © 2016 Tehran University of Medical Sciences. All rights reserved

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis

An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania

Identification and sequencing of candida krusei aconitate hydratase gene using rapid amplification of cdna ends method and phylogenetic analysis

Background: The production and development of an effective fungicidal drug requires the identification of an essential fungal protein as a drug target. Aconitase (ACO) is a mitochondrial protein that plays a vital role in tricarboxylic acid (TCA) cycle and thus production of energy within the cell. Objectives: The current study aimed to sequence Candida krusei ACO gene and determine any amino acid residue differences between human and fungal aconitases to obtain selective inhibition. Materials and Methods: Candida krusei (ATCC: 6258) aconitase gene was determined by 5�Rapid Amplification of cDNA Ends (RACE) method and degenerate Polymerase Chain Reaction (PCR) and analyzed using bioinformatics softwares. Results: One thousand-four hundred-nineteen nucleotide of C. krusei aconitase gene were clarified and submitted in Genbank as a partial sequence and then taxonomic location of C. krusei was determined by nucleotide and amino acid sequences of this gene. The comparison of nucleotide and amino acid sequences of Candida species ACO genes showed that C. krusei possessed characteristic sequences. No significant differences were observed between C. krusei and human aconitases within the active site amino acid residues. Conclusions: Results of the current study indicated that aconitase was not a suitable target to design new anti-fungal drugs that selectively block this enzyme. © 2015, Ahvaz Jundishapur University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License