Preparation and characterization of cyclodextrin nanosponges for organic toxic molecule removal.

Cyclodextrin-based nanosponges (CD-NS) are considered as safe and biocompatible systems for removing toxic molecules from the body. Rapid removal of toxic molecules that are formed in the body from certain food constituents, is relevant especially for patients affected by chronic kidney disease. Within the scope of this study, innovative cyclodextrin polymers were synthesized to form nanosponges able to remove indole, before it could form the toxic indoxyl sulfate in the body. Furthermore, in vivo studies were carried out using the two optimal CD-NS formulations by assessing physicochemical properties, stability, indole adsorption capacity and in vitro cytotoxicity. NS prepared from ß-cyclodextrin cross-linked with toluene diisocyanate was found to be the most effective NS with an in vitro indole adsorption capacity of over 90%. In addition, this derivative was more stable in gastrointestinal media. Animal studies further revealed that oral CD-NSs did not tend to accumulate and damage gastrointestinal tissues and are excreted from the GI tract with minimal absorption. In conclusion, this study suggests that CD-NS formulations are effective and safe in removing toxic molecules from the body. Their potential use in veterinary or human medicine could reduce dialysis frequency and avoid hepatic and cardiac toxicity avoiding the indole formation.

Overexpression of miR-145-5p inhibits proliferation of prostate cancer cells and reduces SOX2 expression.

We aimed to perform functional analysis of miR-145-5p in prostate cancer (PCa) cells and to identify targets of miR-145-5p for understanding its role in PCa pathogenesis. PC3, DU145, LNCaP PCa, and PNT1a nontumorigenic prostate cell lines were utilized for functional analysis of miR-145-5p. Its overexpression caused inhibition of proliferation through apoptosis and reduced migration in PCa cells. SOX2 expression was significantly decreased in both mRNA and protein level in miR-145-5p-overexpressed PCa cells. We proposed that miR-145-5p, being an important regulator of SOX2, carries a crucial role in PCa tumorigenesis.

MicroRNA expression profiling reveals the potential function of microRNA-31 in chordomas.

Chordomas are rare bone tumors arising from remnants of the notochord. Molecular studies to determine the pathways involved in their pathogenesis and develop better treatments are limited. Alterations in microRNAs (miRNAs) play important roles in cancer. miRNAs are small RNA sequences that affect transcriptional and post-transcriptional regulation of gene expression in most eukaryotic organisms. Studies show that miRNA dysregulation is important for tumor initiation and progression. We compared the expression profile of miRNAs in chordomas to that of healthy nucleus pulposus samples to gain insight into the molecular pathogenesis of chordomas. Results of functional studies on one of the altered miRNAs, miR-31, are presented. The comparison between the miRNA profile of chordoma samples and the profile of normal nucleus pulposus samples suggests dysregulation of 53 miRNAs. Thirty miRNAs were upregulated in our tumor samples, while 23 were downregulated. Notably, hsa-miR-140-3p and hsa-miR-148a were upregulated in most chordomas relative to levels in nucleus pulposus cells. Two other miRNAs, hsa-miR-31 and hsa-miR-222, were downregulated in chordomas compared with the control group. Quantification with real-time polymerase chain reaction confirmed up or downregulation of these miRNAs among all samples. Functional analyses showed that hsa-miR-31 has an apoptotic effect on chordoma cells and downregulates the expression of c-MET and radixin. miRNA profiling showed that hsa-miR-31, hsa-miR-222, hsa-miR-140-3p and hsa-miR-148a are differentially expressed in chordomas compared with healthy nucleus pulposus. Our profiling may be the first step toward delineating the differential regulation of cancer-related genes in chordomas, helping to reveal the mechanisms of initiation and progression.

MicroRNA profiling in lymphocytes and serum of tyrosinemia type-I patients.

Tyrosinemia type-I results from lack of fumarylacetoacetate hydrolase (FAH), which is a liver enzyme and also shown to be present in lymphocytes, fibroblasts, and cultured amniotic fluid cells. In young infants, symptoms of untreated Tyrosinemia type-I are restricted to severe liver involvement. Later in the first year; however, it is known to be present with liver and renal tubular dysfunction associated with growth failure and rickets. MicroRNAs are small regulatory RNAs that function post-transcriptionally. They target commonly 3'-UTR of the mRNAs and inhibit protein expression by either blocking the synthesis or causing degradation of the mRNAs. MiRNA deregulation was observed in a variety of pathologic conditions but their roles in metabolic diseases were remained unsolved. We studied 6 patients with classical phenotypes of Tyrosinemia type-I. To identify possible miRNAs targeting FAH transcripts, microarray profiling of 961 miRNAs for lymphocytes and serum is performed. Computational algorithms are used for prediction of putative mRNA-miRNA interactions. A number of deregulated miRNAs, targeting the non-conserved sites on FAH transcripts were found. Besides, there are some miRNAs that are similarly altered both in lymphocytes and serum, possibly contributing to the disease phenotype. Since miRNAs may have an active role in the enzymatic pathway of tyrosine catabolism, characterizing miRNA profile in fibroblasts of tyrosinemia patients is also important because miRNAs would have distinctive role in disease pathogenesis and they are promising for future therapeutic studies.

Identification of a novel mutation in ZAP70 and prenatal diagnosis in a Turkish family with severe combined immunodeficiency disorder.

Protein tyrosine kinases (PTKs) play an important role in T cell development and activation. In vitro and in vivo defects, resulting in variable deficiencies in thymic development and in T cell antigen receptor (TCR) signal transduction, in PTKs have been shown. ZAP70, one of those PTKs, is a 70-kDa tyrosine phosphoprotein and associates with the ζ chain and undergoes tyrosine phosphorylation following TCR stimulation. It is expressed in T and natural killer (NK) cells. Several mutations were shown to lead to an autosomal recessive form of severe combined immunodeficiency disease (SCID). Here, we present a family with a novel mutation in ZAP70. The proband, the second child of the first cousin parents of Turkish origin, was diagnosed with SCID having R514C mutation on homozygous state. She had decreased CD8(+) T and natural killer cells, normal CD4(+) T cells, high serum Ig E level, perivascular dermatitis and ichthyosis. This article presents clinical features of a novel mutation on ZAP70 and the first prenatal molecular diagnosis of ZAP70 deficiency. Different mutations in ZAP70 and related phenotypes reported in the literature are also discussed.

Characterization of cancer stem-like cells in chordoma.

OBJECT: Chordomas are locally aggressive bone tumors known to arise from the remnants of the notochord. Because chordomas are rare, molecular studies aimed at developing new therapies are scarce and new approaches are needed. Chordoma cells and cancer stem-like cells share similar characteristics, including self-renewal, differentiation, and resistance to chemotherapy. Therefore, it seems possible that chordomas might contain a subpopulation of cancer stem-like cells. The aim of this study is to determine whether cancer stem-like cells might be present in chordomas. METHODS: In this study, the authors used gene expression analysis for common cancer stem-like cellmarkers, including c-myc, SSEA-1, oct4, klf4, sox2, nanog, and brachyury, and compared chordoma cells and tissues with nucleus pulposus tissues (disc degenerated nontumorigenic tissues). Differentiation through agents such as all-trans retinoic acid and osteogenic differentiation medium was induced to the chordoma cells. Additionally, U-CH1 cells were sorted via magnetic cell sorting for stem cell markers CD133 and CD15. After separation, positive and negative cells for these markers were grown in a nonadherent environment, soft agar, to determine whether the presence of these cancer stem-like cells might be responsible for initiating chordoma. The results were compared with those of untreated cells in terms of migration, proliferation, and gene expression by using reverse transcriptase polymerase chain reaction. RESULTS: The results indicate that chordoma cells might be differentiating and committing into an osteogenic lineage when induced with the osteogenic differentiation agent. Chordoma cells that are induced with retinoic acid showed slower migration and proliferation rates when compared with the untreated cells. Chordoma cells that were found to be enriched by cancer stem-like cell markers, namely CD133 and CD15, were able to live in a nonadherent soft agar medium, demonstrating a self-renewal capability. To the authors' knowledge, this is the first time that cancer stem-like cell markers were also found to be expressed in chordoma cells and tissues. CONCLUSIONS: Cancer stem-like cell detection might be an important step in determining the recurrent and metastatic characteristics of chordoma. This finding may lead to the development of new approaches toward treatments of chordomas.

The effects of chemotherapeutic agents on differentiated chordoma cells.

OBJECT: Chordoma is a rare type of malignant bone tumor and is known to arise from the remnants of the notochord. Resistance to chemotherapy makes the treatment of chordoma difficult; therefore, new approaches need to be developed to cure this disease. Differentiation therapy, using various differentiating agents, is attracting oncologists as a common therapeutic method to treat other tumors. Based on forcing cells to mature into other lineages, differentiation therapy might be an available method to treat chordomas in addition to conventional therapies. METHODS: In this study a chordoma cell line, U-CH1, was exposed to several chemotherapeutic agents including vincristine, doxorubicin, cisplatin, etoposide, fludarabine, methotrexate, nilotinib, and imatinib mesylate under appropriate conditions. The first group of U-CH1 cells was exposed to drugs only and the second group of cells was exposed to the simultaneous treatment of 1 µM all-trans retinoic acid (ATRA) and chemotherapeutic agents in differentiation therapy. The efficacy of the differentiation method was assessed by measuring the viability of U-CH1 cells. RESULTS: Vincristine, doxorubicin, etoposide, cisplatin, and fludarabine, each at a concentration of 10 µM, decreased the number of chordoma cells when given alone down to 11%, 0%, 30%, 67%, and 3%, respectively. Etoposide and cisplatin, each at a concentration of 10 µM, reduced the percentage of viable chordoma cells in a more effective way when given with 1 µM ATRA simultaneously, reducing the number of viable cells to 14% and 9%, respectively. On the other hand, imatinib and nilotinib, each at a concentration of 3 µM, as well as 10 µM methotrexate, showed no decrease in the number of cancer cells. CONCLUSIONS: The results suggest that chordoma cells may be treated using the differentiation method in a more effective way than when they are treated with chemotherapeutic agents alone. This new approach may be an alternative method to conventional therapies in the treatment of chordoma.

The effect of polymorphisms in the promoter region of the MMP-1 gene on the occurrence and invasiveness of hypophyseal adenoma.

BACKGROUND: The matrix metalloproteinase-1 enzyme (MMP-1, also called collagenase 1) plays a key role in turnover of collagen fibers in the intercellular matrix. Insertion of a guanine residue was found within the promoter region of the MMP-1 gene. We found that MMP-1 levels increased approximately twofold over normal when this insertion was present, enabling MMP-1 to facilitate tumor invasion and metastasis. MMP-1 is also believed to play a role in tumor development. The aim of our study is to investigate the effect of polymorphisms in the promoter region of the MMP-1 gene on the development of benign and invasive hypophyseal adenomas. PATIENTS AND METHODS: Thirty patients with hypophyseal adenomas diagnosed by radiological examination underwent surgical removal, and the diagnosis was confirmed using immunohistochemical staining of the pathology specimens. We found that ten of these patients had invasive adenomas confirmed by radiological examination and immunohistochemical staining. DNA isolation was performed on all specimens, and 5-cc venous blood samples were obtained from all patients as well as 30 volunteers using the Qiagen QIAquick kit. Promoter regions of MMP-1 genes from the DNA samples were amplified using polymerase chain reaction (PCR) and primers designed for the site-directed mutation method. Following PCR, a guanine residue within the promoter region of the MMP-1 gene was identified using the restriction fragment length polymorphism method and the ALU I restriction enzyme. Three genotypes were detected in a genotyping assay: 2G/2G, 1G/2G, and 1G/1G. RESULTS: Of the surgically treated patients, 36.6% had the 2G/2G genotype, 46.6% had the 1G/2G genotype, and 16.6% had the 1G/1G genotype. The 2G allele frequency was found to be 83.4%. In 90% of cases of invasive adenoma, a homozygous 2G/2G genotype was detected. DISCUSSION: The risk for development of hypophyseal adenoma may be greater in patients with the 2G allele. In cases of existing hypophyseal adenoma, those with the homozygous 2G allele tend to be invasive.