ABSTRACT
The present study was performed to analyze molecularly the phylogenetic positions of human-infecting Trichostrongylus species in Mazandaran Province, Iran, which is an endemic area for trichostrongyliasis. DNA from 7 Trichostrongylus infected stool samples were extracted by using in-house (IH) method. PCR amplification of ITS2-rDNA region was performed, and products were sequenced. Phylogenetic analysis of the nucleotide sequence data was performed using MEGA 5.0 software. Six out of 7 isolates had high similarity with Trichostrongylus colubriformis, while the other one showed high homology with Trichostrongylus axei registered in GenBank reference sequences. Intra-specific variations within isolates of T. colubriformis and T. axei amounted to 0–1.8% and 0–0.6%, respectively. Trichostrongylus species obtained in the present study were in a cluster with the relevant reference sequences from previous studies. BLAST analysis indicated that there was 100% homology among all 6 ITS2 sequences of T. colubriformis in the present study and most previously registered sequences of T. colubriformis from human, sheep, and goat isolates from Iran and also human isolates from Laos, Thailand, and France. The ITS2 sequence of T. axei exhibited 99.4% homology with the human isolate of T. axei from Thailand, sheep isolates from New Zealand and Iran, and cattle isolate from USA.
Subject(s)
Animals , Cattle , Humans , Base Sequence , Databases, Nucleic Acid , DNA , France , Goats , Iran , Laos , Methods , New Zealand , Polymerase Chain Reaction , Sheep , Thailand , TrichostrongylusABSTRACT
PURPOSE: Tumor cells have increased turnover of nicotinamide adenine dinucleotide (NAD⁺), the main coenzyme in processes including adenosine diphosphate-ribosylation, deacetylation, and calcium mobilization. NAD⁺ is predominantly synthesized in human cells via the salvage pathway, with the first component being nicotinamide. Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in this pathway, and its chemical inhibition by FK866 has elicited antitumor effects in several preclinical models of solid and hematologic cancers. However, its efficacy in estrogen receptor (ER)-positive and human epidermal growth factor receptor 2-positive breast cancer cells has not been previously investigated. In this study, we aimed to deplete the NAD⁺ content of MCF-7 cells, a model cell line for ER-positive breast cancer, by inhibiting NAMPT in order to evaluate downstream effects on p53 and its acetylation, p21 and Bcl-2-associated X protein (BAX) expression, and finally, apoptosis in MCF-7 breast cancer cells. METHODS: MCF-7 cells were cultured and treated with FK866. NAD⁺ levels in cells were determined colorimetrically. Levels of p53 and its acetylated form were determined by Western blotting. Expression of p21 and BAX was determined by real-time polymerase chain reaction. Finally, levels of apoptosis were assessed by flow cytometry using markers for annexin V and propidium iodide. RESULTS: FK866 treatment was able to increase p53 levels and acetylation, upregulate BAX and p21 expression, and induce apoptosis in MCF-7 cells. Addition of exogenous NAD⁺ to cells reversed these effects, suggesting that FK866 exerted its effects by depleting NAD⁺ levels. CONCLUSION: Results showed that FK866 could effectively inhibit NAD⁺ biosynthesis and induce programmed cell death in MCF-7 cells, suggesting that NAMPT inhibitors may be useful for the treatment of ER-positive breast cancers.