RESUMO
OBJECTIVE: The aim of current study was to provide a proof-of-concept on the mechanism of PLAU and PCDH10 gene expressions and caspases-3, -8, and -9 activities in the apoptotic pathway after treatment of malignant human glioma cell line (U87MG) with cytochalasin H. MATERIALS AND METHODS: In the present experimental study, we have examined cytochalasin H cytotoxic activities as a new therapeutic agent on U87MG cells in vitro for the first time. The cells were cultured and treated with 10-5-10-9 M of cytochalasin H for 24, 48 and 72 hours. The assessment of cell viability was carried out by (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay at 578 nm. The data are the average of three independent tests. mRNA expression changes of PLAU and PCDH10 were then evaluated by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The fluorometric of caspases-3, -8, and -9 activities were carried out. The morphology changes in the U87MG cells were observed by fluorescence microscope. RESULTS: MTT assay showed that cytochalasin H (10-5 M) inhibited the U87MG cancer cells proliferation after 48 hours. Analysis of qRT-PCR showed that the PLAU expression was significantly decreased in comparison with the control (P<0.05). The expression of PCDH10 also showed a significant increase when compared to the control (P<0.001). Fluorescence microscope indicated morphological changes due to apoptosis in U87MG cancer cells, after treatment with cytochalasin H (10-5 M, 48 hours). The fluorometric evaluation of caspase-3, -8, and -9 activities showed no significant difference between the caspases and the control group. CONCLUSION: This study shows the effect of caspase-independent pathways of the programmed cell death on the U87MG cancer cell line under cytochalasin H treatment. Further studies are needed to explore the exact mechanism.
RESUMO
Miscarriage is the most common complication in pregnancy. Considering the importance of the problem thrombophilia in pregnant women and its association with recurrent pregnancy loss (RPL), analysis of polymorphisms of genes involved in thrombophilia can be useful. We investigated the frequency and association between ten polymorphisms of seven thrombophilia genes and RPL in an Iranian population. This case-control study was conducted on 200 women with recurrent pregnancy loss and also on 200 women with at least one successful pregnancy as the control group. Using PCR-RFLP, DNA from samples were analyzed for carrying A5279G, A4070G, and FV Leiden of factor V; FXIII (Val34Leu); FII (A20210G); BF (-455 G/A); ITGB3 (1565T/C); 677C/T and 1298A/C of MTHFR; and PAI-1 (-675 I/D, 5G/4G) polymorphisms. The BF(-455 G/A), MTHFR (677 C/T, 1298A/ C), PAI-1 (-675 I/D,4G/ 5G), FV Leiden, FV (A5279G), FXIII (Val34Leu) polymorphisms, which had shown positive relation, and ITGB3 1565T/C were the polymorphisms with negative relation to RPL. But in this study it is indicated that there is no significant association between FII (A20210G) and FV (A4070G) polymorphism and RPL. All the data acquired from the RPL patients in this experiment illustrate the importance of screening thrombophilia. Nevertheless, more studies on large-scale populations may be needed to identify novel genetic variants. ABBREVIATIONS: ASRM: American Society of Reproductive Medicine; HHCY: hyperhomocysteinemia; MTHFR: methylenetetrahydrofolate reductase; PCR: polymerase chain reaction; PAGE: poly-acrylamide gel electrophoresis; RPL: recurrent pregnancy loss.
