1 1028-852X Pasteur Institute of Iran 1271 Related Fields Downregulation of Kinesin Spindle Protein Inhibits Proliferation, Induces Apoptosis and Increases Chemosensitivity in Hepatocellular Carcinoma Cells Doan Chinh Chung Doan Ngoc Trung Nguyen Quang Huy Nguyen Minh Hoa Do Minh Si Le Van Dong 1 1 2015 19 1 1 16 02 12 2014 02 12 2014 Background: Kinesin spindle protein (KSP) plays a critical role in mitosis. Inhibition of KSP function leads to cell cycle arrest at mitosis and ultimately to cell death. The aim of this study was to suppress KSP expression by specific small-interfering RNA (siRNA) in Hep3B cells and evaluate its anti-tumor activity. Methods: Three siRNA targeting KSP (KSP-siRNA #1-3) and one mismatched-siRNA (Cont-siRNA) were transfected into cells. Subsequently, KSP mRNA and protein levels, cell proliferation, and apoptosis were examined in both Hep3B cells and THLE-3 cells. In addition, the chemosensitivity of KSP-siRNA-treated Hep3B cells with doxorubicin was also investigated using cell proliferation and clonogenic survival assays. Results: The expression of endogenous KSP at both mRNA and protein levels in Hep3B cells was higher than in THLE-3 cells. In Hep3B cells, KSP-siRNA #2 showed a further downregulation of KSP as compared to KSP-siRNA #1 or KSP-siRNA #3. It also exhibited greater suppression of cell proliferation and induction of apoptosis than KSP-siRNA #1 or KSP-siRNA #3 this could be explained by the significant downregulation of cyclin D1, Bcl-2, and survivin. In contrast, KSP-siRNAs had no or lower effects on KSP expression, cell proliferation and apoptosis in THLE-3 cells. We also noticed that KSP-siRNA transfection could increase chemosensitivity to doxorubicin in Hep3B cells, even at low doses compared to control. Conclusion: Reducing the expression level of KSP, combined with drug treatment, yields promising results for eradicating hepatocellular carcinoma (HCC) cells in vitro. This study opens a new direction for liver cancer treatment.
1252 Related Fields Expression of Two Basic mRNA Biomarkers in Peripheral Blood of Patients with Non-Small Cell Lung Cancer Detected by Real-Time RT-PCR, Individually and Simultaneously Karimi Shirin Mohamadnia Abdolreza Nadji Seyed Alireza Yadegarazari Reza Khosravi Adnan Bahrami Naghmeh Saidijam Massoud 1 1 2015 19 1 17 22 08 11 2014 08 11 2014 Introduction: Although extensive research has been conducted on lung cancer markers, a singular clinically applicable marker has not been found yet. The objective of this study was to evaluate the sensitivity and the specificity of carcinoembryonic antigen (CEA) mRNA and lung-specific X protein (LUNX) mRNA biomarkers in peripheral blood to detect lung cancer individually and simultaneously. Methods: Thirty patients affected by lung cancer and 30 healthy individuals were studied in this research. Three vials of cDNA were made from each sample after taking peripheral blood samples and extracting total RNA. Each sample was examined by the real-time RT-PCR technique. The result from each vial was then compared with the sensitivity of overall marker. Results: The CEA mRNA was positive in 24 out of 30 lung cancer patients. Hence, its sensitivity was determined at 80%, differing significantly from that observed in healthy individuals, where 11 positive cases were seen. The overall sensitivity of this marker was significantly associated with positivity in vials 2 and 3 but not in vial 1. The LUNX mRNA was positive in 21 out of 30 patients, indicating 70% sensitivity. This finding significantly differed from that in healthy individuals. The overall sensitivity of this marker was significantly associated with positivity in vials 1 and 3, but not in vial 2. In 93.3% of the patients, at least one positive marker was observed. Conclusions: The mentioned mRNA could be suggested as sensitive and specific markers in peripheral blood for primary diagnosis of lung cancer. 1295 Related Fields Comparison of Mitochondrial-Related Transcriptional Levels of mitochondrial transcription factor A, Nuclear respiratory factor 1 and cytochrome c oxidase subunit 1 Genes in Single Human Oocytes at Various Stages of the Oocyte Maturation Ghaffari Novin Marefat Noruzinia Mehrdad Allahveisi Azra Saremi Aboutaleb Fadaei Fathabadi Fateme Mastery Farahani Reza Dehghani Fard Ali Pooladi Arash Mazaherinezhad Fard Ramin Yousefian Elham 1 1 2015 19 1 23 28 05 01 2015 05 01 2015 Background: The aim of the current study was to assess the mRNA levels of two mitochondria-related genes, including nuclear-encoded NRF1 (nuclear respiratory factor 1), mitochondrial transcription factor A (TFAM), and mitochondrial-encoded cytochrome c oxidase subunit 1 (MT-CO1) genes in various stages of the human oocyte maturation. Methods: Oocytes were obtained from nine infertile women with male factor undergoing in vitro fertilization (IVF)/intra-cytoplasmic sperm injection protocol. Mitochondrial-related mRNA levels were performed by single-cell TaqMan Real-time PCR. Results: the expression level of the target genes was low at the germinal vesicle stage (P>0.05). Although the mRNA level of NRF1 gene remained stable in metaphase I, the mRNA level of TFAM and MT-CO1 increased significantly (P<0.05).In metaphase II, the expression level of all genes increased compared to metaphase I (P<0.05). Conclusion: The overexpression levels of NRF1, TFAM, and MT-CO1 genes are related to the oocyte maturation. Therefore, the current study could be used clinically to improve the success rate of IVF. 1294 Related Fields Neuronal Cell Reconstruction with Umbilical Cord Blood Cells in the Brain Hypoxia-Ischemia Ghaffaripour Hossein Ali Jalali Mehdi Nikravesh Mohammad Reza Seghatoleslam Masoumeh Sanchooli Javad 1 1 2015 19 1 29 34 05 01 2015 05 01 2015 Background: Brain hypoxia-ischemia is a human neonatal injury that is considered a candidate for stem cell therapy. Methods: The possible therapeutic potential of human umbilical cord blood (HUCB) stem cells was evaluated in 14-day-old rats subjected to the right common carotid occlusion, a model of neonatal brain hypoxia-ischemia. Seven days after hypoxia-ischemia, rats received either saline solution or 4 × 105 HUCB cells i.v. Rats in control group did not receive any injection. After two weeks, rats were assessed using two motor tests. Subsequently, rats were scarified for histological and immunohistochemical analyses. Results: Our immunohistochemical findings demonstrated selective migration of the injected HUCB cells to the ischemic area as well as reduction in infarct volume. Seven days after surgery, we found significant recovery in the behavioral performance in the test group (12.7 +/- 0.3) compared to the sham group (10.0 +/-0.05), a trend which continued to day 14 (15.3 ± 0.3 vs. 11.9 ± 0.5, P<0.05). Postural and motor asymmetries at days 7 and 14 in the test group showed a significant decrease in the percentage of right turns in comparison to the sham group (75% and 59% vs. 97% and 96%, P<0.05). Conclusion: The results show the potential of HUCB stem cells in reduction of neurologic deficits associated with neonatal hypoxia-ischemia. 1246 Related Fields Comparative Analysis of CD4+ and CD8+ T Cells in Tumor Tissues, Lymph Nodes and the Peripheral Blood from Patients with Breast Cancer Riazi Rad Farhad Ajdary Soheila Omranipour Ramesh Alimohammadian Mohammad Hossein Hassan Zahir 1 1 2015 19 1 35 44 29 10 2014 29 10 2014 Background: CD4+ and CD8+ T cells are the main types of lymphocytes in cell-mediated immunity and play a central role in the induction of efficient immune responses against tumors. The frequencies of T cell subtypes in the peripheral blood and tumor tissues, and draining lymph nodes (dLN) can be considered as useful markers for evaluation of the immune system in cancers. Methods: In this study, the frequencies of CD4+ and CD8+ T cells in blood, tumor tissues, and dLN samples of breast cancer patients were compared with each other and with similar tissues from normal individuals. Immunophenotyping was carried out by flow cytometry and the expression levels of CXCL10, granzyme B, and mammaglobin were evaluated by real-time PCR. Results: In the peripheral blood, there were no differences in the T cell subsets between the patients and the normal individuals. The frequency of CD8+ T cells was significantly higher in tumor tissue than normal breast tissues while granzyme B expression was similar. Based on mammaglobin expression levels, dLN have been classified into micro- and macro-metastatic dLN. We found significantly lower frequency of CD4+ in macro-metastatic dLN than micro-metastatic dLN. CD8+ frequency was similar in both dLN however, granzyme B expression was higher in micro-metastatic ones. There was not any significant difference in CXCL10 expression between the two types of dLN. Conclusions: Based on our results, although the tumor does not affect the systemic immunity, tumoral cells affect the local immune system in the tumoral tissues and the metastatic dLN. 1270 Related Fields Anticonvulsant Effect of Cicer arietinum Seed in Animal Models of Epilepsy: Introduction of an active Molecule with Novel Chemical Structure Sardari Soroush Amiri Motahareh Rahimi Hourieh Kamalinejad Mohammad Narenjkar Jamshid Sayyah Mohammad 1 1 2015 19 1 45 50 02 12 2014 02 12 2014 Background: Cicer arietinum (Chickpea) is one of the most important harvests in the world with high nutritional value. Lack of essential oils in the seeds of Chickpea is an advantage in search for drug-like molecules with less toxicity. We evaluated anticonvulsant effect of C. arietinum in common animal models of epilepsy. Methods: Dichloromethane extract was obtained from C. arietinum seeds by percolation. Acute toxicity of the extract was assessed in mice. Protective effect of the extract was examined against tonic seizures induced by maximal electroshock (MES 50 mA, 50 Hz, 1 s) in mice, clonic seizures induced by pentylenetetrazole (PTZ 60 mg/kg, i.p.) in mice, and electrical kindling model of complex partial seizures in rats. The extract was fractionated by n-hexane to f1 and f2 fractions. The extract and fractions underwent phytochemical analysis by thin layer chromatography. The active anticonvulsant fraction, f1, was subjected to matrix assisted laser desorption/ionization (MALDI) mass analysis. Results: The crude extract had neither toxicity up to 7 g/kg nor protective activity in MES and kindling models. However, it significantly inhibited clonic seizures induced by PTZ. f1 fraction mimicked protective effect of the extract. Phytochemical screening revealed the presence of considerable amount of alkaloids in the extract and fractions. Moreover, a novel structural class was detected in f1 fraction. Conclusion: Finding an anticonvulsant molecule pertaining to a new structural class in the seeds of C. arietinum promises an effective and inexpensive source of antiepileptic medication. Further studies are needed to identify its mechanism of action and more clues into its structure-activity relationship. 1283 Related Fields Association between p53 Codon 72 (Arg72Pro) Polymorphism and Primary Open-Angle Glaucoma in Iranian Patients Neamatzade Hossein Soleimanizad Reza Atefi Aref Zare-Shehneh Masoud Gharibi Saba Shekari Abolfazl Rahimzadeh Amir Bahman Dept. of Microbial Biotechnology, Payam Noor University, Tehran, Iran 1 1 2015 19 1 51 56 20 12 2014 20 12 2014 Background: Glaucomatous neuropathy is a type of cell death due to apoptosis. The p53 gene is one of the regulatory genes of apoptosis. Recently, the association between the p53 gene encoding for proline at codon 72 and primary open-angle glaucoma (POAG) has been studied in some ethnic groups. This study is the first association analysis of POAG and p53 codon 72 polymorphism in Iranian patients. Methods: A cohort of 65 unrelated patients with POAG (age range from 12-62 years, mean ± SD of 40.16 ± 17.51 years) and 65 unrelated control subjects (without glaucoma, age range of 14-63 years, mean ± SD of 35.64 ± 13.61 years) were selected. In Iranian POAG patients and normal healthy controls, the p53 codon 72 polymorphism in exon 4 was amplified using polymerase chain reaction. The amplified DNA fragments were digested with the BstUI restriction enzyme, and the digestion patterns were used to identify the alleles for the polymorphic site. Results: Comparisons revealed significant differences in allele and genotype frequencies of Pro72Arg between POAG patients and control group. A higher risk of POAG was associated with allele Pro (OR = 2.1, 95% CI = 1.2–3.4) and genotype Pro/Pro (OR = 3.9, 95% CI = 0.13-12.7). Conclusion: The p53 Pro72 allele was more frequent in Iranian POAG patients than in the control group (P<0.05). The present findings show that the individuals with the Pro/Pro genotype may be more likely to develop POAG. However, additional studies are necessary to confirm this association. 1239 Related Fields Simultaneous Analysis of Multidrug Reā€Œsistance 1(MDR1) C3435T, G2677T/A, and C1236T genotypes in Hamadan City Population, West of Iran Saidijam Massoud Mahjub Hossein Shabab Nooshin Yadegarazari Reza 1 1 2015 19 1 57 62 21 10 2014 21 10 2014 Background: One of the limitations in the treatment of common diseases such as cancer chemotherapy is development of multidrug re‌sistance (MDR). Polymorphisms could alter the expression level of MDR1 gene, which plays an important role in MDR. In this research, the frequency of C3435T, C1236T, and G2677T/A polymorphisms of MDR1 gene was investigated in a large group of population from Hamadan city to provide a sample data resource. Methods: Peripheral blood (2 ml) was taken, and DNA extraction was carried out. Multiplexed mutagenically separated PCR, which was followed by polyacrylamide gel electrophoresis and silver staining, was applied to detect the mentioned polymorphisms in 935 individuals. Sequencing performed for confirmation of gel electrophoresis resulted in 10 random cases. In total, alleles and genotypes of 933 persons (776 women and 157 men) were determined. Results: The most frequent alleles of the polymorphisms were: 3435T, C1236, and G2677. The most frequent genotypes were: 3435C/T, 1236C/T, and 2677G/A, and their concurrent presence was also found as the most frequent simultaneous genotypes. There was not any meaningful difference among the prevalence of these genotypes in groups of men and women. Conclusion: Our results were close to those of other studies performed in Iran and compared to the other ethnic groups, which showed more similarity to Asian peoples than Europeans. As an aspect of personalized medicine, it could be used by chemotherapists to improve the routine methods of cancer treatment. 1293 Related Fields Glycoconjugates Distribution during Developing Mouse Spinal Cord Motor Organizers Vojoudi Elham Ebrahimi Vahid Ebrahimzadeh-Bideskan Alireza Fazel Alireza 1 1 2015 19 1 63 68 05 01 2015 05 01 2015 Background: The aim of this research was to study the distribution and changes of glycoconjugates particularly their terminal sugars by using lectin histochemistry during mouse spinal cord development. Methods: Formalin-fixed sections of mouse embryo (10-16 fetal days) were processed for lectin histochemical method. In this study, two groups of horseradish peroxidase-labeled specific lectins were used: N-acetylgalactosamine, including Dolichos biflorus, Wisteria flori‌bunda agglutinin (WFA), Vicia villosa, Glycine max as well as focuse-binding lectins, including tetragonolobus, Ulex europaeus, and Orange peel fungus (OFA). All sections were counterstained with alcian blue (pH 2.5). Results: Our results showed that only WFA and OFA reacted strongly with the floor plate cells from early to late embryonic period of developing spinal cord. The strongest reactions were related to the 14, 15, and 16 days of tissue sections incubated with OFA and WFA lectins. Conclusion: The present study demonstrated that cellular and molecular differentiation of the spinal cord organizers is a wholly regulated process, and &alpha-L-fucose, &alpha-D-GalNAc, and &alpha/&beta-D-GalNAc termi‌nal sugars play a significant role during the prenatal spinal cord development.