Cell Journal (Yakhteh)

253

Authors: Atousa Haghi, Mahdieh Salemi, Mahnaz Mohammadi Kian, Mohsen Nikbakht, Saeed Mohammadi, Bahram Chahardouli, Shaharbano Rostami, Kianoosh Malekzadeh

Objective Acute myeloid leukemia (AML) is a clonal disorder of hemopoietic progenitor cells. The Raf serine/threonine (Ser/Thr) protein kinase isoforms including B-Raf and RAF1, are the upstream in the MAPK cascade that play essential functions in regulating cellular proliferation and survival. Activated autophagy-related genes have a dual role in both cell death and cell survival in cancer cells. The cytotoxic activities of arsenic trioxide (ATO) were widely assessed in many cancers. Sorafenib is known as a multikinase inhibitor which acts through suppression of Ser/Thr kinase Raf that was reported to have a key role in tumor cell signaling, proliferation, and angiogenesis. In this study, we examined the combination effect of ATO and sorafenib in AML cell lines. Materials And Methods In this experimental study, we studied in vitro effects of ATO and sorafenib on human leukemia cell lines. The effective concentrations of compounds were determined by MTT assay in both single and combination treatments. Apoptosis was evaluated by annexin-V FITC staining. Finally, mRNA levels of apoptotic and autophagy genes were evaluated using real-time polymerase chain reaction (PCR). Results Data demonstrated that sorafenib, ATO, and their combination significantly increase the number of apoptotic cells. We found that the combination of ATO and sorafenib significantly reduces the viability of U937 and KG-1 cells. The expression level of selective autophagy genes, ULK1 and Beclin1 decreased but LC3-II increased in U937. Conclusion The expression levels of apoptotic and autophagy activator genes were increased in response to treatment. The crosstalk between apoptosis and autophagy is a complicated mechanism and further investigations seem to be necessary.

273-282

Authors: Sanaz Hasani, Arash Javeri, Asadollah Asadi, Masomeh Fakhr Taha

Objective Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) play important roles in embryonic heart development. Also, two epigenetic modifying molecules, 5ˊ-azacytidine (5ˊ-Aza) and valproic acid (VPA) induce cardiomyogenesis in the infarcted heart. In this study, we first evaluated the role of BMP4 and bFGF in cardiac trans-differentiation and then the effectiveness of 5´-Aza and VPA in reprogramming and cardiac differentiation of human adipose tissue-derived stem cells (ADSCs). Materials And Methods In this experimental study, human ADSCs were isolated by collagenase I digestion. For cardiac differentiation, third to fifth-passaged ADSCs were treated with BMP4 alone or a combination of BMP4 and bFGF with or without 5ˊ-Aza and VPA pre-treatment. After 21 days, the expression of cardiac-specific markers was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, immunocytochemistry, flow cytometry and western blot analyses. Results BMP4 and more prominently a combination of BMP4 and bFGF induced cardiac differentiation of human ADSCs. Epigenetic modification of the ADSCs by 5ˊ-Aza and VPA significantly upregulated the expression of OCT4A, SOX2, NANOG, Brachyury/T and GATA4 but downregulated GSC and NES mRNAs. Furthermore, pre-treatment with 5ˊ-Aza and VPA upregulated the expression of TBX5, ANF, CX43 and CXCR4 mRNAs in three-week differentiated ADSCs but downregulated the expression of some cardiac-specific genes and decreased the population of cardiac troponin I-expressing cells. Conclusion Our findings demonstrated the inductive role of BMP4 and especially BMP4 and bFGF combination in cardiac trans-differentiation of human ADSCs. Treatment with 5ˊ-Aza and VPA reprogrammed ADSCs toward a more pluripotent state and increased tendency of the ADSCs for mesodermal differentiation. Although pre-treatment with 5ˊ-Aza and VPA counteracted the cardiogenic effects of BMP4 and bFGF, it may be in favor of migration, engraftment and survival of the ADSCs after transplantation.

293-301

Authors: Fatemeh Mokhtari-Jafari, Ghassem Amoabediny, Mohammad Mehdi Dehghan, Marco N. Helder, Behrouz Zandieh-Doulabi, Jenneke Klein-Nulend

Objective This study investigated whether short stimulation (30 minutes) of human adipose stem cells (hASCs) with 1,25-dihydroxyvitamin D3(calcitriol or 1,25-(OH)2VitD3), fitting within the surgical procedure time frame, suffices to induce osteogenic differentiation, and compared this with continuous treatment with 1,25-(OH)2VitD3. Materials And Methods In this experimental study, hASCs were pretreated with/without 10 nM calcitriol for 30 minutes, seeded on biphasic calcium phosphate (BCP), and cultured for 3 weeks with/without 1,25-(OH)2VitD3. Cell attachment was determined 30 minutes after cell seeding. AlamarBlue assay, alkaline phosphatase (ALP) assay, ALP staining, real-time polymerase chain reaction (PCR), and protein assay were used to evaluate the effect of short calcitriol pretreatment on proliferation and osteogenic differentiation of hASCs up to 3 weeks. Results Pretreatment with 1,25-(OH)2VitD3enhanced the attachment of hASCs to BCP by 1.5-fold compared to non- treated cells and increased the proliferation by 3.5-fold at day 14, and 2.6-fold at day 21. In contrast, continuous treatment increased the proliferation by 1.7-fold only at day 14. After 2 weeks, ALP activity was increased by 18.5-fold when hASCs were pretreated with 1,25-(OH)2VitD3for 30 minutes but increased only 2.6-fold when compared with its continuous counterpart. Moreover, after 14 days, pretreatment resulted in significant upregulation of the osteogenic markers RUNX2 and SPARC by 3.6-fold and 2.2-fold, respectively, while this was not observed upon continuous treatment. Finally, 30 minutes pretreatment of hASCs with 1,25-(OH)2VitD3increased VEGF189 expression, which may contribute to the process of angiogenesis. Conclusion This study is the first research showing that 30 minutes pretreatment of hASCs with 1,25-(OH)2VitD3, not only enhanced cell attachment to the scaffold at seeding time, but also promoted the proliferation and osteogenic differentiation of hASCs more strongly than continuous treatment, suggesting that short pre-treatment with 1,25-(OH)2VitD3is a promising approach for the regeneration of bones in a one-step surgical procedure.

310-318

Authors: Masoud Vatani, Mohammad-Hossein Beigi, Fatemeh Ejeian, Ahmad Mottaghi, Afshin Yadegari-Naeini, Mohammad-Hossein Nasr-Esfahani

Objective Bioresorbable and titanium plates/screws are considered as a standard treatment for fixation of the bone segments of craniofacial area and paying attention to their biocompatibility is an important issue along with other aspects of application. The purpose of the study was to evaluate the cell viability of two types of plate and screw used in maxillofacial surgeries in contact with gingival fibroblasts and bone marrow stem cells. Materials And Methods In this experimental study after extraction and cultivation of cells from healthy human gingival tissue and alveolar bone of jaw, cytotoxicity of device was evaluated. In direct contact method, samples had near vicinity contact with the both cell lines and in indirect contact method, by-products released, like ions, from samples after 8 weeks were used to assess cytotoxicity. Then cytotoxicity was evaluated on the 2nd, 4th and 6th day with MTS tests and microscopy. The data were analyzed by one-way ANOVA and independent t tests. Results There was a statistically significant difference between the German plate and screw and all the samples studied on day 6 (P<0.05). Furthermore, a statistically significant difference was observed between both metal samples and both bio-absorbable samples on day 6 and both cell lines (P<0.05). Comparisons between the two groups with each other for both cell lines on the 6th day were statistically significant (P<0.05). Conclusion Our results suggest that that cytotoxicity of biomaterial, from different brands, were not similar and some of the biomaterial showed lower degree of toxicity compared to others and specialist using these products showed be aware of this differences. Our investigation indicates more biocompatibility of bioresorbable plates and screws compared to titanium. In addition our results suggest that biomaterials were not completely neutral.

325-333

Authors: Xiaoxiao Yu, Huayang Wang, Hongjia Shao, Cuijuan Zhang, Xiuli Ju, Jie Yang

Objective This study aimed to evaluate the specific roles of polyinosinic:polycytidylic acid (polyI:C) in macrophage chemotaxis and reveal the potential regulatory mechanisms related to chemokine receptor 5 (CCR5). Materials And Methods In this experimental study, THP-1-derived macrophages (THP1-Mφs) induced from THP- 1 monocytes were treated with 25 μg/mL polyI:C. Toll-like receptor 3 (TLR3), Jumonji domain-containing protein (JMJD)1A, and JMJD1C small interfering RNA (siRNAs) were transfected into THP1-Mφs. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TLR3, CCR5, 23 Jumonji C domain-containing histone demethylase family members, JMJD1A, and JMJD1C in THP1-Mφs with different siRNAs transfections. Western blot was performed to detect JMJD1A, JMJD1C, H3K9me2, and H3K9me3 expressions. A transwell migration assay was conducted to detect THP1-Mφ chemotaxis toward chemokine ligand 3 (CCL3). A chromatin immunoprecipitation (ChIP) assay was performed to detect H3K9me2-CCR5 complexes in THP1- Mφs. Results PolyI:C significantly upregulated CCR5 in THP1-Mφs and promoted chemotaxis toward CCL3 (P<0.05); these effects were significantly inhibited by TLR3 siRNA (P<0.01). JMJD1A and JMJD1C expression was significantly upregulated in polyI:C-stimulated THP1-Mφs, while only JMJD1A siRNA decreased CCR5 expression (P<0.05). JMJD1A siRNA significantly increased H3K9me2 expression in THP1-Mφs but not in polyI:C-stimulated THP1-Mφs. The ChIP result revealed that polyI:C significantly downregulated H3K9me2 in the promoter region of CCR5 in THP1- Mφs. Conclusion PolyI:C can enhance THP1-Mφ chemotaxis toward CCL3 regulated by TLR3/JMJD1A signalling and activate CCR5 expression by reducing H3K9me2 in the promoter region of CCR5.

344-349

Authors: Sara Ahmadi, Arfa Moshiri, Mojtaba Mojtahedzadeh, Fatemeh Ettehad Marvasti, Vida Kazemi, Seyed Davar Siadat

Objective The gastrointestinal tract (GI) is colonized by a complex microbial community of gut microbiota. Bacteroides spp. have significant roles in gut microbiota and they host interactions by various mechanisms, including outer membrane vesicle (OMVs) production. In the present study, we extracted and assessed Bacteroides fragilis (B. fragilis) and Bacteroides thetaiotaomicron (B. thetaiotaomicron) OMVs in order to evaluate their possible utility for in vivo studies. Materials And Methods In this experimental study, OMVs extraction was performed using multiple centrifugations and tris-ethylenediaminetetraacetic acid (EDTA)-sodium deoxycholate buffers. Morphology, diameter, protein content, profile, and lipopolysaccharide (LPS) concentrations of the OMVs were assessed by scanning electron microscopy (SEM), nanodrop, Bradford assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the Limulus Amoebocyte Lysate (LAL) test, respectively. Zeta potential (ζ-P) was also assessed. The viability effect of OMVs was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in Caco-2 cells. Results Spherical OMVs with diameters of 30-110 nm were produced. The OMVs had different protein profiles. The LPS concentrations of the B. fragilis and B. thetaiotaomicron OMVs were 1.80 and 1.68 EU/mL, respectively. ζ-P of the B. fragilis OMVs was -34.2 mV and, for B. thetaiotaomicron. it was -44.7 mV. The viability of Caco-2 cells treated with OMVs was more than 95%. Conclusion The endotoxin concentrations of the spherical OMVs from B. fragilis and B. thetaiotaomicron were within the safe limits. Both OMVs had suitable stability in sucrose solution and did not have any cytotoxic effects on human intestinal cells. Based on our results and previous studies, further molecular evaluations can be undertaken to design OMVs as possible agents that promote health properties.

358-366

Authors: Zeynab Mohammadi, Nasim Hayati Roodbari, Kazem Parivar, Mojdeh Salehnia

Objective: The aim of the present study was to evaluate the effects of lysophosphatidic acid (LPA) supplementation of human ovarian tissue culture media on tissue survival, follicular development and expression of apoptotic genes following xenotransplantation. Materials and Methods: In this experimental study, human ovarian tissue was collected from eight normal female to male transsexual individuals and cut into small fragments. These fragments were vitrified-warmed and cultured for 24 hours in the presence or absence of LPA, then xenografted into back muscles of γ-irradiated mice. Two weeks post-transplantation the morphology of the recovered tissues were evaluated by hematoxylin and eosin staining. The expression of genes related to apoptosis (BAX and BCL2) were analyzed by real time revers transcription polymerase chain reaction (RT-PCR) and detection of BAX protein was done by immunohistochemical staining. Results: The percent of normal and growing follicles were significantly increased in both grafted groups in comparison to the non-grafted groups, however, these rates were higher in the LPA-treated group than the non-treated group (P<0.05). There was a higher expression of the anti-apoptotic gene, BCL2, but a lower expression of the pro-apoptotic gene, BAX, and a significant lower BAX/ BCL2 ratio in the LPA-treated group in comparison with non-treated control group (P<0.05). No immunostaining positive cells for BAX were observed in the follicles and oocytes in both transplanted ovarian groups. Conclusion: Supplementation of human ovarian tissue culture medium with LPA improves follicular survival and development by promoting an anti-apoptotic balance in transcription of BCL2 and BAX genes.

375-385

Authors: Tiansheng Guo, Junming Huang, Xiaoyun Wan, Peixi Chen, Shaoshan Du, Qianping Chen

Objective Accumulating evidences indicate that long non-coding RNAs (lncRNAs) play key roles in cancer. This study aims to clarify role of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in non-small cell lung cancer (NSCLC) and uncover the underlying mechanisms. Materials And Methods In this experimental study, MALAT1 and miR-202 expression in tissues and cell lines were detected using quantitative real time polymerase chain reaction (qRT-PCR) assay. Cell transfection was conducted using Lipofectamine 3000. Cell proliferation was determined with CCK-8 assay. MMP2 and MMP9 expressions were measured with Western blot. Cell invasive ability was evaluated by Transwell assay. Starbase 2.0 tool was used to predict targets of MALAT1. Dual luciferase reporter assay, RNA-binding protein immunoprecipitation assay and RNA pull-down assay were conducted to confirm the potential direct interaction between MALAT1 and miR-202. Results MALAT1 was overexpressed in NSCLC samples and cell lines. High expression of MALAT1 was related to large tumor size (>3 cm), poor histological grade, advanced cancer and tumor metastasis in NSCLC. In vitro assays exhibited that knockdown of MALAT1 remarkably decreased A549 cell growth and invasion capacity, while overexpression of MALAT1 significantly enhanced NCI-H292 cell proliferation and invasion ability. Next, we verified that MALAT1 could act as a competing endogenous RNA (ceRNA) by sponging miR-202 in NSCLC and there is a negative correlation between MALAT1 and miR-202. Besides, overexpression of miR-202 inhibited cell proliferation and invasive ability in MALAT1-overexpressed cells. Conclusion This study demonstrated that lncRNA-MALAT1 gets involved in NSCLC progression by targeting miR- 202, indicating that MALAT1 may serve as a novel therapeutic target for NSCLC treatment.

394-400

Authors: Xiu Wang, Yi Han, Guodong Hu, Jianbo Guo, Hongyu Chen

Objective Endoplasmic reticulum (ER) stress causes an adaptive response initiated by protein kinase RNA-like ER kinase (PERK), Ire1 and ATF6. It has been reported that these upstream regulators induce microRNAs. The current study was designed to find a novel microRNA that mediates ER stress components and finally contributes to cell fate decision. Materials And Methods In this experimental study, miR-706 levels were checked under different conditions of ER stress induced by Thapsigargin, Tunicamycin or low glucose media. PERK and ATF4 were knocked-down by administration of lentivirus-mediated short hairpin RNA to explore the effect of ER stress related proteins on miR-706 expression. The effect of miR-706 on caspase activity and apoptosis inhibitor 1 (CAAP1) levels were examined by using mimic-miR-706. The role of CAAP1 in inhibiting cell death (measured by Annexin V staining) and contributing to patient overall survival (measured by Kaplan-Meier estimate) were further confirmed by anti- miR-706 and CAAP1 knock-down. Results We showed that Thapsigargin or Tunicamycin triggered ER stress leading to the induction of miR-706. miR-706 induction is dependent on PERK and its downstream regulator ATF4, as knocking-down of PERK and ATF4 suppressed miR-706 induction in response to ER stress. Knocking-down of miR-706 reduces cell death triggered by ER stress, indicating that miR-706 is pro-cell death microRNA. We further identified CAAP1 as a miR-706 target in regulating ER stress initiated cell death. Conclusion Collectively, our results pointed to an ER signaling network consisting of proteins, microRNA and novel target.