Cell Journal (Yakhteh)

127-134

Authors: Zahra Nouri, M.Sc.1, Fatemeh Karami, Ph.D.2, Nadia Neyazi, M.Sc.3, 4, Mohammad Hossein Modarressi, M.D., Ph.D.2, Roya Karimi, M.Sc.5, Mohammad Reza Khorramizadeh, Ph.D.1, Behrooz Taheri, M.Sc.1, Elahe Motevaseli, M.D., Ph.D.6

Objective: Lactobacilli are a group of probiotics with beneficial effects on prevention of cancer. However, there is scant data in relation with the impacts of probiotics in late-stage cancer progration, especially metastasis. The present original work was aimed to evaluate the anti-metastatic and anti-proliferative activity of lactobacillus rhamnosus supernatant (LRS) and lactobacillus crispatus supernatant (LCS) on the human cervical and colon adenocarcinoma cell lines (HeLa and HT-29, respectively). Materials and Methods: In this experimental study, the anti-proliferative activities of LRS and LCS were determined through MTT assay. MRC-5 was used as a normal cell line. Expression analysis of CASP3, MMP2, MMP9, TIMP1 and TIMP2 genes was performed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), following the cell synchronization. Results: Supernatants of these two lactobacilli had cytotoxic effect on HeLa, however LRS treatment was only effective on HT-29 cell line. In addition, LRS had no side-effect on normal cells. It was shown that CASP3 gene expression has been reduced after treatment with supernatants of two studied lactobacilli. According to our study, LRS and LCS are efficacious in the prevention of metastasis potency in HeLa cells with decreased expression of MMP2, MMP9 and increased expression of their inhibitors. In the case of HT-29 cells, only LRS showed this effect. Conclusion: Herein, we have demonstrated two probiotics which have anti-metastatic effects on malignant cells and they can be administrated to postpone late-stage of cancer disease. LRS and LCS are effective on HeLa cell lines while only the effect of LRS is significant on HT-29, through cytotoxic and anti-metastatic mechanisms. Further assessments are required to evaluate our results on the other cancer cell lines, in advance to use these probiotics in other extensive trial studies. Keywords: Probiotic, HT-29, HeLa, Metastasis, Gene Expression

149-158

Authors: Shubing Zhang, Ph.D.1, 2, Dewen Qiu, M.Sc.2, Jingjiang Liu, M.Sc.2, Zhijian Li, Ph.D.1*

Objective: Gastric cancer is a major health issue worldwide. Using a therapeutic approach, with minor side-effects, is very essential for the treatment of the gastric cancer. Shiraia bambusicola is a parasitic fungus which is widely used in China for curing several diseases with little side-effects. However, the mechanisms are not well understood yet. The aim of this study was to further understand the pharmacological mechanisms of Shiraia bambusicola and investigate whether it can be used for curing gastric cancer. Materials and Methods: In this experimental study, we mainly tested the effect of active components extracted from Shiraia bambusicola on BGC823, A549 and HepG2 cells. We used MTT assay to test cell viability. We also analyzed morphologic changes caused by apoptosis using Hoechst 33342 fluorescence staining, as well as cell cycle status and apoptosis ratio using flow-cytometer. In addition, protein expression level was tested by Western-blotting assay. Results: BGC-823 cell proliferation was specifically inhibited by active components of Shiraia bambusicola. Meanwhile, these active components could induce BGC-823 cells apoptosis and retard the cell cycle in S/G2 phase. We also determined that two critical protein markers cleaved Poly(ADP-ribose) polymerase-1 (PARP-1) and FLICE-inhibitory protein (FLIP), involved in apoptosis process, were regulated by these active components. Conclusion: These data shed light on the treatment of human gastric cancer and conclude that Shiraia bambusicola can be a good therapeutic candidate for treatment of this malignancy. Keywords: Shiraia Bambusicola, Apoptosis, PARP, FLIP, Gastric Cancer

165-178

Authors: Narges Obeidi, Ph.D.1, 2, Ali Akbar Pourfathollah, Ph.D.3, Masoud Soleimani, Ph.D.4, Mahin Nikougoftar Zarif, Ph.D.1*, Fatemeh Kouhkan, Ph.D.5

Objective: MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis. Materials and Methods: In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, β, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14th and 21th after transduction. Results: Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, β, γ, ε and ζ) genes in mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084- and 13.327-fold increse, respectively) (P<0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). Conclusion: Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines. Keywords: MicroRNAs, Mir-451, mESCs, Erythropoiesis, Globin Chains

189-196

Authors: Yousef Mortazavi, Ph.D.1, Fatemeh Sheikhsaran, M.Sc.1, Gholamreza Khamisipour, Ph.D.2*, Masoud Soleimani, Ph.D.3, Ali Teimuri, Ph.D.4, Somayeh Shokri, M.Sc.1

Objective: Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer’s disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs) as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF) and assessed their neural lineage genes. Materials and Methods: In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP) gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293) packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR). Results: We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF), glial fibrillary acidic protein (GFAP) and Microtubule-associated protein 2 (MAP2) genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF) genes reduced. Conclusion: Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this obstacle may be the utilization of differentiated neural stem cells. Keywords: Mesenchymal Stem Cells, Human Nerve Growth Factor, Neural Differentiation, ELISA

205-213

Authors: Behnaz Sadeghzadeh Oskouei, M.Sc.1, Maryam Pashaiasl, M.D., Ph.D.1, 2, Mohammad Hasan Heidari, Ph.D.3, Mohammad Salehi, Ph.D.4, Hadi Veladi, Ph.D.5, Firuz Ghaderi Pakdel, Ph.D.6, Parviz Shahabi, Ph.D.7, Marefat Ghaffari Novin, M.D., Ph.D.4*

Objective: In conventional assisted reproductive technology (ART), oocytes are cultured in static microdrops within Petri dishes that contain vast amounts of media. However, the in vivo environment is dynamic. This study assesses in vitro oocyte maturation through the use of a new microfluidic device. We evaluate oocyte fertilization to the blastocyct stage and their glutathione (GSH) contents in each experimental group. Materials and Methods: In this experimental study, we established a dynamic culture condition. Immature oocytes were harvested from ovaries of Naval Medical Research Institute (NMRI) mice. Oocytes were randomly placed in static (passive) and dynamic (active) in vitro maturation (IVM) culture medium for 24 hours. In vitro matured oocytes underwent fertilization, after which we placed the pronucleus (PN) stage embryos in microdrops and followed their developmental stages to blastocyst formation after 3 days. GSH content of the in vitro matured oocytes was assessed by monochlorobimane (MCB) staining. Results: We observed significantly higher percentages of mature metaphase II oocytes (MII) in the passive and active dynamic culture systems (DCS) compared to the static group (P<0.01). There were significantly less mean numbers of germinal vesicle (GV) and degenerated oocytes in the passive and active dynamic groups compared to the static group (P<0.01). Fertilization and blastocyst formation rate in the dynamic systems were statistically significant compared to the static cultures (P<0.01). There was significantly higher GSH content in dynamically matured oocytes compared to statically matured oocytes (P<0.01). Conclusion: Dynamic culture for in vitro oocyte maturation improves their developmental competency in comparison with static culture conditions. Keywords: In Vitro Oocyte Maturation, Microfluidics, Lab-On-A-Chip Device, In Vitro Fertilization, Glutathione

221-228

Authors: Maryam Peymani, M.Sc.1, Kamran Ghaedi, Ph.D.2, 3*, Shiva Irani, Ph.D.1, Mohammad Hossein Nasr-Esfahani, Ph.D.3*

Objective: Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the PPAR nuclear receptor superfamily. Although PPARγ acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPARγ in order to address its role in cardiac cell differentiation of mouse embryonic stem cells (mESCs). Materials and Methods: In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction (qPCR) was carried out to estimate level of gene expression. Furthermore, the requirement of PPARγ in cardiac differentiation of mESCs, during cardiac progenitor cells (CPCs) formation, was examined by applying the respective agonist and antagonist. Results: The obtained data revealed an elevation in the expression level of PPARγ during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPARγ inactivation via treatment with GW9662 (GW) reduced expression of CPC and cardiac markers. Conclusion: We conclude that PPARγ modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis. Keywords: Embryonic Stem Cell, PPARγ, Differentiation

237-244

Authors: Ali Esfandiary, M.Sc.1#, Zahra Taherian-Esfahani, M.Sc.1#, Atieh Abedin-Do, M.Sc.1, Reza Mirfakhraie, Ph.D.1, Mahdieh Shirzad, Ph.D.2, Soudeh Ghafouri-Fard, M.D., Ph.D.1*, Elahe Motevaseli, M.D., Ph.D.3*

Objective: Hypoxia-Inducible Factor (HIF)-1 plays an essential role in the body’s response to low oxygen concentrations and regulates expression of several genes implicated in homeostasis, vascularization, anaerobic metabolism as well as immunological responses. Increased levels of HIF-1α are associated with increased proliferation and more aggressive breast tumor development. Lactobacilli have been shown to exert anti-cancer effects on several malignancies including breast cancer. However, the exact mechanism of such effect is not clear yet. The aim of this study was to analyze the expression of selected genes from HIF pathway in a triple negative breast cancer cell line (expressing no estrogen and progesterone receptors as well as HER-2/Neu), MDA-MB-231, following treatment with two lactobacilli culture supernatants. Materials and Methods: In this experimental study, we analyzed the expression of HIF- 1α, SLC2A1, VHL, HSP90, XBP1 and SHARP1 genes from HIF pathway in MDA-MB-231 cells, before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants (LCS and LRS, respectively) by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results: Both LRS and LCS had cytotoxic effects on MDA-MB-231 cells, while the former type was more cytotoxic. LRS dramatically down-regulated expression levels of the HIF- 1α, HSP90 and SLC2A1 in the MDA-MB-231 cells. LCS had similar effect on the expression of HSP90, to what was observed in the LRS treatment. The expression level of tumor suppressor genes VHL and SHARP1 were also decreased in LCS treated cells. Conclusion: Although both LCS and LRS had cytotoxic effects on the MDA-MB-231 cells, it is proposed that LRS could be more appropriate for pathway directed treatment modalities, as it did not decrease expression of tumor suppressor genes involved in HIF pathway. Down-regulation of HIF pathway mediated oncogenes by LRS suggests that the cytotoxic effects of this lactobacillus may at least be partly caused by this mechanism. As previous studies have shown that inhibition of HIF-1α and HSP90 expressions have therapeutic impact on cancer treatment, the inhibitory effect of LRS on expression of these genes implies that this lactobacillus can be used in treatment strategies. Keywords: Breast Cancer, Lactobacillus, HIF

255-261

Authors: Maryam Soleimani Farsani, M.Sc.1, Mandana Behbahani, Ph.D.1*, Hamid Zarkesh Isfahani, Ph.D.2

Objective: The genus Thymus L. is a cushion plant that was previously used for the treatment of bronchitis and rheumatism. The present investigation was carried out to study the effects of root, shoot, leaf and seed extracts of five Thymus species and subspecies on peripheral blood mononuclear cells (PBMCs) toxicity and HIV-1 replication. Materials and Methods: In this experimental study, the activity of the Thymus extracts on HIV-1 replication and lymphocytes population were examined respectively using HIV-1 p24 Antigen kit and flow-cytometer. The Thymus species effect was investigated, including Thymus kotschyanus, Thymus vulgaris, Thymus carmanicus, Thymus daenensis subspecies lancifolius and Thymus daenensis subspecies daenensis. Results: The effect of root methanol extracts of all species on PBMCs proliferation was significantly higher than the other extracts. The intensity of CD4, CD3 and CD45 were decreased in the presence of all root extracts. Although the average median fluorescence intensity (MFI) values of CD19 were increased in the cells treated with these extracts. All methanol extracts showed anti-HIV-1 activity at high concentrations (200 and 500 μg/ml). Anti-HIV-1 activity of Thymus daenensis subspecies daenensis was significantly more than the other species. Conclusion: These results demonstrated that root extracts of Thymus species might be a good candidate to investigate anti-HIV infection in vivo. Keywords: Root, Flow-Cytometer, HIV-1, Shoot, Thymus

271-280

Authors: Massoud Seifi, D.D.S., M.S.1, Ali Lotfi, D.D.S., M.S.2, Mohammad Reza Badiee, D.D.S., M.S.3*, Zahra Abdolazimi, D.D.S.4, Parisa Amdjadi, D.D.S.5, Majid Bargrizan, D.D.S., M.S.4

Objective: Orthodontically induced inflammatory root resorption (OIIRR) is an undesirable sequel of tooth movement after sterile necrosis that takes place in periodontal ligament due to blockage of blood vessels following exertion of orthodontic force. This study sought to assess the effect of an angiogenic cytokine on OIIRR in rat model. Materials and Methods: In this experimental animal study, 50 rats were randomly divided into 5 groups of 10 each: E10, E100 and E1000 receiving an injection of 10, 100 and 1000 ng of basic fibroblast growth factor (bFGF), respectively, positive control group (CP) receiving an orthodontic appliance and injection of phosphate buffered saline (PBS) and the negative control group (CN) receiving only the anesthetic agent. A nickel titanium coil spring was placed between the first molar and the incisor on the right side of maxilla. Twenty-one days later, the rats were sacrificed. Histopathological sections were made to assess the number and area of resorption lacunae, number of blood vessels, osteoclasts and Howship’s lacunae. Data were statistically analyzed using ANOVA and Tukey’s honest significant difference (HSD) test. Results: Number of resorption lacunae and area of resorption lacunae in E1000 (0.97 ± 0.80 and 1. 27 ± 0.01×10-3, respectively) were significantly lower than in CP (4.17 ± 0.90 and 2.77 ± 0.01×10-3, respectively, P=0.000). Number of blood vessels, osteoclasts and Howship’s lacunae were significantly higher in E1000 compared to CP (P<0.05). Conclusion: Tooth movement as the outcome of bone remodeling is concomitant with the formation of sterile necrosis in the periodontal ligament following blocked blood supply. Thus, bFGF can significantly decrease the risk of root resorption by providing more oxygen and angiogenesis. Keywords: Root Resorption, Basic Fibroblast Growth Factor, Angiogenesis, Rat

288-288

Authors: Natasa Ristic, Darko Stevanovic, Dejan Nesic, Vladimir Ajdzanovic, Rastko Rakocevic, Ivana Jaric, Verica Milosevic

In this article which was published in Cell J, Vol 17, No 4, Jan-Mar (Winter) 2016, on pages 711-719, the name of the all authors were published incorrectly as: "Ristic Natasa, Stevanovic Darko, Nesic Dejan, Ajdzanovic Vladimir, Rakocevic Rastko, Jaric Ivana, Milosevic Verica". The correct one is "Natasa Ristic, Darko Stevanovic, Dejan Nesic, Vladimir Ajdzanovic, Rastko Rakocevic, Ivana Jaric, Verica Milosevic". This correction was requested by the corresponding author.