Cell Journal (Yakhteh)

612-618

Authors: Shahram Taeb, Mohammad Amin Mosleh-Shirazi, Abbas Ghaderi, Seyed Mohammad Javad Mortazavi, Mahboobeh Razmkhah

Objective: The interaction of tumor cells with surrounding stem cells such as adipose-derived mesenchymal stem cells (ASCs) would be a crucial mechanism of tumor progression. It has been shown that irradiation can affect tumor microenvironment through different mechanisms. Given that, we aimed to examine the bystander radiation-induced effects of ASCs on different cancer cell lines. Materials and Methods: In this experimental study, ASCs were extracted from five healthy donors, cultured and then irradiated with a 5Gy of gamma radiation. Following 72 hours of incubation, irradiated ASCs-conditioned media (IACM) and non-irradiated ASCs-conditioned media (NIACM) were collected. Following incubation of different cell lines, Jurkat, LNCaP, U87-MG, MDA-MB-231 and MCF-7, in different media, DMEM, NIACM, and IACM, ALDEFLUOR assay and wound healing assays, were conducted. Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression of ATP-binding cassette transporter genes, ABCA1 and ABCG2, was measured in these cell lines. Results: NIACM significantly increased ALDH activity in MDA-MB-231 cell (P=0.02), while IACM was associated with significant decrease in the LNCaP and MCF-7 cell lines, respectively P=0.02, P=0.03, compared to DMEM as the control. The area of the scratch site was significantly reduced in MDA-MB-231 cells cultured with NIACM compared to DMEM (P=0.04). Furthermore, ABCA1 mRNA expression was considerably decreased in IACM- but not in DMEMtreated LNCaP line (P=0.01). Conclusion: It seems, after exposing to radiation, ASCs modify to prevent tumor development and metastasis through their radiation-induced bystander effects. Therefore, a better understanding of ASCs function in the tumor microenvironment may provide new insights into therapeutic strategies to surmount radio-resistance in cancer treatment.

626-632

Authors: Zahra Salehi, Masoumeh Beheshti, Bizhan Nomanpour, Pardis Khosravani, Maryam Naseri, Mohammad Ali Sahraian, Maryam Izad

Objective: Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) are believed to involve in multiple sclerosis (MS) pathogenesis. Natural killer (NK) and CD8+ T cells have essential roles in handling viral infections and their phenotypic and functional properties may be influenced following exposure to viral infections. Here, we investigated the association of NK and CD8+ T cells subpopulations frequency with EBV and HHV-6 viral load in MS patients. Materials and Methods: In this case-control study, EBV and HHV-6 viral load were evaluated in plasma of newly diagnosed relapsing-remitting MS (RRMS) patients at relapse phase (n=23), who were not on disease-modifying therapy (DMT), and sex- and age-matched healthy controls (n=19) using real-time polymerase chain reaction (PCR). The frequency of NK and CD8+ T cells subsets were assessed by CD27, CD28, CD45RO, CD56, and CD57 markers using flow cytometry. Results: Despite the increased level of EBV viral load in RRMS patients compared to the control group, there was no statistically significant difference in EBV and HHV-6 copy numbers between the studied groups. In addition, a significant decrease was observed in the percentages of CD56bright CD57- and CD56dim CD57+ CD8low CD45RO- NK cells in RRMS patients in comparison to healthy controls. Analysis of CD8+ T cell subsets showed a substantially high proportion of CD27+ CD28+ CD45RO+ CD57- CD8hi T cells in patients at relapse phase compared to controls. The frequency of NK and T cells subtypes was not associated with EBV and HHV6 plasma viral loads. Conclusion: These findings further highlight the variation of NK and CD8+ T cells subsets frequency in clinically active RRMS patients. Since the composition of cells was not associated with EBV and HHV-6 viral load, perhaps other viral infections may be involved in altered NK and CD8+ T cells subpopulation. Larger cohort studies are needed to confirm these results.

640-649

Authors: Xiaoguang Chen, Yumei Liu, Xuemin Zhu, Qiongxia Lv

Objective: Even a small fragment from the body of planarian can regenerate an entire animal, implying that the different fragments from this flatworm eventually reach the same solution. In this study, our aim was to reveal the differences and similarities in mechanisms between different regenerating fragments from this worm. Materials and Methods: In this experimental study, we profiled the dynamic proteome of regenerating head and tail to reveal the differences and similarities between different regenerating fragments using 2-DE combined with MALDITOF/ TOF MS. Results: Proteomic profiles of head and tail regeneration identified a total of 516 differential expressed proteins (DEPs) and showed a great difference in quantity and fold changes of proteome profiles between the two scenarios. Briefly, out of the 516 DEPs, 314 were identified to be specific for anterior regeneration, while 165 were specific for posterior regeneration. Bioinformatics analysis showed a wide discrepancy in biological activities between two regenerative processes; especially, differentiation & development and signal transduction in head regeneration were much more complex than that in tail regeneration. Protein functional analysis combined with protein-protein interaction (PPI) analysis showed a significant contribution of both Wnt and BMP signaling pathways to head regeneration not but tail regeneration. Additionally, several novel proteins showed completely opposite expression between head and tail regeneration. Conclusion: Proteomic profiles of head and tail regeneration identified a total of 516 differential expressed proteins(DEPs) and showed a great difference in quantity and fold changes of proteome profiles between the two scenarios. Briefly, out of the 516 DEPs, 314 were identified to be specific for anterior regeneration, while 165 were specific for posterior regeneration. Bioinformatics analysis showed a wide discrepancy in biological activities between two regenerative processes; especially, differentiation & development and signal transduction in head regeneration were much more complex than that in tail regeneration. Protein functional analysis combined with protein-protein interaction(PPI) analysis showed a significant contribution of both Wnt and BMP signaling pathways to head regeneration not but tail regeneration. Additionally, several novel proteins showed completely opposite expression between head and tail regeneration.

658-664

Authors: Leila Rezakhani, Morteza Alizadeh, Esmaeel Sharifi, Mostafa Soleimannejad, Akram Alizadeh

Objective: The use of animal or plant exosomes in cancer treatment is promising because of their easy access and low cost. Freshwater crabs are used in traditional Iranian medicine to treat cancer. This study aims to determine the anti-cancer properties of exosomes removed from freshwater crabs on a breast cancer cell line (4T1) compared to bone marrow mesenchymal stem cells (BMSCs). Materials and Methods: In this experimental study, crab haemolymph exosomes were isolated via the precipitation method and characterised by electron microscopy, dynamic light scattering (DLS), and Western blot analysis. The protein concentration and total antioxidant capacity of these exosomes were determined by bicinchoninic acid (BCA) and cupric reducing antioxidant capacity (CUPRAC). The 4T1 cells and BMSCs were treated with exosomes and we assessed the cell survival by the resazurin and MTT assays. The level of nitric oxide (NO) secretion from the 4T1 cells was determined after treatment with the exosomes. Results: Electron microscopy, DLS and Western blot for CD63 confirmed that the isolated exosomes were <100 nm in size and expressed CD63. The total antioxidant capacity in these exosomes was 1.003 μM/ml and the protein concentration was 650 mg/ml. Resazurin and MTT assay results showed a decrease in survival of the 4T1 cells (P≤0.001) after treatment with the exosomes compared to cell growth in the exosome-treated BMSCs. Conclusion: Crab haemolymph contains protein-rich exosomes with antioxidant activities that can have anti-cancer effects on 4T1 cells. These exosomes may be proposed for breast cancer therapeutics.

674-683

Authors: Somayeh Amiri, Azra Rabbani-Chadegani, Jamshid Davoodi, Hoda Gol Fakhrabadi

Objective: Alimta (Pemetrexed) as an antifolate drug has been approved for the treatment of lung cancer. The aim of the present study was to investigate the combination effect of 5-Azacytidine (5-aza) and Alimta on the miR-34a and its target genes expression and induction of apoptotic cell death in non-small lung cancer A549 cells. Materials and Methods: In this experimental study, lung cancer A549 cells were treated with various concentrations of Alimta alone and combined with 5-Aza. Then, viability was assessed by trypan blue and MTT assays. mRNA expressions were performed by real time-polymerase chain reaction (PCR) and western blot. Flow cytometry used to detect apoptotic/ necrotic cells and cell cycle arrest. Results: Alimta alone reduced viability of the cells in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 12 μM. Pretreatment of the cells with 5-aza (5 μM) induced a synergistic cytotoxic effect with IC50 of 3 μM. Sequential exposure of the cells to 5-aza and Alimta enhanced miR-34a expression and significantly downregulated HMGB1, HMGA2 and BCL-2 expressions. Also, it was associated with reduction of nuclear HMGB1 and HMGA2 content. Caspase-3 activation, HMGB1 release into extracellular space and staining of the cells with annexine V/PI suggested that 5-aza reduced late apoptotic/necrotic cell death induced by Alimta. In addition, combination of 5-aza and Alimta arrested the cells at S and sub-G1 phases and inhibited colony formation. Conclusion: 5-aza synergistically enhances Alimta induced apoptotic cell death through HMG proteins regulation, MIR34A gene expression and intrinsic apoptosis mechanism, providing a promising combination therapy in clinical lung cancer therapy.

692-700

Authors: Sahar Esfandyari, Ashraf Aleyasin, Zahra Noroozi, Maryam Taheri, Mahshad Khodarahmian, Mojtaba Eslami, Zahra Rashidi, Fardin Amidi

Objective: Sulforaphane (SFN) is a natural free radical scavenger that can reduce oxidative stress (OS) through mediating nuclear factor (erythroid-derived 2)-like 2 (NF-E2-related factor 2 or NRF2)/antioxidant response element (ARE) signaling pathway and the downstream antioxidant enzymes. Here, we intended to study the role of SFN in OSinduced human granulosa cells (GCs) by investigating the intracellular levels of reactive oxygen species (ROS), cell death, and NRF2-ARE pathway. Materials and Methods: This experimental study was conducted on GCs of 12 healthy women who had normal menstrual cycles with no history of polycystic ovary syndrome (PCOS), endometriosis, menstrual disorders, hyperprolactinemia, or hormonal therapy. After isolation of GCs, the MTT assay was performed to explore GCs viability after treatment with SFN in the presence or absence of H2O2. Flow cytometry was utilized to determine the intracellular ROS production and the apoptosis rate. Evaluation of the mRNA and protein expression levels of NRF2 and phase II enzymes including superoxide dismutase (SOD) and catalase (CAT) was performed by quantitative real-time polymerase chain reaction (PCR) and western blotting. Finally, the data were analyzed by SPSS software using One-way ANOVA and the suitable post-hoc test. Significance level was considered as P<0.05. Results: Pretreatment of GCs with SFN attenuated intracellular ROS production and apoptosis rate in the H2O2-exposed cells. Moreover, SFN treatment increased the mRNA expression level of NRF2, SOD, and CAT. Higher expression of NRF2 and SOD was also observed at the protein level. Conclusion: Our study demonstrated that SFN protects human GCs against H2O2 induced-OS by reducing the intracellular ROS production and the following apoptosis through a mechanism by which NRF2 increases the antioxidant enzymes such as SOD and CAT. This result may have a potential application in assisted reproduction cycles by improving the quality of GCs and the embedded oocyte, especially in PCOS patients.

708-716

Authors: Zahra Samadian, Javad Tolouei Azar, Hamed Rezaei Aghdam, Asghar Tofighi, Mazdak Razi

Objective: The current research was designed to analyze the effect of moderate-intensity exercise training (MEXT), solely and simultaneous with insulin, on the network between oxidative stress and Hsp70 and Hsp90 chaperones after experimental type I diabetes (DM) induction in rats. Materials and Methods: In the experimental study, 36 mature Wistar rats were assigned into control and experimental type I DM-induced groups, and then the diabetic animals were categorized to sedentary type I DM-induced (SDM), exercise training-sole without DM (E), exercise training DM-induced (EDM), insulin-treated sedentary DM-induced (ISDM), and exercise training insulin-treated DM-induced (EIDM) groups. After 6 weeks, Johnson’s score was evaluated to analyze the spermatogenesis ratio. Results: The Hsp70 and Hsp90 expression levels, testicular total antioxidant capacity (TAC), protein peroxidation ratio, testicular DNA fragmentation ratio, and mRNA damage were investigated. The animals in EDM and EIDM groups (solely and simultaneously) represented a significant (P<0.05) improvement in Johnson’s score, spermatogenesis, and TAC ratios versus SDM animals. Moreover, the DM-induced DNA and mRNA damage and protein peroxidation ratio were significantly (P<0.05) recovered in EDM and ISDM groups, which was more remarkable in the EIDM group. The EDM and EIDM groups exhibited significant (P<0.05) increment in Hsp70 and Hsp90 expression levels versus the control and SEDT1 animals. However, the EIDM group exhibited no significant changes compared to the control animals. Conclusion: The EX could ameliorate the EDT1-induced detrimental impact by up-regulating Hsp70 and Hsp90 expressions. Meanwhile, it exerts potentially more effective impact, when it is considered simultaneously with insulin therapy.