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   Table of Contents - Current issue
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November-December 2018
Volume 13 | Issue 6
Page Nos. 476-574

Online since Friday, November 30, 2018

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ORIGINAL ARTICLES  

Effect of freeze drying on stability, thermo-responsive characteristics, and in vivo wound healing of erythropoietin-loaded trimethyl chitosan/glycerophosphate hydrogel Highly accessed article p. 476
Vajihe Akbari, Mahboubeh Rezazadeh, Mohsen Minayian, Marjan Amirian, Azadeh Moghadas, Ardeshir Talebi
DOI:10.4103/1735-5362.245959  
Erythropoietin (EPO) was successfully incorporated into a bioadhesive thermosensitive hydrogel based on trimethyl chitosan (TMC)/β-glycerophosphate (GP) for prevention and treatment of oral mucositis in cancerous patients. The aim of the present study was to evaluate the effect of freeze drying on thermo-responsive property of the hydrogel and structural stability of the loaded protein. The freeze-dried EPO-loaded hydrogel were characterized using various methods. Gelation property by rheological analysis, EPO aggregation in formulations by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein secondary structure by far ultraviolet-circular dichroism (CD), and the antigenic activity of EPO with ELISA techniques. The healing effects of the freeze-dried formulation was also investigated in Sprague-Dawley rats with chemotherapy-induced mucositis and compared with freshly prepared mixture. Finally, the retention time of the gel in the oral cavity was assessed in healthy volunteers. SDS-PAGE, CD, and ELISA confirmed the stability of conformational structure of loaded and released EPO. Severity of mucositis was markedly reduced in animals treated with freeze-dried EPO hydrogel; whereas the group received normal saline did not show any significant healing. EPO salvia level was decreased rapidly following EPO solution compared to the gel application. Approximately, 40% of EPO was maintained on the buccal areas in patients receiving the hydrogel system after 30 min. Therefore, the TMC/GP could preserve EPO stability after freeze drying and has the potential in the treatment of oral mucositis and other oral or subcutaneous wounds.
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Chemopreventive and biological activities of Helicteres isora L. fruit extracts p. 484
Acharaporn Rattanamaneerusmee, Krit Thirapanmethee, Yasushi Nakamura, Bhanubong Bongcheewin, Mullika Traidej Chomnawang
DOI:10.4103/1735-5362.245960  
Helicteres isora L. (H. isora) has been used in traditional medicine in Asia. This study was aimed to determine biological activities of H. isora fruit extracts. Chemopreventive effect was examined by cell proliferation assay and differentiation-inducing effect. Anti-inflammatory activity of extracts was studied on the levels of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), production of prostaglandin E2 (PGE-2), and cyclooxygenas-2 (COX-2). Cell proliferation assay revealed that H. isora extracts and its major compound, rosmarinic acid, showed no cytotoxicity in THP-1 and RCM-1 cells. Methylthio acetic acid from Cucumis melo var.conomon used as a positive control and 80% ethanol extracts demonstrated significant cell differentiation induction. Hexane extract of H. isora could lower the levels of TNF-α, PGE-2, and NO in THP-1 cells with 51.61 ± 0.79%, 69.68 ± 0.017%, and 69.93 ± 9.41% inhibition, respectively. The highest inhibitory effect on COX-2 was obtained from dichloromethane extract. Dexamethasone inhibited the secretion of TNF-α with 95.82 ± 0.50% while celecoxib showed the inhibitory effect on COX-2 and PGE-2 with 100% and 99.86%, respectively. The ethanol extract showed the best antioxidant activity by DPPH and FRAP assays at IC50 of 5.43 ± 1.01 μg/mL and 22.83 ± 0.13 mmol FeSO4/g sample, respectively, while the positive control, trolox, showed the antioxidant activity with IC50 and FRAP values at 4.08 ± 0.85 μg/mL and 10.84 ± 0.04 mmol FeSO4/g sample, respectively. Taken together, H. isora possess chemopreventive and antioxidant activity. Further studies on in vivo activities of this plant are suggested.
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Cadmium induces progesterone receptor gene expression via activation of estrogen receptor in human ovarian cancer cells p. 493
Negar Ataei, Mahmoud Aghaei, Mojtaba Panjehpour
DOI:10.4103/1735-5362.245961  
Cadmium (Cd) as a metalloesterogen may have a role in development of ovarian cancer. One of the critical target genes of estrogens is progesterone receptors (PRs). There are controversial studies on association between Cd, PRs, and cell proliferation. This study investigates the effect of Cd on proliferation of ovarian cancer cell lines, PRA and PRB expression and their relationship. OVCAR3 and SKOV3 cells were treated with CdCl2 (1-100 nM) and cell proliferation was assayed using bromodeoxyuridine (BrdU) method. The mechanism underlying the proliferative effect of Cd mediated by PRs was examined using cell transfection with PR- small interfering RNA (siRNA) and western blot analysis. Our results showed the involvement of PRs in Cd induced proliferation of ovarian cancer cells. Progesterone receptors are involved in proliferative effect of Cd. Moreover, Cd modified the expression of PRA and PRB and induced ovarian cancer cell proliferation through the change of PRA/PRB ratio. In conclusion, there is a mechanistic association between Cd effects on ovarian cancer cell proliferation, estrogen receptors and PRs expression.
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Thymoquinone attenuates hepatotoxicity and oxidative damage caused by diazinon: an in vivo study p. 500
Amir Nili-Ahmadabadi, Parisa Alibolandi, Akram Ranjbar, Leila Mousavi, Hossein Nili-Ahmadabadi, Amir Larki-Harchegani, Davoud Ahmadimoghaddam, Navid Omidifar
DOI:10.4103/1735-5362.245962  
Thymoquinone (TQ) is the main active constituent of Nigella sativa seeds. The objective of this study was to explore the protective effects of TQ on diazinon (DZN)-induced liver toxicity in the mouse model. The animals were divided into five groups of 6 each and treated intraperitoneally. Group 1 received the vehicle, group 2 was given 16 mg/kg DZN, group 3 received 5 mg/kg TQ, and groups 4 and 5 were treated with 1.25 and 5 mg/kg of TQ as well as 16 mg/kg DZN, respectively. Finally, butyrylcholinesterase (BChE), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) serum activity as well as nitric oxide (NO), lipid peroxidation (LPO), total antioxidant capacity (TAC), total thiol molecule (TTM), and histopathological experiments were evaluated in the liver samples. Our findings showed that DZN caused a significant increase in ALT (P < 0.01), AST (P < 0.001), ALP (P < 0.001) serum levels, LPO (P < 0.001) and NO (P < 0.001), the depletion of the TAC (P < 0.05) and TTM (P < 0.001), and structural changes in the liver tissue. Following TQ administration, a significant improvement was observed in the oxidative stress biomarkers in the liver tissue. In addition, our biochemical findings were correlated well to the histopathological examinations. In conclusion, the data from this study indicate that the administration of TQ may prevent liver damage by preventing free radical formation in animals exposed to DZN.
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Exploring the interaction between epidermal growth factor receptor tyrosine kinase and some of the synthesized inhibitors using combination of in-silico and in-vitro cytotoxicity methods p. 509
Rezvan Rezaee Nasab, Mahboubeh Mansourian, Farshid Hassanzadeh, Mohsen Shahlaei
DOI:10.4103/1735-5362.245963  
Quinazoline derivatives are potent inhibitors of human epidermal growth factor receptor (EGFR) as anticancer agents. In this study, the cytotoxic effects of a new series of synthesized quinazoline derivatives were evaluated using MTT assay against MCF-7 and HT-29 cell lines. Using molecular docking, the binding modes of all compounds were analyzed at the binding site of EGFR. Based on the results, the compounds L1, L2, L4, L5, L6, L7, L10, L15, and L18 may be promising EGFR inhibitors based on docking score and hydrogen bonds. Consistent with the experimental data, Met769 is recognized as a key residue in the binding of potential inhibitors. According to the MTT cytotoxicity assays, Lipinski's rule of five (RO5), absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters, and docking studies, three compounds L4, L15, and L10 with IC50 values of 80, 60, and 1 μM against the MCF-7 were selected for further comparative assessments. The dynamics of free EGFR, and selected ligand-EGFR complexes were investigated using molecular dynamics (MD) simulation studies. The results indicated that the three compounds bound to EGFR active site in a stable manner during the simulation through the formation of new hydrogen bonds with Phe699, Leu694, Gly700, Lys721, Met769, Arg817, and Asp831 with the superiority of compound L15. These features can promote future drug candidate designing to produce better derivatives in the search for the anticancer agents.
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The potential effects of hemp seed/evening primrose oils on the mammalian target of rapamycin complex 1 and interferon-gamma genes expression in experimental autoimmune encephalomyelitis p. 523
Soheila Rezapour-Firouzi, Shahram Shahabi, Adel Mohammadzadeh, Ali Asgar Tehrani, Fatemeh Kheradmand, Ebrahim Mazloomi
DOI:10.4103/1735-5362.245964  
The mammalian target of rapamycin (mTOR) has a fundamental role in the metabolism, growth, and regulation of the immune system. The interferon gamma (IFN-γ)derived from T helper 1 (Th1) cells is a prominent pro-inflammatory cytokine in multiple sclerosis (MS) and its animal model, the experimental autoimmune encephalomyelitis (EAE). Due to the exclusive role of rapamycin (RAPA) in mTOR complex 1 (mTORC1) inhibition, essentially Th1 differentiation and IFN-γ production, we evaluated the potential therapeutic effects of hemp seed/evening primrose oils (HSO/EPO) in comparison with RAPA administration in EAE. To evaluate the therapeutic effects of EPO/HSO supplement in comparison with RAPA, EAE was induced using myelin oligodendrocyte glycoprotein (MOG) peptide and complete Freund's adjuvant in C57BL/6 mice. The weight, clinical score, and histological findings were evaluated. Total mRNA was extracted from local lymph nodes and qRT-PCR was used for the purpose of the genes expression level of regulatory associated protein of TORC1 (RAPTOR) and IFN-γ. Our results indicated that the relative expression of RAPTOR and IFN-γ genes were significantly reduced in HSO/EPO, RAPA, and RAPA + HSO/EPO treated groups in comparison with the untreated group. Interestingly, histological findings have shown that the HSO/EPO treated group remarkably regenerated the myelin sheath, but this did not occur in the case of RAPA or combined RAPA and HSO/EPO treated groups. Our findings suggeste that HSO/HPO can be used as a potent immunomodulator and as a good candidate for re-myelination and downregulation of immune response for treatment of MS.
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Anti-melanogenesis and anti-tyrosinase properties of Pistacia atlantica subsp. mutica extracts on B16F10 murine melanoma cells p. 533
Samira Eghbali-Feriz, Akram Taleghani, Hadi Al-Najjar, Seyed Ahmad Emami, Homa Rahimi, Javad Asili, Samira Hasanzadeh, Zahra Tayarani-Najaran
DOI:10.4103/1735-5362.245965  
Pistacia atlantica (P. atlantica) subsp. mutica has been used in traditional medicine and is famous for its medicinal properties. The aim of this study was to evaluate the effect of methanol (MeOH), n-hexane, dichloromethane (CH2Cl2), n-butanol (BuOH), ethyl acetate (EtOAc), water extracts and essential oil of P. atlantica subsp. mutica on melanin synthesis and oxidative stress in B16F10 melanoma cell line. The B16F10 cells viability after treatment with increasing concentrations of different extracts of the plant (0.2-200 μg/mL) was measured using resazurin. Essential oil composition was identified by gas-chromatography-mass spectrometry (GC-MS) analysis and inhibitory effect on synthesis of melanin, mushroom tyrosinase activity, cellular tyrosinase, and oxidative stress were evaluated by the colorimetric and fluorometric methods. The data showed extracts at concentrations 0.2-200 μg/mL, did not show significant toxicity on melanoma cells but concentrations of 200 μg/mL of essential oil had cytotoxic effect. Pistacia atlantica subsp. mutica could inhibit the mushroom tyrosinase activity. Also the amount of melanin in B16F10 cells declined. In addition, the ability of P. atlantica subsp. mutica extracts in decreasing the amount of reactive oxygen species in melanoma cells revealed remarkable antioxidant activity. In addition, all concentrations of essential oil had no significant effect in this study. The melanogenesis inhibitory and antioxidant effects of P. atlantica subsp. mutica on B16F10 cells may suggest the potential whitening activity of the plant for using in dermatological skin care products and for prevention of skin aging in cosmetic industry.
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Effects of thyroid-stimulating hormone on adhesion molecules and pro-inflammatory cytokines secretion in human umbilical vein endothelial cells p. 546
Attabak Toofani Milani, Mohammad Hassan Khadem-Ansari, Yousef Rasmi
DOI:10.4103/1735-5362.245966  
Atherosclerosis is a multifactorial disorder, which affects the arterial wall. It has been reported that, hypothyroidism and thyroid hormone deficiency are related to cardiovascular disorders. Also, endothelial dysfunction plays an essential role in the development of atherosclerosis. We aimed to evaluate the effects of thyroid-stimulating hormone (TSH) on pro-inflammatory tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), angiogenic vascular endothelial growth factor (VEGF) and leukocyte adhesion, intercellular adhesion molecule 1 (ICAM-1) and E-selectin in human umbilical vein endothelial cells (HUVECs). In this study, HUVEC cells were treated with 1 and 2 μM of TSH in different treatment times. The gene and protein expression of ICAM-1, VEGF, and E-selectin were performed by real-time polymerase chain reaction and western blotting, respectively. Likewise, TNF-α and IL-6 protein levels were determined by the ELISA method. VEGF, ICAM-1, and E-selectin as endothelial dysfunction markers and also, TNF-α and IL-6 as pro-inflammatory cytokines were detectable in HUVEC. Besides, the results of this study revealed that TSH treatment down-regulates TNF-α and IL-6. Evaluating the gene and protein expression data revealed the upregulation of ICAM-1, E-selectin, and VEGF in TSH treated cases in different periods of exposure. Considering the multiple actions of TSH, it could be concluded that TSH plays a controversial role in atherogenesis by anti-inflammatory effects and on the other side, angiogenesis and leukocyte adhesion induction which is related to vascular cell proliferation.
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Galectin-9 induces apoptosis in OVCAR-3 ovarian cancer cell through mitochondrial pathway p. 557
Seyyed Mehdi Jafari, Ali Nazri, Mahdi Shabani, Narges Zargar Balajam, Mahmoud Aghaei
DOI:10.4103/1735-5362.245967  
Galectin-9 (Gal-9), a member of animal lectins' family, is implicated in the induction of apoptosis in various cancer cells. Here, we evaluated the anti-tumor effect of Gal-9 in OVCAR-3 ovarian cancer cells. The effect of the Gal-9 on cell viability was evaluated using MTT assays. Apoptosis was assessed using Annexin-V staining. The assessment of mitochondrial membrane potential (ΔΨm) was performed using a JC-1 probe. The activity of caspase-3 and caspase-6 were evaluated with colorimetric assay. The production of reactive oxygen species (ROS) was applied by fluorescent probe. The expression levels of Bax and Bcl-2 were assessed using western blotting. The result showed that Gal-9 inhibits cell viability. Flow cytometry analysis showed that Gal-9 induces apoptosis in ovarian cancer cells. Moreover, Gal-9 decreased ΔΨm and increased the generation of ROS and caspase-3 and caspase-6 activities in ovarian cancer cells. Moreover, Gal-9 induced expression of Bax as well as inhibited expression of Bcl-2. In conclusion, our results indicated that Gal-9 induced apoptosis in ovarian cancer cells through mitochondrial pathway.
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Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies p. 566
Javad Salehinia, Hamid Mir Mohammad Sadeghi, Daryoush Abedi, Vajihe Akbari
DOI:10.4103/1735-5362.245968  
Single chain variable fragment antibodies (scFvs) have attracted many attentions due to their small size, faster bio-distribution and better penetration in to the target tissues, and ease of expression in Escherichia coli. Although, scFv expression in E. coli usually leads to formation of inclusion bodies (IBs). The aim of this research was to improve solubilizing and refolding conditions for IBs of scFv version of pertuzumab (anti-human epidermal growth factor receptor 2 (HER2) antibody). After protein overexpression in E. coli BL21 (DE3), bacterial cells were lysed and IBs were extracted via repeated washing and centrifugation. The effect of different types, concentrations, pHs, and additive of denaturing agents on IBs solubility were evaluated. More than 40 refolding additives were screened and combinations of 10 of the best additives were check out using Plackett-Burman design to choose three refolding additives with the most positive effect on refolding of the scFv. Response surface methodology (RSM) was used to optimize the concentration of adopted additives. The most efficient buffer to solubilize IBs was a buffer containing 6 M urea with 6 mM beta mercaptoethanol, pH 11. The optimum concentration of three buffer additives for refolding of the scFv was 23 mM tricine, 0.55 mM arginine, and 14.3 mM imidazole. The bioactivity of the refolded scFv was confirmed by immunohistochemical staining of breast cancer tissue, a specific binding based method. The systematic optimization of refolding buffer developed in the present work will contribute to improve the refolding of other scFv fragments.
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