In vitro Cytotoxic Activity of Four Plants Used in Persian Traditional Medicine

Purpose: The aim of this study was to investigate in vitro cytotoxic activity of four methanolic crude plant extracts against panel cell lines. Methods: Methanolic extracts were tested for their possible antitumor activity and cytotoxicity using the 3-(4,5-dimetylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay on six cancer cell lines; non-Hodgkin’s B-cell lymphoma (Raji), human leukemic monocyte lymphoma (U937), human acute myelocytic leukemia (KG-1A), human breast carcinoma (MCF-7 cells), human Prostate Cancer (PC3) and mouse fibrosarcoma (WEHI-164) cell lines and one normal cell line; Human Umbilical Vein Endothelial Cells (HUVEC). Results: All species showed dose dependent inhibition of cell proliferation. IC50 values ranging from 25.66±1.2 to 205.11±1.3 μg/ml. The highest cytotoxic activity Chelidonium majus L> Ferulago Angulata DC> Echinophora platyloba DC> Salvia officinalis L, respectively. Conclusion: all extracts demonstrate promising cytotoxicity activity as a natural resource for future bio-guided fractionation and isolation of potential antitumor agents

Induction of CD14 Expression and Differentiation to Monocytes or Mature Macrophages in Promyelocytic Cell Lines: New Approach

Purpose: CD14, one of the main differentiation markers on the surface of myeloid lineage cells, acts as a key role in activation of LPS-induced monocytes. LPS (lipopolysaccharide) binds to LPS-binding protein in plasma and are delivered to the cell surface receptor CD14. In this study, Various stimuli [Dimethyl Sulfoxide (DMSO), active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and LPS], either alone or in combination, have been recognized that have an effect on the level of CD14 expression in the human HL-60 and U937 promonocytic cell lines and therefore induce their terminal differentiation into monocytes or mature macrophages. Methods: U937 and HL-60 cells were cultured in RPMI 1640 supplemented with 10% FBS. For each cell line, 1×106 cells were seeded for 72 hours with DMSO, 14 days with LPS and 18 days with 1, 25-D3 in each well plate; then ELISA method was used to study their responses to the factors by means of anti-CD14. Results: ELISA assay demonstrated that U937 and HL-60 cells were induced by both [1,25(OH)2D3] and DMSO to obtain characteristics of adherent cells and express CD14 protein; moreover, LPS at a low dose increased CD14 expression on surface of this cells. Conclusion: According to the our results, it is speculated that CD14 gene expression may be induced in human U937 and HL-60 cell lines by different factors including 1,25-D3, DMSO and LPS

New Approaches in Immunotherapy of Behçet Disease

Behçet Disease (BD) is an autoimmune disorder with recurrent ocular, vascular, central nervous system, articular, mucocutaneous, and gastrointestinal manifestations with unclear etiology and pathogenesis. The further characterization of inflammatory features of Behçet’s disease may eventually lead to development of better treatment options. Clinical and laboratory observations suggested an important role of IL-17, IL-21 and neutrophil-mediated process in the pathogenesis of BD. New therapeutic modalities target specific and nonspecific suppression of the immune system. The various non-specific immunosuppressive drugs, used either alone or in combinations, frequently fail to control inflammation or maintain remissions. Due to encouraging clinical results (i.e. Antigenic specification, prolonged survival with acceptable levels of toxicity); antibody-based drugs could be effective for the clinical management of Behçet’s disease

Echinophora platyloba DC (Apiaceae) crude extract induces apoptosis in human prostate adenocarcinoma cells (PC 3)

Background: Prostate cancer is the second leading malignancy worldwide and the second prominent cause of cancer-related deaths among men. Therefore, there is a serious necessity for finding advanced alternative therapeutic measures against this lethal malignancy. In this article, we report the cytotoxicity and the mechanism of cell death of the methanolic extract prepared from Echinophora platyloba DC plant against human prostate adenocarcinoma PC 3 cell line and Human Umbilical Vein Endothelial Cells HUVEC cell line. Methods: Cytotoxicity and viability of the methanolic extract were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and dye exclusion assay. Cell death enzyme-linked immunosorbent assay (ELISA) was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and DNA fragmentation gel electrophoresis. Results: E. platyloba could decrease cell viability in malignant cells in a dose- and time-dependent manner. The IC50 values against PC 3 were determined as 236.136 ± 12.4, 143.400 ± 7.2, and 69.383 ± 1.29 μg/ml after 24, 36, and 48 h, respectively, but there was no significant activity in HUVEC normal cell (IC50 > 800 μg/ml). Morphological characterizations and DNA laddering assay showed that the methanolic extract treated cells displayed marked apoptotic characteristics such as nuclear fragmentation, appearance of apoptotic bodies, and DNA laddering fragment. Increase in an early apoptotic population was observed in a dose-dependent manner. PC 3 cell death elicited by the extract was found to be apoptotic in nature based a clear indication of TUNEL assay and gel electrophoresis DNA fragmentation, which is a hallmark of apoptosis. Conclusions: In summary, the E. platyloba extract attenuated the human prostate adenocarcinoma cell proliferation in vitro possibly by inducing apoptosis. E. platyloba is likely to be valuable for the treatment of human prostate adenocarcinoma

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells

Inhibitory and Cytotoxic Activities of Salvia Officinalis L. Extract on Human Lymphoma and Leukemia Cells by Induction of Apoptosis

Purpose: Salvia officinalis L., also known as Maryam Goli, is one of the native plants used to Persian medicinal herbs. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized crude methanol extracts prepared from Salvia officinalis L., on a non-Hodgkin’s B-cell lymphoma (Raji) and human leukemic monocyte lymphoma (U937), Human acute myelocytic leukemia (KG-1A) and Human Umbilical Vein Endothelial (HUVEC) cell lines. Methods: The effect of methanolic extract on the inhibition of cell proliferation and cytotoxic activity was evaluated by Dye exclusion and Micro culture tetrazolium test (MTT) cytotoxicity assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determined whether the mechanism involves induction of apoptosis or necrosis. Results: The present results demonstrated that methanolic extract at 50 to 800 μg/ml dose and time-dependently suppressed the proliferation of KG-1A, U937 and Raji cells by more than 80% (p800 Ag/ml). Nucleosome productions in KG-1A, Raji and U937 cells were significantly increased respectively upon the treatment of Salvia officinalis L. extract. Conclusion: The Salvia officinalis L. extract was found dose and time-dependently inhibits the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway

Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines by Echinophora platyloba DC: In Vitro Analysis

Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared from Echinophora platyloba DC, on mouse fibrosarcoma cell line (WEHI-164). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 μg/mL) when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So the Echinophora platyloba DC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway

Induction of Apoptosis and Cytotoxic Activities of Iranian Orthodox Black Tea Extract (BTE) Using in vitro Models

Purpose: Plant-derivate therapeutic agents can perform cancer chemotherapeutic activity through triggering apoptotic cell death. Our aim was to investigate the cytotoxic effects, induction of apoptosis, and the mechanism of cell death of Iranian orthodox black tea extracts (BTEs) and hydro methanolic purified fractions (40, 60, 80 and 100%) in KB cells (oral squamous cell carcinoma). Methods: In order to analyze the cytotoxic activity of the BTEs, MTT (3-(4, 5- dimetylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide) and Trypan-blue assays were performed in oral squamous cell carcinoma (KB). Furthermore, the apoptosis inducing action of the extracts was determined by TUNEL, DNA fragmentation and cell death detection analysis. Results: Dichloromethane BTE and hydro methanol fractions (40 and 60%) extract showed no cytotoxic effects; however, hydro methanol crude and hydro methanol fractions of BTE (80 and 100%) significantly inhibited cell growth and viability in a dose and time dependent manner. In addition, Cell death assay, TUNEL, and DNA fragmentation indicated induction of apoptosis by hydro methanol 80 and 100% fractions of BTE in KB cells. Statistical significance was determined by analysis of variance (ANOVA), followed by Duncan test and p value ≤0.05 was considered significant. Conclusion: The results from the present study suggests that the hydro methanol crude and hydro methanol fractions of BTE (80 and 100%) are significant source of compounds with the anti proliferative and cytotoxic activities, and this may be useful for developing potential chemo preventive substances

Methanolic Fractions of Ornithogalum cuspidatum Induce Apoptosis in PC-3 Prostate Cancer Cell Line and WEHI-164 Fibrosarcoma Cancer Cell Line

Purpose: The present study, was aimed to assess the cytotoxic effects of Ornithogalum cuspidatum methanolic fractions on PC-3, prostate cancer cells and WEHI-164, Fibrosarcoma cells. Methods: Methanolic fractions of O. cuspidatum were prepared using solid phase extraction and the cells were treated with different concentrations for 12 and 24 hours. Cytotoxicity and cell viability were measured by MTT assay. ELISA was also employed to assess the histone-associated DNA fragments and the involvement of apoptotic mechanisms. Results: 10 and 20% fractions had not significant cytotoxic effects (p>0.05) but other fractions exerted growth inhibition on both cancer cell lines (p<0.05). After 24h of incubation with 40, 60, 80 and 100% fractions, the IC50 values were: 165, 85, 65 and 45μg/ml on PC-3 cells and 200, 96, 76 and 73μg/ml against WEHI-164 cell line, respectively. ELISA results also revealed that, both cell lines had undergone apoptosis. Conclusion: It is deduced that, 80% and 100% methanolic fractions had significant anti-proliferative and apoptotic impacts on PC-3 and WEHI-164 cells in vitro and could be considered for developing chemo-preventive substances