ABSTRACT
Background: Urtica dioica is one of the medicinal herbs with many uses in treating various diseases. In some studies, its antiproliferative and apoptotic effects on cancer cell lines have been shown. Therefore, the evaluation of U. dioica effect was performed on KG-1 cell line for acute myelogenous leukemia [AML] for the first time in this study
Materials and Methods: KG-1 cell line was treated by various extracts [aqueous, hydroalcoholic, chloroform and ethyl acetate] of U. dioica aerial parts and roots in different concentrations. Metabolic activity of extracts on cell line was assessed by MTT assay. To evaluate the percentage of apoptotic cells, the flow cytometry was performed by FITC Annexin V-PI apoptosis detection kit in KG-1 cell line treated with root chloroform [UDC-R] and ethyl acetate [UDE-R] extracts. The results have been reported as percentage of cell viability and IC50
Results: Based on MTT results, the strongest IC50 in KG-1 cell line [219.361microg/ml] was related to UDC-R. The flow cytometric analysis showed that UDC-R and UDE-R in IC50 concentration induced early [53.6% and 57.4%, respectively] and late [27% and 33.2%, respectively] apoptosis in KG-1 cells after 24 hrs. The inhibition of cell proliferation by various extracts of U. dioica was dependent on concentration [p=0.000]
Conclusion: Flow cytometric analysis confirmed that UDC-R and UDE-R extracts affect on proliferation reduction of KG-1 cells by activating the apoptotic pathway
ABSTRACT
Dendritic cells [DCs] are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs [siRNAs] and antisense oligodeoxynucleotides [ODN]s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs
Subject(s)
Down-Regulation , Oligodeoxyribonucleotides, Antisense , Cation Exchange Resins , Dendritic Cells , Genetic Diseases, Inborn/therapyABSTRACT
In order to determine the multidrug resistance [MDR] phenotype due to P-glycoprotein expression in haematological malignancies including acute myeloblastic leukemia [AML], a well-characterized P-gp expressing cell line was required to validate and standardize flow cytometric assays and to calibrate instruments. Therefore, this resistant subline of K562 was established for the first time in Iran in order to study the MDR phenotype due to P-gp expression in some cancers. A resistant subline of K562 [KDI/20] to Doxorubicin from the same parental K562 was derived by stepwise increasing the concentration of Doxorubicin up to 20 ng/ml as a gold standard. For flow cytometric assessment of P-gp expression, 4E3 anti-P-gp was used. The resistant cell line was studied by rhodamine 123 for functional assay of P-gp. MDR1 gene expression was also confirmed using RT-PCR. P-glycoprotein was expressed in final concentration of 20 ng/ml of Doxorubicin on 70% of K562 cells after 120 passages. The Rhodamine 123 influx was 37%. The over-expression of MDR1 gene was observed in a 30-cycle PCR. P-glycoprotein is expressed in human K562 cell line [K562] by continuous exposure to anticancer drug. P-glycoprotein expression is detected by several methods including flow cytometry and RT-PCR, and the number of PCR cycles is very important