ABSTRACT
Aim To study the transport mechanism of bergenin passing through blood-brain barrier ( BBB ) . Methods MTT assay was used to investigate the tox-icity of bergenin on MDCK-MDRl cells. Molecular docking was used to predict the binding mode and effect ability of bergenin with P-gp. In vitro MDCK-MDRl cell monolayer model was used to analyze trans¬port characteristics of bergenin and the effect of con¬centration, time and verapamil (a P-gp inhibitor) on the transport of bergenin. Results Bergenin was non-toxic to MDCK-MDRl cells within the concentration of 5 to 40 jjunol • L . There was hydrogen-bond and hy-drophobic interaction between P-gp and bergenin, and P-gp-bergenin was more stable than P-gp-verapamil. The P value of bergenin transported from AP to BL (PappAP
ABSTRACT
Multidrug resistance (MDR) plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine (Nef) in adriamycin (ADM) resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein (P-gp),γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.
ABSTRACT
To investigate the possibility to enhance resistance of human bone marrow cells to anticancer agents by transfection of mdrl gene. We transferred mdrl gene into human bone marrow cells with plasmid pHaMDR1/A containing human mdrl cDNA by Lipofectin.Pgp,mdrl gene expression product was detected by both flow cytometry and immunohistochemistry.Also,Pgp function was tested by efflux study using rhodamine.The resistance of human bone marrow cells to anticancer agents was assayed by color forming unit culture after exposure to Vincristine,Daunomycin, and Vp16.The results showed that mdrl gene was successfully transferred into human bone marrow cells, and it expressed.Biological activity of Pgp was confirmed by efflux study using rhodamine.Transferred human bone marrow cells possessed resistance to anticancer agents.It suggested that transfection of mdrl gene can increase resistance of human bone marrow cells to anticancer agents.
ABSTRACT
PURPOSE: Multidrug resistance mediated by several drug resistant genes impedes the successful outcome of anti-cancer chemotherapy. In this study, we investigated the expressions of drug resistant genes encoding multidrug resistance (MDR1), multidrug resistance-associated protein (MRP), topoisomerase I (Topo I), topoisomerase II g (Topo II a) in narmal volunteers (n=12) in and patients with myeloid leukemia (n=34). Material and Method: We compared the levels of their transcripts in bone matrow mononuclear cells by semiquantitative RT-PCR. The amount of specific transcripts was represented as the optical density ratio of PCR product of target gene to that of B2- microglobulin (MG). Twenty patients of acute myelogenous leukemia (eight in remission state, twelve in refractory) and fourteen patients of chronic myelogenous leukemia (nine in chronic phase and five in blastic crisis) were examined. Twelve normal healthy persons were compared with leukemic patients. RESULTS: The expression levels of all resistant genes in normal volunteers were relatively high as those of AML patients. Regardless of the disease status including remission status of AML (complete remission versus refractory) and the phase of CML (chronic phase versus blastic phase), the expression levels of all resistant genes in patients with CML were significantly lower than in the patients with AML (p < 0.05). Of interest, the patients with refractary AML did not show any statistical difference in comparison with normal controls and even the patients with AML in complete remission. Among the four drug resistant genes, the optical density ratio of MDRl was significantly lower than that of any other genes (p<0.05). Using HL-60 cell line, we compared the changes of various resistant gene expressions before and after differentiation induced by dimethylsulfoxide. The expressions of resistant genes declined in paralle1 with granulocytic differentiation, suggesting that the induction of cell differentiation might make leukemic cells susceptible to chemotherapeutic agents. CONCLUSION: It is impossibble to explain the mechanism of drug resistance by comparing the level of drug resistant gene expression between nonnal subjects and patients with myeloid leukemias. Therefore, we suppose that longitudinal study of drug resistant gene expression is necessary to demonstrate the development of drug resistant during chemotherapy.
Subject(s)
Humans , Bone Marrow , Cell Differentiation , Dimethyl Sulfoxide , DNA Topoisomerases, Type I , DNA Topoisomerases, Type II , Drug Resistance , Drug Resistance, Multiple , Drug Therapy , Gene Expression , Healthy Volunteers , HL-60 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Leukemia, Myeloid , Leukemia, Myeloid, Acute , Multidrug Resistance-Associated Proteins , Polymerase Chain Reaction , VolunteersABSTRACT
Objective: To investigate the expression of MDR1 and GFP in the human hematopoietic cells mediated by adeno-associated virus. Methods: The GFP gene was transferred into the human hematopoietic cells by AAV vectors and created strong visible fluorescence by purely molecular biological means. Using adeno-associated virus vectors, we have transferred human mdr-1 gene into human hematopoietic cells and investigated the drug resistence of human hematopoietic cells modified with mdr-1 gene. PCR analysis confirmed that mdrl cDNA had been successfully transferred into the human hematopoietic cells. An assay of MTT proved that the human hematopoietic cells modified by mdrl gene had resistance to colchicine. Results: It was about 30% of the hematopoietic cells that expressed the green fluorescent proteins. The resistance of hematopoietic cells was increased parently when the cells were infected by the crude virus stocks. Conclusion: It is conducted that the AAV vector could successfully transfer the foreign gene into the human hematopoietic cells. The cells modified with mdrl gene have increased the resistance to drugs.
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Objective:To investigate the effect of RNA interference targeting mdrl gene with ADM on ovarian cancer resistant strain SKOV 3/ADM.Methods:The expression of mdrlmRNA after transfected pshRNA-mdrl was detected by RT-PCR;MTT test and clone forming test were applied to measure the inhibition combined with adriamycin on proliferation of SKOV 3/ADM strain.Results:These pshRNA-mdrls inhibited the expression of mdrlmRNA and proliferation of SKOV 3/ADM obviously when combined with adriamycin.Conclusion:The short hairpin RNA recombinant plasmid targeting mdrl gene combined with adriamycin inhibits the proliferation of SKOV 3/ADM and increases the sensitivity to chemotherapy.