| ObjectiveTo establish a ADM induced human glioma multidrug resistance cell line SHG44/ADM and study its biological characteristics.Methods1. Stepwise concentration increasing and intermittent administration method was used to construct SHG44/ADM.2. The resistance index to ADM was determined by CCK-8. Cell growth curve was painted and the doubling time was accounted. Cell colony formation assay was tested in soft agar growth.3. The mRNA of MDR1, MRP1 and LRP were detected by RT-PCR.4. The protein expression of MDR1, MRP1, LRP and COX-2, PKC ?were detected by western blotting.5. Flow cytometry was performed to determine cell cycles.6. Fluorescence activated cell analysis was employed to determine the concentration of fluorescence dye of rhodamine123 (Rho123) in the cells for the evaluation of drug-absorptive and expelling capacity.7. Flow cytometry and Fluorescent microscope were performed to determine cell apoptosis ratioResults1. The resistant index of SHG44/ADM was 10.7. Its resistant index (RI) was still maintained at -196℃. But without ADM in culture medium, the RI of SHG44/ADM showed a decrease.2. As compared with the parent cells SHG44/WT, SHG44/ADM exhibited a prolonged doubling time and lower growth rate.3. In soft agar growth test, SHG44/ADM cells formed more cell colonies than SHG44/WT cells.4. The percentages of cells in S and G0/G1 phase were significantly increased in SHG44/ADM in comparison with those in SHG44/WT.5. MDR1 mRNA and protein expression was significantly increased in SHG44/ADM in comparison with SHG44/WT, but MRP1 and LRP weren't changed.6. The concentration of Rho123 in SHG44/ADM was lower than that in SHG44/WT.7. After exposing cell for 48h to 10 g/m1ADM, the cell apoptosis rate of SHG44/ADM was decreased in comparison with SHG44/WT, but pretreatment with MDR1 inhibitor in SHG44/ADM, the cell apoptosis rate of SHG44/ADM was increased in comparison with without pretreatment with MDR1 inhibitor SHG44/ADM.ConclusionThe newly established stable cell line SHG44/ADM possesses multidrug resistant characteristics and its drug-resistant mechanisms can be attributed to MDR1 expression, drug expelling increasing and cell apoptosis decreasing. ObjectiveTo understand the molecular mechanism and effect of PKC and cyclooxygenase-2 on multidrug resistance of human glioma SHG44 cell line.Methods1. The IC50 and RI of SHG44 cell line was determined by CCK-8.2. The live cell ratio was measured with Trypan blue exclusion assays.3. The expression of PKC, COX-2 and MDR-1 were detected by western blotting.4. The production of PGE2 was assayed by enzyme immunoassay.5. The activity of PKC was assayed by PKC kinase assay.6. ADM accumulation was determined using fluorescence spectrometry.7. Flow cytometry was performed to determine cell apoptosis ratioResults1. Comparing with the SHG44/WT cells, in SHG44/ADM, the IC50 /RI and live cell ratio of SHG44/ADM were increased, the protein expression of PKC ?a nd COX-2 and MDR1 were up-regulated, the PKC and MDR1 activity and the production of PGE2 were increased, and the cell apoptosis rate induced by ADM was reduced. After SHG44/ADM cell treated with the PKC inhibitor Staurosporine or the specific COX-2 inhibitor NS398, the IC50/RI and live cell ratio were decreased, the protein expression of MDR1 was down-regulated, MDR1 activity and the production of PGE2 were decreased, and the cell apoptosis rate induced by ADM was enhanced. After treated with Staurosporine, COX-2 protein expression and activity were reduced. But after treated with NS398, PKC ?protein expression and PKC activity weren`t changed.2. After SHG44/WT cell treated with PMA, comparing with the SHG44/WT cells, the IC50/RI and live cell ratio were increased, the protein expression of PKC ?and COX-2 and MDR1 were up-regulated, the PKC and MDR1 activity and the production of PGE2 were increased, and the cell apoptosis rate induced by ADM was reduced. The effect of the PKC inhibitor Staurosporine on the PMA mediated MDR1 expression and activity was assessed, the protein expression of COX-2 and MDR1 were down-regulated, PKC and MDR1 activity and the production of PGE2 were decreased, and the cell apoptosis rate induced by ADM was enhanced. The effect of the specific COX-2 inhibitor NS398 on the PMA mediated MDR1 expression and activity was showed, the protein expression of MDR1 were down-regulated, MDR1 activity and the production of PGE2 were decreased, and the cell apoptosis rate induced by ADM was enhanced, but PKC ?protein expression and PKC activity weren`t changed.3. After SHG44/WT cell tranfected with the COX-2 expression plasmid, comparing with the SHG44/WT cells, the IC50/RI and live cell ratio were increased, the protein expression of COX-2 and MDR1 were up-regulated, the MDR1 activity and the production of PGE2 were increased, and the cell apoptosis rate induced by ADM was reduced, but PKC p?rotein expression and PKC activity weren`t changed. The effect of the PKC inhibitor Staurosporine on the COX-2 mediated MDR1 expression and activity was assessed, comparing with SHG44/WT cell tranfected with the COX-2 expression plasmid, no change was detected. The effect of the specific COX-2 inhibitor NS398 on the COX-2 mediated MDR1 expression and activity was showed, the protein expression of MDR1 were down-regulated, MDR1 activity and the production of PGE2 were decreased, and the cell apoptosis rate induced by ADM was enhanced.ConclusionActivation of PKC pathways represents an upstream event, which enforced expression of COX-2 and addition of products of COX-2 enzymatic activity results in upregulated of expression of MDR1. This prevents cellular accumulation of chemotherapeutic drugs and pumps out apoptotic compounds , thereby inhibiting cellular apoptosis and contributing to the development of multidrug resistance phenotype. |
PDF Full Text Request