| In order to explore new drugs to reverse the multidrug resistance of cancerchemotherapy from clinical existing drugs, the reversal effect against multidrug resistance (MDR) in Doxorubicin(Dox)-resistant erythroleukemic cell line K562/Dox and breast cancer cell line MCF-7/Dox by haloperidol (Hal) and rhTNF-NC and its mechanism were studied. The reversal effects of MDR by the ZYY-6 extract from Chinese herbs and new synthetic compound LY-980503 were also investigated.Using tetrazolium dye assay(MTT) and LDH assay, the cytotoxic effects of various concentrations of Dox at 0-20 nmol/L in K562/Dox and MCF-7/Dox cells were studied. Expression of MDR related genes MDR1, MDR associated protein(MRP),glutathion- S-transferase Pi(GST π) and TopoIIα of K562/Dox cells treated by12.5umol/L Hal or rhTNF-NC 500-5000 u/mlwere assayed by reverse transcriptase-polymerase chain reaction(RT-PCR). Using flow cytometry(FCM), intracellular Dox accumulation in K562/Dox cells treated by 0, 6.25, 12.5 and 25 umol/L Hal or rhTNF-NC and the effects of Hal on cell cycle progression were also observed. MQAE probe was loaded into K562/Dox cells and the cellular fluorescence intensity was determined by Hitachi F4500 fluorescence spectrophotometer for measuring the volume activated chloride channel activity. Intracellular Fura2/AM fluorescence (for monolayer MCF7/Dox cell) was determined for the cell volume change after hypotonicity challenge and the Coulter counter and channelyzer256 was used to determine the cell volume change for cell suspension K562/Dox cells. MCF-7/Dox cells was transfected with antisense PKCa and its effect on chloride channel and cell volume change were investigated.Part I: Experimental results showed that Hal significantly reversed MDR in K562/Dox cells after 12.5, 6.25 and 3.125umol/L Hal treatment, the chemosensitivity to Dox. increased by 9.06,4.33 and 2.19 times respectively. rhTNF-NC also could reverse the MDR by 2.51-5.33 fold at 500 and 5000u/mL respectively. When Hal 3.125umol/L was used in combination with rhTNF-NC 500u/mL the reversal effect was additive. The reversal effect was increased from 2.19 and 2.51 fold by Hal or rhTNF-NC alone to 5.01fold by combination use in k562/Dox and from 2.28 and 2.18 to 5.27 fold in MCF-7/Dox cells. Concentration-effect curve analysis showed that Hal and rhTNF-NC could shift the Dox concentration-effect curve upward in parallel (compared with those not treated by Hal or rhTNF-NC, P < 0.01).Part II: After treatment with Hal 12.5umol/L, MDRl and MRP mRNA expression were gradually downregulated in time-dependent manner in dl-d3. On the d3 MDRl and MRP reached the lowest level (76.29% and 64.63% of the control level, P<0.05) while GST π mRNA level decreased by 66.06% (P<0.05) in d1-d2, and began to recover on d3. Similar effect on MDRl,MRP and GST was also observed after rhTNF-NC treatment. rhTNF-NC also increased the TopoII gene expression When 6.25 umol/L Hal was used in combination with 1000 U/ml rhTNF-NC the inhibition effect on MDRl of two agents was additive, the inhibition rate increased from31.13 % and 33.17% treated by each agent alone to 62.03% by combination use. Similar inhibition effect on MDR1, MRP and GST could also be seen in K562/Dox cells treated by ZYY-6. FCM analysis showed that one hour after Hal 25,12.5 and 6.25 umol/L treatment, inrracellular Dox accumulation increased in a concentration-dependent manner. Hal at 6.25,12.5 and 25 umol/L could increase the Dox accumulation (expressed by fluorescence intensity) from 6.67±1.31 induced by 1.25 umol/L Dox alone to 10.01±2.05, 13.33±2.43, and 17.44±2.69 respectively induced by the combination use, and from 20.12±5.67 induced by 5 umol/L Dox alone to 23.87±5.05, 26.19±7.35, and 30.33±6.81 respectively by combination use. A similar increase of Dox accumulation could also be seen after rhTNF-NC treatment. rhTNF-NC at 1000 and 5000 U/ml could increase the apoptosis rate of k562/Dox cells induced by Dox at 5 umol/L from 3.78% to 4.85 and 6.89%. FCM data also showed that after 48 hou... |
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