Effects Of Six Kinds Of 6,7-Furanocoumarins On The Expression And Activity Of P-glycoprotein

ObjectiveThe purpose of this study:(1)The effects of six furocoumarins on the proliferation of drug-resistant glioma cells U251/TR and non-resistant glioma U251 cells;(2)Six furanones Effects of the combina-tion of imatinoids and temozolomide on the proliferation of U251/TR cells;(3)The effect of six furocoumarins on the function of P-gp;(4)The investigation of six furocoumar com-pounds on the effect of P-gp expression.Therefore,furanocoumarins provide theoretical basis for reversing the resistance of tumor cells.Method1.Effect of anti-tumor activity of six furanocoumarinsA multi-functional microplate reader was used to detect the effect of six furocoumarin co-mpounds of different concentrations on the proliferation of drug resistant cells of U251/TR and non-drug resistant U251 cells by MTT assay;The effect of furocoumarins and tem-ozolomide alone or in combination on the proliferation of drug-resistant 251/TR cells was examined to determine the sensitization effect of furocoumarin compounds on chemothera-peutic drugs.2.Effects of six furanocoumarins on the function of P-glycoproteinTake the six kinds of furocoumarins and verapamil applied to human glioma U251cells and drug-resistant human glioma U251/TR cells respectively.After 24 hours,the drug action was stopped immediately,and 100?l of 10?mol/L rhodamine was added.Two hou-rs after the action of 123,washing with PBS 3 times,and then using a multifunctional mi-croplate reader at the excitation wavelength of 485nm,the fluorescence intensity was dete-cted at the emission wavelength of 535nm.The effect of six furanocoumarin compounds on P-gp function was tested by rhodamine 123 efflux assay to detect the residues of rhod-amine 123 in drug-resistant cells U251/TR and U251 cells.3.Effects of six furocoumarins on the expression of P-glycoproteinSix kinds of furocoumarins and verapamil were used alone to treat human glioma U251/TR cells that had been resistant to human gliomas for 48 hours.Immediately after the drug was stopped,high-performance lysates were added to extract proteins and BCA p-roteins were used.Quantitative methods quantify the protein and adjust the protein conce-ntration.The effect of six furanocoumarins on the expression of P-gp was detected by wes-tern blot.Results:The minimum of IC50 minimum value of six furancoumarins to human brain glioma U251 cells and U251/TR cells was also above 100.The complete medium with concentra-tions of temozolomide is 10~3?M,1.5×10~3?M,2.0×10~3?M,2.5×10~3?M and concentrations of furocoumarin is 25?M,50?M,75?M,and 100?M were administered alone and in co-mbination to effectively increase sensitivity of TMZ to U251/TR cells was a certain degree of concentration dependence(P<0.05).The combined effect index of six furocoumarins was 0.416-0.962(P<0.05),it have a synergistic effects.The combination of piroglitazone,imperatorin,isoimperatorin with fluoxetine had a stronger synergistic effect on U251/TR cells than xanthotoxin,xanthotol,and bergapten.In the accumulation experiment of six fur-ocoumarins exerted a certain inhibitory effect on rhodamine efflux 24 hours after acting on U25/TR cells;compared with the classic drug verapamil,imperatorin and isoimperatorin were 1.14 and 1.19 times that of verapamil.The remaining coumarins were slightly less ef-fective than verapamil(P<0.01).The six furocoumarins and the classical drug verapamil act on U251 cells,after 24 hours,the accumulation of rhodamine in the cells was not seen the difference was that the accumulation volume was significantly higher than that of U251/TR cells(P>0.05).After 48 hours of U251/TR cells treated with imperatorin and isoi-mperatorin,the protein extracted can detect imperatorin and isoimperatorin by western blot and can affect P-gp expression.The results showed that the concentration of P-gp could be decreased in each concentration of imperatorin and isoimperatorin,with the expression of P-gp decreased with the increase of concentration(P<0.05).Conclusions:1.The six furocoumarins could increase the sensitivity of temozolomide to drugres-istant glioma cells in different degrees.2.The six furocoumarins have a certain inhibitory effect on the activity of P-gp.Compared with the classic drug verapamil,imperatorin and isoimperatorin have better effects.3.The imperatorin and isoimperatorin inhibit the activity of P-gp by inhibiting the expression of P-gp,its activity is inhibited,thereby reversing the resistance of tumor cells.