The Reversal Of Multidrug Resistance By Targeting Nanoparticles Carrying Doxorubicin Combined With Trametinib

Background and objective:Multidrug resistance(multidrug resistance,MDR)refers to the cross-tolerance of tumor cells to chemotherapeutic drugs with different structures and targets.It has been reported that overexpression of P-glycoprotein(P-gp)on drug-resistant cell membrane is one of the important reasons for multidrug resistance in tumor.Nano-delivery system has made some achievements in reversing tumor drug resistance,but nanoparticles can reverse tumor reversal to a certain exten through endocytosis and targeting effect.At present,the effective method is to combine P-gp inhibitor to reverse tumor multi-drug resistance.Trametinib is a highly specific inhibitor of mitogen-activated extracellular signal-regulated kinase(ERK)for the treatment of non-resectable or metastatic melanoma.It has been reported that trametinib can reverse multidrug resistance through selective inhibition of P-gp combined with low-dose chemotherapy.At present,there are few reports on the reversal of multidrug resistance of tumor by targeting doxorubicin-loaded nanoparticles combined with trametinib.The effects of trametinib on the biological effects of nanoparticles and the mechanisms of combined effect in reversing multidrug resistance in are still not well studied.We guess that trametinib could enhance the toxicity of targeting doxorubicin-loaded nanoparticles to drug resistant cells by specifically inhibiting P-gp,and further explore the mechanism of combined therapy to reverse multidrug resistance in tumor.Result1.Two types of targeted doxorubicin-loaded nanoparticles were successfully synthesized by transmission electron microscopy(TEM),dynamic light scattering(DLS),1H-NMR,UV spectrophotometry and drug release in vitro.2.The results of Western blot showed that the expression of folate receptor(FR-1)in ovarian cancer cell line was significantly higher than that in colon cancer cell line and the highest expression of folate receptor(FR-1)in A2780 cell line.The expression of P-gp was the main drug resistance protein in resistant ovarian A2780/R cell line and resistant colon HCT-8/R cell line,and the expression of P-gp in A2780/R was higher than that of HCT-8/R.3.The relative survival rate of the four cell lines was free doxorubicin<single ligand doxorubicin-loaded nanoparticles<dual ligand doxorubicin-loaded nanoparticles.With the increasing of concentration,the cell viability of the two drug resistant cell lines remained basically unchanged.The results showed that two drug resistant cell lines were resistant to adriamycin and nanoparticles under these conditions.4.When trametinib was at 1 ?M and verapamil was at 5 ?M,the overall cell survival rate was more than 80%.The reverse concentrations of trametinib and verapamil were determined to be 1?M and 5 ?M.5.The reversal experiments showed that the dual ligand doxorubicin-loaded nanoparticles combined with trametinib had the best inhibitory effect(P<0.01),and the reverse effect mediated by trametinib was better than that by verapamil(P<0.01).6.Flow cytometry of cell uptake assay showed that dual ligand doxorubicin-loaded nanoparticles combined with trametinib had the best uptake effect on two drug-resistant cells,and the cellular uptake mediated by trametinib was better than that by verapamil(P<0.01).7.The laser confocal results showed that dual ligand doxorubicin-loaded nanoparticles combined with trametinib groups had the highest fluorescence intensity in the nucleus,and the cellular uptake mediated by trametinib was better than that by verapamil(P<0.01).8.The results of cellular uptake mechanism showed that the pathway of dual ligand doxorubicin-loaded nanoparticles was more affected by temperature and ?-cyclodextrin(P<0.01);When trametinib combined with two kinds of doxorubicin-loaded nanoparticles,the cellular uptake pathway of the two kinds of targeted doxorubicin-loaded nanoparticles is more dependent on clathrin;Trametinib can obviously change the endocytic pathway of two kinds of targeted doxorubicin-loaded nanoparticles.(P<0.01).9.Western blot analysis showed that the expression of P-gp was not significantly down-regulated by trametinib at 1 ?M for 48 h,but the expression of ERK and pERK was significantly down-regulated(P<0.01).10.The results of ATPase activity test showed that with the increasing of trametinib concentration,the activity of ATPase was obviously increased,and EC50 was 0.892 ?M,which indicated that trametinib was the substrate of P-gp.Conclusion1.The inhibitory effect of single ligand doxorubicin-loaded nanoparticles and dual ligand doxorubicin-loaded nanoparticles were stronger than that of free adriamycin,but it could not completely reverse the multidrug resistance,so it was necessary to overcome the multidrug resistance of tumor by combining with reversal agent.2.The combination of trametinib and two types of targeting doxorubicin-loaded nanoparticles could enhance the toxicity to drug-resistant tumor cells and increase the uptake of two types of targeted doxorubicin-loaded nanoparticles,change the distribution of two types of targeting doxorubicin-loaded nanoparticles in the cell lines to reverse multidrug resistance.3.The specific mechanism of two types of targeting doxorubicin-loaded nanoparticles combined with trametinib in reversing multidrug resistance may be that trametinib changes the endocytosis pathway of two types of targeting doxorubicin-loaded nanoparticles into drug-resistant tumor cells and inhibits the function of P-gp and down-regulate the expression of ERK and pERK.Then the nanoparticles were redistributed in the drug resistant cells,and the drug concentration in the drug resistant cells was increased to overcome multidrug resistance.