P-glycoprotein(P-gp)highly expressed in cancer cells can lead to multidrug resistance(MDR)and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment.I...P-glycoprotein(P-gp)highly expressed in cancer cells can lead to multidrug resistance(MDR)and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment.In this study,we established a label-free and detergent-free system combining surface plasmon resonance(SPR)biosensor with styrene maleic acid(SMA)polymer membrane proteins(MPs)stabilization technology to screen potential P-gp inhibitors.First,P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes(SMALPs).Following that,SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system,and the affinity between P-gp and small molecule ligand was determined.The methodological investigation proved that the screening system had good specificity and stability.Nine P-gp ligands were screened out from 50 natural products,and their affinity constants with P-gp were also determined.The in vitro cell verification experiments demonstrated that tetrandrine,fangchinoline,praeruptorin B,neobaicalein,and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin(Adr).Moreover,tetrandrine,praeruptorin B,and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp.This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system.SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp.The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.展开更多
基金supported by the National Natural Science Foundation of China(No.82173777,81872829,81673386,82174092)the Science and Technology Commission of Shanghai Municipality(21ZR1483000)。
文摘P-glycoprotein(P-gp)highly expressed in cancer cells can lead to multidrug resistance(MDR)and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment.In this study,we established a label-free and detergent-free system combining surface plasmon resonance(SPR)biosensor with styrene maleic acid(SMA)polymer membrane proteins(MPs)stabilization technology to screen potential P-gp inhibitors.First,P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes(SMALPs).Following that,SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system,and the affinity between P-gp and small molecule ligand was determined.The methodological investigation proved that the screening system had good specificity and stability.Nine P-gp ligands were screened out from 50 natural products,and their affinity constants with P-gp were also determined.The in vitro cell verification experiments demonstrated that tetrandrine,fangchinoline,praeruptorin B,neobaicalein,and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin(Adr).Moreover,tetrandrine,praeruptorin B,and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp.This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system.SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp.The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.
