Effects Of Antimicrobial Peptides Temporin-1CEa On Multidrug Resistance Of MCF-7/Adr Cells

Antimicrobial peptide temporin-1CEa is a cationic membrane-active peptide that was extracted from the Chinese forest frog skin secretions.Previous studies have found that most of cationic antimicrobial peptides with positive charge could interact with tumor cell membranes via electrostatic attraction,thereafter play anti-cancer activities without producing drug resistance.We previously reported that drug-resistant breast cancer MCF-7/Adr cells over-expressing multidrug resistance protein P-glycoprotein(P-gP)is sensitive to temporin-1CEa-induced cytotoxicity.In addition,temporin-1CEa can also reverse the resistance of MCF-7/Adr cells and enhance anticancer effect of doxorubicin when being combined with doxorubicin.In the present study,we mainly explored and discussed the mechanisms of resistance-reversal effect of temporin-1CEa against MCF-7/Adr cells.Firstly,we evaluated the influence of temporin-1CEa of MCF-7/Adr cells membrane.The results of morphological observation via laser confocal microscopy and cell membrane potential assay showed that the cell membrane potential and permeability showed no significant changes when incubated with temporin-1CEa of less than 15μM,however,after exposure to temporin-1CEa of higher concentrations(15μM and 20μM),MCF-7/Adr cells began to depolarize together with an increased membrane permeability,as evidenced by the increased nucleus PI fluorescence.In our previous study,10μM temporin-1CEa had significant resistance reversal activity,which suggested that the mechanisms of temporin-1CEa-induced reversal of drug resistance might be non-correlated with membrane permeability but through other molecular mechanism.Therefore,we further evaluated the impacts of temporin-1CEa on efflux function of P-gP on MCF-7/Adr cell membrane;The data of P-gP substrate extrude experiments showed that temporin-1CEa(5-15μM)exposure increased the accumulation of Rho123 and adriamycin in MCF-7/Adr cells;In addition,results of P-gP ATPase activity assayindicated that temporin-1CEa at 5-15μM can stimulate the P-gP ATPase activity;Therefore,temporin-1CEa could inhibit P-gP-mediated efflux function by stimulating P-gP ATPase activity.Finally,we investigated the potential impacts of temporin-1CEa on MCF-7/Adr cells P-gP gene and protein expression levels in different concentrations of Temporin-1CEa and at different times by using real-time quantitative PCR and Western blot assay.The results showed that 1 hour of incubation of temporin-1CEa at 5-15μM can not significantly inhibit the expression of P-gP gene and protein.However,15μM temporin-1CEa can significantly inhibit the expression of P-gP gene and protein with the extended response time.In summary,these results suggested that temporin-1CEa reduces risks of drug resistance via mainly impacting on P-gP efflux function at low concentrations and early phase,and entering cancer cells and affecting the expression of P-gP gene and protein at medium concentrations and late phase.