Effect Of Cationic Antibacterial Peptide L-K6 On Doxorubicin Resistance In Breast Cancer MCF-7/Adr Cells

Chemotherapy is a common method for the treatment of cancer in clinic.However,due to the emergence of MDR（multidrug resistance）,the effect of drug treatment has been seriously limited,which leads to the fail of current clinical chemotherapy.Among many mechanisms that cause MDR,P-glycoprotein（P-glycoprotein,P-gP）is the most widely studied.P-gP can pump the drugs out of the cancer cell,thereafter prevent drugs being effectively delivered to the intracellular drug targets,and ultimately lead to the fail of inhibiting tumor cells proliferation.Cationic antimicrobial peptides（cAMPs）have attracted more and more attention because of their unique mechanisms of action and remarkable selectivity to tumor cells.L-K6 is a cAMP generated from natural antibacterial peptide Temporin-1CEb,according to the transformation of cationic amphiphilic and special amino acids structural features,L-K6 contains 13 amino acid residues and 7 positive charges,and presents amphipathic alpha helical structure.This present study mainly evaluated the anticancer effect and mechanisms of L-K6 against doxorubicin resistance human breast cancer cell line MCF-7（MCF-7/Adr）.MTT assay was performed to detect the antitumor activity of L-K6 against MCF-7/Adr.The results showed that L-K6 was less sensitive to MCF-7/Adr than MCF-7 cells(IC₅₀59.2μM of MCF-7/Adr vs.37.6μM of MCF-7).Moreover,adriamycin（doxorubicin,DOX）as a common clinical chemotherapy drug,showed significantly lower cytotoxicity（near 50 folds）to MCF-7/Adr(IC₅₀258.2μM)than the MCF-7(IC₅₀ 5.33μM).These results indicated that the antitumor activity of L-K6 and DOX alone could be affected by the mechanism of multidrug resistance.However,the combined usage of L-K6 and DOX showed a dramatic reversal of this resistence,as shown by a significantly increased antitumor activity of DOX with the presence of L-K6 of 10,30 and 50μM.Additionally,the fluorescence intensity of DOX was significantly enhanced in the cancer cell nucleus.Among various concentration,the combined usage of 30μM L-K6 and 60μM DOX showed a best synergistic effect with the the index as1.15,indicating that L-K6 and DOX can reduce the drug resistance of MCF-7/Adr cells and play an anti-tumor effect in a synergistic manner.Furthermore,the cell membrane permeability assay indicated that 1h L-K6 exposure had no obvious effect on cell membrane potential and permeability,while after 24h exposure with L-K6 of over 30μM,the MCF-7/Adr cell membrane permeability was significantly increased.In addition,30μM L-K6 can activate P-gP ATPase activity,and significantly increase the intracellular accumulation of DOX and Rhodamine,suggesting that L-K6 can reduce the efflux function of P-gP.At the same time,30μM L-K6 could significantly decrease the expression of P-gP.Taken together,our findings indicated that L-K6 and DOX could inhibit the proliferation of MCF-7/Adr cells in low concentration,and L-K6 may increase the membrane permeability to facilitate the influx of DOX into the target cells;at the same time,L-K6 can inhibit P-gP expression and block the efflux of DOX in MCF-7/Adr cells,further reverse the drug resistance.