The Reversal Effect And Mechanism Of Steroidal Derivative ASC26 On ABCB1-mediated Drug Resistance In Cancer Cells

As one of the fatal diseases which lead to the death of millions of people all around the world,cancer has threatened the health of human for centuries.Multidrug resistance(MDR),is a major cause of chemotherapeutic failures,in which ABC transporters play leading roles.ATP binding cassette subfamily B member 1,ABCB1,is the first identified ABC transporter.Studies have shown that the overexpression of ABCB1 plays a crucial role in MDR.Therefore,it is imperative to find novel compounds which could reverse ABCB1-mediated drug resistance in cancers.In this study,we aimed to discover a selective ABCB1 inhibitor which could significantly reverse ABCB1-mediated drug resistance with low toxicity or side-effects.We screened a series of novel steroidal derivatives,from which we discovered a selective ABCB1 inhibitor,ASC26,and further investigated the underlying mechanisms.Main research works involved in this study are listed here:(1)Based on the ABCB1 overexpressing SW620/Ad300 cells,we screened a series of novel steroidal derivatives using MTT assays.We have discovered a novel compound ASC26,which could significantly reverse drug resistance at 5 ?M,with an inhibition rate of less than 15%.(2)We then investigated the cytotoxicity of ASC26 alone in seven drug resistant cell lines.The results showed that ASC26 at the concentration of 8 ?M had no obvious cytotoxicity on these cell lines,with inhibition rates lower than 15%.(3)The concentration of 8 ?M was used in further mechanistic study.We then performed MTT assay to explore the reversal effect of ASC26 on drug resistant cell lines.The findings demonstrated that ASC26 at none-toxic concentration,could significantly increase the cytotoxicity of anticancer agent paclitaxel(a substrate of ABCB1)in ABCB1 overexpressing cell lines.However,ASC26 could not enhance the anticancer activity of cisplatin,which is not a substrate of ABCB1.Moreover,ASC26 could not reverse none ABCB1-mediated drug resistance,which indicates ASC26 is a selective ABCB1 inhibitor.(4)To explore the effect of ASC26 on the accumulation and efflux of paclitaxel in ABCB1 overexpressing cells,we performed HPLC assay.Compared with the control group,ASC26 could significantly increase the cellular accumulation and inhibit the efflux of paclitaxel in ABCB1 overexpressing cells.(5)We performed Rho123 accumulation assay using flow cytometry and found that ASC26 could significantly increase the cellular accumulation of Rho123 in ABCB1 overexpressing SW620/Ad300 cells.However,ASC26 had no obvious effect on the accumulation of Rho123 in parental SW620 cells.(6)To study the effect of ASC26 on the membrane expression and localization of ABCB1,we performed immunofluorescence assay.The findings demonstrated that ASC26 had no effect on the subcellular localization of ABCB1.(7)Western blot analysis showed ASC26 did not alter the protein expression levels of ABCB1 in SW620/Ad300 cells after treatment for 0,24 h,48 h and 72 h.(8)Docking study was performed to investigate the interaction between ACS26 and homology modeled human ABCB1.Our results showed that ASC26 could bind to the transmembrane domain(TMD)of ABCB1 and the hydrophobic interaction should be the main interaction between ASC26 and human ABCB1.In conclusion,we have discovered a novel steroidal derivative,ASC26,which at none toxic concentration could significantly reverse ABCB1-mediated drug resistance in cancer cells.Further mechanistic study indicated that the reversal effect of ASC26 was via inhibiting the efflux function of ABCB1.This study may provide to be a promising therapy for drug resistant cancer treatment.