Investigation On Mechanism Of ABCB1-and ABCG2-mediated Panobinostat Resistance

Objective: To study the mechanisms of ABCB1-and ABCG2-mediated panobinostat resistance.Research contents: Multidrug resistance in cancer(MDR)remains one of the biggest challenges in cancer treatment.MDR occur when cancer cells are resistant to structure unrelated anticancer drugs,which largely shorten the survival rate of cancer patients.In multiple mechanisms of MDR in cancer,overexpression of ATP-binding cassette(ABC)transporter in cancer cells act as a major factor responsible for MDR.Panobinostat is a second-line FDA-approved drug for treatment of multiple myeloma(MM).In our findings,we investigated that overexpression of ABCB1 or ABCG2 significantly decreased the efficacy of Panobinostat compared with parental cells,indicated that ABCB1 or ABCG2 may act as key factors in panobinostat resistance.In this study,we would study the potential mechanisms of action for ABCB1-and ABCG2-mediated Panobinostat resistance in cancer cells.Methods:(1)MTT assays were performed to detect the antiproliferation effect of Panobinostat in both ABCB1-overexpression cancer cells and parental cells.(2)MTT assays were performed to detect the antiproliferation effect of Panobinostat in both ABCG2-overexpression cancer cells and parental cells.(3)We also used MTT assays to evaluate the efficacy of Panobinostat in both ABCB1-gene-transfected HEK293 cells and its parental cells.(4)To study whether documented ABC transporter inhibitors could reverse Panobinostat resistance in ABC transporter overexpression cancer cells.(5)Western blot assays were conducted to evaluated the effects of Panobinostat on ABCB1 and ABCG2 expression level.Immunofluorescence were conducted to evaluate the impact of panobinostat on ABCB1 and ABCG2 subcellular localization.(6)ATPase assays were conducted to evaluate the effect of panobonostat on ABCB1 and ABCG2 ATPase activities.(7)In silico docking study were performed to find potential binding site of panobinostat on pocket of substrate binding sits of ABCB1 and ABCG2.Results: The anti-proliferation effect of panobinostat could be significantly weaken in the cells overexpression of ABCB1 or ABCG2.Meanwhile,documented ABC transporter reversal agents could re-sensitize the anti-proliferation effects of panobinostat in ABCB1-and ABCG2-overexpression cancer cells.In addition,panobinostat significantly up-regulated the expression level of both ABCB1 and ABCG2 transporters,without impact on the subcellular localization of ABCB1 or ABCG2.Moreover,panobinostat significantly enhance the accumulation level of substrate anticancer drugs,and simulated the ATPase activity of ABCB1 and ABCG2.Docking study predicted the potential binding sites of panobinostat with substrate pocket of ABCB1 and ABCG2.Conclusions: Panobinostat increases the expression level of ABCB1 and ABCG2,without alter the subcellular localization of ABCB1 or ABCG2 in MDR cells.Meanwhile,Panobinostat could stimulate the ATPase activity of both ABCB1 and ABCCG2,binding with substrate binding site,which make Panobinostat easier pumped out by ABCB1 and ABCG2 transporter,and finally cause MDR.Our study provides clues for panobinostat further application in clinic on drug combination strategies.