Reversal Roles And Its Underlying Mechanism Of Gambogenic Acid On P-glycoprotein-mediated Multidrug Resistance In HepG2/Adr Cells

Chemotherapy is the most common approach for the therapy of various cancers in clinics,including liver cancer.However,the overexpression of ATP-binding cassette?ABC?transporters is one of the major obstacles for cancer chemotherapy.Currently,multidrug resistance?MDR?caused by the overexpression of P-gp is one of a major resistance to successful chemotherapy in cancer cells.Gambogenic acid?GNA?,an active ingredient isolated from Gamboge,which possesses diverse antitumor effects in vivo and vitro.Recently,accumulating evidence has demonstrated that GNA has a potential role in the reversal of drug-resistant cells.Previous study of our term found that GNA exhibited the inhibition of the P-gp expression in HepG2 cells in a certain concentration range.Here we were mainly designed to understand the role of GNA in drug resistance in liver cancers cells.Objective:Human liver cancer HepG2/Adr cells were utilized as an in vitro model in the present study to investigate the reversal effect of GNA.Meanwhile,whether the mechanism of drug resistance was related to NF-kB and MAPK pathways were also detected.It could provide a new idea for preventing the occurrence of MDR in drug-resistant cells of liver cancer.Further,it will provide a certain reference value for further enriching the anti-cancer effect of the GNA in drug-resistant cells.Methods:The maximum non-toxic concentration of GNA was screened by MTT method for HepG2/Adr cells,and the drug concentration,at which more than 90%of cells were viable,was used in the reversal experiments.Then,GNA was co-incubated with conventional chemotherapeutic drugs DOX,PTX and CDDP for reversal studies.Finally,Annexin V-FITC was used to detect the percentage of apoptotic cells in HepG2/Adr cells after treatment with GNA.?2?The accumulation of the specific fluorescence substrates DOX and Rho-123 of P-gp in HepG2/Adr cells was determined by flow cytometry and fluorescence microscopy,respectively.ATPase activity assay was performed using the ATPase detection kit in the current study.?3?The effects of GNA on the expression of P-gp were detected by RT-qPCR and Western blotting methods in HepG2/Adr cells,respectively.The P-gp-related proteins NF-kB,ERK1/2,P-ERK1/2,P-p38,p38 were also determined.The aim of the study is to explore whether the NF-kB and MAPK pathways are involved in the reversal of HepG2/Adr cells after treatment with GNA.Results:?1?MTT results showed that GNA,at concentrations of up to 0.8?g/mL,was less toxicity to HepG2/Adr cells when incubated for 48 h,and the cell viability was more than 90%.The IC₅₀ values of the conventional chemotherapy drugs DOX and PTX were decreased in a dose-dependent manner in HepG2/Adr cells after treatment with GNA for 48 h,and the reversal fold were 3.67 and 3.42,respectively.Notably,the IC50 values and the reversal fold of CDDP had no significantly changed?P>0.05?.Moreover,0.8?g/mL GNA could increase the apoptotic rate of HepG2/Adr cells when combined with DOX.?2?Flow cytometry results indicated that GNA could increase the fluorescence intensity of DOX and Rho-123 in HepG2/Adr cells.Similarly,fluorescence microscopy showed that the fluorescence accumulation of DOX and Rho-123 were also increased with increasing concentration of GNA,while the fluorescence intensity of HepG2 cells did not significantly change?P>0.05?.ATPase activity experiments suggested that GNA had no effect on the level of ATPase in HepG2/Adr cells?P>0.05?.?3?RT-qPCR and Western-blotting results revealed that GNA could decrease the mRNA and protein expression of P-gp in HepG2/Adr cells at reverse concentration of 0.4?g/mL and 0.8?g/mL GNA compared with the control group.The expression of P-gp was downregulation in a dose-and time-dependent manner.Additionally,the protein expression of P-gp-related proteins NF-kB,ERK1/2,P-ERK1/2,P-p38,p38 was also performed by western blot method.And,a reduction for the levels of NF-kB and P-ERK1/2 could be found upon treatment with 0.8?g/mL GNA,while had no effect on the protein level of P-p38/p38?P>0.05?.Conclusion:GNA exhibited the reversal effect for HepG2/Adr cells within a certain concentration range,which may be achieved by affecting the function and expression of P-gp in HepG2/Adr cells.Furthermore,the down-regulation of P-gp expression could be contributed to reverse MDR upon a reversal concentration of 0.8?g/mL GNA.Mechanistically,the expression of P-gp was reduced by GNA may result from the inhibition of the NF-kB and MAPK pathway.Collectively,GNA could be a potential inhibitor to reverse P-gp-mediated MDR in liver cancer therapy.The results in this study still give theoretical basis for further exploration of the application of GNA in liver cancer drug-resistant cells.