| ObjectiveCisplatin resistance is the main cause of chemotherapy failure in ovarian cancer.Screening highly effective and low toxic Chinese herbal monomers from Chinese herbal medicines as reversal agents is a new anti-tumor strategy.In this study,we used ovarian cancer resistant cell line A2780/DDP as a model to study the reversal of drug resistance of ovarian cancer resistant cell line A2780/DDP by hesperetin in vitro and to explore the possible mechanism.Methods1.CCK8 assay was used to test the toxicity effect of cisplatin treatment on ovarian cancer cell line A2780/DDP and ovarian cancer cell line A2780 at various concentration gradients,and the IC50 and drug resistance index were calculated.2.CCK8 assay was used to test the toxicity of hesperetin at various concentration gradient to A2780/DDP cells and the reversal of cisplatin resistance.3.Differential expression of autophagy protein LC3 in ovarian cancer cell lines A2780/DDP and ovarian cancer cell lines A2780 detected by Western blot and immunofluorescence.4.Western blot was used to detect hesperetin on ovarian cancer resistant cells A2780/DDP autophagy-related proteins(LC3,Beclin1,P62)and AMPK/m TOR pathway proteins;MDC was used to detect autophagy activity in ovarian cancer resistance A2780/DDP cells.5.Western blot and q RT-PCR were used to detect the effect of hesperetin on the expression of apoptosis proteins(p53,Bcl-2,Bax and caspase3)associated with A2780/DDP cells;Hoechst 33342 fluorescence staining was used to detect the morphological changes of apoptosis of A2780/DDP cells induced by hesperetin.Results1.The IC50 of ovarian cancer cell line A2780 and ovarian cancer resistant cell line A2780/DDP treated with different concentrations(2.5μΜ,5μΜ,10μΜ,20μΜ,40μΜ,80μΜ)of DDP for 24 h were 11.36 and 62.73,respectively.Results show that the fold resistance of ovarian cancer resistant cell line A2780/DDP is 5.52,the IC50 of different concentrations of DDP after 48 hours of intervention is 10.10 and 50.85,respectively,and the resistance index is 4.42.2.Hesperetin inhibited the proliferation of A2780/DDP cells in a time-dependent manner,hesperetin at non-cytotoxic concentrations(less than 20% inhibition)of 50μΜ,100μΜ and 200μΜ was selected for subsequent reversal experiments,and A2780/DDP cells were treated with hesperetin at concentrations of 50μΜ,100μΜ and 200μΜ in combination with the various concentration of DDP for 48 h.The fold resistance reversal was 1.53,1.87 and 2.61,respectively.3.The results of Western blot and immunofluorescence indicated that the expression of autophagy-related protein LC3 was significantly higher in ovarian cancer resistant cells A2780/DDP than in ovarian cancer cells A2780.4.Western blot was used to detect that A2780/DDP cells treated with different concentrations(50μΜ,100μΜ and 200μΜ)of hesperetin for 48 h had decreased LC3 and Beclin1 protein expression and increased p62 protein expression,while it could down-regulate the expression of AMPK/m TOR signaling pathway proteins.The autophagic vacuoles of A2780/DDP cells were observed under a fluorescence microscope with MDC staining,and more MDC-positive fluorescent particles were observed and scattered in the blank control group,and A2780/DDP cells were treated with different concentrations of hesperetin for 48 h.Less MDC-positive fluorescent particles accumulated in the corresponding autophagic occurrence areas of the cytoplasm and the number of MDC-positive fluorescent granule cells gradually decreased with the increase of hesperetin concentration Western blot was used to detect that A2780/DDP cells treated with different concentrations(50μΜ,100Μμ and 200μΜ)of hesperetin for 48 h had decreased LC3 and Beclin1 protein expression and increased p62 protein expression,while it could down-regulate the expression of AMPK/m TOR signaling pathway proteins.The autophagic vacuoles of A2780/DDP cells were observed under a fluorescence microscope with MDC staining,and more MDC-positive fluorescent particles were observed and scattered in the blank control group,and A2780/DDP cells were treated with different concentrations of hesperetin for 48 h.Less MDC-positive fluorescent particles accumulated in the corresponding autophagic occurrence areas of the cytoplasm and the number of MDC-positive fluorescent granule cells gradually decreased with the increase of hesperetin concentration.5.Western blot results showed that different concentrations(50μΜ,100μΜ,200μΜ)of hesperetin dose-dependently increased p53,Bax and caspase3 protein expression,decreased Bcl-2 protein expression,and induced apoptosis in A2780/DDP cells.As the concentration of A2780/DDP cells treated with hesperetin increased,Bax and caspase3 m RNA expression increased and Bcl-2 m RNA expression decreased,and the difference was statistically significant(P<0.05).The results of Hoechst 33342 staining suggested that hesperetin induced apoptosis in A2780/DDP cells in a dose-dependent manner.Conclusion1.HST inhibited the proliferative activity of A2780/DDP cells and could reverse the resistance of A2780/DDP cells to DDP.2.HST acted on A2780/DDP cells to inhibit autophagy and induce apoptosis,which may be mediated by regulating key proteins in the AMPK/m TOR autophagy pathway as well as p53-dependent signaling pathways,thereby increasing cell sensitivity to cisplatin. |
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