NBDHEX Re-sensitizes Adriamycin-resistant Breast Cancer Cell To Adriamycin By Inhibiting Glutathione S-transferase Pi Phosphorylation

Objective: To explore the effect of GSTpi phosphorylation on adriamycin resistance in breast cancer.Also,we studied whether the effect of NBDHEX on GSTpi phosphorylation could reverse adriamycin resistance in breast cancer.Materials and methods: 1、Cell viability of MCF-7 and MCF-7/ADR were detected by CCK-8 assay.The expression of GSTpi was determined by Western Blot and immunofluorescence anlysis.2、The expression of GSTpi was up-regulated by pc DNA3-Flag-GSTpi in MCF-7 and down-regulated by GSTpi-sh RNA in MCF-7/ADR.p Lenti CRISPRv2-GSTpi was used to construct GSTpi knockdown MCF-7/ADR cell lines.Western Blot and immunofluorescence anlysis were used to detect the expression of GSTpi.CCK-8 was used to determine the viability of cells.3、The enzyme activity test was used to detect the effect of NBDHEX on the activity of GSTpi.CCK-8 was used to detect the toxicity of NBDHEX on cells and the sensitivity of cells to adriamycin.Apoptosis rate was detected by flow cytometry,and apoptosis related proteins were detected by Western Blot.4、The phosphorylation sites of GSTpi in MCF-7/ADR were detected by mass spectrometry.Enzyme activity assay was used to detect the effect of phosphorylation on GSTpi activity.CCK-8 assay was used to detect the effect of GSTpi phosphorylation on adriamycin resistance in breast cancer cells.Western Blot was used to evaluate the effect of NBDHEX on GSTpi phosphorylation level.5.Western Blot and immunofluorescence were used to detect the effect of phosphorylation of GSTpi on nuclear translocation.Results: 1.Western Blot and immunofluorescence results showed that GSTpi was not expressed in MCF-7,but was expressed in MCF-7/ADR and enriched in the nucleus.2.Compared with the control group,IC50 increased in MCF-7 overexpressing GSTpi.Compared with the control group,GSTpi-sh RNA decreased the expression of GSTpi and IC50 in MCF-7/ADR.Three GSTpi knockdown MCF-7/ADR cell lines were screened,and their IC50 also decreased compared with the control group.3.The results of enzyme activity showed that NBDHEX could inhibit GSTpi activity.CCK-8 results showed that NBDHEX alone had no inhibitory effect on cell viability,but increased the sensitivity of cells to adriamycin.The results of flow cytometry and Western Blot showed that the co-treatment of NBDHEX and adriamycin could promote the apoptosis of MCF-7/ADR cells.4.Mass spectrometry analysis identified tyrosine-3、7、63、198 were phospho-acceptor residues in GSTpi protein in MCF-7/ADR.The results of enzyme activity and CCK-8 showed that phosphorylation of tyrosine-7 in GSTpi enhanced the activity of GSTpi,and could mediate the resistance of breast cancer cells to adriamycin.Western Blot results suggested that NBDHEX could inhibit the level of GSTpi tyrosine phosphorylation.5,Western Blot and immunofluorescence showed that GSTpi phosphorylation promoted GSTpi nuclear translocation.Conclusions:1.The expression level of GSTpi is positively correlated with the resistance of breast cancer cells to adriamycin.2.The phosphorylation of tyrosine-7 in GSTpi increases its activity and mediates drug resistance.In addition,GSTpi phosphorylation also promotes GSTpi nuclear translocation.3,NBDHEX can inhibit the activity of GSTpi by inhibiting its phosphorylation,thereby reversing the resistance of breast cancer cells to adriamycin.