Effect And Mechanism Of Targeting Glucose-6-phosphate Dehydrogenase To Reverse Adriamycin Resistance In Breast Cancer

Background and ObjectivesOne of the most serious problems in the treatment of breast cancer is the poor prognosis of radiotherapy and chemotherapy.Drug resistance is one of the key reasons for poor prognosis after chemotherapy.Adriamycin,also known as doxorubicin,is a common first-line chemotherapy drug in clinical treatment of breast cancer.But chemotherapy resistance often develops after a period of use,or the tumor being advanced.This poses a great challenge for inhibiting tumor progression and even curing it.Therefore,how to overcome the adriamycin resistance in breast cancer and improve the therapeutic effect has become an urgent problem to be solved.Glucose-6-phosphate dehydrogenase(G-6-PD)is an important oxidoreductase and a key enzyme in pentose phosphate pathway.Recent studies have shown that G-6-PD plays an important role in tumor resistance,but the mechanism is unclear.This study first verified that G-6-PD was one of the targets leading to Adr resistance in breast cancer,and then introduced miR-206 to inhibit the expression of G-6-PD and the activity of G-6-PD inhibitor 6-aminoniacinamide(6-AN)to observe the inhibitory effect on the growth of drug-resistant breast cancer cells(MCF-7/A cells).The combination of 6-AN and Adr to enhance the inhibitory effect of Adr on MCF-7/A cells was further observed,and the related molecular mechanism was discussed.Finally,in vivo experiments demonstrated the efficacy of 6-AN combined with Adr on the growth inhibition of drug-resistant breast cancer cells.MethodsIn this paper,breast cancer cells MCF-7 and Adr resistant breast cancer cells MCF-7/A were used in vitro.In vivo,the SPF-grade BALB/c-nu nude mice were inoculated with MCF7/A cells under the armpit to establish the transplanted tumor model.1.Verification of the effectiveness of G-6-PD as a target for reversing Adr resistance in breast cancerThe MTT method was used to detect the Adr resistance factor of MCF-7/A cells compared to sensitive MCF-7 cells.The Western blot method was used to detect the difference in G-6-PD expression levels between MCF-7/A cells and MCF-7 cells.The inhibitory effect of siG-6-PD transfection on G-6-PD protein expression was then detected by Western blot assay,and the change in sensitivity of MCF-7/A cells transfected with siG-6-PD to Adr was detected by MTT assay.2.Study on the enhancement of miR-206 on the proliferation inhibition of Adr on MCF7/A cellsMiRNA overexpression technology was used to upregulate miR-206 expression in MCF7/A cells.The efficiency of miR-206 overexpression was detected by fluorescent real-time quantitative PCR(qRT-PCR),and the difference in miR-206 expression between MCF-7/A cells and MCF-7 cells was detected.The effect of miR-206 alone on MCF-7/A cells was detected by MTT assay,and the difference in G-6-PD expression levels between transfected MCF-7/A cells and NC group was detected by Western blot method.The change in sensitivity of MCF-7/A cells transfected with miR-206 to Adr was detected by MTT assay.3.Detection of the reversal effect of 6-AN on adriamycin resistance in MCF-7/A cells when used in combinationThe MTT assay was used to investigate the inhibitory effect of 6-AN alone on proliferation of MCF-7/A and MCF-7 cells.The G-6-PD detection kit was used to detect the G-6-PD inhibitory effect of different concentrations of 6-AN on MCF-7/A cells.The MTT assay was used to evaluate the enhanced inhibitory effect of 6-AN in combination with Adr on MCF-7/A cell proliferation.The Hochest 33324 staining method was used to detect the effect of 6-AN in combination with Adr on apoptosis of MCF-7/A cells.The Western blot assay was used to investigate the effects of 6-AN in combination with Adr on apoptosis proteins Bcl-2 and Bax.Flow cytometry was used to evaluate the cell cycle distribution of MCF-7/A cells treated with PI-stained 6-AN and Adr in combination.The β-galactosidase staining method was used to detect the effect of 6-AN combined with Adr on the senescence of MCF-7/A cells.4.Study on the mechanism of 6-AN reversal of Adr resistance in MCF-7/A cellsFlow cytometry was used to detect ROS levels in MCF-7/A cells and MCF-7 cells.ROS levels of MCF-7/A cells were changed after siG-6-PD/miR-206 inhibited G-6-PD expression.The effects of different concentrations of 6-AN on ROS levels of MCF-7/A cells;The effect of 6-AN combined with Adr on ROS level of MCF-7/A cells;Immunofluorescence assay was used to detect the effect of 6-AN and Adr on DNA damage in MCF-7/A cells.The effects of 6-AN and Adr on AKT/mTOR pathway were detected by Western blot.5.Detection of the combination of 6-AN and Adr on the growth of MCF-7/A xenografts in nude miceThe MCF-7/A cell transplanted tumor model was established in nude mice,and the inhibitory effect of 6-AN and Adr on the growth of MCF-7/A cell transplanted tumor was detected.Western blot assay was used to determine whether the protein expression level in vivo was consistent with that in vitro.Experimental results1.The epigenetic difference between resistant MCF-7/A cells and sensitive MCF-7 cells showed that the adriamycin resistance fold of MCF-7/A cells was 206.59 compared with sensitive cells.G-6-PD expression in MCF-7/A cells was significantly higher than that in MCF7 cells.ROS levels were lower than those of MCF-7 cells without drug treatment.The adriamycin IC50 values of MCF-7/A cells transfected with 100 nM or 200 nM siG-6-PD at 117.00 μM and 51.88μM,decreased to 69.08 μM,39.07 μM,respectively.The resistance was reversed at 1.69 times and 1.32 times,respectively.2.The prediction results showed that miR-206 was a miRNA that could target the inhibition of G-6-PD expression.The miR-206 level of MCF-7 cells was significantly higher than that of MCF-7/A cells.miR-206 levels were significantly increased in MCF-7/A cells transfected with miR-206 mimics.In MCF-7/A cells,miR-206 was targeted to inhibit G-6-PD expression.The adriamycin IC50 values of MCF-7/A cells transfected with 100 nM or 200 nM siG-6-PD at 117.00 μM and 51.88 μM decreased to 52.17 μM,34.04 μM,respectively.The resistance ratio was reversed 2.24 times and 1.52 times,respectively.3.6-AN alone inhibited the proliferation of MCF-7 cells and MCF-7/A cells in a concentration-dependent and time-dependent manner.The activity level of G-6-PD was inhibited also in a concentration-dependent manner.After the combination of 6-AN and Adr in MCF-7/A cells,the Adr IC50 of MCF-7/A cells decreased from 75.33 μM to 12.98 μM and 9.75μM,respectively,when the combination of 5 μM and 10 μM 6-AN,and the reversal resistance times were 5.80 times and 7.72 times,respectively.6-AN promoted ADR-induced apoptosis of MCF-7/A cells,G2/M phase arrest and cell senescence.4.ROS level of MCF-7 cells without drug treatment was higher than that of MCF-7/A cells;The ROS level of MCF-7/A cells which were treated with siG6PD/miR-206 significantly increased.ROS levels of MCF-7/A cells were significantly increased after 6-AN treatment.The ROS level of MCF-7/A cells treated with 6-AN and Adr was higher than that of Adr alone,which was roughly the same as that of 6-AN alone.The combination of 6-AN and Adr promoted ADR-induced DNA damage and the inhibition of AKT/mTOR pathway in MCF-7/A cells.5.The combination of 6-AN and Adr significantly inhibited the growth of MCF-7/A transplanted tumor in nude mice.In addition,the AKT/mTOR pathway is inhibited in the tumor,and the expression of Bcl-2 is decreased and Bax is increased.ConclusionAdriamycin resistance in breast cancer can be reversed by inhibiting the expression or activity of G-6-PD.Treatment with the inhibitor 6-AN was more effective in reversing adriamycin resistance than miR-206 and siG6PD.6-AN combined with Adr can promote MCF7/A cell apoptosis,G2/M phase arrest and cell senescence,and play a synergistic inhibitory effect on the growth of MCF-7/A cells in vivo and in vitro.The action mechanism is related to 6-AN enhancing DNA damage by increasing ROS levels in MCF-7/A cells and enhancing the inhibition of AKT/mTOR pathway.