Inhibition Of Mitochondrial Glutaminase Activity Reverses Adriamycin Resistance In Breast Cancer

Objectives Chemotherapy is one of the most effective methods of the treatment of malignant tumors so far,but it isn't optimistic,because the tumor cells produce drug resistance in the treatment process,leading to tumor recurrence and metastasis.Adriamycin is one of the most representative antitumor drugs.Overcoming the ADR resistance of tumor will lead to a breakthrough in the treatment of multidrug resistance.Therefore,it is urgent to find a new agent to overcome Adriamycin resistance.Studies have shown that the metabolism of tumor cells is different from that of normal tissues and cells.So is it possible to inhibit the action of some metabolic pathway of tumor to reverse the Adriamycin resistance? In this study,we used MCF7/ADR as the basic model to study whether the inhibitor of key enzymes in the process of tumor metabolism could reverse the Adriamycin resistance,and to study the reversal mechanism preliminarily.Methods1.Intracellular ATP content was detected in MCF-7 and MCF-7/ADR after treated with different concentrations of ADR,and then calculated the IC50 value,respectively.2.A suitable concentration of cerulenin,Orlistat,compound 968 and EGCG were used to treat MCF-7/ADR combined with Adriamycin,respectively.3.MCF7 and MCF7/ADR cells were treated with different concentration of compound 968 for 48 h and then intracellular ATP content was detected.4.MCF7 and MCF7/ADR cells were treated with compound 968(10u M),or Adriamycin(5ug),or compound 968 combined with Adriamycin for 48 h.5.The fourth experiment was repeated for different time.6.MCF7/ADR cells were treated with Adriamycin(5ug)combined with increasing concentrations of compound 968 or the two exchanged for 48 h or untreated.7.MCF7 and MCF7/ADR cells were cultured in RPMI 1640 medium with glutamine or without for 5 days.The intracellular ATP content was detected every day.8.MCF7/ADR cells were treated with Adriamycin combined with compound 968, adding Glu,?-KG,Pyr,Oaa,Asp,Asn,respectively.ATP was detected after 48 h.9.MCF7/ADR cells were transfected with control si RNA or si RNA1 and si RNA2 targeting GAC.Detect the expression of GAC by western blot.MCF7/ADR cells and the above three kinds of cells were treated with Adriamycin(5ug)or untreated.Results1.The IC50 value of Adriamycin for MCF-7/ADR was about 300 times of MCF7.2.Compound 968 can reverse Adriamycin resistance in MCF-7/ADR cells.3.Compound 968 had inhibitory effects on the growth of MCF7 and MCF7/ADR cells,and the similar efficacy can be seen in the concentration range of 10-80 u M.4.Compound 968 could reverse Adriamycin resistance in MCF-7/ADR cells.Moreover,the combination of compound 968 and Adriamycin potently inhibited the growth of MCF7/ADR cells in both time and dose-dependent manner.However,there was no synergistic effect of the combination of the two drugs in MCF7 cells.5.The growth of both MCF7 and MCF7/ADR cells has dependency on glutamine.6.Adding Glu or Pyr to MCF7/ADR cells which were treated with compound 968 and Adriamycin can obtain rescue efficiency.7.Downregulation the level of GAC expression can reverse Adriamycin resistance.Conclusions The tumor cells have an abnormal metabolism of glutamine.In addition to providing nutrition and energy for the rapid proliferation of tumor cells,glutamine catabolism may also have other biological functions.The results suggest that the metabolic balance of glutamine is essential for the rapid growth of breast cancer cells.It was also found that compound 968 was able to reverse ADR resistance.Moreover,the combination of compound 968 and ADR potently inhibited the growth of MCF7/ADR cells in both time and dose-dependent manner.A preliminary study of this process suggests that reducing the activity of glutaminase reverses ADR resistance,and that glutamate and pyruvate can play a role in the rescue.