GSTP1 Enhances Resistance Of Breast Cancer Cells To Adriamycin Through Promoting Autophagy

The resistance of cancer cells to chemotherapeutic drugs is the main cause of chemotherapy failure.Although the mechanism of drug resistance in tumor cells is very complicated,studies have shown that some key enzymes play an important role in this process,among which glutathione S-transferase PI is one of the most resistance-related enzymes of interest to researchers.GSTP1 is highly expressed in many resistant tumor cells,and the high expression of GSTP1 is closely related to the poor prognosis of cancer patients.Therefore,the study of GSTP1 has attracted much attention in the research field of tumor drug resistance.Since GSTP]catalyzes the electrophilic binding of reduced glutathione(GSH)to heterologous biomass,which in turn promotes cell detoxification,most studies have linked GSTP1’s transferase activity to its role in drug resistance.However,some chemotherapeutic agents(such as Adriamycin,ADR)are not GSTP1 enzyme substrates,and therefore cannot explain the mechanism of GSTP1 leading to drug resistance in some tumor cells using the GSTP1 transferase enzyme activity.In recent years,foreign and our laboratories have reported that GSTP1 can regulate cell signaling pathways,such as Mitogen-activated protein kinase(MAPK)signaling pathway,through non-transferase activity and play an important role in signal regulation of tumor cell apoptosis.These studies suggest that GSTP1 may lead to tumor cell resistance through other non-enzymatic pathways,but the mechanism by which GSTP1 promotes drug resistance in tumor cells has not been well understood.MCF-7 is a human breast cancer cell line.It has very low levels of GSTP1 in ADR-sensitive cells(MCF-7)and a high level of GSTP1 in ADR-resistant MCF-7 cells(MCF-7/ADR),the GSTP1 content in the two cells varies greatly.Based on the studies of MCF-7/ADR cells and MCF-7 cells we found a new mechanism of GSTP1 leading to drug resistance by promoting autophagy.Our experimental results are as follows.We overexpressed GSTP1 in MCF-7 and knocked down GSTP1 in MCF-7/ADR,verifying that high expression of GSTP1 could lead to MCF-7 cell resistance.By using GSTP1 mutant variant GSTP1(Y7F)and GSTP1 inhibitor 6-(7-Nitro-2,1,3-benzoxadiazol-4-thiohexanol)(NBDHEX),we proved that GSTP1 transferase activity did not play a leading role in MCF-7 resistant to ADR,and it also did not depend on the regulation of c-Jun N-terminal kinase(JNK).The process of drug resistance in tumor cells is usually accompanied by a high level of autophagy,and autophagy is considered to be a cell physiology approach to maintain cell homeostasis and is believed to promote tumor cell resistance.We first compared autophagy levels of MCF-7/ADR and MCF-7 cells.Electron microscopic observations,Western blots,and other experimental results showed that autophagy level of MCF-7/ADR was significantly increased under ADR stimulation and was higher than MCF-7 cells.Overexpression of GSTP1 in MCF-7 cells and RNA interference experiments in MCF-7/ADR demonstrated that GSTP1 could positively regulate the autophagy level of cells.Meanwhile,Western blotting results showed that the activity of GSTP1 transferase play a minor role in regulating autophagy.The use of autophagy inhibitors to inhibit GSTP1 enhanced autophagy can significantly reduce the resistance of MCF-7/ADR cells.The above results indicate that GSTP1 can promote cell resistance by regulating autophagy.By comparing the autophagy upstream pathways of MCF-7 and MCF-7/ADR cells stimulated by ADR,we found that the activation of PI3K-Akt-mTOR pathway in MCF-7/ADR cells was significantly lower than that of MCF-7 cells.The level of ERK activation was the same in the both cells,suggesting that GSTP1 may regulate autophagy through the PI3K-Akt-mTOR pathway.Further GSTP1 overexpression and RNA interference experiments in MCF-7/ADR and MCF-7 cells showed that under ADR stimulation,GSTP1 did regulate autophagy by inhibiting the PI3K-Akt-mTOR pathway.The results of co-immunoprecipitation and mass spectrometry showed that GSTP1 binds to the catalytic subunit p110a of PI3K but not to the regulatory subunit p85a of PI3K and downstream Akt and mTOR.GSTP1 binding to p110α inhibits the phosphorylation of p110α,which in turn blocks the activation of the PI3K-Akt-mTOR pathway and eventually upregulates autophagy levels.Molecular docking simulations showed that Pro 123,Leu 160,and GIu163 of the GSTP1 C-terminal alpha helix may be key amino acids for the formation of the GSTP1-p110α complex.We constructed GSTP1 plasmids with Pro 123,Leu 160 and Glu163 mutations.Co-immunoprecipitation experiments showed that the binding of GSTP1 variants with Pro 123,Leu 160,and Glu163 mutations to p110α was significantly reduced in MCF-7 cells and the GSTP1 variant does not enhance cell autophagy and cell resistance toADR.These results indicate that Pro 123,Leu 160,and Glu163,which located in the GSTP1 alpha-helix,are the key amino acids for GSTP1 binding to p110 of P13K.The bended structure of Pro123 with adjacent Gly124 and the GSTP1 substrate binding pocket with Leu 160 and Glu163 located in provide a conformational space for the binding of GSTP1 and p110α.The function of GSTP1 to promote cell resistance by regulating autophagy was also confirmed in another breast cancer cell line,MDA-MB-468.In summary,GSTP1 binds to the PI3K catalytic subunit p110α via its ligand binding activity at its C-terminal a-helical domain,inhibits the PI3K-Akt-mTOR signaling pathway,and subsequently promotes autophagy,leading to breast cancer cell resistance to ADR.Innovative outcome obtained in this study1.The first discovery of GSTP1 can promote breast cancer cell resistance to ADR by promoting autophagy,which is a new mechanism for GSTP1 to promote tumor tolerance to chemical drugs.2.The molecular mechanism of GSTP1 regulating autophagy by inhibiting the PI3K-Akt-mTOR signaling pathway by binding to the PI3K catalytic subunit p110α was elucidated.Namely:GSTP1 inhibits the activation of downstream signaling molecules through its C-terminal a-helical domain interacting with p110α,which further promotes autophagy of breast cancer cells by affecting the mTOR signaling pathway and promotes its tolerance to ADR.