The Impact Of Glycerin Kinase On Cisplatin-antagonizing Effect Of Genistein In Breast Cancer

As a broad-spectrum antitumor drug,Cisplatin(CIS)is widely used in the treatment of various malignant tumors including breast cancer.The recurring of Cisplatin resistance,which significantly interferes with the chemotherapy of cancer,is an urgent clinic problem to be sorted.Glycerol Kinase(GK)catalyzes phosphorylation of free glycerol and directly regulates the utilization of free glycerol in cells.Our previous study found that GK expression in breast cancer cells was significantly up-regulated during the process of dietary genistein antagonizing cisplatin chemotherapy.The purpose of this study was to clarify the role of GK in the antagonism of genistein against cisplatin,and to explore the potential effect of triglyceride metabolism on the sensitivity of breast cancer to chemotherapy drugs.Objective:1.The expression changes of key enzymes in lipid metabolism antagonized by genistein during cisplatin chemotherapy were investigated,the key targets and corresponding key signal pathways were screened,and the expression correlation between GK and the target sites of classical cisplatin resistance was analyzed.2.Investigated the expression level and activity of GK protein.3.Investigated the effect of GK on genistein antagonistic cisplatin effect in breast cancer cells.4.Investigated the effect of GK on the sensitivity of breast cancer cells to cisplatin chemotherapy.Methods:1.Breast cancer xenograft tumor tissues in our former animal experiment was analyzed via RNA-Seq,differentially expressed genes and main targeted signaling pathways were screened by bioinformatics analysis,including KEGG annotation and enrichment analysis.The differentially expressed key enzymes in lipid metabolism were examined associated with genistein’s cisplatin antagonizing activity,and double checked via q RT-PCR.Moreover,the expression correlation between GK and various classic targets in cisplatin resistance relevant pathway were also investigated.2.Breast cancer MCF-7 cells were assigned into blank control group,cisplatin treated group,genistein treated group and cisplatin+genistein co-treated group.The storage of lipid droplet were observed via Nile Red staining.The expression level and activity of GK protein were investigated by Elisa and glycerol utilization ability assay.3.GK specific inhibitor 1-thiolglycerol was used.Cells were divided into 5groups(Blank control group,cisplatin treated group,cisplatin-genistein co-treated group,GK inhibitor treated group,GK inhibitor-cisplatin-genistein tri-treated group.MTT assay to evaluate the effect of cisplatin chemotherapy,Nile Red staining was applied to observe the storage of lipid droplet.the protein level of PCNA,Cleaved Caspase3,p Akt/Akt and other important target proteins were detected by Western Blot.4.Lentivirus infection was used to establish GK overexpressed breast cancer cell model.Supplemented with GK specific inhibitor 1-thiolglycerol.Blank control group,cisplatin treated group,GK inhibitor treated group,GK inhibitor-cisplatin treated group were respectively set up for both GK OE breast cancer cells and its corresponding vehicle cells.Cell viability changes after treatment were detected by MTT assay,cell cycle distribution was analyzed by flow cytometry,lipid droplet storages were detected by Nile Red staining,glycerol metabolism was detected by glycerol content kit.Key targets,such as PCNA,Cleaved Caspase3,BAX,Bcl-2,p Akt/Akt and other proteins were detected by Western Blot.Reusults:1.RNA-seq analysis of breast cancer xenograft tumors tissues showed revealed significant differences in expression level of 13 key enzymes in glycerides metabolism.There is high correlation between GK and other key enzymes in lipid metabolism,and also and classical targets in cisplatin resistance signaling pathways(such as PI3K-Akt).2.Along with the genistein’s cisplatin antagonizing activity,intracellular GK expression was up-regulated,the utilization of free glycerol was significantly enhanced,and intracellular lipid droplet storage was reinforced.Once the GK specific inhibitor was applied,the intracellular lipid droplet storage induced by genistein was reduced,the activation of AKT pathway was blocked,and the antagonistic effect of genistein against cisplatin chemotherapy was weakened in both aspects of cell proliferation and apoptosis.3.The over expression of GK in breast cancer MCF-7 cells leaded to an enhanced the storage of intracellular lipid droplets,and a decrease in cellular sensitivity to cisplatin-induced proliferation inhibition and apoptosis induction.The application of GK specific inhibitors significantly subdued the effect of GK overexpression,enhanced the sensitivity of cells to cisplatin anti-tumor effects.Reusults:The present work was engaged in investigating the role of GK in genistein’s anti-cisplatin activity via RNA-seq analysis and a series of cellular experiments.It was proved that GK regulated the utilization of free glycerol,encouraged the storage of lipid droplets.Besides play an important role in the process of genistein’s interference with cisplatin chemotherapy,GK showed a very close relationship with the cisplatin chemotherapy tolerance in breast cancer cells.Due to its sensitivity to dietary induction,GK can be one of the important potential targets for dietary adjuvant tumor chemotherapy.