Construction Of Screening Model For GLDC Expression Inhibitors And Screening Effective Constituent From Traditional Chinese Medicine

GLDC(glycine decarboxylase)is one of the four components of the mitochondria-located enzyme system for glycine cleavage.It is responsible for catalyzing the deamination and decarboxylation of glycine.Recent studies showed that GLDC possesses oncogenic function which is relevant to cell metabolism.GLDC is highly expressed in cancer stem cells(CSCs)and critical for CSCs survival and tumorigenesis.Overexpression of GLDC in normal cells can induce cellular transformation and tumorigenesis.In cancer patients,GLDC is aberrantly expressed in a variety of tumor tissues and closely related with the poor prognosis,which implies it as a new potential cancer therapeutic target for anti-tumor drug development.Therefore in this study,we tried to construct a screening system for screening GLDC expression inhibitors using GLDC promoter – luciferase system with which we screened GLDC expression modulators from Chinese medicine originated compounds.Then we further elucidated the possible mechanism for inhibiting GLDC expression of obtained natural compounds and investigated their anti-tumor effects.The main contents include:(1)Construction of GLDC promoter dependent luciferase reporter vector.GLDC promoter fragment spanning from-2186 to-113 upstream of transcription start site was amplified from the genome of 293 T cells by PCR and then inserted into the pGL3-Basic vector to obtain GLDC promoter dependent luciferase reporter vector,designated as pGL3-GLDCP.Reporter assay confirmed that the GLDC promoter had high transcriptional activity.(2)Natural compounds screening based on GLDC promoter-luciferase system.Through primary and secondary screening cycle,natural compounds,TI13 and TI102 were identified to be able to efficiently inhibit GLDC promoter activity from more than 300 compounds.Their inhibitory effect was further confirmed by detecting the mRNA and protein levels of GLDC in ovarian cancer cell line A2780.(3)Elucidating the molecular mechanisms for the inhibition of TI13 and TI102 on GLDC expression.By using a series of different transcription factor-responsive luciferase reporter vectors,we found that TI13 and TI102 can inhibit ERK/MAPK or PI3K/Akt responsive luciferase activities respectively.Subsequently,TI13 was shown to inhibit significantly the phosphorylation of ERK,while TI102 obviously inhibitedAKT phosphorylation.Furthermore,we demonstrated that ERK/MAPK and AKT pathways were involved in regulation GLDC transcription,which implied that TI13 and TI102 might downregulate GLDC expression by inhibiting ERK/MAPK or AKT pathway respectively.(4)Investigating the anti-tumor effects of TI13 and TI102.First,compound TI13 and TI102 were shown to obviously inhibit the cell viability of A2780 cells in MTT assay.Then they were demonstrated to significantly inhibit the anchor-independent colony formation of A2780 cells on soft agar.Flow cytometry showed that TI13 and TI102 could decrease the number of CD44,the marker of CSC,positive A2780 cells.Finally,TI13 and TI102 were found to reduce the lactic acid production of cancer cells,probably by inhibiting GLDC-mediated glycolysis.Taken together,we successfully constructed a luciferase-based drug screening system for screening GLDC expression inhibitors and two natural compounds TI13 and TI102 were identified to suppress GLDC expression through inhibiting ERK/MAPK and AKT pathways respectively and elicit their anti-tumor effects likely by targeting GLDC and CSCs.These studies provided a new idea and experimental evidence for anti-tumor drug research and development.