Experimental Study Of Lactate Dehydrogenase A Promotes The Invasion And Proliferation Of Pituitary Adenoma

Pituitary adenoma(PA)which is mostly originated from the anterior lobe of the pituitary gland is a common intracranial neuroendocrine tumor(accounting for about 10-15% of all intracranial tumors).The clinical symptoms of PA patients are caused by tumor compression and tumor-induced abnormal hormone secretion.PA is benign tumor of monoclonal origin.Despite their histologically benign status,some tumors which are defined as invasive PA can invade surrounding tissues.Invasive PA have a tendency toward malignant biological behavior and have strong proliferation ability.And patients of invasive PA are poor prognosis for the tumor is difficult to be completely removed and easy to relapse after surgery.Therefore,it is very significant to study the molecular mechanism of PA invasion and to explore the new method of treating invasive PA.Tumor cells have higher level of glucose metabolism than normal cells and tend to produce energy by anaerobic glycolysis even in the presence of sufficient oxygen.This abnormal metabolic phenomenon of tumor cells is called "Warburg Effect".Lactate dehydrogenase A(LDHA)which are highly expressed in a variety of malignant tumors is one of the key enzymes of glycolysis.It can regulate proliferation,invasion and some other malignant biological behaviors of tumor cells.It has been reported that the glucose uptake capacity of PA is significantly higher than pituitary,and the capacity of pituitary macroadenoma is also significantly higher than pituitary microadenoma.These evidence strongly suggested that the proliferation and invasion of PA may be closely related to abnormal glucose metabolism.In order to explore the role and mechanism of LDHA in the development of invasive PA,we first detected LDHA gene and protein expression in PA clinical pathological specimen.And we analysed the correlation between invasion and proliferation of PA tumors and the expression of LDHA protein.In addition,we explored the effect and corresponding mechanisms of LDHA in PA cell line and confirmed the role of LDHA in PA nude mice model.We investigated the effect of oxamate which is a specific chemical inhibitor of LDHA in PA cell line,animal model and primary cultured PA cells to explore the treatment effect to invasive pituitary adenoma.Finally,we found metformin which is an anti-diabetic drug commonly used in clinical significantly down-regulated LDHA gene expression in the PA cell line GH3.Therefore,we further use GH3 cell line and primary PA cells to explore the role of metformin in PA proliferation.According to above research work,we initially elucidated the role of LDHA in the development of invasive PA and explored new approaches to the treatment of invasive PA so that we canopened up new areas and new directions in clinical diagnosis and treatment of invasive PA.METHODS:Part I.The expression of LDHA gene and protein in tumor specimens were detected by q RT-PCR and immunohistochemistry.And the correlation between LDHA protein expression and PA invasion and proliferation were analyzed according to clinical data.Part II.GH3 cells were transfected with lentivirus to up-regulate LDHA expression.Detecting the production of lactate in GH3 cellswith or without overexpressed LDHA by colorimetric.Transwell and CCK8 were used to detect the GH3 cell invasion and proliferation of each group.GH3 cell apoptosis and cell cycle distributions of each group were detected by flow cytometry.Detecting the expression of related proteins in GH3 cells of each group by western-blot.Part III.Treating GH3 cells with different concentrations of sodium oxamate which is a LDHA-specific chemical inhibitor,andthen we detected the production of lactate and glucose uptakeof GH3 cells in different groupsby colorimetric and 2-NBDG.Transwell and CCK8 were used to detect the GH3 cell invasion and proliferation of different groups.GH3 cell apoptosis and cell cycle distributions of different groups were detected by flow cytometry.The production of reactive oxygen species(ROS)in different groups were determined by ROS assay kit.The changes of mitochondrial membrane potential in different groups were detected by JC-1 staining.Detecting the expression of mitochondrial apoptosis-related proteins in different groups by western-blot.Exploring the effect of oxamate on the growth of PA in tumor-bearing nude mice.We cultured and identifed primary PA cells derived from patients after transsphenoidal resection.Detecting the invasion and proliferation of primary cultured cells after treated with oxamate.Part IV.GH3 cells were treated with different concentrations of metformin,and cell proliferation of different treatment groups were detected by CKK-8.The cell cycle distribution and apoptosis of different treatment groups were detected by flow cytometry.We conducted the microarray analysis using the GH3 cells with or without the overexpressed LDHA.According to the result of microarray analysis,we investigated the mechanism of metformin-induced apoptosis of GH3 cells.Finally,we evaluated the effect of metformin on cell proliferation of primary PA cells.RESULTS:1.LDHA is highly expressed in invasive PA and is positive associated withtumor proliferation(1)The mRNA and protein expression of LDHA in invasive PA were significantly higher than that in noninvasive PA.(2)LDHA protein expression was positive correlated with Ki-67 index(p <0.05).2.LDHA promotes GH3 cells glycolysis,invasion and proliferation(1)Overexpression of LDHA in GH3 cellspromoted lactate production and the protein expression of glucose transporter-1(GLUT-1).(2)Overexpression of LDHA in GH3 cells promoted cell invasion and expression of related regulatory protein(MMP2).(3)Overexpression of LDHA in GH3 cells activated AKT,inhibited the activity of GSK3β,upregulated the expression of Cyclin D1 and promoted the cell cycle progression.While it had no effect on GH3 cell apoptosis.3.The effect of oxamate which is a specific LDHA inhibitor on the glycolysis,invasion and proliferation of GH3 cells,animal model and primary cultured cells(1)Treating GH3 cells with oxamate significantly reduced lactate production,glucose uptake and the protein expression of GLUT-1.(2)Treating GH3 cells with oxamate significantly reduced cell invasion and expression of related regulatory protein(MMP2).(3)Treating GH3 cells with oxamate significantly reduced cell proliferation.(4)Treating GH3 cells with oxamate significantly reduced the activity of AKT,activated GSK3β,downregulated the expression of Cyclin D1 and arrested cell cycle at G0/G1 phase.(5)Treating GH3 cells with oxamate significantly induced apoptosis of GH3 cells,promoted intracellular ROS production,decreased mitochondrial membrane potential,upregulated the expression of Bax and Cleaved-Caspase3 and inhibited the expression of Bcl-2.ROS scavenger NAC reversed the pro-apoptotic effect of oxamate.(6)Upregulation of LDHA in GH3 cells promoted cell proliferation and oxamate significantly inhibited tumor growth in nude mice.(7)Upregulation of LDHA in GH3 cells significantly promoted the expression of CyclinD1 and MMP2 in vivo,and oxamate-treated tumors exhibited decreased Cyclin D1 and MMP2 expression and increased Cleaved-Caspase3 expression.(8)Treating primary PA cells with oxamate significantly reduced cell invasion and proliferation.4.Treating GH3 and primary PA cells with metformin significantly reduced cell proliferation(1)Treating GH3 cells with metformin significantly reduced cell proliferation.(2)ATF3 knockdown signifcantly suppressed the metformin-induced expression of cleaved caspase-3.(3)Treating primary PA cells with oxamate significantly reduced cell proliferation.CONCLUSIONS:1.LDHA is usually upregulated in invasive PA,and the expression of LDHA is positively correlated with the proliferative activity of adenoma.2.Forced expression of LDHA can promote GH3 cell glycolysis and invasion,and could promote the proliferation of GH3 cells by regulating AKT-GSK3β-Cyclin D1 signaling pathway.3.Oxamate can inhibit the glycolysis and invasion of GH3 cells effectively.It can also arrest cell cycle progression by regulating AKT-GSK3β-Cyclin D1 signaling pathway.And treating GH3 cells with oxamate can induce apoptosis via ROS-mediated mitochondrial apoptosis pathway.4.Metformin can effectively inhibit proliferation of PA cells.