Experimental Study On Drp1 Inhibiting The Growth Of Pituitary Adenomas By Regulating Mitochondrial Metabolism

BackgroundPituitary adenomas(PA)are the common type in brain tumors,which accounts for about10-15%.The 2017 WHO classification of PA has emphasized the pathologic evaluation of tumor proliferative potential for the identification of clinically aggressive adenomas.As proliferative activity increases,a subset of PA cases gradually shows malignant features such as rapid local growth and tumor extension,which make total resection difficult and result in early recurrence,resulting in poor prognosis.However,according to the WHO classification of PA in 2017,there is no specific definition and classification of this type of PA.Some scholars named it as "High proliferation index PA"(HI-PA).Accordingly,it is crucial for developing novel strategies to attenuate the proliferative activity of PA to improve patient prognosis.Metabolic change is a hallmark of tumor,which is closely related to tumorigenesis.Reprogramming of cellular metabolism is an important characteristic of tumors.Aberrant hormone secretion is conspicuous in most PA,which causes significant metabolic reprogramming in PA cells.In turn,these metabolic alterations may influence the progression of PA.In a previous study,we demonstrated the role of lactate dehydrogenase in regulating abnormal glucose metabolism in PA.As the vital organelle for glucose metabolism,mitochondria play a pivotal role in tumor cellular energy supply through oxidative phosphorylation(OXPHOS).In addition,mitochondria not only provide building blocks for tumor anabolism,control redox,and calcium homeostasis but also participate in the regulation of oncogenic transcription and tumor cell death.Therefore,mitochondria constitute promising targets for the development of novel antitumor therapies in studies of various tumors.However,there are few studies on the role of mitochondrial metabolic homeostasis in PA progression.The homeostasis of mitochondrial fission and fusion is the key process to maintain mitochondrial health and regulate cell metabolism.The homeostasis is regulated by many molecules.One of them is the dynamin-related protein 1(Drp1),which plays a key role in regulating mitochondrial fission in many mammalian cells and permits renewal and redistribution of metabolites inside the cells.Whether Drp1 can affect the progression of PA by directly regulating mitochondrial metabolism is unclear.PurposeTo explore the effects and mechanisms of Drp1 on regulating PA mitochondrial metabolic homeostasis and PA growth,so as to supply laboratory evidences to mitochondrial metabolic-targeted treatment of PA.Contents1.Differences of Drp1 expression levels in PA samples with different proliferative indexs were compared.2.Effects of Drp1 on proliferation and apoptosis of PA cells were explored in wild-type PA cell lines,Drp1-overexpression(OE)cell model of GH3 cells,and tumor-bearing model of nude mice.3.Effects and mechanisms of Drp1 on the mitochondrial structure and function of PA cell lines were explored.Accordingly,the mechanism of Drp1 regulating cell proliferation of PA was explored.4.Mechanisms of Drp1 on regulating apoptosis of PA cells were explored in PA cell lines and tumor-bearing nude mice model.5.Effects and mechanisms of Drp1 on the mitochondrial structure and function of PA cell lines were explored.Methods1.Study on the expression levels of Drp1 in PA samples: PA samples were grouped according to their proliferative capacity.Expression levels of Drp1 in PA samples were detected and analyzed by q RT-PCR and immunohistochemical(IHC),respectively.2.Effects of Drp1 on proliferation and apoptosis of PA cells: PA cell lines(GH3,MMQ,At T20)were used for cell culture in vitro.Lentivirus vector was used to construct GH3 cell model with stable Drp1 overexpression,and the transfection efficiency was tested by q RT-PCR and western blotting,respectively.Effects of Drp1 on PA cell proliferation were evaluated by CCK-8.Effects of Drp1 on PA cell apoptosis were detected by flow cytometry.Effects of Drp1 on PA growth in vivo were evaluated by the tumor-bearing model of nude mice.3.Effects and mechanisms of Drp1 on mitochondrial structure and function and the proliferation of PA cells: Effects of Drp1 on mitochondrial structure of GH3 cells was observed by transmission electron microscopy(TEM).After stained with JC-1,the effects of Drp1 on mitochondrial membrane potential(MMP)of GH3 cells were detected by laser confocal microscope.Oxygen consumption rate(OCR)and extracellμlar acidification rate(ECAR)of GH3 cells were examined by Seahorse XF96 energy metabolism detector.Effects of Drp1 on total ATP production of GH3 cells were detected by luminometer counter.Effects of Drp1 on reactive oxygen species(ROS)in GH3 cells were examined by flow cytometry.The above mitochondrial function indicators were detected in GH3 cells with Mito-TEMPO(a mitochondrial ROS scavenger)treatment,respectively.And the effects of Mito-TEMPO on GH3 cell proliferation were also observed.4.Mechanisms of Drp1 on promoting the apoptosis of PA cells: Effects of Drp1 on the distribution of cytochrome c in GH3 cells were observed by immunofluorescence(IF).Activation of Drp1 on mitochondrial apoptotic pathway was evaluated in GH3 cells by western boltting.Mdivi-1,Mito-TEMPO and Cyclosporin A(Cs A)were used to intervene the pathway at different stages,then the levels of activation were evaluated.Expression levels of cytochrome c protein in GH3 cells were knocked down by sh RNAs trasfection.After transfection efficiency was examined by q RT-PCR and western blotting,the activation of downstream proapoptotic molecules were explored by western blotting.Flow cytometry was used to detect the effects of the above different intervention methods on PA cell apoptosis rate.Effects of Drp1 on the mitochondrial cytochrome c release of GH3 cells were evaluated in tumor-bearing nude mice model.5.Effects of autophagy on Drp1 regulation of PA cell growth: Effects of Drp1 on the regulation of autophagosomes in GH3 cells were observed by TEM and analysed.Activation of Drp1 on AMPK-ULK1 pathway in GH3 cells were examined by western boltting.Activation of AMPK-ULK1 pathway was evaluated after Compound C(CC)treatment with the vetor and Drp1-OE group cells,respectively.Effects of Drp1 on the cell autophagy were explored in vivo using tumor-bearing nude mice model.Effects of CC and3-Methyladenine(3-MA)on GH3 cell proliferation were explored by CCK-8.Effects of3-MA on GH3 cell apoptosis were evaluated by flow cytometry.Effects of 3-MA on mitochondrial cytochrome c release in GH3 cells were evaluated by western blottingResults1.Drp1 expression was negatively correlated with PA proliferative capacity.(1)Compared with PA samples,the expression of Drp1 m RNA was higher in normal pituitary RNA standard,significantly.(2)Compared with HI-PA(high proliferation index PA),expression of Drp1 m RNA was increased in LI-PA(low proliferation index PA)samples,significantly.(3)Compared with HI-PA,expression of Drp1 protein was increased in LI-PA samples,significantly.2.Drp1-OE inhibited cell proliferation and promoted cell apoptosis in PA cells.(1)Mdivi-1(the Drp1 inhibitor)significantly promoted the proliferation of PA cell lines.(2)Drp1-OE significantly inhibited the proliferation of GH3 cells.(3)Drp1-OE significantly promoted apoptosis of GH3 cells,which could be reversed by Mdivi-1.(4)Drp1-OE significantly inhibited the tumor-forming ability in tumor-bearing model of nude mice,and the inhibitory effect of Drp1 could be reduced by Mdivi-1,significantly.3.Drp1 damaged mitochondria through the overproduction of ROS and caused energy deficiency,which inhibited proliferation of PA cells.(1)Drp1-OE exhibited obvious structural damage in mitochondria of GH3 cells.(2)Drp1-OE treatment increased monomeric JC-1 in GH3 cells,which indicated MMP loss.(3)OCR was significantly reduced by Drp1-OE treatment in GH3 cells.(4)ROS level in the Drp1-OE group GH3 cells was obviously increased compared with that in the control group.(5)Decreased MMP,OCR in the Drp1-OE treatment groups were all rescued by using Mito-TEMPO.(6)Drp1-OE led to a significant decrease in total ATP production in GH3 cells.(7)Mito-TEMPO significantly restored the impaired capacity of total ATP production and proliferation of GH3 cells caused by Drp1 overexpression.4.Drp1 promoted the cytochrome c release from damaged mitochondria and induced apoptosis of PA cells by activating downstream proapoptotic proteins.(1)Drp1-OE treatment significantly upregulated the cytochrome c release from mitochondria in GH3 cells,which then activated downstream proapoptotic proteins,significantly promoted cell apoptosis and inhibited cell proliferation.(2)Mito-TEMPO significantly attenuated the inhibitory effect of Drp1 on cell apoptosis in GH3 cells.(3)The inhibitory effect of Drp1 on cell apoptosis in GH3 cells was obviously attenuated by using Cs A.(4)Cycs sh RNAs treatment obviously downregulated the cytochrome c expression,which alleviated the suppressed effect of Drp-OE on GH3 cell apoptosis.(5)Drp1-OE treatment significantly promoted the release of cytochrome c from mitochondria in the tumor-bearing model of nude mice.5.Drp1-induced energy stress activated protective autophagy by activating the AMPK-ULK1 pathway in PA cells.(1)Drp1-OE led to a significantly increased number of autophagosomes in GH3 cells.(2)Drp1-OE induced autophagy processes by activating the AMPK-ULK1 pathway,which was alleviated when Compound C(CC)was added.(3)CC significantly rescued the suppressed proliferative capacity of the Drp1-OE group GH3 cells.(4)3-MA treatment dramatically inhibited cell proliferation and promoted apoptosis induced by Drp1-OE treatment in GH3 cells,and obviously enhanced the level of cytochrome c release from mitochondria in the Drp1-OE group GH3 cells.(5)Drp1-OE treatment significantly enhanced autophagy level in the tumor-bearing model of nude mice.Conclusion1.The expression of Drp1 was negatively correlated with the proliferation index of PA.2.Drp1-OE inhibited cell proliferation and promoted cell apoptosis in PA cells.3.Upregulation of Drp1 damaged mitochondria through the overproduction of ROS and caused energy deficiency,which inhibited proliferation of PA cells.4.Drp1 overexpression led to cytochrome c release from mitochondria in PA cells,which induced cell apoptosis by activating downstream proapoptotic proteins.5.Drp1-induced energy stress activated protective autophagy by activating the AMPK-ULK1 pathway in PA cells.