Investigation Of The Role Of Abnormal Chondriokinesis On The Invasion And Proliferation Of Growth Hormone-Secreting Pituitary Adenomas Cell

Background and purposeGrowth hormone-secreting pituitary adenomas(GHPAs)is a refractory intracranial tumor.GHPAs secreted excessive growth hormone can give rise to megalakri,hypertrophic cardiomyopathy and the risk of intestinal tumors or thyroid tumors,also crush or invade optic nerve,cavernous sinus and lateral normal pituitary which cause cranial nerve damage and pituitary hypofunction.Surgical resection is the most effective treatment at present.However,it is difficult to excise the invasive growth hormone-secreting pituitary adenomas(IGHPAs)completely and result in residual tumor thereafter,which further worsen its prognosis.The mechanism of the development and invasion of GHPAs is still unclear by now.The electron microscopic images of GHPAs for clinical pathological diagnosis suggested that there may be differences in the number and morphology of mitochondria between IGHPAs and non-invasive growth hormone-secreting pituitary adenomas(NIGHPAs).Lactate dehydrogenase A(LDHA)is a key rate limiting enzyme that inhibits aerobic respiration and enhances anaerobic glycolysis.The high expression of LDHA suggest that there is an abnormal change in energy metabolism and mitochondria in the tumor.Previous studies showed it was positively correlated between LDHA and GHPAs invasion.The invasion and metastasis of breast cancer was closely related to mitochondria.Therefore,we hypothesized that the number and morphology of mitochondria in GHPAs may be associated with the development and invasion of which.Here we utilized stereology combining morphology with statistics to analyze the differences of mitochondrial number and morphology in IGHPAs and NIGHPAs.The quantity and morphology of mitochondria are mediated respectively by Drp1 and Fusion protein Mfn2.By interfering with the expression of Drp1 and Mfn2 proteins,it has become a major method of regulating mitochondrial number and morphology in trials.STAT3 closely relate to the occurrence and development of tumor including two important subunits,Y705 and S727.Previous studies showed that JAK phosphorylated subunit Y705 that activted STAT3 signaling pathway to regulate polygenic transcription related to tumor invasion.It has also been reported that the activation of STAT3 by phosphorylation of subunit S727 enhanced mitochondrial oxidative respiration and promoted tumor proliferation.However,there were a little available information on the effect of subunit S727 in mitochondrion.Cell matrix metalloproteinase 2 and 9(MMP2/9),the important downstream molecule of STAT3,mediate tumor invasion.Previous studies showed that it was positively correlated between the activity levels of MMP2/9 and invasion of pituitary adenomas(PAS).This study will detect the difference on number and morphology of mitochondria,as well as Drp1 and Mfn2 protein expression between IGHPAs and NIGHPAs.The correlation between mitochondrial division and STAT3 activation will be also discussed.That will indicate the correlation between energy metabolism and tumorigenesis and tumor behavior,and contributed to IGHPAs drug treatment.Methods1.The volume density(Vv),number density(Nv)and surface area density(Sv)of mitochondria in invasive(group IGHPA)and noninvasive growth hormone pituitary adenomas(group NIGHPA)were analyzed by stereological methods.2.The expressions of Drp1,Mfn2,LDAH,STAT3,p-STAT3(Y705)and p-STAT3(S727)in group IGHPA and group NIGHPA were detected and compared by RT-q PCR and western blotting(WB).3.Lentivirus transfection was utilized to up-regulate the expression of Drp1 in GH3 cells,Transwell and cell proliferation experiments were used to detect the differences in invasion and proliferation.4.The immunohistochemistry(IHC-P)was used to detect the expression of p-STAT3(Y705)and p-STAT3(S727)in group IGHPA and group NIGHPA,in situ fluorometric kit was used to detect the activity of MMP2/9.HO-3867 and Cryptotanshinone(Cry)acted as inhibitors of STAT3.5.Primary cells of GHPAs were cultured to discuss the correlation between invasion and proliferation of GHPAs and chondriokinesis by Transwell and cell proliferation experiments.Results1.The number of mitochondria decreased in group IGHPA,and the expression of LDHA protein increased.Compared with NIGHPAs,the number of mitochondria decreased significantly in IGHPAs,but the membrane mitochondrial remain intact,and the cristae of mitochondrial showed slightly indistinct.Nv,Vv and Sv decreased significantly.The expression of LDHA protein in IGHPAs was higher than that in NIGHPAs.The results suggested that the number of mitochondria decreases with abnormal changes in energy metabolism in IGHPAs.2.Inhibition of chondriokinesis by mdivi-1 promoted GH3 cells invasiveness and proliferation.Compared with NIGHPAs,the expression of Drp1 reduced in IGHPAs,but there was no difference in the expression of Mfn2 protein.The results showed that the invasiveness of GHPAs was related to the morphology of mitochondrial fission.GH3 cell invasiveness was enhanced when chondriokinesis was inhibited by mdivi-1 in vitro experiments.Compared with the control group,the number of cells increased in group inhibited Drp1,but decreased in group up-regulated Drp1.Cell cycle analysis showed that the proportion of G0 and G1 increased in group inhibited Drp1 and group up-regulated Drp1,and the proportion of G2 and S decreased.The apoptosis was inhibited in group inhibited Drp1 and promoted in group up-regulated Drp1.The mitochondrial membrane potential(MMP),reactie oxygen species(ROS)and the expression of Bax and Caspase3 protein decreased in group inhibited Drp1,and the expression of Bcl-2 protein increased.MMP,ROS and the expression of Bax and Caspase3 protein increased in group up-regulated Drp1,and the expression of Bcl-2 protein decreased.The results showed that the proliferation of GH3 cells was mainly due to the inhibition of apoptosis,when chondriokinesis was inhibited.3.STAT3-MMP2/9 in GH3 cells was activated when chondriokinesis was inhibited,and the activation of STAT3 reduces intracellular ROS and MMP to inhibit mitochondrial apoptosis pathways.The expression of STAT3,p-STAT3(Y705)and p-STAT3(S727)increased in IGHPAs,and decreased in NIGHPAs.The expression of STAT3,p-STAT3(Y705)and p-STAT3(S727)increased when Drp1 was inhibited by mdivi-1 and vice versa.The activity of MMP2/9 was also enhanced.The result suggestsed that inhibition of chondriokinesis was associated with activation of the STAT3-MMP2/9 signal in GH3 cells.Aggressiveness was inhibited when HO-3867 or Cry was added to group inhibited Drp1.The results suggested that inhibition of chondriokinesis was associated with activation of the STAT3-MMP2/9 signal and mediated invasion of GH3 cells.GH3 cell proliferation was inhibited by HO-3867 in group VE+Mdivi-1,rather than Cry.The result showed that inhibition of STAT3 could block the proliferation of GH3 cells mediated by inhibiting chondriokinesis.In this process,both S727 and Y705 play an important role,while inhibition of Y705 phosphorylation alone had no effect on cell proliferation.No significant difference in cell cycle was observed in group inhibited Drp1 when STAT3 phosphorylation was inhibited by HO-3867 or Cry.Apoptosis,MMP,ROS,Bax/Bcl-2,and the expression of Caspase3 protein increased in group VE+Mdivi-1+HO-3867,and decreased in group VE+Mdivi-1+Cry.No significant changes in apoptosis occurred between group VE+Mdivi-1 and group VE+Mdivi-1+Cry.However,MMP,Bax/Bcl-2 and the expression of Caspase3 decreased,and ROS increased significantly in group VE+Mdivi-1+Cry.These results suggested that inhibition of chondriokinesis was associated with STAT3 activation,in which subunit Y705 may regulate ROS metabolism and S727 was critical for stabilizing MMP to inhibit apoptosis.4.Inhibition of chondriokinesis promoted invasion and proliferation of PAPCs.Compared with the control group PAPC,aggressiveness enhanced in group PAPC+ Mdivi-1 was inhibited by HO-3867 and Cry.Proliferation was promoted in group PAPC+Mdivi-1 and PAPC+Mdivi-1+Cry,but was noly inhibited in group PAPC+Mdivi-1+ HO-3867.The results suggested that aggression and proliferation were enhanced by inhibition of chondriokinesis,which was associated with activation of STAT3 in PAPCs.Invasion was mainly related to phosphorylation of STAT3(Y705),and proliferation was more related to phosphorylation of STAT3(S727).Conclusions1.The invasiveness and proliferation of GHPAs were negatively correlated with chondriokinesis and number,and were positively related to the expression of LDHA.2.Inhibition of chondriokinesis promoted the invasiveness of GHPAs and was accompanied with activation of the STAT3(Y705)-MMP2/9.3.Proliferation which was promoted by inhibition of chondriokinesis was related to the inhibition of apoptosis mediated by STAT3 in GH3 cells.4.The activation of STAT3 was negatively correlated with chondriokinesis in GHPAs.Activation of STAT3(Y705)promoted aggressiveness and negatively regulated ROS.The activation of STAT3(S727)regulated negatively MMP and inhibited apoptosis mediated by ROS.The coordination of STAT3(Y705)and STAT3(S727)inhibited apoptosis to promote GHPAs cell proliferation.