1.The Regulation Mechanism Of NOK Oncogene In Glucose Metabolism 2.The Molecular Mechanism Of Interferon-α-induced Apoptosis In Hela Cells

Glucose metabolism is the major source of energy to meet cell proliferation.In normal tissue cells,glucose is first converted to pyruvate through glycolysis,hereafter,in the aerobic environment,pyruvate will enter into the mitochondria to generate acetyl-CoA,which is used as a substrate for the TCA cycle to finally produce H2O and CO2 by thorough oxidative decomposition;in anaerobic environment,pyruvate will be mainly metabolized into lactate and secreted out of the cells.However,in tumor cells,unrestricted rapid proliferation makes their demands for energy and nutrients more urgent,so most of the tumor cells will reprogram the glucose metabolism to meet their rapid proliferation,such as aerobic glycolysis,it is so-called Warburg effect,in which cells still use glycolysis as the main way for energy production and almost all up-taken glucose will be converted into lactate even in the presence of sufficient oxygen.NOK,the Novel Oncogene with Kinase-domain,is isolated from human tonsil cancer cells in 2004 and belongs to the receptor protein tyrosine kinase family.It has been shown to promote cell proliferation and transformation in vitro as well as tumorigenesis and metastasis in vivo.Therefore,NOK oncogene may be able to cause changes in cellular energy metabolism.However,the regulation of NOK-mediated tumor energy metabolism is currently unknown.We aim to explore the new mechanism of NOK in promoting tumor development from the perspective of glucose metabolism.In this study,NOK transiently expressed cell lines 293T and NIH3T3,as well as NOK stably expressed cell line BaF3 were used as the research models.We systematically investigate the molecular mechanism responsible for NOK mediated glucose metabolism and the effects of NOK on mitochondrial functions for the first time.The results demonstrate that NOK oncogene could dramatically enhance aerobic glycolysis in these cell lines and this phenomenon was also validated in the glucose/lactate analysis using serum from NOK transgenic mice.Afterwards,expression levels of glucose transporters GLUT1-4 and various glycolytic enzymes were measured by using Western Blot,the data show that GLUT4 may be the primary transporter for NOK-mediated increase in glucose uptake,meanwhile,NOK could selectively promote the expression of certain key enzymes,and there exist diversity between different cell lines.In view of the fact that the glycolysis process is closely related to the downstream mitochondrial function,we further speculate whether NOK has effects on the TCA cycle and oxidative phosphorylation.Immunostaining analysis demonstrated the co-localization of NOK protein with mitochondria.Although NOK has a minimal effect on TCA cycle,it could markedly inhibit the process of electron transport and oxidative phosphorylation processes,and this inhibition depends on the mitochondrial localization of NOK.Interestingly,NOK could not only promote the mitochondrion-nuclear translocation of pyruvate dehydrogenase complex(PDC),but also enhance the histone acetylation in cell nucleus,which indicates that NOK mediated changes in glucose metabolism might lead to the transcriptional activation of target genes via nuclear PDC associated histone acetylation,which ultimately promotes cell proliferation and malignant transformation.Overall,our researches reveal the mechanism of NOK-mediated tumorigenesis and metastasis from the metabolism perspct for the first time,this will to some extent provides new ideas for drug development and biotherapy of cancers targeting cell metabolism.Interferon a(IFN-a)not only plays an important role in regulating the immune system and inducing antiviral innate immune responses,but also has potential application value in anti-tumor therapy.It has been reported that IFN-α is able to promote the apoptosis of certain tumor cells,and often used as a sensitizing agent for the treatment of various malignancies such as hepatocellular carcinoma(HCC),malignant melanoma,and renal cell cancer(RCC).However,the effect of IFN-a on cervical cancer and its molecular mechanism remain unknown.In this study,HeLa cells were used as a testing model for the treatment of IFN-a on cervical cancer.The preliminary results indicate that IFN-a markedly inhibits the proliferation and induces the apoptosis of HeLa cells.The activation of caspase 3,the up-regulation of both pro-apoptotic protein Bim and cleaved poly(ADP-ribose)polymerase(PARP)1,the down-regulation of anti-apoptotic protein Bcl-xL,as well as the release of cytochrome c from mitochondria were significantly induced upon IFN-a treatment,indicating that the intrinsic apoptotic pathway could be activated by IFN-a stimulation.In addition,caspase 4,which is involved in the endoplasmic reticulum(ER)stress-induced apoptosis,was activated in response to IFN-a treatment.Knocking down caspase 4 by small interfering RNA(siRNA)markedly reduced the IFN-a-mediated cell apoptosis.However,no significant changes in the expressions of caspase 8 and 10 were observed upon IFN-a treatment,indicating the apoptosis caused by IFN-a might be independent of the extrinsic apoptotic pathway.Our data demonstrate that IFN-a-mediated HeLa cell apoptosis may involve both of the intrinsic and caspase 4-related ER-stress-induced apoptotic pathways.All in all,our researches elaborate the molecular mechanism of IFN-α-mediated HeLa cell apoptosis for the first time,revealing that IFN-a may possess anti-cervical cancer capacity.This study may provide clues for the follow-up animal experiment and clinical application.