1.The Effect And Mechanism Of CNS And Its Analogs In Treating Cancer Cachexia 2.Research Of The Mechanism Of NEK2 Kinase Regulating Biological Function In Gastric Cancer

Part 1 The Effect and Mechanism of CNS and Its Analogs in Treating Cancer CachexiaBackground: Cancer cachexia is a progressive and systemic wasting syndrome often with metabolic disorder and weight loss,which can not be completely reversed by nutritional support.It is mainly characterized by skeletal muscle atrophy,fat lipolysis and loss of appetite.However,the mechanism of cancer cachexia is still not fully understood.One possible mechanism is that Cancer cachexia is caused by inflammatory factors secreted by the tumor and body,such as TNF-α、IL-1、IL-6 and ZAG,and more research focus on TNF-α.It is well known that NF-κB signaling pathway contributes to the occurrence and development of cancer cachexia.TNF-αactivates the NF-κB signaling pathway in the tumor environment.On the one hand,NF-κB enters the nucleus to initiate the transcription process of the transcription factor FOXO,induces the expression of Mu RF-1and Atrogin-1,which causes muscle protein degradation.On the other hand,the activation of the NF-κB pathway promotes the phosphorylation of HSL,enhances the protein activity of ATGL,and regulates fat degradation as a result.Due to the complex and diverse pathogenesis of cancer cachexia,there is no effective therapeutic drugs been approved for marketing in the clinic.Therefore,new drug development of cancer cachexia therapy is urgent.Carnosol(CNS)is a polyphenolic compound derived from a variety of plants in the family of labiatae such as Rosmarinus officinalis and Salvia japonica Thunb.However,the effect and molecular mechanism of CNS and its analogs in treating cancer cachexia are still unrevealed,and the further research is urgently needed.Purpose: In this thesis,C26 was used to establish C2C12 muscle atrophy models and3T3-L1 lipolysis models in vitro,and C26 tumor-bearing mice were used to establish cancer cachexia models in vivo.we are aimed to reveal the role of CNS and its analogs in treating cancer cachexia and the possible mechanism through using Oncology,Cell Biology,Molecular Biology,Biochemistry and so on technology.Methods:(1)The supernatant of C26 cells was used to induce C2C12 muscle atrophy and 3T3-L1 fat degradation model in vitro;(2)MTT assay was used to detect the cell viability;(3)Cell H&E staining was used to measure C2C12 myotube diameter;(4)The glycerol and triglyceride detection kits were used to detect the glycerol and triglyceride content in adipocyte cells and the serum of mice;(5)Oil red O staining was used to evaluate lipid droplet size;(6)C26 tumor-bearing mice were used to establish a cancer cachexia model in vivo to evaluate the treatment effect of CNS and its analogues on cancer cachexia;(7)H&E staining of tissue was used to count muscle bundle and fat area;(8)The content of inflammatory factors TNF-α and IL-6 in mouse serum were detected by ELASA kit;(9)Protein levels in cells and tissues was detected by Western Blot.Results:(1)The NO.14 rosemary extracts finally screened from 14 natural plant extracts have a relief effect on the C2C12 cell muscle atrophy model induced by C26 medium in vitro,and the muscle atrophy reversion rate is significantly concentration dependent.(2)Rosmarinic acid,Carnosic acid 和 Carnosol(CNS)are three mainly components in NO.14 rosemary extracts.Among these three components,CNS has the strongest relief effect on the muscle atrophy in vitro,which is concentration dependent as well.(3)CNS and its analogs can effectively relieve C2C12 myotube atrophy induced by C26 medium,with the increasing concentration of administration,the diameter of C2C12 myotubes increased significantly compared with the C26 model group,and the reversion rate of muscle atrophy increased significantly,which were concentration dependent.and its possible mechanism may has two parts.On the one hand,CNS and its analogs inhibit NF-κB initiating the transcription process of FOXO,expression of Mu RF-1and Atrogin-1,which led to muscle protein degradation inhibition.On the other hand,CNS and its analogs promote the phosphorylation of AKT,upregulate the expression of MHC and Myo D,and enhances the protein synthesis;(4)CNS and its analogs can effectively relieve 3T3-L1 fat lipolysis induced by C26 medium,with the increasing concentration of administration,the fat degradation is significantly and concentration dependent decreased.and its mechanism may be targeted inhibition of the NF-κB、AMPK and HSL signaling pathways;(5)CNS and DMCNS have a certain therapeutic effect on C26 cancer cachexia.After CNS treatment,the weight and tumor-free weight of C26 cachexia mice significantly improved.CNS has a better rescue effect on adipose tissue than muscle tissue.Similarly,after CNS treatment,both weight and tumor-free weight of C26 cachexia mice improved significantly as well,and both the weight of muscle tissue and adipose tissue are rescued with the same effect..Furthermore,the effects of CNS and DMCNS were studied in vivo,and the results were similar to those in vitro.Conclusion: CNS and its analogs may treat cancer cachexia through targeting the NF-κB signaling pathway,as a widely existing natural polyphenolic compound,CNS is the first time that has been used to alleviate cancer cachexia.In addition,the extended research on its analogs in this paper also proved for the first time that compounds of this sort can also relieve cancer cachexia.The research results have been patented and they can provide a theoretical basis for further drug development and clinical treatment.Part 2 A Research of the Mechanism of NEK2 Kinase Regulating Biological Function in Gastric CancerBackground: NEK2 is a cell cycle and cell mitosis related serine / threonine protein kinase,which mainly involved in centrosome assembly and separation,chromatin condensation and separation,and spindle assembly checkpoint signal regulation to maintain the stability of genomic.More and more studies have proved that NEK2 is highly expressed in various cancer,which results in tumorigenesis and development,tumor invasion and metastasis promotion,drug resistance and poor prognosis.Therefore,NEK2 may be an important potential molecular target of cancer.In previous studies,we designed and synthesized several NEK2 inhibitors which have great effect on a variety of cancer cells such as gastric cancer,liver cancer,and breast cancer both in vitro and in vivo.However,the effect is not only related to NEK2 expression level,but also KDM5 B expression level.NEK2 inhibitors can simultaneously downregulate the protein levels of NEK2 and KDM5 B,suggesting that there may direct or indirect regulatory pathways in NEK2 and KDM5 B,which is unreported.Purpose: We are aimed to study the biological effects of NEK2 on the occurrence and development of gastric cancer by constructing NEK2 silenced and overexpressed stable transgenic cells,to confirm the regulatory role of NEK2 on KDM5 B in gastric cancer and its specific molecular mechanism,to find out new molecular pathways which contribute to the development of gastric cancer.We also want to provide new directions and ideas for clinical anti-cancer treatment research.Methods:(1)The tet-on and tet-off systems were used to establish NEK2 silenced and overexpressed monoclonal stable clones of gastric cancer,respectively;(2)CCK-8and clone formation assay were used to detect proliferation of cancer cells;The migration ability of cancer cells was detected by Transwell assay;(4)Cell cycle was detected by flow cytometry based on PI staining;(5)Protein levels in cells and tissues was detected by Western Blot.;(6)The interaction between NEK2 and KDM5 B was detected by Co-IP test;(7)Nude mouse xenograft model experiments were used to observe tumor growth under NEK2 silencing and overexpression.Results:(1)NEK2 and KDM5 B are highly expressed in most gastric cancer cells;(2)Silencing NEK2 inhibits the proliferation and migration of MGC-803,and causes cycle arrest,which result in tumor growth inhibition in vivo;(3)Silencing NEK2 in MGC-803 resulted in down-regulation of NEK2 protein level,down-regulating the protein of KDM5 B and increasing H3K4me3 on the contrary;(4)Overexpression of NEK2 promoted the proliferation and migration of BGC-823 in vitro and tumor growth in vivo;(5)Overexpression of NEK2 in BGC-823 led to up-regulation of NEK2 protein level,up-regulating the expression of KDM5 B and reducing the expression of H3K4me3 reversely;(6)Co-IP assay showed that NEK2 does not directly regulate KDM5 B,but regulates KDM5B/H3K4me3 through activationβ-catenin/Myc signaling pathway,which means,NEK2 promotes the expression of c-Myc by activating β-catenin,further promotes the increase of KDM5 B expression,and decreases of H3K4me3 expression.(7)The upstream and downstream relationships of the above signal pathways were verified by c-Myc and KDM5 B inhibitors.(8)The molecular mechanism of NEK2 regulating KDM5B/H3K4me3 through β-catenin/c-Myc was further verified on nude mice xenograft tumor models.Conclusion: We revealed the role of NEK2 in the development of gastric cancer.Silencing NEK2 inhibited the proliferation,migration,and cell cycle of gastric cancer,while overexpression of NEK2 promoted the proliferation and migration of gastric cancer.It is the first time that NEK2 regulates KDM5B/H3K4me3 through β-catenin/Myc signaling pathway is revealed by our laboratory.It is innovative and has a positive effect on clinical research of anti-tumor drugs.