Inhibitive Effects Of Enriched Environment On Mice Tumors And Related Integrative Biological Research

Cancer is a systemic disease.Although cancer is characterized by abnormal growth of local tissues and cells,the nervous,immune and endocrine systems of the body are involved with corresponding changes and play important roles in carcinogenesis and cancer development.The impact of environmental factors on tumor limited traditionally to physical and chemical research.As one kind of eustress,the enriched environment is widely used to study the impact of the environment on brain function,including the study of normal animal models as well as a variety of pathological models relating to central nervous system.The effect of enriched environment on genesis and development of tumor has been rarely reported at home and abroad.This project is to carry out pioneering work in the following two aspects:1.Establish and optimize the rearing conditions of enriched environment,and screen for effective mouse tumor models in enriched environment;2.On the basis of an effective mouse model and by means of integrative biological research,discover the pathways relating to the mechanisms through which the enriched environmental can exert anti-tumor effect.Part One Establishment of tumor model of mice in the enriched environment Objective: To establish rearing conditions of enriched environment,and observe the effect of mental behavior factors on the growth of mice tumors.Methods: Establish both enriched and standard rearing environment.Twenty-four male C57BL/6 mice(Shanghai Lab Animal Center,CHINA),aged 3 weeks,were divided randomly and equally into two groups,namely the Enhanced Environment group(EE)and Standard Environment group(SE).There were four mice per cage(SE)or twelve per cage(EE).The EE consisted of an polypropylene rat cage(60*40*20cm)equipped with one or two running wheels,one shelter,two to four tunnels,one bath box and one climbing ladder.The SE consisted of standard polypropylene cages(30*18*25 cm)with only wooden padding.Mice were left in their respective environments(EE or SE)for 3 weeks,with the exception of weighing and timely routine cage cleaning.The housing room were kept on a 12-h light/dark cycle(lights were automatically turned on at 06:00 and were turned off at 18:00).Room temperature was controlled 21-23 centigrade degree)and humidity around 60%.The mice were given ad libitum access to food and tap water.All experimental procedures were approved by the Shanghai Cancer Institute Animal Care Committee and met the guidelines of the Chinese Council on Animal Care.Three weeks later,they were implanted subcutaneously with tumor cells.Panc02 pancreatic cancer,B16F10 melanoma and Lewis lung cancer model in EE and SE were then established.Models of Panc02 pancreatic cancer and B16F10 melanoma in C57 BL / 6 female mice,and models of CT-26 and CT-26 sci colon cancer in BALB / c male mice were established using the same method.Activities of mice were observed and tumor growth curves were graphed regularly.When the animal experiment was achieved,mice were euthanatized and the appropriate samples were taken for further analyses.Tumors were weighed and tumor inhibitory rates were calculated.Result: In subcutaneous xenograft models of C57 BL / 6 male mice,tumor inhibition rates of EE for Panc02 pancreatic cancer,B16F10 melanoma and Lewis lung cancer were 58.2%(EE 0.23 ± 0.12 g v.s.SE0.55 ± 0.14 g,P<0.001),43.1%(EE 2.01±2.11 g v.s.SE 3.53±2.24 g,P=0.05)and 36.5%(EE 2.51±0.25 g v.s.SE3.95±0.26 g,P<0.01)respectively.PCNA immunohistochemistry was carried out and the result proved that the EE tumor cell proliferation activity was lower than the SE tumors.No significant tumor-inhibitive effect was observed in tumor models of C57 BL / 6 female mice.Tumor inhibition rates of EE for Panc02 pancreatic cancer and B16F10 melanoma were1%(EE 0.94±0.21 vs.SE 0.96±0.23 g,P>0.05)and 16.3%(EE 1.28±0.27 g vs.SE 1.53±0.25 g,P=0.497)respectively.This result suggests that the inhibitive effect of EE on mice tumor might be gender-specific.No significant tumor-inhibitive effect was observed in tumor models of BALB/c male mice.Tumor inhibition rates of EE for CT26 colon cancer and CT26-sci colon cancer were insignificant(EE 3.67±1.14 g vs.SE 3.16±1.02 g,P=0.382;EE 4.11 ± 0.92 g vs.SE 3.40 ± 1.19 g,P=0.105).This result suggests that the inhibitive effect of EE on mice tumor might be strain-specific.In subcutaneous xenograft models of C57 BL / 6 male mice,tumor inhibition rates of Mere Activity,Mere Social and EE-Member group for Panc02 pancreatic cancer were 15.5%、17.4% and 14.8% respectively.This result suggests that the inhibitive effect of EE on mice tumor was a complex of physical and social activities.Conclusion: EE has comprehensive and significant effects on inhibition of subcutaneous xenografts of C57 BL / 6 male mice.The effects may be gender and strain-specific.Part 2 Study of EE on Panc02 pancreatic cancer with microarray and quantitative mass spectrometry analysesPancreatic cancer is the fourth leading cause of cancer death with a median survival of 6 months and a dismal 5-year survival rate of 3–5% and this figure has remained relatively unchanged over the past 30 years.Even for patients diagnosed with local disease,the 5-year survival rate is only 15%.Mice tumor is significantly inhibited in EE,especially for Panc02 panreatic cancer.Here perform genomic and proteomic analyses on Panc02 tumor in order to disclose the tumor-inhibitive mechanism of EE.Objective: To investigate the effect of EE on Panc02 pancreatic cancer using microarrray and mass spectrometry analyses.Methods: Total RNA and protein of Panc02 pancreatic cancer were extracted and purified respectively.Agilent Whole Genome Microarry 4×44K and i TRAQ-labeled mass spectrometry analyses were then performed to discover differentially expressed genes and proteins.Microarray data were processed with Gene Spring11.5 and protein information was explored using Proteinpilot 4.0.DAVID online software was used to perform gene ontology(GO)and KEGG pathway analysis.Results:Microarrray analysis suggested that tissues of Panc02 pancreatic cancer had comprehensive and significant changes on the gene-transcriptive level in EE and the changes tended to be down-regulated.Biological process of GO in EE were mainly enriched in the terms of adhesion,development and motion.The terms of cellular component of GO in EE mainly enriched in of junction,cytoskeleton and membrane.While the terms of molecular function mainly enriched in link and molecular activity.These terms were closely related to the growth,migration and invasion of tumors,and might involve in the inhibitive mechanism of EE on mice tumors.KEGG pathway analysis showed that differentially expressed gene of Panc02 pancreatic cancer in EE enriched on Parkinson disease,Alzheimer’s disease and Huntington’s disease pathways.Theses genes were located mainly between the area of two-fold change and five-fold change.One thousand six hundred and five-eight proteins were obtained in the quantitative analysis of Panc02 pancreatic cancer protein by mass spectrometry.Among them,242 proteins were upregulated(Fold change>1.5)and 380 were downregulated(Fold change>1.5).These differentially expressed proteins were enriched on Parkinson disease,Alzheimer’s disease and Huntington’s disease pathways.Intersection analysis of differential genes and proteins further proved the enrichment of Parkinson disease,Alzheimer’s disease and Huntington’s disease pathways in Panc02 pancreatic cancer in EE.Conclusion: Tissues of Panc02 pancreatic cancer had comprehensive and significant changes in EE.These differentially expressed genes were enriched on Parkinson disease,Alzheimer’s disease and Huntington’s disease pathways.Part 3 Bioinformatic study of the influence of EE on neuro-endocrine system.Objective: To investigate influence of EE on neuro-endocrine system and to explorethe interaction between brain and tumor using bioinformatic measures.Methods:EE and SE were established respectively.Extract and purify total RNA of hippocampus,hypothalamus,pituitary gland and adrenal gland of non-tumor burden mice and tumor-burden mice for whole genome microarray analyses.Gene Spring11.5,DAVID6.7 were used to perform differential expressed gene screening,GO and KEGG pathway analyses for hippocampus and non-tumor burdened hypothalamus,pituitary and adrenal gland.Gene set enrichment analysis(GSEA)were used to investigate tumor-burden hypothalamus-pituitary-adrenal axis in EE and SE.Results:Microarray analyses revealed that the commonly upregulated genes of both tumor-burden and non-tumor burden mice in EE were enriched on Neuroactive ligand-receptor interaction pathway,while the commonly downregulated genes enriched on G-protein coupled signaling pathway.There were 520 genes which differentially expressed in EE.Among them,224 were upregulated and 296 were downregulated.Among the upregulated genes,198 were not induced by EE in the non-tumor burden mice,indicating they might involved specificly in the inhibitive mechanism of EE on mice tumors.KEGG pathway analyses revealed that these genes were enriched on Erb B signaling pathway and Adherens junction pathway.Among the downregulated genes,264 were not induced by EE in the non-tumor burden mice.KEGG pathway analyses revealed that these genes were enriched on Vascular smooth muscle contraction pathway.Brain-derived neurotrophic factor(BDNF)was frequently reported to be significantly upregulated in EE.In our microarray study,we observed that the fold change(vs SE)was 1.80,2.17 and 2.57 respectively in non-tumor EE,Tumor-burden SE and Tumor-burden EE.This indicated that BDNF might mediate the tumor-inhibitive effect of brain in EE.GSEA revealed that in 165 KEGG gene sets,52 were upregulated in HPA microarray in EE.Among them,FDR of 3 genes sets was lower than 25%.These gene sets were involved in Glycosphingolipid biosythesis,Glycosylphosphatidylinositol synthesis and basal transcription.While in SE,113 genes set revealed upregulation in HPA microarray.Among them,FDR of 6 genes sets was lower than 25%,involving in mitosis,recombination and RNA polymerase,etc..Conclusion: Hippocampal BDNF was significantly upregulated in EE,indicating hippocampus may involve in the tumor-inhibitive mechanism of EE through BDNF.Differential genes of hippocampus and HPA axis were enriched on neuroactive ligand-receptor interaction pathway,indicating which may be a potential way involved in the inhibition of mice tumor by EE.