Eustress Induces The Growth Inibition And Chemosensitivity Enhancement In Tumor

With the deepening of multidisciplinary research,people come to realize that cancer is a systemic disease,the body's mental state,neuvous,immune and endocrine systems have an important impact on the occurrence,development and prognosis of cancer.Stress can be divided into eustress and distress.Strees can produce significant impact on the host systems,which process includes a series of mental change and body function.Currently the significance relationship between psychological stress and cancer has been seriously recognized more than ever.The clinical application of psychotropic drugs relieve pain caused by depression,improve quality of life for cancer patients has become an important part of supportive therapy.EE is a eustress model has been widely accepted,and its improvement in mice for behavioral and psychological aspects are manifold.EE can improve depression,reduce anxiety behavior in mice,and the social,memory in mice and so on.Mental behavior is an important part of psychological stress,the question we can adress is what is the real impact on cancer?Our lab has previously confirmed that EE significantly inhibited subcutaneous xenografts of Panc02 pancreatic cancer,B16F10melanoma cancer and Lewis lung cancer in C57BL/6 male mice.To explore this question,the present study intends to conduct research in the following three aspects:1.Based on the research of EE inhibited subcutaneous tumor of our lab,established mouse MC38 subcutaneous transplantable colon adenocarcinoma model,mouse Panc02 orthotopic transplanted pancreatic adenocarcinoma model,mouse APC^Min/+gene mutation induced spontaneous intestinal adenomatosis model.Research the effect of EE for tumorigenesis.2.we conducted the study on the impact of EE to the chemosensitivity of the tumor for drug treatment,and further study its mechanism.3.We established a mouse behavior analysis platform to study the impact of EE to behavior of mice,further investigate behavioral effects on tumor development.These studies explored the effects on tumor occurrence and development process of the enriched environment.For the first time,we discovered the role of EE in cancer chemotherapy,and the putative drugs for improvement of neurobehavior to cancer translational medicine.Our result will put some new insight on new aspects of cancer translational research.Part One EE affect tumor occurrence and developmentObjective:To establish mouse MC38 subcutaneous transplantable colon adenocarcinoma model,mouse Panc02 orthotopic transplanted pancreatic adenocarcinoma model,mouse APC^Min/+gene mutation induced spontaneous intestinal adenomatosis model to observe the effect of EE to the development of tumors in vivo.Methods:3-week-old male C57BL/6 mice were randomly placed in EE?12/cage?or standard environment?SE,4/cage?for three weeks,then were inoculated subcutaneously with MC38 colon cancer cell or orthotopic pancreatic cancer of Panc02.After 3-5 weeks when experiments terminated,weigh and calculate the inhibition rate.Additional,3 weeks APC^Min/+mice were randomly placed in EE or SE environment,the mice were sacrificed till 20 weeks of age,the number and volume of intestinal adenomas were statisticed.Results:EE inhibition rate for subcutaneous xenografts tumor of MC38 colon cancer?SE group 0.85±0.14g vs EE group 0.41±0.08g,P<0.01?and orthotopic pancreatic cancer of Panc02?SE group 0.15±0.02g vs EE group 0.09±0.01g,P<0.01?were reached to 52.2%and 43.3%.In APC^Min/+mouse model,EE significantly inhibited the number of intestinal adenomas?SE group 24.17±1.19/mouse vs EE group was 18.08±1.75/mouse,P<0.01?and the volume of intestinal adenomas?SE group 139.07±13.57mm³ vs EE group 86.70±10.32mm³,P<0.01?,the inhibition rate was 25.2%and 37.7%.Conclusion:A series of experiments illustrate that EE can extensive and significantly inhibite tumorigenesis in C57BL/6 mice.Part Two EE influence on tumor chemosensitivityObjective:To research the influence of EE on tumor chemosensitivity in vivo,and to explore its possible mechanism.Methods:3-week-old male C57BL/6 mice were randomly divided into four groups:SE-PBS group,SE-5FU group?Standard Environment,4/cage?and EE-PBS group,EE-5FU group?Enriched Environment rearing,12/cage?.3 weeks after,mice were subcutaneous inoculated Panc02 pancreatic.When tumor volume>1mm3,mice were treatment with 5-fluorouracil?5FU?by intraperitoneal injection,CTRL group were intraperitoneal injected PBS.5 weeks later,mice were sacrificed and the tumor weighed.In further experiments,by observing the EE for Panc02 pancreatic cancer xenografts on Gemcitabine?,GEM?and MC38 colon cancer xenografts for 5FU chemosensitivity,ditto experimental methods.To further confirm the synergistic effect of chemotherapy drugs and EE,the Panc02tumor of SE and EE group were primary cultured and to detect the inhibitory concentration(IC₅₀)to GEM and 5FU.To investigate the effects of EE on tumor chemosensitivity,total RNA of tumor of EE group and SE group were extracted for genome microarray expression profiling.The chip results were verificated by Real-time PCR and Western Bolt.By overexpression or RNA interference,reserch the expression of genes affected by EE on tumor chemosensitivity in vitro and in vivo.Results:In experiment of EE enhanced chemosensitivity of Panc02 for 5FU in vivo,SE-PBS group,SE-5FU group,EE-PBS group,EE-5FU group tumor weight was0.629±0.027g,0.530±0.04g,0.375±0.021g,0.203±0.015g;EE susceptibility in vivo experiments Panc02-5FU in,SE-PBS group,SE-5FU group,EE-PBS group,EE-5FU group of mice subcutaneous tumor weight was 0.629±0.027g,0.530±0.04g,0.375±0.021g,0.203±0.015g;which was no significant difference between SE-5FU group and EE-PBS group?P=0.082?,the remains pairwise comparisons were significantly different?P<0.05?.The inhibition rate was analyzed,with same dose of5FU,effect in EE increased from 15.8%to 45.9%.Extensive validation of EE synergy with chemotherapy drugs,5FU in the EE environment for subcutaneous xenograft of MC38 colon cancer,tumor inhibition rate increased from 17.22%to 34.38%?P<0.05?.While GEM on subcutaneous xenograft of Panc02 pancreatic tumor inhibition rate was 50.21%,compared to SE environment?29.28%?was significantly improved?P<0.05?.Successfully establish ABCA8A,ABCA8B overexpressing Panc02 cell lines?Panc02-ABCA8A and Panc02-ABCA8B?,in vitro and in vivo experiments showed ABCA8A can significantly reduce the sensitivity to chemotherapeutic drugs.After5FU(concentration of 10^-5M)or GEM(concentration of 10^-6M)treatment for 48hours,Panc02-ABCA8A cell survival rate?5FU group:58.86±3.23%;GEM group:58.16±4.37%?,significantly higher than control cells?5FU group:37.56±0.47%,P<0.05;GEM group:39.25±1.18%?.However,Panc02-ABCA8B 5FU and GEM treatment in cell viability?5FU group:47.57±0.75%;GEM group:50.58±1.97%?,has no significant difference compared to control cells.After the C57BL/6 mice were inoculated subcutaneously ABCA8A,ABCA8B overexpression cell lines and control cells were treated with 5FU by intraperitoneal injection,Panc02-ABCA8A 5FU group inhibition rate?20.44%?was significantly less than the control group?57.19%,P<0.05?;while inhibition rate of Panc02-ABCA8B group was 33.74%,had no significant difference compared with the control group?P>0.05?Conclusion:EE enhanced tumor sensitivity to chemotherapy,and ABCA8 as a transmembrane transporter proteins may have sensitizing effect of cancer chemotherapy targets.Part Three The behavioral changes in the enriched environment and its effect on tumorObjective:To study enriched environment impact on mouse behavior in order to understand the influence of behavioral changes on tumor development in vivo.Methods:Established behavioral tests and screening methods.Establish mouse behavioral testing methods to anxiety,depression,using a international standard test methods.Among them,the elevated plus maze?EPM?test may reflect anxiety and depression;the open field?OF?test reflects the explore ability of mice;forced swimming?FS?reflected despair in mice experiments.Data analysis by supermaze software.To study the influence of EE on the anxiety-like behavior in the C57BL/6 mice,EE and SE mice were observe in 4 weeks,7 weeks and 9 weeks of age,to observe the indicators of openarm activity time and percentage of openarm activity distance.To study the effects of behavioral on pancreatic cancer growth,the 4-week-old male C57BL/6 mice were test in EPM,OF and FS.At the time of 7 weeks old pancreatic cancer cells of Panc02 were inoculated subcutaneously.5 weeks later,the tumor was weighed and the behavioral indicators associated with tumor weight were analysised.To study the effects of behavioral on pancreatic cancer growth,the 4-week-old male C57BL/6 mice were test in EPM,OF and FS.At the time of 7 weeks old pancreatic cancer cells of Panc02 were inoculated subcutaneously.5 weeks later,the tumor was weighed and the behavioral indicators associated with tumor weight were analysised.To further verify the anxiety-like behavior in mice on the growth of pancreatic cancer,the 4-week-old male C57BL/6 mice is divided by the openarm time in high openarm group,the moderate openarm group and the low openarm group.3 weeks after feeding in SE environment,Panc02 pancreatic cancer cells were inoculated subcutaneously,10 days after inoculation volume were measured every 5 days.Meanwhile,in the time of inoculation and vaccination two weeks EPM test performed again.To further confirm the impact of anxiety-like behavior in mice the growth of pancreatic cancer,we observed the effects of the anti-anxiety drugs fluoxetine and quetiapine change the anxiety-like behavior in mice.4-week-old male C57BL/6 mice were randomly divided into control group,fluoxetine group and quetiapine group and the control group,while the fluoxetine group and quetiapine in drinking water were added to the appropriate dose of psychotropic drugs.3 weeks after treatment mice were subcutaneously inoculated Panc02 pancreatic cancer cells,10 days after inoculation volume measured every 5 days in mice.Meanwhile,in the time of inoculation and two weeks after vaccination EPM tested again.Results:Behavior in EPM has a significant correlation?P<0.05?with Panc02pancreatic tumor,while the open field and forced swim test and behavioral tests showed no correlation?P>0.05?.Our results show that mice with open arms activities and the growth of pancreatic cancer showed a significant negative correlation?P<0.05?,prompted anxiety-like behavior may promote the growth of pancreatic cancer.The open field test and forced swimming test in almost no correlation.With mice age grownn,the open arms of active time for each group decrease.At7 weeks and 9 weeks of age,the high activity time on open arms in mice remains active significantly longer than the moderate active group?P<0.01?and the low activity group?P<0.01?.However,the open-arm activity time although longer than low activity group,but the difference was not statistically significant?P>0.05?.Tumor growth consistent with the expected results,Panc02 high activity of pancreatic tumor growth in mice group was significantly slower in the low activity and the active group?P<0.01?.However,the tumor volume in mice is slightly less than the activity of the low activity group,no significant difference?P>0.05?between the two groups of tumor growth.In behavioral correction experiments,psychotropic drugs Fluoxetine Quetiapine treatment after tumor inoculation,can reduce anxiety-like behavior in mice?shown as open-arm activity in mice prolonged in the elevated maze test?.Analysis of tumor growth,fluoxetine and quetiapine significantly reduced tumor volume than the PBS group?Fluoxetin group 98.93±10.92mm³&Quetiapine group 117.57+29.41 mm³vs control group 220.28+34.34 mm³,P<0.01?.The cytology in vitro experiments showed that the two drugs have no direct killing of tumor cells in vivo drug concentrations.Conclusion:EE have significantly improved mental behavior in mice,and this improvement can be simulated by mental and behavioral drugs and achieve a similar inhibitory effect.