The Therapeutical Effects Of Recombinant Lactococcus Lactis With Linoleate Isomerase On The Primary Colorectal Tumor In Mice

BackgroundFollowing the development of economy and quality of life,people common diet contains excess fatty liver,mainly saturated fatty acid and ?-6 fatty acids.They are the sources of many mediators of inflammation and acting in atherosclerosis,diabetes and inflammation and even promoting cell proliferation and tumors.In current,the ?-6 fatty acids are excess and ?-3 severely insufficient under present diet condition,following with sharply rise of disease rate such as fat,cardiovascular,diabetes and malignant tumors in our country for decades.Among these,the incidence cases of colorectal cancer(CRC)was near 137 million all over the world in 2014,and its incidence was the third in all malignant tumors.Notably,?-3 fatty acids are not only related to incidence and development of CRC but also have effects on inhibiting it.To keep healthy,the ?-6/?-3 rate of ideal diet oil is about 5-8:1.However,co-3 fatty acids are far from enough of standard.Many sea fish oils containing ?-3 fatty acid are expensive and with bad effects,at the same time,available clinical therapies of CRC are expensive and with individual differences,so patients bear multiple stress from economy,body and psychology an so on.Therefore,we want to find a effective functional food content to treat colorectal tumors.Linoleic acid(LA)is a common ?-6 fatty acid and rich in food oil,in which some oils contain 30%and even 60%-70%.Conjugated linoleic acid(CLA)is a collective term for positional and geometrical isomers of LA.The major dietary sources of CLA are beef and dairy products,in which mainly contain about 90%c9t11-CLA,about 10%t10c12-CLA and the rest of CLA isomers is vary few.Studies found that CLA had a variety of functions such as anti-cancer,anti-inflammation,anti-obesity,anti-atherosclerosis,anti-diabetes and so on,in which it is the c9t11-CLA that act as anti-cancer.A lot of researches suggested that c9t11-CLA inhibited breast cancer,prostatic cancer and colorectal cancer by decreasing of cell proliferation and inducing the apoptosis.Linoleic acid isomerase(LAI)from many bacteria and Eubacterium can transform the LA into various CLA.The LAI from Lactobacillus,Clostridium,Butyrivibrio and Eubacterium has c9t11-CLA isomerase,and those LAI from Propionibacterium has t10c12-CLA isomerase.Lactobacillus is a kind of Gram-positive bacteria and probiotics in intestinal tract of human and other animals,and it is not only a kind of probiotics but also dominant bacterial colony in human intestinal tract.It is broadly applied for food fermentation,medicine production,health medicine and feed ingredients and so on.Recently,many studies focus on the new drug-delivery way that transformed Lactobacillus was as carrier to express interest proteins.Our group had obtained transformed Lactobacillus with Oxyntomodulin and Glucagon Like peptide-1(GLP-1)successfully to treat obesity and diabetes mellitus effectively.So,the Lactobacillus was selected to be as the host of recombinant vectors in this study.In current study,LZ-LAI plasmid with LAI from Lactobacillus reuteri was constructed,and then was transformed into Lactococcus lactis.And the Lactococcus lactis with LZ-LAI plasmid was used to treat APCmin/+mice model with colorectal cancer by oral administration.ObjectiveTo construct LZ-LAI plasmid and transform it into food grade Lactococcus lactis.And the LZ-LAI was used to treat mice with primary colorectal tumors by oral administration and then analyze its effects of LZ-LAI.Methods(1)Construction of plasmids:The interesting genes were obtained with NCO I and Xba I sites by PCR pLADs-GFP and pLADs-LAI-Flag,and then inserted into pLZ vector obtaining pLZ-GFP and pLZ-LAI-Flag.(2)Electransformation and identification:The above combinant plasmids were transferred into Lactococcus lactis by elec-transformation.And then positive colonies with pLZ-GFP and pLZ-LAI-Flag were selected by bromcresol purple obtaining LZ-GFP and LZ-LAI separately.Morphology of positive colonies were observed under microscope,and then their 16s rDNAs were identified.In the later,their plasmids were extracted and conducted PCR to indentify the intercsting genes.(3)Selection of conditions:Confirmed LZ-GFP were cultured in EM medium with lactose until OD600 attained 0.6,and then induced using nisin and photographed under fluorescence microscope;Western Blot was used to detect the expression of GFP and Flag in the supernatant and bacteria of LZ-GFP and LZ-LAI separately.The different concentrations of nisin(0,0.5,1,2,3,4,5ng/mL)was used to induce LZ-GFP for 6h to select optimum induction concentration of nisin by microplate reader;and best concentration of nisin was used to induce LZ-GFP for 0,3,6,9,12,24h to select optimum induction time.(4)Measuring activity of recombinants in vitro:The LZ-GFP(6 X 109/mL)were added into artificial intestinal fluid and gastric fluid and cultured for 12 and 24h,and then stained using CTC,and the live cells contents were measured using flow cytometry(FCM).LZ-LAI were transformed the LA into c9t11-CLA using optimum concentration of nisin at optimum induction time,and then the induction liquid were collected to extract fat acid,and the fat acid contents were detected using Gas Chromatography-Mass Spectrometer(GC-MS)machine after methylation.And the transformation efficiency was also be calculated.(5)Measuring activity of recombinants in vivo:The 12 male C57BL mice aged 6 weeks and weighted for 18-20g were selected,and 9 mice were randomly divided into three groups including blank control group(NC),LZ-GFP group(LG)and LZ-GFP+nisin group(LGN),each group had 3 mice.After fasting for 4d,three group were fed with 0.1 mL LZ+nisin(6×109/mL),0.1 mL LZ-GFP(6×109/mL),0.1 mL LZ-GFP+nisin(6×109/mL)separately for best induction time;another 3 mice were divided into blank control group(NC),LZ-LAI group(LL)and LZ-LAI+nisin group(LLN).After fasting for 4d,three group were fed with 0.1 mL LZ+nisin(6×109/mL),0.1mL LZ-LAI(6×109/mL),0.1 mL LZ-LAI+nisin(6×109/mL)separately for best induction time.One mice of each group were executed to collect their intestinal contents and indentify the GFP expression using Western Blot;one mice of each group were used to analyze the bacteria site in vivo by Animals living imager;the rest mice were executed to obtain their intestinal contents and measure the survival rate of LZ-GFP after CTC stain under fluorescence microscope.(6)Animal experiments:The 18 successful APCmin/+ model mice aged 6 to 8 weeks and weighted for about 18-20g were randomly divided into control group(Control group),LZ group(LZ group)and LZ-LAI group(CLA group),each group had 6 mice.Three groups were fed with 0.1mL saline+nisin,0.1mL LZ+nisin(6×109/mL),0.1mL LZ-LAI+nisin(6×109/mL)separately by gavage.All groups were administrated once a day,freely eating and drinking,in which the food was with high-fatty,and mice were weighted each 3d.Mice were executed when their situation was the worst,and their peripheral blood was obtained to measure the cytokines levels indcluding TNF-?,IL-2,IL-4 and IL-6.Dissected mice and obtained and weighted their organs.Spleens were used to extract lymphocyte cells,and then the CD3+,CD4+and CD8+lymphocyte cells percentage were measured using FCW.The colorectum were longitudinal cut,in which the tumor numbers and diameters were measured.The tumor tissues from three groups were analyzed using HE stained;immunohistochemical analysis of ?-cantenin,Ki-67;TUNEL of apoptosis and Real-time PCR of COX-2,p53,EGFR and PPARy.Results(1)Consturction of recombined plasmids and Lactococcus Lactis:The pLZ-GFP and pLZ-LAI plasmids were successfully constructed,and transformed into Lactococcus Lactis obtaining LZ-GFP and LZ-LAI positive colonys.It were confirmed by Gram stain,the sequencing of 16srDNA and PCR.(2)Selection of conditions:The LZ-GFP could express GFP protein and only in cells,while the LAI could be secreted into the surpernant of LZ-LAI.The best induction centration of nisin and its best induction time were 3ng/mL and 18h separately.(3)Measuring activity of recombinants in vitro:The survival rate of LZ-GFP after treating with artificial gastrointestinal juice for 0.5h and 1h were as following:PBS were 33.5%and 32.9%;artificial gastric juice were 51.6%and 99.8%;artificial intestinal juice were 28.2%and 24.6%;artificial colon juice were 43.2%and 25.0%;artificial colon juice containing lOg/L NH3 were 35.0%and 25.6%;artificial colon juice containing 20g/L NH3 were 47.0%and 39.5%.Results suggested that lots of Lactococcus Lactis survived after treating with artificial gastric for 0.5 and 1h.GC-MS analysis detected the c9t11-CLA in the products of LZ-LAI,and its tranformtion efficiency was 42,59%.(4)Measuring activity of recombinants in vivo:Animal imaging system results showed that recombinants were located in colorectal site of mice.And the Western blotting results of intestinal contents suggested that recombinants could express target proteins in intestinal of mice.The fluorescence results of intestinal contents dyed by CTC proved that transformed Lactococcus Lactis could survive in mice intestinal and succeed in expressing objective protein normally.(5)Animal experiments:? Comparison of each organ index of mice,we found that there was no significant difference between Control and LZ group,LZ and CLA group in these organ indexes,while differences between CLA group and Control gorup were statisitically significant in liver index(p<0.001).Besides,we found that lest organ indexes of CLA group were lower than other groups except lung,expecially spleen.?Results showed that LZ-LAI significantly decreased the tumor numbers,in which there were extremely significant differences between CLA group and Control group as well as CLA group and LZ group in total tumor numbers(p<0.001,p<0.05);although no difference was found between three groups in diameter of<2mm and>2mm groups,CLA group had a trend decreasing tumor diameter,which revealed that CLA group had effective roles in colorectal mice.Interestingly,the LZ group also tended to decrease the tumor numbers and diameter;?Histopathological analysis results proved the benign effects of CLA group to the colorectal cancer.CLA group had significant differences with Control group in survival time of mice(p<0.001),which obviously prolonged the mice survival time treated with LZ-LAI.And the LZ group also showed the same effects.The immunohistochemical and TUNEL results showed that CLA group could inhibit the proliferation of intestinal tumor and promote apoptosis of tumor cells;?Results of flow cytometry showed that the total percentage of mice lymphocyte and CD4-CD8+T lymphocyte cells increased in CL A group.ELISA results found that there was no significant difference except that was between LZ group and Control group as well as LZ and CLA group in TNF-? level(p<0.01).But the CLA group had a potential effect to increasing IL-2.The Real-time PCR results revealed that CLA group significantly decreased the expression of COX-2 gene compared with LZ group;LZ and CLA group dramatically increased the expression of p53.Furthermore,CLA group significantly inhibited the expression of EGFR gene comparing with Control group(p<0.05).And the LZ group had no significant difference with Control group,but potentially inhibited the expression of EGFR.The differences among three groups showed no statistical significance in the expression of PPARy,while the CLA group tended to decrease its expression.ConclusionThe transformed LZ-GFP and LZ-LAI were successfully constructed;GFP was only expressed in LZ-GFP cells except their surpernant,while the LAI could be secreted into surpernant.The best induction concentration of nisin and its best induction time were 3ng/mL and 18h separately;GC-MS analysis results suggested that LZ-LAI could succeed transforming LA into c9t11-CLA,and its transformation efficiency was 42.59%;bacteria could survival in mice colorectal and express objective proteins;LZ-LAI could prolong the mice survival period,inhibit the proliferation of tumor and induce its apoptosis,improve mice immune function,increase lymphocyte and Tc cells percentage.Besides,it could promote the expression of p53,as well as inhibit the expression of COX-2 and EGFR.It was interesting that LZ group also showed certain inhibiting effects to colorectal tumors.In this study,the Lactococcus Lactis was considered as the vector to secrete LAI in mice intestine and transformed the LAinto c9t11-CLA,which can not only decrease co-6 level but also effectively cure colorectal tumors.It might be a new therapy to colorectal cancer.