The Inhibition Effect Of High Adhesive Recombinant KiSSl Target-delivering Lactococcus Lactis On Colorectal Cancer Cell

Oral administration is a convenient and effective method for disease treatment.However,some proteinaeous bioactive substances and drugs are susceptible to the harsh conditions of the human gastrointestinal tract and thus less effective.In recent years,lactic acid bacteria(LAB)have been verified to be a promising delivery vector for susceptible functional proteins and drugs.Among these,Lactococcus lactis has been demonstrated to be an ideal expression vector,mainly because this bacterium is a noninvasive and nonpathogenic organism with fewer secretary proteins and extracellular proteases produced.Though great advances have been made for the expression of bacterial and viral antigens in L.lactis,the use of L.lactis as vectors for delivering functional anti-cancer proteins has not been widely investigated.The transfer inhibition effect of KiSS1,a suppressor of tumor metastasis,has been verified in various types of cancer.However,studies targeting the expression of this functional protein in LAB as cancer treatment strategy have not been reported.This study is mainly focused on exploring the feasibility of L.lactis as a KiSSl delivery vector.To improve targeting and inhibition effects for colorectal cancer treatment,we first tried to increase the adhesion ability of L.lactis by expressing adhesion protein cwaA from Lactobacillus plantarzum,and then modified KiSS1 protein with RGD peptide.This study provides new evidences for delivering functional protein using L.lactis as vector to treat cancer.This study firstly sequenced the whole genome of L.plantarum NL42,a strain with high adhesion ability stored in our lab.Form this strain,we identified a novel adhesion protein CwaA using comparative genomic analysis.We demonstrated that the expression of cwaA in L.lactis significantly increased its adhesion ability,providing foundations for further using L.lactis expressing CwaA as a functional protein delivery vector.KiSSl from Homo sapiens was then successfully expressed in L.lactis.We verified that KiSSl protein can inhibit the proliferation and migration of human colon adenocarcinoma HT-29 cells.We also showed that the KiSS1 exerting function by not only inducing the apoptosis of HT-29 but also activing the ERK-MAPK pathway to downregulate the expression of MMP9.To achieve target delivering,we modified the KiSSl protein using RGD peptide,resulting in the successfully expression of KiSSl-RGD fusion protein in L.lactis.After modifying KiSSl with RGD,The modification of KiSSl with RGD increased the uptake of KiSSl in HT-29 cell,and thus enhanced the effects of inhibition and migration.Caco2 cell momolayer permeability assay was used to evaluate the ability of KiSSl and KiSSIRGD transporting through the intestinal barrier,and the results showed that both KiSS1 and KiSSIRGD can transport the intestinal barrier.These results suggested that the probiotic L.lactis can be engineered to secrete bioactive KiSSl and RGD modified KiSSl using for partial and whole body delivery treatment.Finally,we expressed the RGD modified KiSSl using the adhesion ability improved L.lactis.Compared to the parent strain,the effects of inhibition was further strengthened,suggesting its potential for oral delivery treatment in the future.This study demonstrated that the probiotic L.lacti can be engineered to secrete bioactive RGD modified KiSS1 and facilitate its transport through the intestinal barrier,providing a novel strategy for oral functional protein delivery using L.lactis.