The Research Of Lampreys LIP On Recognition,killing Mechanism To Human Lung Cancer Cells

An immune protein(Lamprey Immune Protein,LIP)with lectin domain(Jacalin?like)and Aerolysin domain(Aerolysin correlation)that selectively recognizes and kills tumor cells was isolated from lamprey tissues in our laboratory,LIP proteins target tumor cells by identifying the ends of breast cancer cells(MCF-7)surfaces sugar-type structures that were modified through Neu5 Gc sialic acid,to form deposition on the surface of cell membrane.Because of no Neu5 Gc modified sugar-type structure in normal cell surface,thus LIP proteins do not recognize,bind and kill normal tissue cells(MCF-10A).Lung cancer is one of the malignancies with the highest morbidity and mortality in China and in the world.Advanced lung cancer mainly depends on drug therapy.During the course of treatment,it also has a killing effect on normal cells,that is,side effects of drugs,because of the side effects of drugs,the choice of drugs for lung cancer treatment has become one of the concerns of clinicians.so it is vital for exploring LIP how to identify and kill small lung cancer and non-small lung cancer and to reveal its mechanism.First,23 tumor cell lines from different tissue sources of cancer were screened by LIP protein,LIP had different recognition ability and killing effect on tumor cell lines from different tissue sources.It was demonstrated that LIP has specific selection and certain broad spectrum for tumor cell killing,and has no combination or killing effect on normal tissue cells.The killing of LIP to human small cell lung cancer NCI-H446,lung squamous cell carcinoma cells NCI-H520,human lung adenocarcinoma A549 and normal lung epithelial cell lines L132 was detected by high imaging analysis system and lactate dehydrogenase(LDH)method,cell death results indicate that as LIP concentrations increased,the viability of NCI-H446,NCI-H520 and A549 cells decreased.A549 has the lowest mortality rate,26.7percent;NCL-H520 has the highest mortality rate,61.3 percent.Compared with cisplatin,paclitaxel,5-FU uracil,low-dose lip protein can also achieve the killing efficiency of high-dose chemotherapy drugs,which makes cell micro clusters collapse,break up and no longer aggregate into clusters.The results of cell migration shows that LIP significantly inhibits the migration of three lung cancer cell lines,on the contrary,it does not inhibit the migration of L132 cells.The mitochondria,endoplasmic reticulum and cytoskeleton were observed by fluorescence probe,immunofluorescence combined with confocal microscope.It was found that LIP caused the mitochondria of lung cancer cells to become short and shrink into spherical shape,the number of mitochondria decreased significantly,and the fluorescence disappeared;LIP caused endoplasmic reticulum swelling and injury,vacuolization,and severely destroyed cellular homeostasis,then finally caused the leakage of cell contents and apoptosis;LIP caused the collapse of cytoskeleton system and microtubule depolymerization.LIP proteins directly affect the cytoskeleton structure,protein synthesis and energy synthesis sites of lung cancer cells,then kill tumor cells.Annexin V-FITC method was used to detect the effect of LIP on apoptosis of lung cancer cells.The results showed that LIP prompted the valgus of phosphatidylserine in three tumor cells.The result of flow cytometry showed that 2 ?M and 4 ?M LIP treated L132 cells for 24 h,cells were relatively stable.The apoptosis rate of NCI-H446,NCI-H520,A549 cells that were treated with the same dose LIP significantly increased after 24 h.At the same time,LIP destroyed mitochondria and reduced mitochondrial membrane potential.The confocal microscope images showed that the level of reactive oxygen in three lung cancer cells treated with LIP was higher than those untreated control group,indicating that the LIP mediated mitochondrial destruction of tumor cells was related to reactive oxygen species(ROS).In order to explore the mechanism of LIP inducing apoptosis of lung cancer cells,after LIP treated NCI-H446,NCI-H520 and A549 cells,the expression of PARP1,GRP94,Bip,Caspase-12,Caspase-3,CHOP and p21 was significantly upregulated by immune blotting,in time-and dose-dependent manner.The endoplasmic reticulum-induced apoptosis depends on IRE-1,ATF-6 and PERK pathways,leading to upregulation of downstream CHOP expression.The expression of PARP1,GRP94,Bip,Caspase-12,Caspase-3,CHOP and p21 was significantly decreased after 48 h under the use of endoplasmic reticulum stress inhibitor4-PBA.Together,lamprey immune protein LIP mediates the apoptosis of lung cancer cells through endoplasmic reticulum stress signaling pathway.The passage through building a tumor-bearing model of lung cancer mice and direct injection of LIP protein at the tumor,LIP can significantly delay the growth rate of the tumor,the tumor tissue is significantly reduced,but there is no obvious side effect on the normal tissue cells of mice.Through the immune histochemical test,LIP can selectively recognize human lung cancer tissue samples,but have no effect on adjacent cancer.