Initial Studies On The Effects Of HAI-TMIP Antibody On Human Non-small Cell Lung Cancer-derived Vascular Endothelial Cells And Its Related Mechanism

Recent progress in molecular biology has enabled us to better understand the molecular mechanism underlying pathogenesis of human malignancy including lung cancer.Sequencing of human genome has identified many oncogenes and tumor suppressor genes,giving us a better understanding of the molecular events leading to the formation,progression,metastasis,and the development of drug resistance in human lung cancer.In addition,many signal transduction pathways have been discovered that play important roles in lung cancer.Novel strategy of anti-cancer drug development now involves the identification and development of targeted therapy that interrupts one or more than one pathways or cross-talk among different signal transduction pathways.In addition, efforts are underway that combine the traditional cytotoxic(non-targeted) agents with the biological(targeted)therapy to increase the response rate and survival in patients with lung cancer,especially advanced non-small cell lung cancer(NSCLC).Lung cancer is the leading cause of cancer related death in both men and women.Despite decades of basic and clinical research,the 5-year survival of lung cancer is still low.The treatment of advanced NSCLC is largely palliative in order to achieve symptom control,better quality of life,and a small survival gain.Platinum based combination chemotherapy remains the current standard treatment,with a response rate of 30 to 40%,median survival of 8 to 9 months and 1-year survival of 35%in patients with advanced disease,despite the use of newer agents such as taxanes,gemcitabine and vinorelbine.Manv randomized phaseⅢclinical trials have failed to demonstrate the superiority of any one platinum doublet over another.Therefore the focus of drug development in the treatment of lung cancer has shifted in recent years to the development of targeted biological therapy.This includes agents that affect the function of the epidermal growth factor receptor(EGFR),angiogenesis,signal transduction pathways,immunotherapy, vaccines and gene therapy.Targeted therapy has generated new hope that better treatment with higher efficacy and lower toxicities in specific patient population will ultimately lead to increased survival in patientscially advanced NSCLC.Microvessels in malignant tumors exhibit different features.Newly formed microvessels in different tumors vary in number,morphology,structure and architecture. Due to the lack of defined criteria for assessing the morphology and distribution of tumor microvessels,pathologists often ignore tumor vessels that in contrast may serve as markers for differential diagnosis.The wall of newly formed microvessels can be either thick or thin.At different stages of the progression of a single tumor,vessels usually undergo a series of morphological changes which become even more evident when metastasis occurs.Endothelial cells derived from tumor vessels(TECs) exhibit differences from those from surrounding normal tissues as demonstrated by a global gene profiling study despite their expression of EC-specific markers.Immunotherapy targeting tumor vascular EC antigens is emerging as a promising antiangiogenesis therapy.The isolation and culture of TECs have been not established up to now.In the study of angiogenesis in vitro,normal vascular endothelial cells or endothelial cell-like cell lines were used.So,culturing NSCLC-derived vascular endothelial cells(NSCLC-VECs) has an important value in studying tumorigenesis,tumor infiltration,metastasis of NSCLC and developing new drugs.So,we established the method for isolating and culturing NSCLC-VECs and to observe their characteristic in vitro studied the effects of HAI-TMIP antibody on human NSCLC-VECs and its related mechanism.Maybe,it will turn out a new potential antibody to target for the treatment of non-small cell lung cancer.ObjectiveTo establish the method for isolating and culturing NSCLC-VECs and to observe their characteristic in vitro.To detect the expression of ATPaseF1αin NSCLC-VECs.To investigate the suppressing effect of Human Angiostatin Interacting and Tumor Metastasis Invovling Protein(HAI-TMIP) antibody on proliferation activity,metastasis activity of NSCLC-VECs and vascular tube formation in the chick embryo chorioallantoic membrane (CAM) assay,and to study the enhancing effect of HAI-TMIP antibody on apoptosis of NSCLC-VECs.To discuss the related mechanism of the effects of HAI-TMIP antibody on NSCLC-VECs.Methods1.Enzyme method was used to isolate mierovascular fragments from human non-small cell lung cancer tissue,and tissue nubbles cultivation was used to isolate mierovascular endothelial cells,then purified by local digestion and differential conglutination.NSCLCDECs were identified by optical microscope, immunocytochemistry and immunofluorescence.2.The membrane expression level of ATPaseF1αwas examined by immunofluorescence in NSCLCDECs.The expression of ATPaseF1αmRNA of NSCLCDECs was exhibited by real time PCR.The expression of ATPaseFla peptide on the pericellular membrane of NSCLCDECs was observed by immunocytochemistry technique.We utilized sucrose density gradient centrifugalization in order to dissociate plasma membrane fraction and extracted pericellular membrane protein by the way of Pierce—Kit reforming method.The extracted protein was validated by COX-1 MoAb so as to deplete the interference of mitochondrial membrane proteinum.Then the distinction of the pericellular membrane expression of ATPaseF1αbetween NSCLCDECs and normal tracheal epithelium cell line was detected by Western blotting assay.3.The suppressing effect of HAI-TMIP antibody on proliferation of NSCLCDECs is evaluated by CCK-8 assay,and the similar effect on metastasis activity is surveyed by Wound-healing assay.FACS was used to detect the enhancing effect of HAI-TMIP antibody on apoptosis of NSCLC-VECs.The suppressing effect of HAI-TMIP antibody on vascular tube formation in the chick embryo chorioallantoic membrane (CAM) assay were identified by optical microscope.Results1.The NSCLCDECs expressed FⅧ-RAg,CD34.They grew well found by optical microscope and could be passaged in vitro.Its growth curve show S-shape.2.Immunofluorescence analysis showed midrange and higher expression of ATPaseF1αin the membrane of NSCLCDECs.Positive ATPaseF1αmRNA expression was confirmed by RT-PCR in NSCLCDECs.The amplification production electrophoresis of RT-PCR displayed two distinct strap:β-actin and ATPaseF1α.The conspicuous positive expression of ATPaseF1αpeptide on the pericellular membrane of NSCLCDECs was irrefutably observed by immunocytoehemistry technique.The extracted protein was validated by COX-1 MoAb so that we confirm the authenticity of the above extraction of pericellular membrane proteinum.Western blot assay showed that there is positive expression of ATPaseF1αin the pericellular membrane protein of NSCLCDECs while negative expression in the normal tracheal epithelium cell.3.Compared with blank control group,the result of CCK-8 assay demonstrate that HAI-TMIP antibody showed predominance depressant effect on cell proliferation.The result of Wound-healing assay revealed that HAI-TMIP antibody compared with blank control group obviously restrain the migration of NSCLCDECs(P＜0.05).The proliferation activity and metastasis activity would lysis along with the augmentation of HAI-TMIP antibody concentration(P＜0.05).It presented a dose-dependent effect(P＜0.05).The apoptosis rate of NSCLCDECs in HAI-TMIP antibody group was higher than blank control group(P＜0.05).There were no significant differences between HAI-TMIP antibody group and positive control group(P＞0.05).4.Compared with blank control group,HAI-TMIP antibody obviously blocked vascular tube formation in the chick embryo chorioallantoic membrane(CAM) assay. ConclusionsA practical method was first established for isolating NSCLCDECs and is useful in the studies of tumor angiogenesis.The pericellular membrane expression of ATPaseF1αin NSCLCDECs is relatively higher while negative expression in the normal tracheal epithelium cell.HAI-TMIP antibody may repress the proliferation and migration of NSCLCDECs and make them undergo apoptosis.HAI-TMIP antibody also can inhibit vascular tube formation.HAI-TMIP antibody can be studied for further antiangiogenic characteristics in vitro and in vivo and could be used to prevent tumor angiogenesis.Maybe, it will turn out a new potential antibody to target for the treatment of non-small cell lung cancer.