Chimeric Antibody Targeting SRPK-1 In The Treatment Of Non-small Cell Lung Cancer

Objective Non.small cell lung cancer(NSCLC)is one of the most common types of cancer in humans,and is characterized by rapid growth,migration,invasion and reoccurrence.Evidence has indicated that the protein and m RNA levels of serine.arginine protein kinase-1(SRPK-1)are upregulated in NSCLC tissues.However,the functions of SRPK-1 and targeted therapy for SRPK-1 in the progression and treatment of NSCLC remain to be fully elucidated.The aim of this study is to construct a chimeric antibody of SRPK-1(Chan SRPK-1)by antibody genetic engineering technology and to confirm the increase of SRPK-1 expression in NSCLC by in vitro and in vivo studies.Evaluate the neutrality of Chan SRPK-1 by constructing and synthesizing The therapeutic effect of NSCLC was investigated whether the migration and invasion of NSCLC were inhibited after Chan SRPK-1treatment.It is expected that this study will explore the mechanism of action of Chan SRPK-1 and find a new therapeutic idea for the diagnosis and treatment of NSCLC.Methods(1)Screening the antibody targeting of SRPK-1 by conventional methods,in order to enhance the stability and half-life of SRPK-1 antibody,the chimeric antibody Chan SRPK-1 targeting SRPK-1 was successfully constructed and stabilized in E.coli expression system.Expression secreted by Chan SRPK-1.(2)Chan SRPK-1was determined by SDS-PAGE gel electrophoresis.In addition,the affinity of Chan SRPK-1 for SRPK-1 was determined using ELISA and western blot analysis.(3)In vitro effects of Chan SRPK-1 on the expression of SRPK-1 in NSCLC cells.The levels of SRPK-1 expression in NSCLCDVECs and MRC-5 cells were detected by using RT-q PCR and ELISA.(4)Cell viability was analyzed in vitro effects of Chan SRPK-1 on the growth of NSCLCDVECs by MTT colorimetry.(5)NSCLCDVECs and MRC-5 cells were treated with different concentrations of SRPK-1 and Chan SRPK-1 for analyzing cell viability and measuring migration and invasion and confirming the effect of SRPK-1 on migration and invasion and apoptosis.(6)The expression of migration-related promoting protein of Chan SRPK-1in NSCLC model mice was determined by ELISA and western blot analysis.Inclouding the expression levels of Chan SRPK-1 on tumor cell cytochalasin D,G-actin and the effect of phosphorylation GSK3-?.(7)The tumor-bearing mice were intravenously injected with Chan SRPK-1(400 mg),with the same volume PBS injected in the control group mice.The treatment was continued for 14 days(one injection per day).After 14 days after continuous treatment,we observed for 25 days to 120 days.The tumor volumes were calculated according to a previous study.Results(1)The molecular weight of ChanSRPK-1 was determined to be about 67.5k Da by SDS-PAGE gel electrophoresis.Further,ELISA and western blot analysis were used to confirm that Chan SRPK-1 maintains high affinity with SRPK-1 and has the ability to bind to SRPK-1.(2)NSCLCDVECs and MRC-5 cells were analyzed using RT-q PCR analysis and ELISA.The results showed that the expression of SRPK-1 was upregulated in the NSCLCDVECs,compared with that in the MRC-5human lung cells.In addition,Chan SRPK-1 treatment neutralized the expression of SRPK-1 in a dose-dependent manner(200-2,000 mg/ml).Chan SRPK-1 treatment also neutralized the expression of SRPK-1 in a time-dependent manner(24,48,72 and 96 h).Chan SRPK-1(400 mg/ml)suppressed NSCLCDVECs growth from 48 h.(3)To investigate the inhibitory effects of Chan SRPK-1 on NSCLC,cell viability,migration and invasion were analyzed.The viability of NSCLCDVECs was significantly decreased in the Chan SRPK-1 treated groups(200,400 and 1,000 mg/ml for 48 h),compared with that of the MRC-5 cells.The presence of SRPK-1 markedly promoted NSCLCDVECs migration and invasion at doses of 200,400 and 1,000mg/ml for 24 h,compared with the untreated group.By contrast,it was demonstrated that Chan SRPK-1 markedly inhibited the migration,invasion and apoptosis of NSCLCDVECs.(4)The m RNA expression levels of ?CF,MMP,CT1 and FBC in NSCLCDVECs were significantly higher than those in Chan SRPK-1 treated cells.Using paired t-test(P <0.01),the difference in expression of migration-promoting proteins between Chan SRPK-1 group and the control group was also determined to be statistically significant(P <0.01).it was found that the downregulation in the expression of Cytochalasin-D and G-actin,and the phosphorylation of GSK3-? were significantly different in the Chan SRPK-1 treated tumor cells,compared with those in the control.In addition,the tumor size of the NSCLC bearing mice in the Chan SRPK-1 treated group was significantly inhibited compared with the control group,after 120 days of treatment with Chan SRPK-1,the long-term survival was beneficial efficacy compared with the control mice,Chan SRPK-1 treatment(N= 10 in each group)prolonged survival of NSCLC-bearing mice.Chan SRPK-1 treatment significantly protected tumor-bearing mice.Chan SRPK-1 inhibited NSCLC tumor metastasis compared to PBS-treated mice.Conclusion(1)The results indicate that the chimeric antibody Chan SRPK-1targeting SRPK-1 is constructed by a conventional approach and was secretesd stably in Escherichia coli expression system.Chan SRPK-1 had specific binding ability to SRPK-1.(2)The results show that Chan SRPK-1 significantly inhibited the growth,migration and invasion of NSCLC tumors in vivo and in vitro.Chan SRPK-1 could control tumor metastasis,prolong survival time and increase eradication rate.