| As a serious threaten to public health,cancer is gradually changing the way modern people live.As one of the most deadly malignancies,gastric cancer(GC)accounts for millions of new cases every year,and 40% of these cases came from China.Surgical resection combined with chemotherapy and radiotherapy is the conventional therapy strategy of GC,among which surgery is still the best choice for radical treatment.However,not all GC patients have the opportunity to receive surgery,and metastasis and recurrence are easy after resection.The prognosis of advanced GC patients after conventional treatment is poor,and the average 5-year survival rate is less than 30%.Although adjuvant therapy is of great significance for cure of GC patients,it has huge side effects and can cause great pain for patients.Therefore,molecular targeted therapy has become a hot topic in the field of tumor therapy.Compared with chemoradiotherapy,molecular targeted therapy kills tumor cells based on the heterogeneity of tumor by targeting specific molecules and their downstream signal transduction.This study aimed to comprehensively evaluate the clinical application value of CCKBR as a therapeutic target of recombinant toxin in GC by analyzing its expression level,biological function and specific killing activity of recombinant toxin which mediated by CCKBR.The relative expression levels of CCKBR were detected by online database,RT-PCR and Western-blotting methods in pan-cancer,30 tumor cell lines,and 59 groups of GC tumor samples and their corresponding normal stomach tissue samples which were collected from China-Japan Union Hospital of Jilin University from 2016 to 2017;Recombinant lentivirus was used to construct CCKBR knockdown and overexpression GC cell lines,and the effects of CCKBR expression level on proliferation,migration and invasion and tumorigenesis of GC cells were analyzed by cytotoxicity assay,clone formation assay,scratch healing assay,Transwell chamber assay and tumorigenesis assay;Recombinant toxins which have a gastrin 17 peptide(G17)as the targeting domain were constructed based on the interaction between CCKBR and its natural ligand.CCK-8,flow cytometry,nude mice model of GC and other methods were used to evaluate targeted killing activity of recombinant toxins against GC cells followed by expression and purification;At last,clinical samples were classified according to CCKBR expression level,and the relationship between CCKBR level and clinical pathological characteristics was analyzed statistically in combination with clinical diagnosis of the patients,and a screening strategy for clinical potential cases for CCKBR targeted therapy was established on this basis.The results showed that CCKBR was overexpressed in a variety of gastrointestinal tumor cells,especially in GC cell lines BGC-823 and SGC-7901;The CCKBR level of GC tissue samples was significantly higher than that of normal tissue samples in both m RNA transcription level(P=0.007)and protein expression level(P=0.037);Prognostic analysis results showed that the prognostic risk of GC patients increased with the up-regulation of CCKBR level,and the overall survival of GC patients with low CCKBR expression level significantly prolonged(4.5years vs 1.6 years,P=0.005).Biological functions of CCKBR were analyzed using knockdown and overexpression of GC cell lines,the results showed that proliferation,monoclonal formation,migration and invasion rate of GC cell lines BGC-823 and SGC-7901 were significantly decreased after CCKBR knockdown(P<0.05);After overexpression of CCKBR in GC cell line MKN-1,opposite trends were observed in the proliferation,monoclonal formation rate,migration rate and invasion rate(P<0.05);The results of flow cytometry showed that BGC-823 and SGC-7901 cells exhibited G2/M phase arrest(P < 0.001)and S phase arrest(P < 0.001)after CCKBR knockdown,respectively;In addition,the down-regulation of CCKBR level significantly inhibited the tumorigenicity of BGC-823 cells in vivo.These results confirmed the important role of CCKBR in the pathogenesis,development and prognosis of GC,which also provided a theoretical basis for the establishment of new therapy targeting CCKBR for GC.The prepared recombinant toxin FQ17 P could specifically bind to CCKBR on the surface of GC cells,and exhibited CCKBR expression dependent targeted activity;FQ17P treatment could significantly increase the apoptosis rate of GC cell lines BGC-823(P<0.0001),SGC-7901(P<0.0001)and MGC-803(P<0.01);In addition,FQ17 P can kill primary GC cells with high CCKBR expression,and inhibit tumor growth by intraperitoneal injection in vivo.After 10 days of 2 mg/kg FQ17 P treatment,tumor weight was significantly reduced(P < 0.001);and the tumor weight of mice in 8 mg/kg FQ17 P group was significantly lower than that in 2 mg/kg FQ17 P group(P<0.001).The clinical samples were classified according to CCKBR level and their correlation with clinicopathological factors was analyzed.The results showed that the screening threshold of CCKBR/GAPDH of samples for FQ17 P was 0.493;The applicability of FQ17 P was closely correlated with EGFR expression level(?2=13.26,P=0.004)and differentiation degree(?2=13.59,P=0.001).The serum minimally invasive CCKBR assay has low sensitivity but high accuracy,with an area under curve 0.7108 of ROC(P<0.001),which means it can be used as a preliminary aid for screening potential cases for CCKBR-targeted treatment.Combined with the above results,a screening strategy for potential clinical cases was established.In conclusion,we evaluated the clinical application value of CCKBR as a target of recombinant toxins therapy for GC from three aspects,the CCKBR expression abundance,biological function of overexpressed CCKBR and targeted killing activity of recombinant toxins mediated by CCKBR,and established a potential case screening strategy for CCKBR targeted therapy by combining with clinical GC tissue and serum samples.This study not only provides a reference for anti-CCKBR targeted therapy for GC,but also provides valuable clinical data support for the further application of CCKBR-targeted recombinant toxin,which is expected to improve the treatment status of GC patients. |
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