| Colorectal cancer,the third most common malignancy and the second most common cause of cancer-related mortality,emerges as a major public health issue.Symptoms of early-stage colorectal cancer are not always obvious or visible.Oftentimes when diagnosed in patients and animals,cancer has grown into the advanced stage.Surgical treatment remains the first-line treatment for primary colorectal cancer,which will achieve a desirable therapeutic effect and will not encounter the issue of biological resistance.But such therapy carries higher risks and will inflict greater surgical trauma.Radiotherapy and chemotherapy are often employed as adjuvant therapy for surgical treatment,but they can provoke severe toxicity and adverse drug reactions.They often don’t work well for population with poor physical fitness,serious disease build-up,and metastatic and recurrent diseases.As diagnostic performances and targeted therapy technology advance,more and more attention has been paid to the search for significantly indicative molecular targets for colorectal cancer.At present,the most common tumor markers include proteins,DNAs,RNAs and compositions of intestinal microorganisms.Among them,some receptors have been found to hold satisfactory potential for targeted therapy.By far,the target drugs for colorectal cancer marketed in China mainly act on EGFR and VEGF.Although these drugs have displayed desirable clinical efficacy and safety,they can only work on single targets.In this case,only a small number of patients with colorectal cancer will respond to those drugs.In clinical practices,the existing diagnostic markers and therapeutic regimens are far from enough to meet the growing needs of colorectal cancer patients.Cck2 r,a member of the G protein-coupled receptor superfamily,has been reported to be highly expressed in pancreatic cancer,gastric cancer,and colorectal cancer.Gastrin and CCK are two natural ligands of CCK2 R.They work in synergy to achieve physiological functions and play different roles in different cancers.A growing body of evidence has demonstrated that gastrin,by binding to CCK2 R,has a role to play in stimulating the growth and invasion by cancer cells and therefore facilitates the occurrence and progress of cancer.However,the role CCK plays amid CCK2R’s regulation of carcinogenesis and development remains unclear.The main purpose of this study is to analyze the biological functions of CCK2 R and its ligands in colorectal cancer and explore the therapeutic potential and in vivo safety of the antiCCK2 R targeting recombinant toxin constructed and optimized thereof according to the relevant mechanism of action,and so as to provide a theoretical basis for preclinical research and clinical trials.Firstly,the methods of bioinformatics,RT-q PCR,and immunohistochemistry were employed to validate the expression level of CCK2 R in the clinical samples of colorectal cancer patients.The results showed that CCK2 R expression was significantly higher in colorectal cancer tissues than in adjacent healthy tissues.The high expression of CCK2 R indicates a shorter overall survival time for patients with colorectal cancer,which is an independent prognostic factor in the development of colorectal cancer.Then,the model of CCK2 R knockdown and overexpression was constructed with lentiviral packaging and overexpression technology.By performing on varying cells,the in-vitro toxicity assay,clone formation assay,cell cycle assay,wound healing assay,Transwell assay,and xenograft assay in nude mice,the biological function of CCK2 R in colorectal cancer was studied.The results indicated that CCK2 R overexpression enhanced the proliferation and clone formation ability of colorectal cancer cells and increased the proportion of cells in the S phase;Conversely,CCK2 R knockdown suppressed the proliferation of colorectal cancer cells in vivo and in vitro,and the cell cycle was arrested in G0/G1 phase.In addition,CCK2 R overexpression enhanced the migration and invasion of rectal cancer cells while CCK2 R knockdown exerted reverse effects on most colorectal cancer cells.Those results revealed that CCK2 R played a crucial role in the proliferation and clonogenesis of colorectal cancer cells and an important role in the migration and invasion of most colorectal cancer cells.To explore the functions and mechanism of actions of CCK2 R and its ligands in the occurrence and development of colorectal cancer,the colorectal cancer cell model was treated with amidated gastrin 17 peptide and CCK octapeptide,and the proliferation and migration of cancer cells were analyzed through Cell Counting Kit-8 assay,Ed U assay and wound healing assay.The results showed that both G-17 and CCK-8 could bind to CCK2 R.In this process,G-17 promoted the proliferation of colorectal cancer cells in a CCK2R-dependent manner while CCK-8,in the same manner,exerted reverse effects.CCK-8 could inhibit tumor progression by competing with autocrine gastrin for the CCK2 R receptor.Furthermore,the molecular mechanism underlying the inhibition of colorectal cancer by the CCK/CCK2 R axis was analyzed with the RNA SEQ technique.The results demonstrated that CCK-8 stimulation and CCK2 R knockdown shared the same downstream molecular expression pattern,which further proved that CCK-8 functioned in a CCK2R-dependent manner.In addition,as indicated in the KEGG analysis and Western blot analysis,CCK/CCK2 R axis may exert effects by inhibiting MAPK signal transduction in the tumor cell.Based on the above observations,efforts were made to apply this mechanism to the construction of recombinant toxins for colorectal cancer.The results showed that the fusion expression of CCK-8 and PE toxin might hinder its binding to CCK2 R while that of reverse CCK-8 and PE toxin retained the binding characteristics with CCK2 R.To analyze whether CCK-8 activity was affected after fusion expression,the technology of point mutation was used to inactivate PE38.The results indicated that reverse CCK-8-based recombinant toxin expressed in E.coli retained the binding ability to CCK2 R but lowered its tumor inhibitory activity.To make up for this loss,the linker and toxin units of the recombinant toxin were optimized by constructing a linker library and random mutation.After screening,the anti-CCK2 R targeting recombinant toxin characterized by high stability,cytotoxic activity,and yield was obtained and named GD9 P.Highly expressed in E.coli,the recombinant toxin reached 92.1% with respect to purity after two-step purification,and the affinity with CCK2 R on the surface of colorectal cancer cells was78.3 n M.To further analyze the in-vivo half-life,efficacy,and safety of GD9 P in mice,the pharmacokinetic profile of GD9 P was checked with an ELISA-based assay and the acute toxicity of GD9 P was validated in the ICR mice.The results indicated that the pharmacokinetic parameters of recombinant toxin GD9 P were satisfactory with a half-life of 69.315 min.The acute toxicity test showed that the drug did not induce gastrointestinal toxicity or systemic toxicity.The xenotransplantation model of nude mice was established receiving 10-day therapy to test the drug effect.The results showed that medium doses of GD9P(4 mg/kg)could prevent tumor progression while high doses of GD9P(8 mg/kg)or co-administration could almost induce a complete disappearance of the tumor.After treatment,no obvious abnormality arose in the sections of GD9P-treated mice while liver,kidney,and lung impairment of a certain degree occurred in the oxaliplatin-treated group.However,the co-administration of GD9 P and oxaliplatin could reduce the side effects due to chemotherapy.The above results demonstrated that GD9 P exhibited satisfactory therapeutic efficacy and safety in the in-vivo targeted therapy.Through the above analyses,this study demonstrates that CCK2 R has a promoting role in the occurrence and development of colorectal cancer.CCK-8 inhibits the development of colorectal cancer through CCK2 R,and the CCK-8-based recombinant toxin possesses desirable tumor-killing activity and safety.The specific manifestations are as follows: CCK2 R is differentially expressed in colorectal cancer tissues and adjacent healthy tissues which plays an important role in the proliferation of,migration of,and invasion by colorectal cancer cells;Amidated G-17 and CCK-8 promote and inhibit separately the proliferation of colorectal cancer cells in the CCK2R-dependent manner where CCK-8 inhibits colorectal cancer cell proliferation by competing for gastrin and impairing MAPK signaling;After optimization and screening,efficient CCK-8-based anti-CCK2 R recombinant toxin GD9 P was successfully obtained;The drug exhibits desirable pharmacokinetic profile,antitumor activity,and safety in mice. |
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