Silkworm Carboxypeptidase Inhibitor Inhibits Gastric Cancer Cells Proliferation Through EGF/EGFR Signaling Pathway

Cancer is a non-negligible enemy that threatens the health and life of all human beings.With the increasing incidence,cancer is increasingly invading people’s lives.Gastric cancer is a common malignant tumor originating from the epithelium of the gastric mucosa and ranks first in the incidence of digestive system tumors.More than half of the new cases of gastric cancer in the world each year come from the East Asia region where China is located.The annual number of gastric cancer cases and deaths in China is close to half of the world.Surgery,radiotherapy and chemotherapy are used in the treatment of gastric cancer currently.Early gastric cancer has a better prognosis after treatment,while advanced gastric cancer has a worse prognosis.In the treatment of gastric cancer,patients are usually accompanied by the pain caused by the highly toxic side effects of chemotherapy drugs.Therefore,it is urgent to search for the development of new novel drugs and treatments with low or no toxic side effects.With more than one million known species,insects are the most numerous animal group on earth.Many of these insects are used for treat or assist in the treatment of certain diseases,and there are more than 300 species of medicinal insects recorded in Chinese medicine in China.With the development of more modern biological research techniques,people have conducted more in-depth research on various parts and metabolites of insects and started to develop the medicinal value of insects in a more scientific and rational way.It has been shown that insects contain antibacterial peptides,zebularin and other bioactive substances that can play anti-cancer functions,and these bioactive substances have less toxic side effects than chemotherapy drugs.In recent years,more and more attention has been paid to the medicinal value of the natural active substances of the silkworm,and there is great potential to explore their medicinal value.A large number of studies have shown that protease inhibitors are widely used as adjuvant drugs in cancer,but no studies have been reported on the application of silkworm carboxypeptidase inhibitors as antitumor drugs.As a typical representative of the Lepidoptera,the silkworm(Bombyx mori),is an economic insect that is constantly being studied scientifically in order to understand its physiological processes such as metamorphosis regulation and innate immune response.The integument and blood of the silkworm are rich in natural active substances,such as proteases and protease inhibitors,which play an important role in various physiological and biochemical processes of the silkworm,including food digestion and defense against pathogenic microorganisms.Scientists have done a lot of research on serine proteases,cysteine proteases and their inhibitors by using genomics and proteomics methods.However,the distribution and functions of Silkworm carboxypeptidases(Bm CP)and Silkworm carboxypeptidases inhibitor(Bm CPI)in silkworm are unclear.In this study,carboxypeptidase inhibitors were identified for the first time,and their functions are yet to be developed.Nature is a treasure trove for screening effective lead compounds,and many active ingredients of natural origin have been used as lead compounds and eventually become drugs.With a wide variety of natural active ingredients and rich content,the silkworm is a very promising library for screening lead compounds for drug development.A lead compound is a chemical structure with some biological activity obtained by various methods or means that can serve as a lead substance for the development of modern new drugs.After further structural modification,drugs that meet the requirements for clinical use may be obtained.In order to gain a comprehensive understanding of the carboxypeptidase and carboxypeptidase inhibitors of the silkworm,and whether the inhibitors of the silkworm can inhibit the proliferation of gastric cancer cells and have potential as lead compounds for subsequent drug development,we conducted the present study,including the following four parts.1.Identification and expression characterization of carboxypeptidase and carboxypeptidase inhibitors in the silkwormLike other organisms,the silkworm has a variety of proteases and protease inhibitors involved in its life activities.Understanding the structures of proteases and inhibitors and studying and developing their functions in other fields can better apply them to human production and life.Based on the sequencing data of the silkworm genome,we identified a total of 48 members of the silkworm carboxypeptidase gene family,of which 34 belong to metallo-carboxypeptidases(9 M2 domain metallo-carboxypeptidases and 25 M14 domain metallo-carboxypeptidases)and 14 belong to serine carboxypeptidases(5 S10 domain serine carboxypeptidases and 9 S28 domain serine carboxypeptidases).The phylogenetic tree constructed with several species showed that M2 and M14 structural domain metallo-carboxypeptidases could be clearly divided into two clusters.M2 structural domain metallo-carboxypeptidases are angiotensin converting enzymes and their functions in insects are currently unclear.M14 structural domain metallocarboxypeptidases mainly function in processes such as digestion of food.In addition,the S10 structural domain serine carboxypeptidase and S28 structural domain serine carboxypeptidase are also clearly divided into two clusters.Serine carboxypeptidases are thought to be involved in yolk protein degradation in insects.Analysis of silkworm genome database microarray data combined with fluorescence quantitative PCR showed that silkworm carboxypeptidases were differentially expressed in different tissues.Some of the carboxypeptidase genes were highly expressed in the testis of the silkworm,presumably involved in the reproductive development of the male silkworm.The other genes were highly expressed in the midgut,which might be related to the digestive process of the silkworm.Additional genes were expressed in various tissues,and we hypothesized that carboxypeptidase may also be involved in other physiological and biochemical activities of the silkworm.Subsequently,by making microarrays analyzing different periods in both sexes,we found that the genes specifically expressed in the midgut were all expressed in the fifth instar and not in the pupal stage.The genes specifically expressed in the midgut produced corresponding expression differences after starvation stimulation,indicating that the silkworm carboxypeptidase genes are involved in the digestive process in the midgut.Using bi-directional electrophoresis and mass spectrometry,we identified the silkworm carboxypeptidase inhibitor for the first time.The silkworm carboxypeptidase inhibitor is a 113-amino acid protein with a molecular weight of about 12.5 k Da.The recombinant protein with biological activity was obtained by using prokaryotic expression technique,and the antibody was prepared.We found that the inhibitor had high thermal stability and acid-base stability after treatment with different temperatures or p H environments.By fluorescence quantitative PCR and Western blot,we found that the silkworm carboxypeptidase inhibitors are abundant in the silk glands of the silkworm.The expression of the silkworm carboxypeptidase inhibitor in different regions of the silk gland showed a significant temporal correlation.The expression of silkworm carboxypeptidase inhibitor was high in the posterior silk gland in the middle and early fifth instars,while it was high in the middle and anterior silk gland in the late fifth instars.The stable activity of the silkworm carboxypeptidase inhibitor,as a bioactive substance present in the silkworm,is an important prerequisite for the medicinalization of the silkworm.2.Carboxypeptidase inhibitors of the silkworm inhibit the proliferation of gastric cancer cellsWith the advancement of scientific research,the medicinal value of insects is gradually being explored.Biologically active components of insect origin have the potential to provide clues for the search of effective novel anticancer drug candidates.In order to investigate whether the insect carboxypeptidase inhibitor can inhibit the proliferation of gastric cancer cells,three gastric cancer cell lines,MKN45,SGC7901 and HGC27,were selected as the experimental subjects.At the cellular level,the growth of MKN45 and SGC7901 gastric cancer cell lines was significantly inhibited by the silkworm carboxypeptidase inhibitor,accompanied by a significant dose-and timedependent effect.Gastric cancer cells were cultured with 10 μg/m L carboxypeptidase inhibitor,and the inhibition rate of gastric cancer cells could reach 25%.The anti-gastric cancer cell activity was measured after treatment with different temperature or p H environments,and it was found that it could stably inhibit the proliferation of gastric cancer cells after treatment with different conditions.The plate cloning assay showed that the addition of Silkworm carboxypeptidase inhibitor reduced the ability of gastric cancer cell to form clones,and the clone size was significantly smaller than that of the control group.3.Carboxypeptidase inhibitors inhibit gastric cancer cell proliferation via the EGF/EGFR signaling pathwayAutophosphorylation of epidermal growth factor receptors in tumor cells is an important marker of their rapid proliferation.We found by Western blot that both MKN45 and SGC7901 were able to undergo significant autophosphorylation of epidermal growth factor receptor.Adding EGF can significantly promoted the proliferation rate of gastric cancer cells,and this trend was curbed by the addition of carboxypeptidase inhibitor.By detecting the MAPK/ERK pathway-related proteins associated with the epidermal growth factor receptor,it was found that EGF,as a signaling pathway transduction initiator,activated the expression and phosphorylation of downstream-related proteins,while the addition of carboxypeptidase inhibitor could inhibit the cascade reaction activity triggered by EGF.By examining the expression of all the proteins related to MAPK/ERK pathway,we found that carboxypeptidase inhibitor could down-regulate the expression of proteins on this pathway in a dose-dependent manner.The expression of c-Myc was downregulated by the MAPK/ERK pathway and overexpression of c-Myc slowed down the inhibitory effect of carboxypeptidase inhibitor on gastric cancer cells.Subsequently,we found that the expression of four cycle proteins,including CDK2,CDK4,Cyclin D1 and Cyclin E1,was down-regulated by the addition of carboxypeptidase inhibitor in gastric cancer cells.The above results suggest that carboxypeptidase inhibitor inhibits the expression of proto-oncogene c-Myc through MAPK/ERK pathway initiated by EGF/EGFR,which in turn affects the expression of related cell cycle proteins and inhibits the proliferation of gastric cancer cells.4.The design of tumor lead compounds based on the effective peptide fragments of carboxypeptidase inhibitorsThe discovery of lead compounds from the active ingredients of natural products is one of the important sources for the development of modern new drugs.Based on our experimental results,we explored whether the active peptide fragments based on silkworm carboxypeptidase inhibitor could be used as a lead compound for tumor therapy.By using computer simulation of molecular docking technology,we have docked the carboxypeptidase inhibitor to the kinase region of the epidermal growth factor receptor and found that some peptides of the carboxypeptidase inhibitor overlap with the reported small molecule inhibitor Tak-285 at the epidermal growth factor receptor.Based on the docking results,we designed the peptide fragment for CCK-8 cell proliferation assay,and found that the effective peptide of the silkworm carboxypeptidase inhibitor could effectively inhibit the proliferation of gastric cancer cell lines.The peptide fragment also played the inhibitory function through MAPK/ERK pathway,but the inhibitory efficiency was not as good as Tak-285,indicating that there is still room for further optimization of the peptide fragment.The plate cloning assay revealed that the cell clones were significantly smaller and less numerous after the addition of the effective peptide fragment of carboxypeptidase inhibitor compared with the clones formed by the control cells.In summary,these studies showed that there were 48 carboxypeptidase and one carboxypeptidase inhibitor in silkworm.These carboxypeptidases are involved in digestion in the midgut and in male reproductive development in the testis,and different carboxypeptidases play important functions in different parts of the body at different times.Carboxypeptidase inhibitors are abundant in the silk glands of the silkworm.As a bioactive component,the silkworm carboxypeptidase inhibitor was able to inhibit the proliferation of gastric cancer cells.Further,we found that the silkworm carboxypeptidase inhibitor inhibits the expression of proto-oncogene c-Myc through MAPK/ERK pathway initiated by EGF/EGFR,and inhibits the proliferation of gastric cancer cells.c-Myc abnormally high expression is a common feature of gastric cancer patients with poor prognosis.Based on the silkworm carboxypeptidase inhibitor can inhibit c-Myc expression,we designed the silkworm carboxypeptidase inhibitor.Based on the silkworm carboxypeptidase inhibitor can inhibit c-Myc expression,we designed silkworm carboxypeptidase inhibitor as a tumor therapy lead compound to explore the potential of silkworm carboxypeptidase inhibitor as a therapeutic agent for gastric cancer and provide clues for subsequent clinical applications.