Inhibitory Effect And Molecular Machenism Of Bioactive Peptide P11 On Human Thyroid Carcinoma Cells

BackgroundThyroid cancer is the most common malignancy of the endocrine system.Although thyroid cancer can occur at any age,the prevalence of thyroid cancer is more common among young and middle-aged women.Thyroid cancer accounts for a low proportion of total malignant tumors,its incidence has gradually increased in recent years,and the mortality rate has gradually increased.In some research reports,it can be seen that thyroid cancer has risen to the fifth place in the United States in terms of female malignancy.In China,thyroid cancer has gradually risen with the development of society as the seventh malignant tumor.Therefore,the attention of the community to thyroid cancer has gradually increased.The active prevention and treatment of thyroid cancer has important clinical and scientific value.Biologically active peptides are also known as functional peptides because of their physicochemical properties and their high biological activity.A generic term containing more than 10 but not more than 50amino acid residues is referred to as a polypeptide.A number of anti-tumor peptides have been identified from the phage display library,including CYYGQSKYC(P6)and LSPPRYP(P9).P6 could bind to vascular endothelial growth factor receptor 3(VEGFR-3)and selectively inhibiting vascular endothelial growth factor C(VEGF-C)binding to VEGFR-3,which could suppress the migration and invasion of cancer cells.P9 could act as an effective basic fibroblast growth factor(bFGF)/fibroblast growth factor receptor(FGFR)antagonist and inhibit the growth of the murine melanoma B16-F10 cells in mice.In the present study,we connected P6 with P9 via a flexible linker Gly-Gly-Gly(GGG)to reconstruct a novel peptide P11,CYYGQSKYCGGGLSPPRYP.In this study,cell proliferation,migration,invasion,apoptosis and the signaling pathway was detected in vitro and human thyroid carcinoma xenografts were established and the angiogenesis was examined to determine the effect and machenism of peptide P11 on human thyroid carcinoma cells.ObjectiveTo study the inhibitory effect and molecular mechanism of biologically active P11 peptide on human thyroid cancer cells.Methods1.In vitro experiment1.1 MTS experimentThe MTS assay was performed to induce thyroid cancer cell lines TT,ARO,and TPC-1 cells to be treated with 200μmol/L peptide P6,peptide P9,peptide P6+P9,and peptide P11 for 24 hours.The OD values of each subgroup were accurately measured on a microplate reader to determine the effect of the biologically active peptide P11 on the viability of thyroid cancer cells.1.2 Cell migration and invasion assaysCell scratching experiments,migration,and invasion experiments were performed.Human thyroid cancer cell lines TT,ARO,and TPC-1 were treated with 200μmol/L peptide P6,peptide P9,peptide P6+P9,and peptide P11 for 24 hours.Then the cells were photographed at 0 h,12 h,and 24 h,respectively,and the effects of the bioactive peptides on the migration and invasion of thyroid cancer cells were examined by analyzing the number of penetrated cells and the healing of cell scratches.1.3 Western BlotHuman thyroid cancer cell lines TT,ARO,and TPC-1 were treated with 200μmol/L peptide P6,peptide P9,peptide P6+P9,and peptide P11 for 24 hours.Total protein was extracted from TT,TPC-1,and ARO cells.Western blotting was applied to detect the expression levels of cleaved caspase-3,8,9,Cleaved PARP,AKT,p-AKT,PI3K,p-PI3K,m-TOR,and p-mTOR.β-actin was used as control.1.4 EDU experimentHuman thyroid cancer cell lines TT,ARO,and TPC-1 were treated with 200μmol/L peptide P6,peptide P9,peptide P6+P9,and peptide P11 for 24 hours.Through specific reaction with Apollo567fluorescent dyes,the tumor cells were photographed under an inverted fluorescence microscope in the dark to detect the effect of bioactive peptides on the differentiation and proliferation of tumor cells.Cell proliferation rate=(EdU positive cells)/(total cell number)×100%.2.In vivo experiment2.1 Establishment of human thyroid carcinoma xenograftsBuild a tumor model:a subcutaneous injection was performed in the right armpit of each nude mouse.Human thyroid cancer cell lines TT,ARO,and TPC-1 were subjected to a sterile subcutaneous injection of5×10 ~6 cells per nude mouse.After 24 hours,tumor formation was observed.The success rate of tumor inoculation was 100%.2.2 Determination of tumor inhibition rateExperimental grouping and administration:after inoculation 24h,the nude mice of the inoculated human thyroid cancer TT,ARO and TPC-1 cells were randomly divided into 5 groups by body weight.Peptides(dissolved in normal saline)were administrated subcutaneously(near the implanted tumor)for four weeks(0.1 ml/10 g):group 1 with normal saline(control),group 2 with peptide P6(320 mg/kg/day),group 3 with peptide P9(320 mg/kg/day),group 4 with peptide P6+P9(320 mg/kg/day),and group 5 with peptide P11(320 mg/kg/day).During the experiment,the nude mice were allowed to eat and drink freely.The tumor volumes were calculated as volume=a×b~2/2,where a is the longest dimension parallel to the skin surface and b is the dimension perpendicular to a and parallel to the surface.At the end of the experiment,mice were sacrificed and tumors were excised and weighted to measure the inhibition rate(IR).The IR of tumor growth was calculated as IR(%)=[(A-B)/A]×100,where A is the average tumor weight of the control group,and B is that of the treatment group.Results1.MTS and EDU results showed that peptide P11 could significantly reduce the proliferation and viability of human thyroid carcinoma cells compared with peptides P6,P9,and P6+P9(P<0.05).2.The results of migration and invasion assays showed that peptide P11 could significantly reduce the migration and invasion of human thyroid carcinoma cells compared with peptides P6,P9,and P6+P9(P<0.05).3.The results of Western Blot showed that peptide P11 could significantly increase the protein expression of cleaved caspase-3,cleaved caspase-8,cleaved caspase-9,and cleaved PARP compared with peptides P6,P9,and P6+P9 in human thyroid carcinoma cells(P<0.05).4.The results of Western Blot showed that peptide P11 could significantly reduce the protein expression of p-PI3K、p-AKT、p-mTOR compared with peptides P6,P9,and P6+P9 in human thyroid carcinoma cells(P<0.05).5.Compared with peptides P6,P9,and P6+P9,peptide P11 could significantly inhibit the growth of human thyroid carcinoma xenografts.6.Compared with peptides P6,P9,and P6+P9,peptide P11 could significantly down-regulate the expression levels of CD31and Ki67 in human thyroid carcinoma xenografts.Conclusion1.Bioactive peptide P11 could inhibit the proliferation,migration,and invasion of human thyroid carcinoma cells.2.Bioactive peptide P11 could induce apoptosis by blocking the PI3K-AKT-mTOR signaling pathway.3.Bioactive peptide P11 could suppress the growth of human thyroid carcinoma xenografts by inhibiting angiogenesis.