| Allicin is a natural food-derived bioactive ingredient.Diallyl trisulfide（DATS）is the highest content of organic sulfide in allicin,with multiple biological activities,of which the anti-tumor activity of DATS is the most concerned.Anaplastic thyroid carcinoma（ATC）is a very aggressive tumor.The high incidence of metastasis is an important reason for the difficult treatment and poor prognosis of ATC.Therefore,we aim to explore the activity of DATS in inhibiting the metastasis of ATC cells and its related mechanism,to expand the understanding of DATS anti-tumor activity,and to provide theoretical basis for the adjuvant treatment of ATC with DATS.The results of trypan blue staining showed that 5-20mM and 10-40mM of DATS had no significant effect on the cell viability of ATC cells 8505C and C643.The metastasis experiment can be further studied in this low cytotoxic concentration range.Through wound healing assay and cell invasion assay,we found that with the treatment of DATS for 24 h,the migration ability of 8505C and C643 cells decreased by 50.4%and 63.6%,and the invasion ability decreased by 57.6%and 63.0%.The results of Western Blot showed that DATS could regulate the expression of key proteins of epithelial-mesenchymal transformation in 8505C and C643 cells.The expression of E-cadherin was up-regulated to 1.89 and 1.81 times,leading to the reversal of epithelial-mesenchymal transformation.Meanwhile,collagen,concanavalin A and poly-lysine were used to simulate the different interactions between cells and the extracellular matrix to carry out cell adhesion and spreading experiments.The results showed that DATS could reduce the adhesion rate of 8505C and C643 cells on collagen matrix by 42.2%and 22.5%,and inhibit the spreading of cells on collagen.It demonstrated that DATS could play an anti-tumor role in the migration,invasion,adhesion and spreading of ATC cell metastasis cascade.On this basis,we explored the target of DATS inhibiting ATC metastasis.Through the bioinformatics analysis of the thyroid carcinoma patients tissue data from the public database,it was found that the expression level of integrinα2 increased 2.92 times in the tissues of ATC patients,and the expression of its paired integrin subunitβ1 also increased correspondingly.The expression level of integrinα2 in tumor tissues with extrathyroidal extension and lymph node metastasis increased by 67.2%and 81.0%.The results of RT-PCR and Western Blot analysis on two ATC cell models showed that DATS could down-regulate the m RNA and protine expression of integrinα2 by 43.2%and 57.5%,and the m RNA and protine expression level ofβ1 by 75.4%and79.1%.In addition,by separating and extracting cell membrane proteins,we found that DATS could reduce the membrane localization of integrinα2β1.These results showed that DATS inhibits the expression and function of integrinα2β1.DATS can inhibit tumor through H2S dependent or non-H2S dependent pathways.Cells were treated with H2S fast releasing donor Na HS and slow releasing donor GYY4137.And the results of wound healing assay showed that the release of H2S had no significant effect on the migration ability of 8505C and C643 cells.Moreover,we blocked the H2S release of DATS with iodoacetamide（IAM）,and found that IAM could not restore the down-regulation of integrinα2β1expression induced by DATS.It demonstrated that DATS inhibits the metastasis of ATC cells and the expression of integrinα2β1 through a non-H2S dependent pathway.Transforming growth factor-b（16）（TGF-b（16））is an important cell regulator to promote tumor metastasis and integrin expression.Through the analysis of the tissues data from public database,we found that the expression level of TGF-b（16）increased by 1.29 times in tissues of ATC patients.And the high expression of TGF-b（16）was significantly correlated with the pathological characteristics of extrathyroidal extension,lymph node metastasis and distant metastasis.Adding TGF-b（16）to simulate the state in the tumor microenvironment,wound healing assay and cell adhesion assay were used to detect the metastasis of ATC cells.We found that TGF-b（16）promoted the migration and cell adhesion of 8505C and C643 cells,while DATS could down-regulate the migration ability of 8505C and C643 cells by TGF-b（16）by 24.1%and 43.9%,and the adhesion ability by 22.9%and 14.0%.The results of Western Blot showed that TGF-b（16）could promote the phosphorylation activation of Smad3 in ATC cells and correspondingly regulate the expression of integrinα2β1.While DATS could inhibit the phosphorylation activation of Smad3 induced by TGF-b（16）（11）and down-regulate the expression level of integrinα2 by 68.6%and 62.4%,and expression level ofβ1 by 71.7%and 63.3%.The results showed that DATS could exert the biological activity of inhibiting ATC metastasis by interfering with TGF-b（16）-Smad3 signal pathway and targeting integrinα2β1.In conclusion,low cytotoxic concentration of DATS inhibited the migration,invasion,adhesion and spreading of ATC cells by targeting integrinα2β1.And DATS played the role of metastasis inhibition through non-H2S dependent pathway.The metastasis of ATC cells and the expression of integrinα2β1 were promoted by TGF-b（16）in the tumor microenvironment.DATS inhibited the expression of integrinα2β1 by interfering with TGF-b（16）-Smad3 signal pathway,thereby weakening the metastasis ability of ATC cells and exerting the anti-tumor activity of inhibiting the progression of ATC metastasis.DATS is expected to become a food-derived bioactive ingredient for adjuvant treatment of ATC by targeting tumor metastasis. |
