The Mechanisms Of Diallyl Trisulfide Inducing Apoptosis And Re-differentiation Of Anaplastic Thyroid Cancer Cell

Diallyl trisulfide?DATS?,a garlic-derived organosulfur compound,is one of the efficient biologically active components in garlic.With the incidence of cancer increasing worldwide year by year,the potential antitumor effect of DATS has attracted more and more attention.Anaplastic thyroid carcinoma?ATC?is the most aggressive thyroid cancer with the characteristics of undifferentiated,highly invasion and chemotherapy-resistant.ATC patients have poor prognosis after traditional strategies treatments.Whether DATS can inhibit the development of ATC has not been reported.The study aimed to explore the inhibitory effect of DATS on ATC 8505C cells and the underlying mechanisms.Our results not only provide the theoretical basis for DATS in ATC therapy,but also broaden the application of DATS in anti-cancer therapy.Firstly,the study explored the inhibition effect of DATS on 8505C cells.Cell morphology,SRB assay and colony formation assay all showed that DATS could inhibit cell viability of 8505C cells in a dose-and time-dependent manner.Meanwhile,the results of SRB showed that DATS could also inhibit the other two ATC cell lines,ARO and FRO.Furthermore,we found that DATS could induce p-H2A.X in a dose-and time-dependent manner,indicating that DATS could induce DNA damage in 8505C cells.And DATS-induced DNA damage depending on an increase in reactive oxygen species was mediated by ATM rather than ATR.DNA damage can trigger a series of physiological responses in the cell.On this basis,PI staining and flow cytometry showed that DATS could induce G2/M cell cycle arrest.The results of western blot showed that DATS could promote the translocation of Cdc25C from the nucleus to the cytoplasm followed by the inhibition of its downstream cdc2/cyclin B1 complex.On base of the results above,we further explored the specific ways in which DATS induced 8505C cell death.The Hoechst 33342/PI and Annexin V/PI staining showed that DATS could induce apoptosis in 8505C cells.After treatment with DATS,the expression of pro-apoptosis protein Bax was increased.In contrast,the expression of Bcl-2 family proteins including Bcl-2 and Bcl-X_L was downregulated.The results suggested that DATS could induce apoptosis in 8505C cells through the mitochondrial pathway.Meanwhile,the cleavage of caspase-3,caspase-8,caspase-9 and PARP indicated that DATS could trigger a caspase-cascade mediated apoptosis in 8505C cells.The stemness capacity of ATC cells is the main reason leading to its dedifferentiation.Also,the iodine uptake of highly dedifferentiated ATC cells is difficult,which increases the difficulty of treatment.3D culture and sphere formation assay showed that DATS could induce re-differentiation of 8505C cells.We also found that DATS could increase the glycosylation of NIS,which enhanced the iodine uptake ability of 8505C cells.The results of fluorescent probe P3 assay and ELISA showed that DATS could release H₂S and promote the increase of H₂S level in 8505C cells.Additionally,two different H₂S donors,NaHS and GYY4137,could also inhibit the growth of 8505C cells and promote re-differentiation of8505C cells.Excessive activation of stemness associated transcription factors is closely related to stem cell growth and differentiation.The results of qPCR showed that DATS could directly downregulate mRNA levels and protein expression of three stemness-associatied genes including SOX2,OCT4 and Nanog.In addition,the results showed that H₂S could also downregulate the protein expression of SOX2.Then H₂S could further inhibit the stemness of ATC cell 8505C and finally induce re-differentiation of 8505C cells.In summary,DATS can promote the production of ROS in 8505C cells and then induce DNA damage followed by G2/M cell cycle arrest and apoptosis.In addition,DATS can inhibit the expression of the stemness-associated gene SOX2 and then induce cell re-differentiation by releasing H₂S.Therefore,DATS is promising to become a potential food-borne drug for the treatment of ATC and further increases the survival rate of ATC patients.