The Inhibitory Effect And Related Mechanisms Of Punicalagin On Papillary Thyroid Carcinoma BCPAP Cells

Thyroid cancer belongs to endocrine malignant tumor and its incidence is increasing in recent years.Among different types of thyroid carcinomas,papillary thyroid carcinoma has the highest incidence.Although various strategies have been applied to thyroid cancer therapy,there are still risks of recurrence,metastasis and deterioration of thyroid cancer and even some of treatments will bring about side effects.Polyphenol compounds possess antitumor activity and as the representatives of natural compounds,they have wide sources,low toxicity and diverse targets in cancer.Punicalagin,which is derived from pomegranate,belongs to the macromolecular substances in polyphenol compounds and has triggered the attentions of scientists due to its anticancer effect.However,the roles of punicalagin in thyroid cancer remained unclear.Our paper mainly investigated the inhibitory effect of punicalagin on papillary thyroid carcinoma BCPAP cells and probed the underlined mechanisms.Our results not only provide scientific proofs for the application of punicalagin in thyroid cancer therapy,but also give new prospects for further exploring the new therapeutic targets in thyroid cancer.We first investigated the toxic effect of punicalagin on BCPAP cells.The SRB and MTT results showed that punicalagin suppressed the survival of BCPAP cells in a dose-and time-dependent manner,and compared with normal thyroid cells Nthy-ori 3-1,punicalagin inhibited BCPAP cells more obviously and significantly.Trypan blue and PI staining results showed that punicalagin destroyed the integrity of BCPAP cell membrane in a concentration-dependent manner.Based on the above,we discussed the ways through which punicalagin induced BCPAP cell death.The results of PI/Hoechst 33342 double staining and western blot showed that punicalagin failed to induce cell apoptosis.AO staining and autophagy-related protein detection showed that punicalagin induced cell autophagy and moreover,we found that punicalagin promoted the autophagic flux in BCPAP cells after being combined with autophagy inhibitors 3-MA/Baf A1.MTT results showed that 3-MA significantly reversed the cell death effect induced by punicalagin.Western blot results further illustrated that punicalagin may induce autophagy through positively regulating ERK and p38 pathways and negatively regulating mTOR pathway,thereby promoting BCPAP cell death.The third part discussed the effect of punicalagin on DNA damage in BCPAP cells and explored the related mechanisms.The results showed that punicalagin enhanced the phosphorylation of H2 A.X(a marker of DNA damage)and caused DNA breaks in BCPAP cells but did not alter the DNA conformation,and the punicalagin-induced DNA damage was independent of the production of reactive oxygen species.Punicalagin also enhanced the phosphorylation of DNA damage response-related protein ATM without affecting the phosphorylation of ATR.ATM inhibitor KU-55933 reversed the inhibitory effect of punicalagin on BCPAP cell viability,suggesting that ATM mediated punicalagin-induced BCPAP cell death.The final part mainly investigated the effect of punicalagin on BCPAP cell senescence and uncovered the related mechanisms.The results of cell particle diameter determination,cell granularity analysis,?-galactosidase activity detection,cell cycle detection and related protein levels detection indicated that punicalagin induced cell senescence in a dose-dependent manner.After analyzing the mRNA levels of senescence associated secretory phenotype IL-8,IL-6,IL-1?,we found that punicalagin dose-dependently upregulated the expression levels of IL-6 and IL-1? without inducing the expression of IL-8.Besides,punicalagin failed to induce endoplasmic reticulum stress compared to the positive control of thapsigargin.Further study demonstrated that punicalagin activated NF-?B pathway and positively regulated the expression of IL-6 and IL-1? via NF-?B.NF-?B also mediated punicalagin-induced BCPAP cell senescence and cell death.In conclusion,our study proved that punicalagin inhibits the survival of BCPAP cells through inducing autophagic cell death,DNA damage and cell senescence and MAPK,mTOR,ATM and NF-?B pathways are involved in the punicalagin-induced BCPAP cell death.Our results showed that punicalagin is promising to be an emerging drug for thyroid cancer therapy.