Effect Of Digitoxin On Expression Of NIS Protein And Autophagy-related Proteins In Papillary Thyroid Carcinoma Of Nude Mice

ObjectiveThyroid cancer(TC)is the most common endocrine malignant tumor.The incidence of thyroid carcinoma has been rising in recent years.The most common pathological classification is Papillary Thyroid cancer(PTC).Through Surgical treatment combined with radioactive iodine(RAI)treatment and TSH suppression treatment,most patients have significant effects.However,some patients have recurrence or distant metastasis due to tumor dedifferentiation.And the efficacy of RAI treatment has weakened or even disappeared.The main reason is reduced or absent expression of sodium/iodide symporter(NIS)protein.Promoting thyroid cancer redifferentiation and restoring its iodine uptake ability is a potential therapeutic strategy.Recent studies have shown that autophagy plays an important role in the dedifferentiation of thyroid cancer.Cardiac glycosides(CGs)can induce autophagy.Cardiac glycosides can inhibit the proliferation of tumor cells in vitro,but the specific mechanism is still unclear.In this article,cardiac glycosides digitoxin was applied to nude mice with papillary thyroid carcinoma to study its effect on the expression of autophagy-related proteins Beclin-1,LC-3,and NIS in cancer tissues.To explore the possible mechanism of autophagy induced by digitoxin.The aim is to further verify the anticancer effect of digitoxin in vivo.MethodsAfter nude mice models were established,they were divided into 5 groups: blank control group(normal saline),positive control group(rapamycin,1.5mg/kg),low,medium and high dose groups of digitoxin(Respectively,0.2mg/kg,1mg/kg,5mg/kg),with 8 nude mice in each group.Intraperitoneal injection was performed on every other day.After 15 days of administration,tumor volume was analyzed.Then tumor was removed.The digitoxin group with strong antitumor effect and moderate dose was selected as experimental group.Hematoxylin-eosin staining(HE stain),Western blot detection and Real-time fluorescence quantification PCR were performed in blank control group,positive control group and digitoxin group.The expression of NIS,Beclin-1,LC-3 protein and m RNA in each group of papillary thyroid carcinoma tissues were detected.The phosphorylation level of PI3K/ AKT/ mTOR pathway proteins in each group of papillary thyroid carcinoma tissues was detected.ResultsObserving the tumor volume of nude mice,it was found that after 15 days of administration,compared with blank control group,the tumor volume was significantly decreased in positive control group,low dose group of digitoxin,medium dose group of digitoxin and high dose group of digitoxin(P < 0.01).Compared with positive control group,the tumor volume of nude mice was increased in low dose group of digitoxin,but there was no statistical significance(P >0.05),and the tumor volume of nude mice was significantly decreased in medium dose group of digitoxin and high dose group of digitoxin(P < 0.01).Compared with low dose group of digitoxin,the tumor volume of nude mice was significantly decreased in medium dose group of digitoxin and high dose group of digitoxin(P < 0.01).Compared with medium dose group of digitoxin,the tumor volume of nude mice was decreased in high dose group of digitoxin.But there was no statistical significance(P>0.05).HE stainIn some areas of digitoxin group and positive control group,The cytoplasm of tumor cells was lightly stained.The tumor cells’ nuclei was lightly stained and shrunk.The nuclear edge was not clear.The ratio of nucleus to cytoplasm was decreased.Degeneration was observed in some areas of digitoxin group and positive control group.Western blotCompared with blank control group,the relative expression of NIS,Beclin-1,and LC-3 protein were significantly increased in digitoxin group(P <0.01).Compared with blank control group,the relative expression of Beclin-1 and LC-3 protein were increased(P <0.05),and the relative expression of NIS protein was significantly increased in positive control group(P <0.01).Compared with positive control group,the relative expression of NIS,Beclin-1,and LC-3 protein were significantly increased in digitoxin group(P <0.01).Real-time fluorescence quantification PCRCompared with blank control group,the relative expression of NIS m RNA,Beclin-1 m RNA,and LC-3 m RNA were significantly increased in digitoxin group and positive control group(P <0.01).Compared with positive control group,the relative expression of NIS m RNA,Beclin-1 m RNA,and LC-3 m RNA were significantly increased in digitoxin group(P <0.01).The autophagy-related pathway PI3 K/AKT/mTOR phosphorylation levelCompared with blank control group,the ratios of p-PI3 K/PI3 K,p-AKT/AKT,and pmTOR/mTOR were significantly decreased in digitoxin group and positive control group(P <0.01).Compared with positive control group,the ratios of p-PI3 K/PI3 K,p-AKT/AKT,and p-mTOR/mTOR were significantly decreased in digitoxin group(P <0.01).ConclusionsDigitoxin can inhibit the growth of papillary thyroid carcinoma tissues in nude mice.Digitoxin can increase the expression levels of NIS protein and m RNA related to iodine metabolism in papillary thyroid carcinoma tissues of nude mice,and can also increase the expression levels of Beclin-1,LC-3 protein and m RNA related to autophagy.Digitoxin can inhibit the autophagy-related PI3 K/AKT/mTOR signaling pathway.