Study On Mechanism Of Morusin-induced Apoptosis Of Papillary Thyroid Cancer TPC-1 Cells

Background:Thyroid cancer（TC）is a disease arising from the variation of follicular epithelial cells or parafollicular C cells,and is the most common tumor in the endocrine field.Follicular cell-derived TCs are divided into four histological types:papillary thyroid cancer（PTC,80%～85%）,follicular thyroid cancer（10%～15%）,poorly differentiated thyroid cancer（<2%）and anaplastic thyroid cancer（<2%）.Although the incidence of PTC is increasing year by year,it is an indolent tumor clinically.Surgical resection and postoperative thyroid-stimulating hormone suppression therapy or radioactive iodine therapy are generally adopted.Although most PTCs have a good prognosis and low rates of local invasion,recurrence,or metastasis,long-term follow-up found that after conventional treatment,many adverse reactions occurred,which reduced the quality of life of PTC patients.Data have shown that there is a small proportion of patients with heterogeneous cancer tissue and more aggressive mutations,resulting in more than 25%of PTC patients recurring during follow-up.Therefore,it is of great significance to seek new therapeutic strategies for PTC.Flavonoids are a kind of natural polyphenols that widely exist in the plant kingdom,and have attracted extensive attention due to their various biological activities such as anti-inflammatory and anti-tumor.Morusin,a typical prenylated flavonoid,has been shown to exhibit anticancer effects on various types of tumor cells.It has been reported that morusin can exert anti-cancer activity against renal cell carcinoma by activating the MAPK signaling pathway;another study has reported that morusin can induce apoptosis of lung cancer cells by increasing intracellular ROS levels and inhibiting PI3K/Akt pathway.However,the effect of morusin on the apoptosis of PTC cells and its mechanism remain to be studied.In this study,the PTC cell line TPC-1 was used as the research object to investigate the effect of morusin on the apoptosis of TPC-1 cells,and further analyze its mechanism of action,in order to provide potential new drugs for the clinical treatment of PTC.Objective:This paper mainly explores the effects of morusin on apoptosis of papillary thyroid cancer（PTC）cells.To explore whether its mechanism is related to ROS-mediated mitochondrial apoptosis pathway.Method:Using the research methods of cell experiments in vitro,TPC-1 cells were treated with different concentrations（0,2.5,5,10,20,40,80μM）of morusin for24 h and 48 h,and the cell proliferation activity was detected by MTT and the IC₅₀value was calculated;TPC-1 cells were treated with different concentrations（0,5,10,20μM）of morusin or 20μM morusin combined with 5 m M reactive oxygen species（ROS）scavenger NAC for 24 h,and Hoechst33258 staining was used to observe the nuclear morphological changes;Annexin V-FITC/PI method was used to detect the apoptosis of PTC-1 cells;The levels of MDA,SOD and GSH-Px in cells was detected by colorimetric method;The level of ROS in cells was detected by DCFH-DA staining method;The change of mitochondrial membrane potential in cells was detected by JC-1 staining method;Western blot to detect the expression levels of apoptosis-related proteins Bax,Bcl-2,cleaved-caspase-3 and p38MAPK/p53axis-related proteins p-p38MAPK（Thr180/Thr182）,p38MAPK,MDM2,p53 and PUMA,as well as the expression level of Cyt C in cytoplasm and mitochondria;Finally,all the data were performed with SPSS 22.0 statistical software,and P<0.05was regarded as a statistically significant difference.Result:1.MTT test results showed that the proliferation activity of TPC-1 cells was gradually decreased after the treatment of morusin,and with the prolongation of the drug intervention time,the cell proliferation activity was gradually decreased,the difference was statistically significant(F_time=129.653,P<0.001;F_{concentration}=664.610,P<0.001;F_{time×concentration}=12.704,P<0.001);In addition,the IC₅₀values of TPC-1 cells treated with different concentrations of morcine for 24 h and 48 h were 24.523μM and 13.135μM,respectively.2.Hoechst33258 staining showed that after treatment with different concentrations（0,5,10,20μM）of morusin for 24 h,the number of cells with dense nuclear staining in TPC-1 cells were gradually increased,indicating morphological changes of apoptosis.3.Flow cytometry with Annexin V-FITC/PI showed that the apoptosis rate of TPC-1 cells was gradually increased after treatment with different concentrations（0,5,10,20μM）of morusin for 24 h,and the difference was statistically significant（P<0.05）.While the apoptosis rate of TPC-1 cells was significantly decreased after combined treatment with NAC（P<0.05）.4.The results of colorimetric method showed that after treatment with different concentrations（0,5,10,and 20μM）of morusin for 24 h,the content of lipid peroxide MDA in TPC-1 cells was gradually increased（P<0.05）,while the activities of antioxidant indexes SOD and GSH-Px were gradually decreased,but the MDA in TPC-1 cells was significantly decreased after NAC intervention（P<0.05）,while the SOD and GSH-Px activities was significantly increased（P<0.05）.5.The results of DCFH-DA staining showed that the ROS level was significantly increased in TPC-1 cells after treatment with different concentrations（0,5,10,and 20μM）of morusin for 24 h（P<0.05）,while the ROS level was significantly decreased in TPC-1 cells after the combined NAC intervention（P<0.05）.6.JC-1 staining showed that the mitochondrial membrane potential was significantly decreased after treatment with different concentrations（0,5,10,and 20μM）of morusin for 24 h（P<0.05）,while the mitochondrial membrane potential was significantly increased in TPC-1 cells after combined NAC intervention（P<0.05）.7.Western blot results showed that after intervention with different concentrations（0,5,10,20μM）of morusin for 24 hours,the protein expression levels of Bax,cleaved-caspase-3,p-p38MAPK/p38MAPK ratio,p53 and PUMA in TPC-1 cells were gradually increased（P<0.05）,while the protein expression levels of Bcl-2 and MDM2 were gradually decreased（P<0.05）.However,the protein expression levels of Bax,cleaved-caspase-3,p-p38MAPK/p38MAPK ratio,p53 and PUMA were significantly decreased in TPC-1 cells after combined NAC intervention（P<0.05）,the protein expression levels of Bcl-2 and MDM2 protein were significantly increased（P<0.05）.Conclusion:1.Morusin can induce the apoptosis of papillary thyroid carcinoma TPC-1 cells;2.Morusin can promote the production of endogenous ROS in TPC-1cells,and then directly or indirectly activate the mitochondrial apoptosis pathway through the p38MAPK/MDM2/p53 axis to induce the apoptosis of TPC-1 cells.