Study On The Anti-thyroid Cancer Effect And Mechanism Of Parthenolide

Object Thyroid carcinoma is common malignant tumor of head and neck,accounting for 5.11% of head and neck malignant tumors.Studies have shown that thyroid cancer includes thyroid papillary carcinoma(PTC),thyroid follicular carcinoma(FTC),poorly differentiated thyroid carcinoma(PDTC),undifferentiated thyroid carcinoma(ATC)and so on.About 95% thyroid cancers originate are from thyroid follicular epithelial cells.Thyroid papillary carcinoma is the most common,accounting for about 70-80% of all thyroid cancers.The survey showed that the prevalence of PTC has increased at an average rate of nearly 4% a year in recent years.Studies at home and abroad suggest that the majority of patients with new thyroid cancer are PTC,which is the main cause of the incidence of thyroid cancer.Studies have showed that postperative cervical lymph node metastasis rate of some patients with PTC is as high as 5.4-13%,although early surgery and chenoradiotheraph are effective.Therefore,to further strengthen the study of papillary thyroid carcinoma,active prevention and treatment of papillary thyroid carcinoma is an urgent task facing the medical community.Parthenolide(PTL)is a naturally occurring sesquiterpene lactone,extracted from the herbal plant feverfew(Tanacetum parthenium).PTL has been proved to exhibit anti-inflammatory,antioxidant and it always used to treat fever,headhedach,rheumatoid arthritis.Recently,studies have found that PTL could inhibit stats3 activity,inhibit NF-k B,mediate oxidative stress and inhibit mitochondrial activity,block cell cycle,inhibit cell proliferation and other mechanisms to play an anticancer role in various tumors such as liver cancer,ovarian cancer,breast cancer,and it has become one of the hot spots in anti-tumor drugs research.However,there are few reports about the anti-thyroid cancer effect of PTL.The mechanism of the anticancer treatment of thyroid cancer is not fully elucidated if PTL has anti-thyroid cancer effect.In this study,the effect and mechanism of the anti-thyroid mechanism of small white chrysanthemum were studied.Method In this study,human thyroid papillary carcinoma cells(TPC-1)were first cultured on RPMI-1640 medium,then different concentrations of Parthenolide(0,4,6,8,10,12 ?M)were added to the medium and continued to culture for 24,48 h.The value of the maximum half inhibition rate(IC50)was detected and calculated by using the MTS method.TPC-1 cells were treated with different concentrations of PTL in(0,4,8,10 ?M),and the apoptosis rates of TPC-1 cells were detected by Hoechst 33258 staining and Annexin V-FIC/PI double staining.JC-1 was applied to determine mitochondrial membrane potential(MMP)changes.Detection of cellular reactive oxygen species(ROS)was used DCFH-D fluorescence probe flow cytometry.Expression of Bcl2,Bax,related marker proteins of apoptosis was detected by Western blotting.The changes of endogenous metabolites in papillary thyroid carcinoma cells were analyzed by using metabonomics combined with multivariate statistical method,and the potential biomarkers of anti-thyroid carcinoma were found.This experiment examined the Nrf2-HO-1 pathway associated with cell metabolism,oxidative stress,ELISA was used to detect SOD,MDA and GSH,and Western blotting was used to detect Nrf2,HO-1 and NQO1 to further elucidate the mechanism of the anti-thyroid cancer effect of PTL.Results Part ?: 1.PTL had an inhibitory effect on thyroid cancer cell viabilityIn this study,MTS was used to detect the inhibitory of PTL on the thyroid carcinoma proliferation.We applied a series of concentrations(0,4,6,8,10,12 ?M)of PTL to TPC-1 cells for 24 or 48 h.Half maximal inhibtor concentration(IC50)value was estimated to be 8.42 ?M and 5.26 ?M at 24 h and 48 h,respectively.2.PTL induced apoptosis in TPC-1 cellsThe results of Hoechst 33258 staining showed that TPC-1 cells treated with different concentrations of PTL(4,8,10 ?M)were compared with those of control group(0.1% DMSO treated TPC-1 cells),TPC-1 cells showed bright fluorescence after Hoechst 33258 staining in different groups.It showed the characteristic of dense and concentrated stained cell apoptosis,and with the increase of drug concentration.The control group was observed fewer apoptotic cells with nuclear enrichment.Flow cytometry analysis showed that the apoptosis induced by PTL was significantly higher than that of the control group.the apoptosis rate induced by 10 ?M PTL at 24 h was 70.37%,whereas the normal cells were lower(3.34%).3.Effect of PTL on ROS in TPC-1 cellsIn comparison with the control group,treatment of TPC-1 cells with PTL at concentrations of 4 and 10 ?M increased the ROS levels to 44.51% and 70.44%,respectively.4.Effect of PTL on MMP in TPC-1 cellsTreatment of TPC-1 cells with PTL at concentrations of 4,8,10 ?M reduced the MMP levels.Compared with the control,the red-green fluorescence is reduced,thus suggesting a loss of membrane potential.5.Effect of PTL on Bcl-2 and Bax expression in TPC-1 cellsCompared with the control group,the expression of Bcl-2,anti-apoptosis related indexes,was decreased and the expression of Bax,apoptosis related indexes was increased with the increase of the concentration of PTL.Part ?: Cell metabonomics analysisThe metabolite of VIP>1.5 and P < 0.05 was used as the differential metabolite between the PTL group and the control group.These differential metabolites through HMDB and MTELIN database were searched for structural identification and naming.As a result,31 metabolic disorders were obtained,of which 23 increased,including Hydroxyglutaric acid,Nicotinamide,cytidine,5-diphosphocholine,pipecolic acid,L-pyroglutamic acid,N-phenylacetylglutamine,DL-sstachydrine,gamma-thiomethy 1 glutamate and so on,and 8 decreased,including Lysophosphatidylinositol,Uracil,?-aminoadipic,orotic acid,3-Hydroxypicolinic acid,Homo-L-arginine and so on.Feature selected by SAM were ploted as Cluster-heatmap using pearson distance.The identified biomarkers associated with anti-thyroid cancer of PTL were enriched by KEGG,and eight metabolic pathways were obtained :(a)Pyrimidine metabolism,(b)Cysteine and methioine metabolism,(c)Alanine,aspartate and glutamate metabolism,(d)Nitrogen metabolism,(e)Glycerphospholipid metabolism Part ?: 1,Effects of PTL on Oxidative Stress Related IndicatorsWe found an augmented level of MDA and reduced status of antioxidant enzymes(GSH and SOD)in PTL treated TPC-1 cells when evaluated to control cells.At the same time,these changes are related to the concentration of PTL.2,Effect on the expression of Nrf2/HO-1Compared with the control group,the expression of Nrf2,HO-I and NQO-1 related indexes,was decreased with the increase of the concentration of PTL.Conclusion In conclusion,this study showed that PTL could inhibit the growth of thyroid carcinoma TPC-1 cells,increase the content of ROS in mitochondria and induce apoptosis of TPC-1 cells.Through cell metabonomics analysis,the amino acid metabolism,choline metabolism and lipid metabolism of TPC-1 cells were changed after treated with PTL,which affected the energy metabolism and tricarboxylic acid cycle of the cells.Thus,the proliferation of tumor cells was inhibited and the apoptosis of tumor cells was promoted.PTL can play an anti-tumor role through multiple channels and targets.This study not only provides a new clue for the study of the anti-tumor effect of PTL from the perspective of metabonomics,but also can be used as an effective analytical method to study the mechanism of action of traditional Chinese medicine by cell metabolism method based on LC-MS/MS.By downregulating Nrf2,HO-1 expression,PTL reduced the activity of SOD,decreased the content of GSH,and increased the content of MDA.These results provide a new understanding of potential interventional strategy for PTL inducing of thyroid cancer's apoptosis through promoting oxidation activity in tumors.