| Background: thyroid cancers are the most common endocrine malignant tumor,and their incidence has been increasing in recent years.Thyroid cancers include differentiated thyroid cancer(>95%)(papillary thyroid carcinoma,follicular thyroid carcinoma)and anaplastic thyroid cancer.Anaplastic thyroid cancer accounts for less than 5 percent of clinically diagnosed thyroid malignant cancers,but accounts for more than half of thyroid-cancer deaths,with a mortality rate of over 90 percent and only 6months of mean survival time after diagnosis.Currently,the main treatment methods for thyroid cancers include surgical resection,radioactive iodine ablation of residual tissue and chemotherapy,but due to the dedifferentiated phenotype and high invasiveness of anaplastic thyroid cancer,it's efficacy and prognosis are poor.Therefore,an effective chemotherapy drug with low toxicity is urgently needed to improve this situation.Resveratrol,a natural compound found in grapes,peanuts,berries and tea,has a wide range of biological properties,including anti-glycation,anti-oxidation,anti-inflammation,neuroprotection,anti-cancer and anti-aging.A large number of studies have shown that resveratrol has a clear anti-tumor effect and has no obvious toxicity and side effects compared with commonly used chemotherapy drugs(such as docetaxel and cisplatin).In early research results of our laboratory,after resveratrol treatment of three cell lines of human anaplastic thyroid cancer(THJ-16 T,THJ-21 T and THJ-11T)found that resveratrol can inhibit the proliferation of the cell line(THJ-16 T and THJ-21T)of human anaplastic thyroid cancer and accelerate it's apoptosis excluding THJ-11 T,also a cell line of human anaplastic thyroid cancer.A large number of studies have shown that STAT3 signaling pathway is one of the targets of resveratrol.To explore the causes of sensibility differences of the three cell lines of human anaplastic thyroid cancer to resveratrol,we tested the effect of resveratrol to STAT3 signaling pathway's activity in these three cell lines,found that resveratrol can inhibit STAT3 phosphorylation in the cells of THJ-16 T and THJ-21 T,but increase the STAT3 phosphorylation in THJ-11 T cells.Therefore,we speculated that the abnormal activation of STAT3 might be the cause of resveratrol-resistance of human anaplastic thyroid cancer THJ-11 T cells.The aim of this study is to elucidate the mechanism of resveratrol resistance in human anaplastic thyroid cancer THJ-11 T cell line,and to further explore the reasons for the activation of the STAT3 signaling pathway by resveratrol.Methods: Human anaplastic thyroid cancer THJ-11 T cell line was formed in 2010.The cell line was transferred by authorization of professor Liu Qiang,Cancer Stem Cell Research Institute,Dalian Medical University.In this study,the human anaplastic thyroid cancer THJ-11 T cell line(resveratrol-resistance)was selected and treated with resveratrol(100?M)?AG490(80?M,JAK2/STAT3 signaling pathway specific inhibitors)alone or in combination respectively.The mechanism of resveratrol-resistance of THJ-11 T cell line of human anaplastic thyroid cancer was elucidated through the following biological methods:(1)proliferation of THJ-11 T cells was detected by MTT experience and cell morphology was observed by H/E staining.(2)TUNEL staining and Flow Cytometry are used to detection apoptosis.(3)RT-PCR,immunocytochemical staining and Western blot were used to detect the phosphorylation level of STAT3,the expression of the upstream activators(IL-6,LIF)of STAT3 signaling pathway,negative regulators(SOCS3,PIAS3)and their downstream target genes(survivin,Bcl-2),after treatment with different drugs.Results:(1)MTT results showed that there was no significant difference in the OD value of THJ-11 T cell line treated with resveratrol at 24 h and 48 h compared with the normal group(P>0.05).The OD value of THJ-11 T cells treated with AG490 decreased(P<0.05).However,the OD value of THJ-11 T cells treated with resveratrol combined with AG490 showed a decreasing trend at 24 h and 48 h,and the decrease was more obvious at 48h(P<0.01).H/E staining showed that after AG490 was treated with THJ-11 T cells alone and in combination with resveratrol,the number of cells decreased significantly and some cells shrank.(2)TUNEL and Flow Cytometry showed that THJ-11 T cells were not significantly apoptotic after being treated with resveratrol alone for 48h(9.57%).THJ-11 T cells were treated with AG490 alone for 48 h,and partial apoptosis occurred,with a apoptosis rate of 27.93%.However,the combined treatment showed significant apoptosis,and the apoptosis rate increased to 64.07%.(3)RT-PCR,immunocytochemical staining and Western blot results showed that after 48 h of resveratrol treatment of THJ-11 T cells,the phosphorylation of STAT3(p-STAT3)increased significantly,especially in the nucleus,while the phosphorylation of STAT3 in AG490 alone or in combination significantly decreased.However,resveratrol did not cause further increase of survivin and Bcl-2 levels of its downstream target genes,which may be related to the initial high expression level of survivin and Bcl-2 in THJ-11 T cells,while the expression level of survivin and Bcl-2 in AG490 alone or in combination with resveratrol was significantly decreased compare with normal group and resveratol-treated group.(4)To explore the mechanism of abnormal activation of STAT3 after resveratrol treatment of THJ-11 T cells,we detected the expression of the upstream activators(IL-6 and LIF)and their negative regulators(SOCS3 and PIAS3)in the STAT3 signal transduction pathway.The results showed that the expression levels of LIF and IL-6 increased after resveratrol treatment of THJ-11 T cells,and the expression levels of SOCS3 decreased,while the expression levels of PIAS3 did not change significantly.Conclusions:(1)resveratrol neither can inhibit the proliferation of human anaplastic thyroid cancer THJ-11 T cells nor promote their apoptosis;(2)JAK2/ STAT3-specific inhibitor AG490 can inhibit the proliferation and promote the apoptosis of THJ-11 T cells;(3)resveratrol activates the STAT3 signaling pathway in human anaplastic thyroid cancer THJ-11 T cell line;(4)the increased expression levels of IL-6 and LIF,the upstream activators of STAT3 signal transduction pathway,and the decreased expression levels of SOCS3,the negative regulator,may be important factors for the activation of STAT3 signaling pathway caused by resveratrol.(5)activation of STAT3 is the key mechanism of resveratrol resistance human anaplastic thyroid cancer THJ-11 T cells,and AG490 can reverse the resistance of THJ-11 T cells to resveratrol. |
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