Research On The Effect And Mechanism Of Bisphenol A And Estradiol On Papillary Thyroid Carcinoma

Thyroid cancer is one of the most common endocrine-related malignancies,and also is the fastest growing malignancy in the worldwide,with papillary thyroid carcinoma(PTC)accounting for more than 80%of them.Although PTC tumors are seen as slow-growing tumors,the primary treatment is partial or complete resection of the thyroid,followed by the elimination of residual thyroid cancer cell tissue with radioactive iodide(131I,RAI),which can cure most of the TC patient.Patients postoperative need thyroid hormone replacement therapy for life,about 20-30%of patients develop local recurrence or distant metastasis,especially when the tumor becomes demoneralized and lose the capacity of intake iodine,as well as distant metastasis occurs in the early stages of small papillary carcinoma,the risk of death greatly increased.Of course,there is a part of the tiny cancer that grows very slowly and may grow for many years without being found.At present,the pathogenesis of thyroid cancer is not clear,and actively looking for pathogenic cause and risk factors,in-depth study of its pathogenesis for prevention and treatment of the disease is essential.Studies have shown that estrogen on the occurrence and development of thyroid cancer plays an important role.Our previous study of the group also showed that estrogenic activity of endocrine disrupting bisphenol A can also affect the proliferation of thyroid cancer cells.But what is the effect of estrogen and bisphenol A on migration and cell cycle of thyroid papillary carcinoma?What does this role play through the receptor and signaling pathways?Are there any differences in the role of the two?These questions are our interest.The results suggest that the relationship between bisphenol A and estradiol and thyroid cancer will provide a theoretical basis for the prevention and treatment of the disease.Part Ⅰ Effects of bisphenol A and estradiol on the proliferation of papillary thyroid carcinoma cells BackgroundThe international epidemiological survey showed that the prevalence of papillary thyroid cancer in women of childbearing age higher than the same age men 3-4 times,indicating that the occurrence of thyroid cancer may be affected by female sex hormones,especially estrogen.The pathogenesis of Thyroid tumor is complex,common causes may be related to family history,iodine intake,ionizing radiation and BRAF,RET,NTRK gene mutation.The history of radiation exposure and thyroid cancer family history,as well as BRAF,RET/PTC and NTRK which are seen as common somatic mutations can not explain the significant gender differences in the incidence of thyroid cancer,Iodine is an important component of thyroid hormone synthesis,and lack of it can lead to hypothyroidism,low intelligence and short stature.At present,it is generally believed that the pathological type of thyroid cancer is related to the intake of iodine.High iodine intake can induce the thyroid tissue of the rat to develop papillary thyroid carcinoma but this also does not explain the increase in the number of women with thyroid cancer,and the involvement of sex hormones in the occurrence of thyroid papillary carcinoma appears to be a reasonable hypothesis.The previous studies have made it clear that estrogen can affect cancer cell growth and is considered susceptible to thyroid cancer due to prolonged exposure to a higher estrogen level in the female menstrual cycle,during pregnancy and breastfeeding.17β-estradiol(E2)is one of the most potent estrogens in humans and has a high affinity for estrogen receptors present in benign and malignant thyroid cells.E2 also promotes estrogen target organs such as breast and endometrial cancerous lesions.Studies have shown that breast cancer patients with a higher incidence of thyroid nodular swelling,indicating that estrogen-related organs,there is a co-stimulatory factor involved in the pathological process of thyroid tumors.With the widespread use of environmental estrogens in people’s daily life,the impact of environmental factors on thyroid tumors has received increasing attention.Endocrine disrupting substances(EDCs)with estrogenic properties may also be involved in the development of thyroid cancer.Bisphenol A(BPA)is a representative of environmental endocrine disruptors,composed of polycarbonate plastic and epoxy resin,and is present in thousands of consumer products such as plastics,pesticides,pharmaceuticals,detergents and cosmetics.Animal studies have shown that BPA can compete with endogenous hormones receptor to interfere with endocrine signaling pathways that disrupt the synthesis and secretion of endogenous hormones in humans and other organisms and simulate endogenous hormones,destroy the normal function of the endocrine system,interfere with the normal cell differentiation and embryonic development.Since BPA is structurally similar to E2,it may compete with genomic and non-genomic estrogen receptors through estrogen-dependent pathways that mediate the development of estrogen-responsive organ cancer and promote the proliferation and migration of cancer cells in mammary glands and ovaries.There is little research on the relationship between bisphenol A and thyroid cancer.Therefore,this study focuses on elucidating the role of BPA with estrogenic activity in papillary thyroid carcinoma and how to promote the proliferation of thyroid cancer cells,and the differences between BPA and E2 to promote estrogen receptor expression,explore the exact role of estrogen receptor in the pathogenesis of thyroid cancer and provide theoretical basis for non-operative treatment of thyroid papillary carcinoma.Purpose:1.To investigate the effects of different concentrations of bisphenol A and estradiol on the proliferation of papillary thyroid carcinoma cells(BHP10-3).2.To clear the effects of the optimal concentration of bisphenol A and estradiol on the migration and cell cycle of BHP10-3 cells.3.To detect whether the proliferative effect of bisphenol A and estradiol on BHP10-3 cells is based on the expression of estrogen receptor(ERa/ERβ)and membrane receptor(GPR30).Method:1.(BHP10-3)were treated with different concentrations of bisphenol A(10-3-10-8M)and estradiol(10-4-10-9M)at different time points(24h,48h,72h)The proliferation of BHP10-3 cells was detected by CCK-8 assay.The cell growth curve was observed and the number of viable cells in BHP10-3 cells was measured to determine the optimal concentration and time of BP A and E2 which are promote cell proliferation.2.The effect of bisphenol A and estradiol on the BHP10-3 cells migration was evaluated by cell scratches.3.Using the optimal concentration of bisphenol A and estradiol interferd with BHP10-3 cells,at different time points(24 h,48 h,72 h),respectively,using flow cytometry to detect cellular DNA content and calculate cell proliferation index,analysis with the influence of BPA and E2 on cell cycle phase;4.The expression and location of estrogen receptor(ERα/ERP)and membrane receptor(GPR30)in human papillary thyroid carcinoma BHP10-3 cells were determined by immunofluorescence.Result:1.(BHP10-3)was stimulated with different concentrations of bisphenol A(BPA)and estradiol(E2),BPA and E2 stimulated cells in a dose-dependent manner,but produced a U-shaped dose-response curve,which was considered "nonmonotonic".2.Cell scratch test showed that BHP10-3 cells under the action of bisphenol A and estradiol,the capacity of cell movement and migration were increased.3.Flow cytometry showed that bisphenol A and estradiol promoted the progress of BHP10-3 cells from GO/G1 phase to S phase,and 24h is the best time.4.Immunofluorescent labeling and confocal microscopy revealed that ERa,ERβ,and GPR30 co-expressioned in BHP10-3 thyroid cancer cells(green for GPR30 and ERa and red for ERP).ERa was mainly observed within the nucleus,and some on the nuclear membrane,while ERβ was located thoroughly within the nucleus.The estrogen membrane receptor GPR30 was mainly distributed in the cell membrane,partially concentrated in the cytoplasm or endoplasmic reticulum,exposure to BPA and E2 increased the specific immunofluorescence density of ERa,GPR30,and ERβwithin 24 h.Conclusion:1.Bisphenol A has significant estrogenic activity,low doses of bisphenol A(BPA,10-7M)and estradiol(E2,10-8M)can play a significant biological activity to promote thyroid cancer cells proliferation,migration.2.Estrogen receptor ERa,ERβ and GPR30 were co-expressed in human papillary thyroid carcinoma cells(BHP10-3),Bisphenol A and estradiol promoted the proliferation of papillary thyroid carcinoma cells(BHP10-3)by estrogen receptor-dependent pathway.Part Ⅱ Effects of bisphenol and estradiol on mERα and GPR30 induced in papillary thyroid carcinoma and its effect on AKT/mTOR pathway BackgroundThe PI3K/Akt/mTOR pathway has long been recognized as a basic intracellular signaling pathway involved in cell physiology and pathology,controlling cell growth in vivo,proliferation and survival,cellular metabolism and autophagy.The role of PI3K/Akt pathway in carcinogenesis has been extensively explored and reported,and recent studies have confirmed that this pathway is closely related to TC,especially with the pathogenesis of FTC and ATC.The activation of the PI3K/Akt/mTOR pathway has multiple ways,and more and more subclinical or clinical data support this pathway may represent a potential target for the treatment of TC,whereas PI3K/Akt/mTOR has been successful for oral administration and treatment of a’variety of malignant tumors,is gradually being widely used.Especially when thyroid tumors are resistant to conventional radioactive iodine therapy,that is,the treatment of refractory thyroid cancer,the efforts are needed to determine the possible therapeutic goals in these pathways.Studies have shown that the development of estrogen-induced estrogen-responsive organ malignancies may be due to activation of PI3K/Akt/mTOR pathway by binding to ERs.Due to the high incidence of papillary thyroid tumors and significant gender differences,we hypothesized that estrogen receptors are involved in its pathogenesis,and it is not clear which specific receptors are involved in the activation of the PI3K/Akt/mTOR pathway.Since BPA is structurally similar to E2,it is also possible to activate the pathway via the estrogen receptor-dependent pathway.Whether the use of a single target or dual receptor inhibitor can inhibit the activation of the pathway is also the focus of this paper.Purpose:1.To test the changes of estrogen-related receptor(ERa,ERβand GPR30)gene and protein expression in BHP10-3 cells treated with bisphenol A(10-7M)and estradiol(10-8M);2.To clarify the role of PI3K/Akt/mTOR pathway in the pathogenesis of papillary thyroid carcinoma and to determine whether bisphenol A is similar to estradiol to activate PI3K/Akt/mTOR pathway;3.To investigate whether selective estrogen receptor antagonist ICI182,780(10-6M)and specific GPR30 antagonist G15(10-8M)can block the activation of PI3K/Akt/mTOR(PAM)pathway in BHP10-3 cells by inhibiting the expression of estrogen receptor;4.Comparison of the role of bisphenol A and E2 in promoting tumor progression.Method:1.To detect the change of gene and protein of estrogen-related receptor ERα,ERβ,GPR30 in human papillary thyroid carcinoma cells(BHP10-3)by real-time PCR and Western blotting,which are treated with bisphenol A(BPA)and estradiol(E2)at 24 h or 48 h.2.Real-time PCR and Western blot were used to verify the change of estrogen-related receptor(ERα,ERβ,GPR30)in BHP10-3 cells after pretreated by estrogen receptor antagonist ICI(10-6M)and G15(10-8M)for 1h.3.BHP10-3 cells were treated with the best concentration of BPA(10’7M)and E2(-10-8M)at different time points(0,5,15,30,60,90min).Western blot was used to detect Akt/mTOR molecule phosphorylation level,and to clear the time point which was reached the maximum effect.4.Pretreatment BHP10-3 cells with estrogen receptor-related antagonists ICI(10-6M)and G15(10-8M)for 1 h.then stimulated the cells with BPA and E2 for a certain period of time and detected AKT/mTOR phosphorylation levels by Western blot technique.Result:1.The results showed that bisphenol A(10-7M)and estradiol(10-8M)could significantly enhanced the expression of ERa,ERβ and GPR30 in BHP 10-3 cells.2.After pretreatment of BHP 10-3 cells with selective estrogen receptor antagonist ICI(10-6M),ICI showed the ability that could inhibit the expression of ERa protein and gene induced by BPA and E2,and selective inhibited the level of E2-induced ERβ protein or gene,but the effect on ERβ protein and gene stimulated by BPA was uncertain.3.In the absence of BPA and E2 interference,ICI could enhance the expression of GPR30 protein and gene in BHP10-3 cells.However,the specific estrogen receptor antagonist G15(10-8M)cound significantly inhibited GPR30 level stimulated by E2,but had no significant effect on BPA.4.Specific estrogen receptor agonist G1(10-8M)acts similarly to BPA or E2,specifically enhancing GPR30 expression.5.Bisphenol A(10-7M)and estradiol(10-8M)could rapidly stimulate the phosphorylation of Akt/mTOR in thyroid papillary carcinoma(BHP10-3),the best time is 30 minutes.6.BPA and E2 could significantly increased the level of p-Akt/p-mTOR,and pretreated with G15 in BHP10-3 cells for 1h,the levels of p-Akt and p-mTOR protein in E2 group were significantly decreased,that is,G15 could block E2 the effect on the expression of p-Akt/p-mTOR,but for the BPA no effect.Pretreatment with ICI +G15 in BHP10-3 cells could further enhance the inhibitory effect than G15 alone,But the inhibitory effect on BPA is still not obvious.7.Specific estrogen receptor agonist G1 similar to the role of BPA or E2 could activate Akt/mTOR pathway,enhanced Akt/mTOR protein phosphorylation levels.8.There was no significant difference between BPA and E2 for the expression of ERa,ERβ and GPR30.Conclusion:1.PI3K/Akt/mTOR signaling pathway involved in the development of thyroid papillary carcinoma,and promote the growth of tumor cells.2.Activation of the Akt/mTOR pathway may be based on overexpression of the upstream estrogen receptor.3.ICI can significantly inhibit the expression of ERa in BHP10-3 induced by BPA and E2.Because of the different roles of ERa and ERβ,ICI is used alone,and its antagonism is uncertain.4.G-15 can significantly inhibit the expression of E2-induced estrogen receptor and downstream pathway molecules,so as to achieve anti-tumor cell proliferation,but have no inhibition to BPA;ICI and G15 are synergistic for E2-induced expression of GPR30 and downstream pathway molecules.5.BP A and E2 similar to the role of specific agonist G1 can significantly promote the proliferation of thyroid papillary carcinoma cells,and there is no significant difference between them.6.BPA can promote the progression of papillary thyroid tumors by genomic or non-genomic pathways,but the way of activating the estrogen receptors and downstream pathway is not exactly the same as E2,which may not be completely mediated by estrogen-dependent receptors pathway.