Effect Of Chidamide On Development And Chemotherapy Sensitization Of Anaplastic Thyroid Carcinoma

Thyroid cancer is one of the most common endocrine malignancies,with the fastest incidence growth in solid tumors.Anaplastic thyroid carcinoma(ATC),an undifferentiated thyroid cancer subtype,is rare,aggressive,rapidly progressing and fatal,which accounts for only 1 to 2 percent of thyroid cancers.However,ATC is considered the most deadly form of thyroid cancer,and its related deaths accounts for 38 to 50 percent of all thyroid cancer ones.ATC patients’ median survival is poor(3-10 months).During the development of ATC,it can rapidly invade the important nerve vessels and viscera of human,which causes a great challenge for ATC treatment.So far,there has been no treatment model which can significantly change the progression or prognosis of the disease.Therefore,there is an urgent need to develop specific treatment options for ATC.Patients with ATC have poor prognosis and respond poorly to conventional treatment.Although some patients may respond or remain stable,single-drug chemotherapy is not very effective.Combination of radiotherapy and other drugs may be more effective for treatment of ATC.Currently,doxorubicin is the only FDA approved drug for ATC.Doxorubicin,a DNA topoisomerase II inhibitor,belongs to the anthracycline family of anticancer drugs and is used to treat a variety of solid and hematologic malignancies.However,the reduced sensitivity of cancer cells to doxorubicin has become a major obstacle to the treatment of ATC.At present,the roles of cancer cell proliferation,autophagy and apoptosis in tumor sensitivity to adriamycin and their mechanisms have become one of the research focuses.It is a promising therapeutic strategy to reverse adriamycin resistance by regulating cell functions such as proliferation and autophagy.Understanding the mechanism of drug sensitivity regulation and developing molecular targeted modulators will provide a promising therapeutic strategy for overcoming adriamycin resistance in cancer treatment.Chidamide(CS055/HBI-8000)is a new type of innovative drugs of China’s independent research and development,which aims at to selective inhibition of activity of histone acetylation enzyme HDAC1,2,3,10.Approved by China food and drug administration(CFDA)in December 2014,chidamide could be used to treat recurrent or refractory Peripheral t-cell lymphoma(PTCL).According to the research,chidamide has the therapeutic effect on a variety of solid tumors such as lung cancer,pancreatic cancer,liver cancer and refractory blood malignancy.However,it is still not clear whether chidamide can treat ATC,whether chidamide combined with adriamycin can be used to treat ATC,and its potential molecular mechanism.The exploration of those problems is of great significance for the formulation of effective treatment methods for ATC.The purpose of this topic is to explore the effects of chidamide on development and chemotherapy sensitization of ATC.First of all in vitro MTT,flow cytometry assay,scratch assay,Transwell assay,ChanDan sulfonyl cadaverine(MDC)fluorescent staining assay were determined to detect the effect of chidamide/adriamycin treatment separately and combined treatment on ATC cells proliferation,apoptosis,migration,invasion and autophagosome.qRT-PCR and Western blot assays were carried out to test the effect of chidamide/adriamycin treatment separately and combined treatment on the expression of autophagy related gene and protein in ATC cells;Secondly,8505c cells were injected into mouse to construct ATC nude mouse transplantation tumor models.The effect of chidamide/adriamycin treatment separately and combined treatment on the ATC transplantation tumor growth was analyzed.HE staining was determined to detect the effect of chidamide/adriamycin treatment separately and combined treatment on the histologic morphology of the ATC transplanted tumor.qRT-PCR,Western blot and immunohistochemistry assays were performed to detect the effect of chidamide/adriamycin treatment separately and combined treatment on the expression of autophagy-related genes and proteins such as BECN1,Atg5,LC3-II and LC3-I in ATC transplanted tumors;In addition,in order to study the mechanism of the effect of chidamide combined with adriamycin on ATC,Western blot was performed to detect the effect of chidamide/adriamycin treatment separately and combined treatment on the expression of related molecules in the PI3K-AKT-mTOR signaling pathway in ATC cells and ATC transplanted tumors.This paper is divided into three parts:Part I:Effect of chidamide on biological function of adriamycin-mediated ATC cells;Part II:Effect of chidamide on tumor growth and adriamycin sensitivity in ATC nude mice;Part III:Study on mechanism of inhibitory roles of chidamide combined with Adriamycin in ATC.main content:Part I:Effect of chidamide on biological function of adriamycin-mediated ATC cellsMethods1.The effect of chidamide combined with adriamycin on proliferation of 8505C and HTh74 cells was determined by MTT assay.2.The effect of chidamide combined with adriamycin on apoptosis of 8505C and HTh74 cells was detected by Flow cytometry assay.3.The effect of chidamide combined with adriamycin on the migration of 8505C and HTh74 cells was detected by scratch experiment.4.The effect of chidamide combined with adriamycin on invasion of 8505C and HTh74 cells was detected by Transwell assay.5.The effect of chidamide combined with adriamycin on the formation of autophagosomes in 8505C and HTh74 cells was observed by MDC fluorescence staining.6.The effect of chidamide combined with adriamycin on the expression of autophagy related genes BECN1 and Atg5 in 8505C and HTh74 cells was measured by qRT-PCR.7.The effect of chidamide combined with adriamycin on was used to the effect of sidabemine on the expression of autophagy related proteins BECN1,Atg5,LC3-Iand LC3-Ⅱ in 8505C and HTh74 cells was detected by Western blot.ResultsAfter treatment with adriamycin or/and chidamide,the proliferation,invasion and migration of ATC cells were significantly inhibited,and apoptosis and autophagy was promoted.Compared with the doxorubicin or citabemine treated group alone,the combination treatment singificantly suppressed proliferation,invasion,migration of ATC cells and promoted apoptosis and autophagy.Part II:Effect of chidamide on transplanted tumor formation and adriamycin sensitivity in ATC nude miceMethods1.8505C cells was injected into back of nude mice to build ATC transplantation tumor,and the mice were treated with chidamide or/and adriamycin,then transplanted tumors volume was regularly detected,and mice were euthanized at day 30,and the weight of the transplanted tumor was determined.2.HE staining was performed to detect the effect of chidamide combined with adriamycin on tissue morphology of ATC transplanted tumor.3.qRT-PCR was performed to detect the effect of chidamide combined with adriamycin on BECN1 and Atg5 mRNA expressions in ATC transplanted tumors.4.Western blot was carried out to determine the effect of chidamide combined with adriamycin on the expression of BECN1,Atg5,LC3-I and LC3-II proteins in ATC transplanted tumors.5.Immunohistochemical assay was carried out to detect the effect of chidamide combined with adriamycin on the expression of BECN1,Atg5,LC3-I and LC3-II proteins in ATC transplanted tumors.Results1.Compared with the control group,after treatment with adriamycin or chidamide,the growth of ATC transplanted tumor was significantly inhibited,and the volume and weight were significantly reduced.Compared with the groups treated with adriamycin or chidamide alone,the tumor volume and weight of ATC transplantation after treatment with adriamycin and chidamide were significantly decreased.2.HE staining results showed that,compared with the control group,the malignant degree of ATC transplanted tumor tissue was significantly reduced after adriamycin or chidamide treatment.Compared with chidamide or adriamycin alone,the malignant degree of the transplanted tumor was significantly reduced after both treatments.3.qRT-PCR analysis showed that compared with the control group,BECN1 and Atg5 mRNA expression were significantly increased in the adriamycin or chidamide treated group.The mRNA expressions of BECN1 and Atg5 were significantly increased after the simultaneous treatment of chidamide and adriamycin comparing with that of chidamide or adriamycin treatment.4.Immunohistochemical and Western blot analysess exhibted that compared with the control group,BECN1,Atg5 protein expression and LC3-Ⅱ/LC3-Ⅰ ratio were significantly increased in the adriamycin or chidamide treated group in ATC transplanted tumor tissues.BECN1,Atg5 protein expression and LC3-Ⅱ/LC3-Ⅰ ratio were obviously increased after the simultaneous treatment of chidamide and adriamycin compared with that of chidamide or adriamycin treatment.Part III:Study on mechanism of inhibitory roles of chidamide combined with adriamycin in ATCMethodsWestern blot was performed to detect the effect of chidamide combined with adriamycin on the expression of PI3K,AKT,p-PI3K,p-AKT and mTOR molecules related to the PI3K-AKT-mTOR signaling pathway in ATC cells and ATC transplanted tumors.ResultsIn ATC cells and transplanted tumors,p-PI3K,p-AKT and mTOR protein levels were significantly reduced after treatment with chidamide or adriamycin,and there was no significant change in the expression of PI3K and AKT.Compared with chidamide or adriamycin treated alone,the decrease levels of p-PI3K,p-AKT and mTOR were more significant after the simultaneous treatment of chidamide and adriamycin,while the expressions of PI3K and AKT did not change significantly.ConclusionChidamide could enhance the inhibitory roles of adriamycin in ATC cell proliferation,invasion,migration and the activity of the PI3K-AKT-mTOR signaling pathway,as well as the promotive roles of adriamycin in cell apoptosis and autophagy,and thus inhibited the development of ATC.