Suberoyl Bis-Hydroxamic Acid Activates Notch-1 Signaling And Induces Apoptosis In Anaplastic Thyroid Carcinoma Through P53

Objective: Anaplastic thyroid cancer(ATC)is a disease with low survival rate due to uncontrolled systemic metastasis and with less effective treatment options.It is resistant to standard chemotherapy,external beam radiation,and radioiodine treatment,thus new treatments are urgently desired.The Notch cascade is a highly evolutionally conserved molecular cascade that plays an important role for cell fate determination,proliferation,differentiation and survival in development,neurogenesis and homeostasis.Suberic bis-hydroxamic acid(SBHA)is a new histone deacetylase(HDAC)inhibitors,and it can activate the Notch-1 cascade and lead to a decrease in growth in many human cancers.This study firstly investigated SBHA could activate Notch1 signaling cascade,inhibit tumor cell proliferation and induce apoptosis in anaplastic thyroid cancer cells.Secondly,the study investigated the relationship between Notch1 and P53 cascades in anaplastic thyroid cancer cells after using SBHA.Methods:1.Anaplastic thyroid cancer tissues were obtained from 16 patients who were admitted to Tianjin Medical Uinversity Cancer Institute and Hospital between January 2010 and February 2013.These tissues were immunohistochemical detection for Notch1 and P53.And we used Western blot to detect Notch1 in normal thyroid cells,thyroid papillary carcinoma cells and anaplastic thyroid cancer cells.2.We performed Real-time PCR and Western blot to detect Notch1 in anaplastic thyroid cancer cells after using different dosage of SBHA and Notch1 were efficiently knocked down by siRNA.MTT assay and flow cytometry assay were used to detect the proliferation and apoptosis of cells.And western blot was used to detect the markers of apoptosis.3.We performed Real-time PCR and Western blot to detect P53 and P21 in anaplastic thyroid cancer cells after using different dosage of SBHA and P53 were efficiently knocked down by shRNA.And co-immunoprecipitation was used to detect the relationship between Notch1 and P53.Results: 1.The immunohistochemical staining demonstrated that positive stainimg of Notch1 and P53 was occurred in only one was of 16 samples,and it was the same ATC sample.All other slides show double negative staining.The results of Western blot showed that Notch1 was normally expressed in normal thyroid cells,less in thyroid papillary carcinoma cells and no in anaplastic thyroid cancer cells.2.SBHA treatmentled to a dose-dependent increase of Notch-1 signaling cascade compared with non-detectable NICD proteins in CAL-62 cells without the treatment of SBHA and it could significantly inhibit cell viability in a does dependent manner detected with MTT assays.Apoptosis cells were identified by flow cytometer as the positive staining with Annexin V after treatment SBHA for 24 to 48 hours.Notably,we demonstrated SBHA could induce cell apoptosis detected with apoptosis markers cleaved caspase-3,cleaved PARP and Bax.3.SBHA treatmentled to a dose-dependent increase P53 and P21 compared with cells without the treatment of SBHA and the same to Notch1.The result of coimmunoprecipitation and P53 knocking down by shRNA were showed SBHA activated Notch-1 signaling and induced apoptosis in anaplastic thyroid carcinoma through P53.Conclusion: 1.There were little Notch1 proteins in ATC samples and no Notch1 proteins in ATC cells.2.SBHA could activate the Notch-1 signaling cascade,inhibit tumor cell proliferation and induce apoptosis in anaplastic thyroid cancer cells with time and dose dependence.3.SBHA activated Notch-1 signaling and induced apoptosis in anaplastic thyroid carcinoma through P53.