Hesperetin Activates The Notch1 Signaling Cascade, Induces Cellular Differentiation, And Causes Apoptosis In Anaplastic Thyroid Cancer

Abstract

Introduction: Anaplastic thyroid cancer (ATC) is characterized by aggressive growth and extremely poor prognosis, for which there is no standardized therapy. In addition, the undifferentiated feature of ATC in which sodium iodide symporter (NIS) expression is reduced makes treatment such as radioactive iodine ineffective. Therefore, there is an urgent need for strategies that sensitize ATC cells to iodine treatment. It has been reported that the Notch1 signaling pathway, which affects cell proliferation and differentiation, is inactivated in ATC. However, it is unknown whether activation of Notch1 could yield therapeutic effects in ATC. Therefore, in this study we evaluated whether Hesperetin, a naturally occurring flavanone and potential Notch activator, effects ATC cell proliferation and possibly up-regulates thyroid-specific differentiation markers such as NIS. Methods: In-vitro studies were carried out to evaluate the drug effects using a human derived ATC cell line (HTH7). To determine whether Hesperetin activates Notch1 in ATC, expression of Notch1 mRNA and protein was evaluated upon treatment. In addition, mRNA levels of Notch1 response genes (Hes1 and Hey1) were measured, and the Notch1 activity was assessed using CBF-1-luc binding assay. Cellular proliferation was measured by viable cell counts every 24 hours post-treatment (0-200mM) for 72 hours, and the underlying mechanism was investigated by western blot. Expression of thyroid-specific differentiation markers including Thyroid Transcription Factor 1 (TTF-1), Thyroid Transcription Factor 2 (TTF-2), Paired Box Gene 8 (PAX-8), Thyroid Stimulating Hormone Receptor (TSHR), and NIS were also measured. Results: Hesperetin caused activation of the Notch1 signaling pathway. Treated ATC cells displayed elevated Notch1 protein and increased mRNA levels of Hes1 and Hey1, important Notch1 response genes. Treatment induced a dose-dependent increase in Notch1 activity as evidenced by increased luciferase activity. Hesperetin treatment also resulted in a time and dose-dependent decrease in cell proliferation: at 72 hours, 50mM and 100mM Hesperetin resulted in a 27% and 47% reduction respectively compared to control. Protein expression of apoptotic markers cleaved PARP, cleaved Caspase-3, and BAD increased with treatment. Importantly, Hesperetin up-regulated NIS mRNA levels, a thyroid-specific marker responsible for sensitivity to radioactive iodine. Other thyrocyte markers (TTF-1, TTF-2, PAX-8 and TSHR) were also increased, further evidence that Hesperetin induces differentiation in ATC. Conclusions: Hesperetin activates the Notch1 signaling cascade and has a significant antiproliferative effect on ATC that can be largely attributed to apoptosis. Hesperetin also induces differentiation in ATC, and by up-regulating markers such as NIS, could be useful in combination with radioactive iodine for treating ATC. Further investigation to elucidate the in-vivo effects of Hesperetin on ATC is warranted.