Novel withanolides target medullary thyroid cancer through inhibition of both RET phosphorylation and the mammalian target of rapamycin pathway

Abstract

Background Despite development of current targeted therapies for medullary thyroid cancer (MTC), long-term survival remains unchanged. Recently isolated novel withanolide compounds from Solanaceae physalis are highly potent against MTCs. We hypothesize that these withanolides uniquely inhibit RET phosphorylation and the mammalian target of rapamycin (mTOR) pathway in MTC cells as a mechanism of antiproliferation and apoptosis. Methods MTC cells were treated with novel withanolides and MTC-targeted drugs. In vitro studies assessed cell viability and proliferation (MTS; trypan blue assays), apoptosis (flow cytometry with Annexin V/PI staining; confirmed by Western blot analysis), long-term cytotoxic effects (clonogenic assay), and suppression of key regulatory proteins such as RET, Akt, and mTOR (by Western blot analysis). Results The novel withanolides potently reduced MTC cell viability (half maximal inhibitory concentration [IC50], 270–2,850 nmol/L; 250–1,380 nmol/L for vandetanib; 360–1,640 nmol/L for cabozantinib) with induction of apoptosis at <1,000 nmol/L of drug. Unique from other targeted therapies, withanolides suppressed RET and Akt phosphorylation and protein expression (in a concentration- and time-dependent manner) as well as mTOR activity and translational activity of 4E-BP1 and protein synthesis mediated by p70S6kinase activation at IC50 concentrations. Conclusion Novel withanolides from Physalis selectively and potently inhibit MTC cells in vitro. Unlike other MTC-targeted therapies, these compounds uniquely inhibit both RET kinase activity and the Akt/mTOR prosurvival pathway. Further translational studies are warranted to evaluate their clinical potential.

Antiproliferative withanolides from several solanaceous species

“…Preliminary results revealed 1 as a promising chemotherapeutic candidate for antitumor therapy, and that further translational evaluation of 1 was warranted (Grogan et al 2013; Samadi et al 2012). However, difficulties arose in obtaining sufficient quantities of V. breviflora biomass needed to isolate the required amounts of 1 for such studies.…”

“…Furthermore, mechanistic studies showed that 1 inhibits proliferation by inducing a dose-dependent G2/M cell cycle arrest while promoting cell death through both intrinsic and extrinsic apoptotic pathways (Grogan et al 2013; Samadi et al 2009; Samadi et al 2010; Samadi et al 2012). …”

ENDOCRINE TUMOURS: Progressive metastatic medullary thyroid carcinoma: first- and second-line strategies

“…This kinase inhibitor showed inhibitory effects on both MZ-CRC and TT cell proliferation, although no effects on RET autophosphorylation were observed, suggesting that the PI3K/mTOR activation is not RET related in this specific cell line. Novel experiments with anolide derivatives have also demonstrated an inhibitory effect on RET phosphorylation as well as on the mTOR signalling in MTC-TT and DRO 81-1 cells (50). The fact that RET activation stimulates mTOR signalling could imply that the observed inhibition of mTOR signalling is the direct result of the inactivation of RET.…”

The molecular basis for RET tyrosine-kinase inhibitors in thyroid cancer

“…2004; Akeno-Stuart et al 2007; Brandt et al 2010; Konings et al 2010; Samadi et al. 2012; Frett et al 2014; Sun et al 2014; Schwartz et al 2015; Dunna et al.…”