withaferin-a and Thyroid-Neoplasms

Sorafenib (SO), a multikinase-targeted inhibitor in clinical trials for papillary and anaplastic cancers, shows limited efficacy with moderate toxicity. Withaferin A (WA), a natural withanolide, shows potent preclinical anticancer activity in thyroid cancers through multiple cytotoxic mechanisms including heat-shock protein inhibition. We hypothesized that combination therapy (WA + SO) would have a synergistic effect against anaplastic and papillary carcinoma cells at lower sorafenib doses.. Human papillary (BCPAP) and anaplastic (SW1736) thyroid cancer cell lines were evaluated after treatment with SO, WA, or their combination at different doses. Proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and trypan blue exclusion; apoptosis and cell-cycle arrest was measured by flow cytometry. Western analysis confirmed apoptosis (Poly ADP ribose polymerase [PARP] and caspase-3 cleavage) and Raf inhibition. Experiments were repeated in triplicate and were evaluated statistically with significance set at a P value of less than .05.. The concentration of drug at which 50% of the cells are inhibited (IC(50)) in BCPAP were 6.3 μmol/L (SO), .155 μmol/L (WA), and .055 μmol/L (IC(50)WA + 50% IC(50)SO), whereas in SW1736 cells the concentration was 7.6 μmol/L (SO), 2.5 μmol/L (WA), and 1.4 μmol/L (IC(50)WA + 50% IC(50)SO). Combination (WA + SO) at IC(50) decreased cell viability to 19% (from 50% individually). Apoptosis levels on flow cytometry in anaplastic cells increased significantly from 0% to 2% (SO or WA alone) to 89% (combo at IC(50), P < .001). Combination therapy apoptosis (PARP cleavage and caspase-3 inactivation) and BRAF/Raf-1 down-regulation were dose-dependent starting at 50% IC(50) levels. Cell-cycle modulation was significant with combination treatment (35% increase in G2 arrest at 50% IC(50)SO + WA and 70% increase at 75% IC(50)SO + WA; P < .01).. Combination therapy with sorafenib + withaferin showed synergistic efficacy in papillary and anaplastic cancers in vitro with significant induction of apoptosis. This combination achieved potent anticancer activity with lower overall doses of sorafenib, indicating a potential strategy to decrease sorafenib toxicity in future translational studies.

Topics: Antineoplastic Agents; Apoptosis; Benzenesulfonates; Blotting, Western; Carcinoma; Carcinoma, Papillary; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; Humans; Inhibitory Concentration 50; Niacinamide; Phenylurea Compounds; Pyridines; Sorafenib; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Withanolides

Most medullary thyroid carcinomas (MTC) recur or progress despite curative resection. Current targeted therapies show promise but lack durable efficacy and tolerability. The purpose of this study was to build on previous in vitro work and evaluate withaferin A (WA), a novel RET inhibitor, in a metastatic murine model of MTC.. A total of 5 million DRO-81-1 human MTC cells injected in the left posterior neck of nu/nu mice generated metastases uniformly to the liver, spleen, and/or lungs. Treatment with WA (8 mg/kg/day, intraperitoneally, for 21 days) was started for neoplasms > 100 mm(3). Endpoints were survival, neoplasm > 15,00 mm(3), decreased body weight, or body score (all measured three times/wk).. All controls (saline; n = 5) died or deteriorated from metastatic disease by 7 weeks postinjection. All treated animals were alive (WA; n = 5), having tumor regression and growth delay without toxicity or weight loss at 6 weeks posttreatment (P < .01). Tumor cells treated with WA demonstrated inhibition of total and phospho-RET levels by Western blot analysis in a dose-dependent manner (almost complete inhibition with treatment of 5 μM WA) as well as potent inhibition of phospho-ERK and phospho-Akt levels.. WA is a novel natural-product RET-inhibitor with efficacy in a metastatic murine model of MTC. Further long-term efficacy/toxicity studies are warranted to evaluate this compound for clinical translation.

Topics: Animals; Antineoplastic Agents; Biopsy, Fine-Needle; Calcitonin; Carcinoma, Neuroendocrine; Humans; Mice; Mice, Nude; Neural Crest; Proto-Oncogene Proteins c-ret; Thyroid Neoplasms; Transplantation, Heterologous; Withanolides