BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.