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 (IC₅₀) in BCPAP were 6.3 μmol/L (SO), .155 μmol/L (WA), and .055 μmol/L (IC₅₀WA + 50% IC₅₀SO), whereas in SW1736 cells the concentration was 7.6 μmol/L (SO), 2.5 μmol/L (WA), and 1.4 μmol/L (IC₅₀WA + 50% IC₅₀SO). Combination (WA + SO) at IC₅₀ 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 IC₅₀, P < .001). Combination therapy apoptosis (PARP cleavage and caspase-3 inactivation) and BRAF/Raf-1 down-regulation were dose-dependent starting at 50% IC₅₀ levels. Cell-cycle modulation was significant with combination treatment (35% increase in G2 arrest at 50% IC₅₀SO + WA and 70% increase at 75% IC₅₀SO + 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.