title: A novel combination of withaferin A and sorafenib shows synergistic efficacy against both papillary and anaplastic thyroid cancers.
abstract: 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.
authors.affiliation: Department of Surgery, University of Kansas Medical Center, 3901 Rainbow Blvd., MS 2005, Murphy Building, Room 4008, Kansas City, KS 66160, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Cohen
authors.forename: Ridhwi
authors.initials: R
authors.lastname: Mukerji
authors.forename: Barbara N
authors.initials: BN
authors.lastname: Timmermann
authors.forename: Abbas K
authors.initials: AK
authors.lastname: Samadi
authors.forename: Mark S
authors.initials: MS
authors.lastname: Cohen
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001557:Benzenesulfonates / Q000008:administration & dosage / Q000494:pharmacology*; D015153:Blotting, Western; D002277:Carcinoma / Q000188:drug therapy*; D002291:Carcinoma, Papillary; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005434:Flow Cytometry; D006801:Humans; D020128:Inhibitory Concentration 50; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D011725:Pyridines / Q000008:administration & dosage / Q000494:pharmacology*; D000077157:Sorafenib; D000077273:Thyroid Cancer, Papillary; D065646:Thyroid Carcinoma, Anaplastic; D013964:Thyroid Neoplasms / Q000188:drug therapy*; D054358:Withanolides / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D001557:Benzenesulfonates; D010671:Phenylurea Compounds; D011725:Pyridines; D054358:Withanolides; D009536:Niacinamide; D000077157:Sorafenib; C009684:withaferin A