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The non-canonical Hh inhibitor GANT61 decreased not only cell growth, but also the CSC population in TNBC cells. GANT61 enhanced the anti-cell growth activity of paclitaxel in these cells. These results suggest for the first time that GANT61 has potential as a therapeutic agent in the treatment of patients with TNBC.

Estradiol (E2) increases not only the cell growth but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)‐positive breast cancer cells. It has been suggested that the non‐canonical hedgehog (Hh) pathway activated by E2 plays an important role in the regulation of CSC proportion in ER‐positive breast cancer cells. We studied anti‐CSC activity of a non‐canonical Hh inhibitor GANT61 in ER‐positive breast cancer cells. Effects of GANT61 on the cell growth, cell cycle progression, apoptosis and CSC proportion were investigated in four ER‐positive breast cancer cell lines. CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. Expression levels of pivotal molecules in the Hh pathway were measured. Combined effects of GANT61 with antiestrogens on the anti‐cell growth and anti‐CSC activities were investigated. E2 significantly increased the cell growth and CSC proportion in all ER‐positive cell lines. E2 increased the expression levels of glioma‐associated oncogene (GLI) 1 and/or GLI2. GANT61 decreased the cell growth in association with a G1‐S cell cycle retardation and increased apoptosis. GANT61 decreased the E2‐induced CSC proportion measured by the mammosphere assay in all cell lines. Antiestrogens also decreased the E2‐induced cell growth and CSC proportion. Combined treatments of GANT61 with antiestrogens additively enhanced anti‐cell growth and/or anti‐CSC activities in some ER‐positive cell lines. In conclusion, the non‐canonical Hh inhibitor GANT61 inhibited not only the cell growth but also the CSC proportion increased by E2 in ER‐positive breast cancer cells. GANT61 enhanced anti‐cell growth and/or anti‐CSC activities of antiestrogens in ER‐positive cell lines.

Ethylene glycol monomethyl ether (EGME) is a widely used industrial solvent known to cause adverse effects to human and other mammals. Organs with high metabolism and rapid cell division, such as testes, are especially sensitive to its actions. In order to gain mechanistic understanding of EGME-induced toxicity, an untargeted metabolomic analysis was performed in rats. Male rats were administrated with EGME at 30 and 100 mg/kg/day. At days 1, 4, and 14, serum, urine, liver, and testes were collected for analysis. Testicular injury was observed at day 14 of the 100 mg/kg/day group only. Nearly 1900 metabolites across the four matrices were profiled using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Statistical analysis indicated that the most significant metabolic perturbations initiated from the early time points by EGME were the inhibition of choline oxidation, branched-chain amino acid catabolism, and fatty acid β-oxidation pathways, leading to the accumulation of sarcosine, dimethylglycine, and various carnitine- and glycine-conjugated metabolites. Pathway mapping of these altered metabolites revealed that all the disrupted steps were catalyzed by enzymes in the primary flavoprotein dehydrogenase family, suggesting that inhibition of flavoprotein dehydrogenase–catalyzed reactions may represent the mode of action for EGME-induced toxicity. Similar urinary and serum metabolite signatures are known to be the hallmarks of multiple acyl-coenzyme A dehydrogenase deficiency in humans, a genetic disorder because of defects in primary flavoprotein dehydrogenase reactions. We postulate that disruption of key biochemical pathways utilizing flavoprotein dehydrogenases in conjugation with downstream metabolic perturbations collectively result in the EGME-induced tissue damage.