Costunolide

Cells were treated with Bevacizumab (Avastin®), provided by Hoffmann-La Roche Ltd. (Switzerland), at 5 ng/ml and 100 μg/ml, telomerase inhibitors BIBR-1532 (10 μM) and costunolide (10 μM), PI3K inhibitor PI 828 (10μM), AKT inhibitor GSK 690693 (100 nM), mTOR inhibitor Rapamycin (200 nM), and HIF-1α inhibitor KC7F2 (40 μM) from Tocris (Tocris Bioscience, United Kingdom).

Cysteine conjugation was performed as described by Liu et al.^{13 (link)}. In brief, cysteine (150 mM) in 7 µL potassium phosphate buffer (100 mM; pH 6.5) and standards (30 mM) in 7 µL ethanol were added to 1000 µL potassium phosphate buffer (100 mM; pH 6.5). The reaction was initiated by adding 7 µL of glutathione S-transferase (GST) (1 g L⁻¹, in 100 mM potassium phosphate buffer; pH 6.5) into the mixture. Complete assay mixtures with(out) GST enzyme or either of the substrates were used as controls which was analysed by LC-Orbitrap-FTMS as described above. Samples were incubated for 30 min at room temperature and were kept at −20 °C until analysis. Costunolide was purchased from TOCRIS Bioscience (United Kingdom). Parthenolide, 3β-hydroxyCostunolide and 3β-hydroxyparthenolide, isolated from dried aerial parts of feverfew plants, were provided by Dr. Justin T. Fischedick from the PRISNA company^{9 (link)}. Kauniolide, 1β,10β-epoxy-kauniolide (arglabin), 1α,10α-epoxy-kauniolide and 3β,4β-epoxy- kauniolide were kindly provided by Professor Yue Chen (State Key Laboratory of Medicinal Chemical Biology, Nankai University, China)^{15 (link)}.