Licochalcone a

2′-hydroxy-4,4′,6′-trimethoxychalcone (HCH) with ≥98% purity, cardamonin (CA) with ≥98% purity, xanthohumol from hop (Humulus lupulus) (XN) with ≥96% purity, isobavachalcone (IBC) with ≥98% purity and licochalcone A (LIC) with ≥96% purity were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Methanol, ethanol, ethylene glycol, DMSO, acetonitrile, THF and n-hexane were of analytical grade.

Licochalcone A, TRAM-34, and PAP-1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). IP₃ was purchased from Merck Millipore (Billerica, MA, USA), and BTP2 was obtained from Tocris (Bristol, UK). All chemicals, except IP₃, were dissolved in dimethyl sulfoxide, which was prepared in distilled water.

The chemicals for molecular biology and buffer preparation, such as Tris, NaCl, SDS and licochalcone A (>98%), were obtained from Sigma. Osimertinib, PD98059 and MK2206 were purchased from Selleck Chemicals. Antibodies against ERK1/2 (#9102), p‐ERK1/2‐Thr202/Tyr204 (#4370EGFR (#4267), PARP (#9532), Akt (#4691), β‐actin (#3700), survivin (#2808), 4E‐BP1 (#9644)), p‐EGFR‐Tyr1068 (#3777), p‐Akt‐Ser473 (#4060), Bcl‐2 (#15071), cleaved caspase 3 (#9664), Bcl‐xL (#2764) and Mcl‐1 (#94296) were purchased from Cell Signaling Technology, Inc. Anti‐eIF4E (LS‑B12932) antibody was obtained from LifeSpan BioSciences, Inc. Human NSCLC cells, including H3255 (EGFR L858R), HCC827 (EGFR Del E746‐A750), H1975 (EGFR L858R/T790M) and A549 (EGFR WT), and immortalized lung epithelial or fibroblast cells, such as HBE, MRC5 and NL20, were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cell culture was performed following the standard protocols provided by ATCC. All cells were authenticated and cytogenetically tested before being frozen. The foetal bovine serum (FBS) and cell culture medium were products of Thermo Fisher Scientific. The Ba/F3 cell was purchased form Cell Engineering Division/RIKEN BioResource Center and maintained according to the instructions provided.

Human NSCLC cell line A549 and H460 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in RPMI 1640 medium (Gibco, Eggenstein, Germany) containing 10% heat-inactivated fetalbovine serum (Gibco, Eggenstein, Germany), 100 units/mL penicillin, and 100 ug/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO₂ at 37 °C. Antibodies including anti-MDM2 (sc-965, 1:200), anti-Cdc2 (sc-54, 1:200), anti-Cdc25C (sc-13138, 1:200) anti-Cyclin B1 (sc-245, 1:200), anti-Bcl-2 (sc-7382, 1:200), anti-Bcl-xL (sc-7382, 1:200), anti-cleaved PARP-1 (sc-56196, 1:200), anti-GAPDH (sc-293335, 1:1000), goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies including anti-ATF4 (#11815, 1:1000), anti-p-EIF2α (#3398, 1:1000) and anti-EIF2α (#9722, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), Licochalcone A and DMSO (dimethyl sulfoxide) were purchased from Sigma (St. Louis, MO, USA); FITC Annexin V apoptosis Detection Kit I and PI (propidium iodide) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA).

Benzyl isothiocyanate (BI) and licochalcone-A (LICO) were obtained from Sigma-Aldrich. Chelidonine (CHE), evodiamine (EVO), isoliquiritigenin (ISO) and phenethyl isothiocyanate (PI) were obtained from Chromadex (Los Angeles, CA, USA). Salinomycin (SAL) was obtained from AdooQ Bioscience (Irvine, CA, USA). Glycogen Synthase Kinase-3 inhibitor X (GSK3i) was purchased from Calbiochem (San Diego, CA, USA). All phytochemicals were prepared by dissolving in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at 10 mg/mL stock.

COX enzymatic activity was measured using the arachidonic acid assay, as described previously [63 (link)]. For COX-1 activity, cells were plated in 24-well plates, and after 24 h, medium was removed and replaced with serum-free medium. Licochalcone A (0.1, 0.5, 1, and 2.5 μM) or the selective inhibitor of COX-1 SC560 [(1 μM); Sigma-Aldrich GmbH, Taufkirchen, Germany] were added and left for 15 min. Then, arachidonic acid (15 μM; Sigma-Aldrich GmbH, Taufkirchen, Germany) was applied for another 15 min. Finally, supernatants were collected and used for the determination of PGE₂ as described above.
For COX-2 enzymatic activity, the assay was conducted as for COX-1, but with pre-incubation with LPS (10 ng/mL) for 24 h to induce COX-2 synthesis and using diclofenac sodium [(10 μM); Sigma-Aldrich GmbH, Taufkirchen, Germany] as commercial COX-2 preferential inhibitor.