Gefitinib

To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x10⁵ cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na₃VO₄, Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [22 (link)]; using Catalase, Sigma, Cat. no. C1345 and H₂O₂ Sigma, Cat. no. 31642) for 1 hour and lysed. Following concentrations of inhibitors were used: DMSO (Sigma-Aldrich, Cat. no. D2438, solvent control), 10 μM erlotinib (Tarceva, Molecula; Cat. no. 89983631), 0.5 μM afatinib (Selleckchem, Cat. no. S1011); 5 μM gefitinib (CellSignaling; 4765), 5 μM lapatinib (Selleckchem, Cat. no. S1028), 300 nM flavopiridol (Selleckchem, Cat. no. S1230), 5 μM arry380 (Gentaur Europe, Cat. no. A8366), 10 μM Nu7441 (Selleckchem, Cat. no. S2638) and 10 μM ruxolitinib (Selleckchem, Cat. no. S1378).

Gefitinib (#G-4408), erlotinib (#E-4007), trametinib (#T-8123), crizotinib (#C-7900) and paclitaxel (#P-9600) were purchased from LC Laboratories (Woburn, MA, USA). Cisplatin (#479306) was purchased from Sigma Aldrich (St. Louis, MO, USA). 2D and 3D cultures were incubated in the presence of the drugs for 72 hours. Cell viability was determined using the commercially available alamarBlue^® assay kit (Thermo Fisher Scientific, Madison, WI, USA). Apoptosis was analyzed using the Caspase-Glo^® 3/7 assay multiplexed with the MultiTox-Fluor GF-AFC life stain (Promega, Madison, WI, USA). Luminescence and fluorescence intensities were measured using the Paradigm detection platform (Molecular Devices, Sunnyvale, CA, USA). Statistical analysis was done using the GraphPad Prism Software 7.03 (GraphPad Software Inc., La Jolla, CA, USA). For the in situ analyses of apoptosis in cocultures the cells were incubated in the presence of 50 nM Gefitinib or 20 nM crizotinib for 24 hours and processed for immunofluorescent microscopy using the cleaved caspase-3 antibody (Cell Signaling Technology, #9661) that specifically detects apoptotic cells.

Oleanolic acid was isolated from an industrial by-product obtained during the process of mistletoe herb essence production. Spectral data of the resulting chemicals were consistent with the data from the literature [63 ]. The semisynthetic OA derivatives, methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate (HIMOXOL) and 12α-bromo-3-hydroxyimonoolean-28→13-olide (Br-HIMOLID) were synthesized as described in the earlier studies [64 (link),65 (link)] at the Department of Organic Chemistry, Poznan University of Medical Sciences, Poland (Figure 8). Before use in the experiment, the derivatives were dissolved in DMSO and stored at 4 °C.
The cell culture medium, McCoy’s 5A, and fetal bovine serum were purchased from Lonza (Lonza, Verviers, Belgium). Rapamycin, DMSO, camptothecin, propidium iodide, RNAse, and MTT were obtained from Sigma-Aldrich (Sigma-Aldrich, Munich, Germany). Gefitinib was purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).

Metformin (1,1-dimethylbiguanide hydrochloride) and gefitinib were purchased from Aladdin chemistry Co. Ltd and Selleck-Biotool Co. Ltd, respectively. Metformin was diluted across a range of concentrations in culture media and gefitinib was dissolved in DMSO to prepare the stock solution of 5 mM. Antibodies for the protein characteristics were against total EGFR, phosphor-EGFR, total Akt, phosphor-Akt(Ser473), total Erk1/2, phosphor-Erk1/2, phosphor-Acetyl-CoA Carboxylase (Ser79), phosphor-p70 S6 kinase(Thr389), phosphor-AMPKα (Thr172) and β actin (Cell Signaling, Beverly, MA, USA) and they were purchased from Cell Signaling Technology. Apoptosis Detection kit (FITC Annexin V) was purchased from BD Pharmingen (San Diego, California, USA).

The antibody against EGFR was purchased from Abcam (Cambridge, UK; ab2430) and the antibody against actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; I-19). Gefitinib was obtained from Cell Signaling Technology (Danvers, MA, USA) and AZD9291 (osimertinib) was from Selleckchem (Houston, TX, USA).