Doxorubicin

The culture media used in the experiments were the following: cation-adjusted Mueller–Hinton broth (MHB II; Sigma-Aldrich, St. Louis, MO, USA and Biokar Diagnostics, Allone, Beauvais, France), Luria–Bertani broth (LB-B; Sigma, St. Louis, MO, USA), Tryptic Soy broth (TSB; Scharlau Chemie S. A., Barcelona, Spain), and Tryptic Soy agar (TSA; Biokar Diagnostics, Allone, Beauvais, France) were purchased. The modified Luria–Bertani agar (LB*-A), used for the quorum sensing (QS) inhibition assays, was prepared in-house, according to the formula: 1.0 g of yeast extract (Merck, Darmstadt, Germany), 10.0 g of tryptone (Biolab, Budapest, Hungary), 10.0 g of NaCl (Molar Chemicals, Halásztelek, Hungary), 1.0 g of K₂HPO₄ (Biolab, Budapest, Hungary), 0.3 g of MgSO₄∙7H₂O (Reanal, Budapest, Hungary), 5 mL of Fe-EDTA stock solution and 20.0 g of bacteriological agar (Molar Chemicals, Halásztelek, Hungary) per 1 L of media. S. aureus ATCC 29213 was purchased from ATCC and the mouse embryonic fibroblast cell line (NIH/3T3) was purchased from Sigma.
DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS; pH 7.4), EB, reserpine, CCCP, PMZ and crystal violet (CV) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Doxorubicin (2 mg/mL) was purchased from Teva Pharmaceuticals, Budapest, Hungary.

In the experiments described herein, the culture media used were the following: cation-adjusted Mueller–Hinton broth (MHB II; Sigma-Aldrich, St. Louis, MO, USA and Biokar Diagnostics, Allone, Beauvais, France), Luria–Bertani broth (LB-B; Sigma, St. Louis, MO, USA), Tryptic Soy broth (TSB; Scharlau Chemie S. A., Barcelona, Spain), and Trypto-Casein Soy agar (TSA; Biokar Diagnostics, Allone, Beauvais, France). For the QS inhibition assays, the agar medium used was Luria–Bertani with a few modifications (LB*-A) and was prepared in house. The composition was as follows: 1.0 g yeast extract (Merck, Darmstadt, Germany), 10.0 g tryptone (Biolab, Budapest, Hungary), 10.0 g NaCl (Molar Chemicals, Halásztelek, Hungary), 1.0 g K₂HPO₄ (Biolab, Budapest, Hungary), 0.3 g MgSO₄·7 H₂O (Reanal, Budapest, Hungary), 5 mL Fe-EDTA stock solution and 20.0 g of bacteriological agar (Molar Chemicals, Halásztelek, Hungary) per liter of media.
The chemicals used—DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS; pH 7.4), ethidium bromide (EB), reserpine, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), PMZ, PAβN, and CV—were bought from Sigma-Aldrich Chemie GmbH (Steinheim, Germany), and doxorubicin (2 mg/mL) was purchased from Teva Pharmaceuticals, Budapest, Hungary.

Doxorubicin (2 mg/mL, Teva Pharmaceuticals, Budapest, Hungary) was serially diluted horizontally in 100 µL as previously described, starting with 8.6 µM. The resistance modifier was subsequently diluted vertically in 50 µL; the starting concentration was determined based on the IC₅₀. After resuspending the cells in culture medium, they were distributed into each well in 50 µL containing 6 × 10³ cells, with the exception of the medium control wells, to a final volume of 200 µL per well. The checkerboard plates were kept for 72 h at 37 °C in a CO₂ incubator, and at the end of the incubation period, the cell growth was determined by MTT staining method, as described earlier. Drug interactions were evaluated using Calcusyn software [36 ]. Each dose–response curve (for individual agents as well as combinations) was fit to a linear model using the median effect equation in order to obtain the median effect value (corresponding to the IC₅₀) and slope (m) [28 (link),29 (link)]. The goodness-of-fit was assessed using the linear correlation coefficient, r, and only data from analysis with r > 0.90 were presented. The extent of interaction between drugs was expressed using the combination index, in which a CI value close to 1 indicates additivity, while a CI < 1 is defined as synergy and a CI > 1 as antagonism.

As compounds 1–3 were found to exert very low cytotoxicity activity on each cell line (see below), the activity of 50 μM of compound on the IC₅₀ of doxorubicin (Teva), paclitaxel (Mayen) cisplatine (Teva), or vincristine was tested using the same protocol as described above to the cytotoxicity testing for the respective cell lines. In each case, statistical analysis was carried out by one-way ANOVA with Bonferroni's post hoc test, and differences were considered significant at ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. In order to prevent any possible false positive results and strengthen the relevance of our data for possible in vivo studies, we also set up a stricter criterion: statistically significant potentiation was considered relevant only when at least a two-time decrease in the IC₅₀ of the chemotherapeutic agent was observed. Such measures were not applied in case of an antagonistic effect (see results for cisplatin).

The primary antibodies MAPK (Erk) (#9102, 1:1000), Akt (1:1000, #9272), Phospho-EGF Receptor (Tyr1068) (1:1000, #2234), p(Thr308)-Akt (1:1000, #9275), Phospho-Akt (Ser473) (1:1000, #9271), Phospho-MAPK (pERK) (1:1000, #9101), β-Actin (1:10000, #4967) were purchased from Cell Signaling (Leiden, The Netherlands) and anti-EGFR (1:1000, sc-03-G) was purchased from Santa Cruz Biotechnology(Texas, USA). Cetuximab (ERBITUX) was ordered from Merck (Dietikon, Switzerland). Gefitinib (Iressa) was bought from Sigma (Zwijndrecht, The Netherlands); sunitinib was purchased from LC Laboratories (Woburn, USA). Entinostat and SAHA were purchased from Selleckchem (Munich, Germany). Staurosporine and cisplatin were purchased from Sigma-Aldrich (Zwijndrecht, Nederland). Doxorubicin was purchased from Teva Pharmaceuticals. All drugs were aliquoted in DMSO and stored at -20°C. The human epidermal growth factor (hEGF) and platelet-derived growth factor (PDGF) were purchased from Sigma-Aldrich (Zwijndrecht, Nederland).