TY - JOUR
T1 - Molecular mechanisms and modulation of key pathways underlying the synergistic interaction of sorafenib with erlotinib in non-small-cell-lung cancer (NSCLC) cells
AU - Giovannetti, E
AU - Labots, M
AU - Dekker, H
AU - Galvani, E
AU - Lind, J S W
AU - Sciarrillo, R
AU - Honeywell, R
AU - Smit, E F
AU - Verheul, H M
AU - Peters, G J
PY - 2013
Y1 - 2013
N2 - Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key pathways in NSCLC progression such as EGFR, K-Ras and VEGFR. The sorafenib-erlotinib combination showed clinical activity and acceptable safety. Therefore, we evaluated mechanisms underlying sorafenib-erlotinib interaction in seven NSCLC cell lines selected for their heterogeneous pattern of EGFR and Raf-kinase-inhibitor protein (RKIP) expression, and EGFR/K-Ras mutations. Pharmacologic interaction was studied using MTT/SRB assays and the combination index (CI) method, while effects on EGFR, Erk1/2 and Akt phosphorylation, cell cycle and apoptosis were studied with western-blot, ELISA, and flow cytometry. Intracellular drug concentrations were measured with LC-MS/MS, whereas kinase activity profiles were generated on tyrosine kinase peptide substrate arrays. Synergism was detected in all cell lines, with CIs < 0.6 in K-Ras mutated A549, SW1573 and H460, as well as in H1975 (EGFR-T790M) cells. Sorafenib slowed cell cycle progression and induced apoptosis, which was significantly increased in the combination. Moreover, sorafenib reduced Akt/ERK phosphorylation in erlotinib-resistant cells, associated with significant RKIP up-regulation. No direct drug interaction was detected by LC-MS/MS measurement, while lysates from A549 and H1975 cells exposed to erlotinib+sorafenib showed a significant inhibition in the phosphorylation of 16 overlapping peptides, including sites from RAF, VEGFR2, PDGFR, CDK2 and SRC, suggesting new markers to identify NSCLC patients who are likely to respond to this treatment. In conclusion, several mechanisms, including apoptosis-induction, modulation of expression/phosphorylation of RKIP and crucial kinases contribute to erlotinib-sorafenib synergistic interaction and should be evaluated in future trials for the rational development of this combination in NSCLC.
AB - Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key pathways in NSCLC progression such as EGFR, K-Ras and VEGFR. The sorafenib-erlotinib combination showed clinical activity and acceptable safety. Therefore, we evaluated mechanisms underlying sorafenib-erlotinib interaction in seven NSCLC cell lines selected for their heterogeneous pattern of EGFR and Raf-kinase-inhibitor protein (RKIP) expression, and EGFR/K-Ras mutations. Pharmacologic interaction was studied using MTT/SRB assays and the combination index (CI) method, while effects on EGFR, Erk1/2 and Akt phosphorylation, cell cycle and apoptosis were studied with western-blot, ELISA, and flow cytometry. Intracellular drug concentrations were measured with LC-MS/MS, whereas kinase activity profiles were generated on tyrosine kinase peptide substrate arrays. Synergism was detected in all cell lines, with CIs < 0.6 in K-Ras mutated A549, SW1573 and H460, as well as in H1975 (EGFR-T790M) cells. Sorafenib slowed cell cycle progression and induced apoptosis, which was significantly increased in the combination. Moreover, sorafenib reduced Akt/ERK phosphorylation in erlotinib-resistant cells, associated with significant RKIP up-regulation. No direct drug interaction was detected by LC-MS/MS measurement, while lysates from A549 and H1975 cells exposed to erlotinib+sorafenib showed a significant inhibition in the phosphorylation of 16 overlapping peptides, including sites from RAF, VEGFR2, PDGFR, CDK2 and SRC, suggesting new markers to identify NSCLC patients who are likely to respond to this treatment. In conclusion, several mechanisms, including apoptosis-induction, modulation of expression/phosphorylation of RKIP and crucial kinases contribute to erlotinib-sorafenib synergistic interaction and should be evaluated in future trials for the rational development of this combination in NSCLC.
KW - Apoptosis/drug effects
KW - Blotting, Western
KW - Carcinoma, Non-Small-Cell Lung/drug therapy
KW - Cell Cycle/drug effects
KW - Cell Line, Tumor
KW - Chromatography, Liquid
KW - Drug Synergism
KW - Enzyme-Linked Immunosorbent Assay
KW - Erlotinib Hydrochloride
KW - Flow Cytometry
KW - Gene Expression Regulation, Neoplastic
KW - Humans
KW - Lung Neoplasms/drug therapy
KW - Molecular Targeted Therapy
KW - Niacinamide/administration & dosage
KW - Phenylurea Compounds/administration & dosage
KW - Phosphatidylethanolamine Binding Protein
KW - Phosphorylation/drug effects
KW - Protein Kinase Inhibitors/administration & dosage
KW - Quinazolines/administration & dosage
KW - Tandem Mass Spectrometry
M3 - Article
C2 - 22973961
VL - 19
SP - 927
EP - 939
JO - Current Pharmaceutical Design
JF - Current Pharmaceutical Design
SN - 1381-6128
IS - 5
ER -