Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells.
OBJECTIVE
Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been demonstrated to exhibit its antitumor activity by the blockade of EGF receptor, the role of signaling pathways downstream of EGFR in gefitinib sensitivity remains unknown. In this study, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity.


METHODS
Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively.


RESULTS
Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Gefitinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-src-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially reversed the gefitinib resistance, with concomitant inhibition of Akt and Erk.


CONCLUSIONS
Our results suggest that activated Ras and Src could induce gefitinib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationale for assessment of these specific signaling molecules downstream of EGFR to customize treatment.
First Department of Internal Medicine and Department of Biosystemic Science of Medicine, Graduate School of Medicine, Kyushu University, 3-1-1 Maidashi, 812-8582, Higashi-Ku, Fukuoka, Japan.
Baoli
B
Qin
Hiroshi
H
Ariyama
Eishi
E
Baba
Risa
R
Tanaka
Hitoshi
H
Kusaba
Mine
M
Harada
Shuji
S
Nakano
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D005704:Gallbladder / Q000378:metabolism / Q000473:pathology; D000077156:Gefitinib; D006801:Humans; D020128:Inhibitory Concentration 50; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology*; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism
D000970:Antineoplastic Agents; D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D000077156:Gefitinib