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Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells

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Abstract

Purpose: 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. Conclusion: 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.

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Abbreviations

EGFR:

Epidermal growth factor receptor

EGF:

Epidermal growth factor

RTK:

Receptor tyrosine kinase

MAPK:

Mitogen activated protein kinase

Erk:

Extracellular signal-regulated kinase

PI-3K:

Phosphatidylinositol 3’-kinase

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Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture of Japan

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Authors and Affiliations

  1. 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 Qin, Hiroshi Ariyama, Eishi Baba, Risa Tanaka, Hitoshi Kusaba, Mine Harada & Shuji Nakano

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  1. Baoli Qin
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  2. Hiroshi Ariyama
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  3. Eishi Baba
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  4. Risa Tanaka
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  6. Mine Harada
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  7. Shuji Nakano
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Correspondence to Shuji Nakano.

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Baoli Qin and Hiroshi Ariyama contributed equally to this work.

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Qin, B., Ariyama, H., Baba, E. et al. Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells. Cancer Chemother Pharmacol 58, 577–584 (2006). https://doi.org/10.1007/s00280-006-0219-4

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