Tyrphostin AG 1024 modulates radiosensitivity in human breast cancer cells

Abstract

Summary Insulin-like growth factor-1 (IGF-1) plays an important growth-promoting effect by activating the PI3K/Akt signalling pathway, inhibiting apoptotic pathways and mediating mitogenic actions. Tyrphostin AG 1024, one selective inhibitor of IGF-1R, was used to evaluate effects on proliferation, radiosensitivity, and radiation-induced cell apoptosis in a human breast cancer cell line MCF-7. Exposure to Tyrphostin AG 1024 inhibited proliferation and induced apoptosis in a time-dependent manner, and the degree of growth inhibition for IC20 plus irradiation (4 Gy) was up to 50% compared to the control. Examination of Tyrphostin AG 1024 effects on radiation response demonstrated a marked enhancement in radiosensitivity and amplification of radiation-induced apoptosis. Western blot analysis indicated that Tyrphostin AG 1024-induced apoptosis was associated with a downregulation of expression of phospho-Akt1, increased expression of Bax, p53 and p21, and a decreased expression of bcl-2 expression, especially when combined with irradiation. To our knowledge, this is the first report showing that an IGF-1 inhibitor was able to markedly increase the response of tumour cells to ionizing radiation. These results suggest that Tyrphostin AG 1024 could be used as a potential therapeutic agent in combination with irradiation. 2017-2021© 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.2171, available online at http://www.idealibrary.com on http://www.bjcancer.com 2 These authors contributed equally to the work.(EGFR), insulin-like growth factor receptor (IGF-1R) in a selective manner (Gazit et al, 1989).Tyrphostin AG 1024, one tyrosine kinase inhibitor specifically targeting IGF-1 receptor, is metabolized intracellularly, creating substances with increased activity towards the receptor and downregulated activity of Akt kinase. The objective of this study was to examine the potential therapeutic purpose of Tyrphostin AG 1024 in combination with irradiation to counteract tumour cell proliferation and to modulate cellular radiosensitivity and apoptosis in a human breast cancer cell line, MCF-7. MATERIALS AND METHODS MaterialsAll materials and chemicals were purchased from Sigma (St Louis, MC, USA) unless noted otherwise. The inhibitor of IGF-1R, Tyrphostin AG 1024 was purchased from Alexis Biochemicals. Akt, phospho-Akt, and NIH-3T3 PDGF-treated protein were purchased from New England Biolabs, Inc. Bax and bcl-2, and p21 antibodies were purchased from Santa Cruz Biotechnology, Inc.Horseradish peroxidase-linked anti-rabbit and anti-mouse antibody were from Jackson ImmunoResearch Laboratory Inc.; molecular markers and fetal bovine serum were from Biological Industries and ECL was purchased from Amersham Biotech Com. ApopTag™ Plus apoptosis Detection Kit-Fluorescence was purchased from Oncor Inc. Cell lineMCH-7 was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin and streptomycin, 2 mM L-glutamine at 37˚C in a humidified atmosphere of 5% CO 2 . IGF1R inhibitor treatmentThe ant...

Ionizing radiation activates IGF-1R triggering a cytoprotective signaling by interfering with Ku-DNA binding and by modulating Ku86 expression via a p38 kinase-dependent mechanism

“…Used in a concentration that specifically inhibited IGF-1R, AG1024 increased radiosensitivity of U1810 cells. Downstream consequences of IGF-1R activation were examined by selectively inhibiting PI3-K and p38 kinase, previously identified as IGF-1R mediators of cytoprotective responses after radiation (Heron-Milhavet et al, 2001;Wan et al, 2001;Wen et al, 2001). In accordance with other studies, we found that both p38 kinase and PI3-K inhibition induced radiosensitivity in IGF-1-stimulated U1810 cells, indicating that these proteins were part of IGF-1R signaling in response to radiation.…”

“…Previously, we described the IGF-1R involvement in cell protection from radiation-induced cell death (Cosaceanu et al, 2005). Blocking of IGF-1R activity by tyrosine kinase inhibitors or by anti-IGF-1R-neutralizing antibodies was shown to increase radiosensitivity in several types of human tumor cell lines (Wen et al, 2001;Cosaceanu et al, 2005). Nevertheless, the effect of radiation on IGF-1R activation is poorly understood.…”

BCR-ABL- and Ras-independent activation of Raf as a novel mechanism of Imatinib resistance in CML

“…Cells were lysed with 20 mM Tris, pH 7.5, 0.27 M sucrose, 1 mM EdTA, 1 mM EGTA, 1% Triton X-100 and protease/phosphatase inhibitors. Anti-B-Raf or c-Raf antibodies (1 µg) was added to the cell lysates and extracts were incubated in the cold for 1 h with mixing (21). Protein-G-Sepharose bead suspension (40 µl) was added to each vial and reactions were incubated at 4˚C in a head-to-head mixer for 2 h. Precipitates were washed using lysis buffer supplemented with 0.6 M NaCl.…”

The GH–IGF-I axis and breast cancer

“…Selective inhibitors of the IGF-1 receptor also inhibit proliferation and enhance apoptosis by increasing the response of tumour cells to ionising radiation 165 The IGF-1 receptor can also be targeted by small molecular inhibitors of the IGF-1R tyrosine kinase, and molecular agents such as antisense and small interfering Somatostatin analogues exert antiproliferative effects both in vivo and in vitro, in part by inhibiting GH release and therefore reducing IGF-I levels, eg octreotide reduces breast cancer growth by 50% in mice 3 .…”