Enhanced anti-angiogenic effect of E7820 in combination with erlotinib in epidermal growth factor receptor-tyrosine kinase inhibitor-resistant non-small-cell lung cancer xenograft models.
Bottom Line: Treatment with E7820 (50 mg/kg) with erlotinib (60 mg/kg) showed a synergistic antitumor effect in three xenograft models.Immunohistochemical analysis indicated that combined treatment with E7820 and erlotinib significantly decreased microvessel density and increased apoptosis of tumor-associated endothelial cells compared with use of only one of the agents.This combination increased apoptosis in HUVECs through activation of both intrinsic and extrinsic apoptosis pathways in vitro.
Affiliation: Tsukuba Research Laboratory, Eisai Co., Ltd., Tsukuba, Japan.Show MeSH
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Mentions: To confirm the efficacy of E7820 in combination with erlotinib in terms of decreased MVD and enhanced apoptosis of tumor-derived endothelial cells in the NSCLC A549, H1975, and H1650 xenograft models, we next examined the growth-inhibiting and apoptosis-inducing effects of E7820 in combination with erlotinib on cultured HUVECs and also tumor cells (A549, H1975, and H1650). The HUVECs and A549, H1975, and H1650 cells were treated with E7820 and/or erlotinib at the indicated doses for 48 or 96 h. E7820 in combination with erlotinib significantly inhibited HUVEC growth (P < 0.05; Fig. 4). Annexin V–PI two-color flow cytometry revealed significantly enhanced apoptosis in HUVECs treated with E7820 in combination with erlotinib compared with either single agent alone or control cells (P < 0.05; Fig. 5a,b). The enhancement of apoptosis in HUVECs after combination treatment was confirmed by significantly increased MMP loss (P < 0.05) and cytochrome c release, which are markers of apoptosis (Fig. 5c,d). Furthermore, the results of annexin V–PI two-color flow cytometry showed that the pan-caspase inhibitor zVAD-fmk significantly inhibited apoptosis in HUVECs that is induced by combination treatment with E7820 and erlotinib (P < 0.05; Fig. 5e). Caspase-3 activity was also significantly increased in HUVECs treated with E7820 in combination with erlotinib compared to single agent or control (P < 0.05), indicating that E7820 in combination with erlotinib induced apoptosis in a caspase-dependent manner (Fig. 5f). Blockade of pro-apoptosis signals by the capase-3 inhibitor Ac-DEVD-CHO at a point where the intrinsic and extrinsic apoptosis pathways meet, by the caspase-9 inhibitor Ac-LEHD-CHO at a point downstream of the mitochondria in the intrinsic pathway, or by the caspase-8 inhibitor Ac-IETD-CHO at a point in the extrinsic pathway, resulted in inhibition of the apoptosis induced by combination treatment (Fig. 5e). In terms of tumor cells, E7820 in combination with erlotinib significantly inhibited H1650 cell growth (P < 0.05; Fig. 4) and enhanced H1650 cell apoptosis (Figs. 5a,S1). We observed neither inhibition of cell proliferation nor induction of apoptosis in both cell lines (A549 and H1975) after combination treatment (Figs. 4, 5a,S1). These data showed that E7820 in combination with erlotinib inhibited cell proliferation and enhanced apoptosis through activation of both the intrinsic and extrinsic apoptosis pathways in cultured HUVECs.
Affiliation: Tsukuba Research Laboratory, Eisai Co., Ltd., Tsukuba, Japan.