Abstract
BackgroundIn the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells.Material and methodsColorectal cancer cell lines (HCT-15, HT-29, SW480) were subjected to BV6 treatment (0–4 μM) with or without irradiation (2–8 Gy, single dose) followed by MTT, Caspase 3/7 activity, γH2AX/53BP1 foci assays, AnnexinV staining, cell cycle analysis, 3D colony forming assays and Western blotting (cellular IAP1 (cIAP1) and cIAP2, Survivin, X-linked IAP (XIAP)).ResultsBV6 treatment decreased cell viability and significantly increased irradiation-induced apoptosis as analyzed by Caspase 3/7 activity, AnnexinV-positive and subG1 phase cells. While basal 3D clonogenic survival was decreased in a cell line-dependent manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced γH2AX/53BP1-positive foci. Western blot analysis revealed a markedly reduced cIAP1 expression at 4 h after BV6 treatment in all cell lines, a substantial reduction of XIAP expression in SW480 and HT-29 cells at 24 h and a slightly decreased cIAP2 expression in HCT-15 cells at 48 h after treatment. Moreover, single or double knockdown of cIAP1 and XIAP resulted in significantly increased residual γH2AX/53BP1-positive foci 24 h after 2 Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells.ConclusionThe SMAC mimetic BV6 induced apoptosis and hampered DNA damage repair to radiosensitize 3D grown colorectal cancer cells. Our results demonstrate IAP targeting as a promising strategy to counteract radiation resistance of colorectal cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0507-4) contains supplementary material, which is available to authorized users.
Highlights
In the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells
We aimed to examine cytotoxicity, apoptosis, cell cycle distribution, expression of IAPs Survivin, cellular IAP1 (cIAP1), cIAP2 and X-linked IAP (XIAP) and used the more physiologic three-dimensional (3D) clonogenic survival assay in three colorectal cancer cell lines, pretreated with BV6 followed by exposure to different doses of ionizing radiation
A 24 h BV6 treatment reduced SW480, HT-29 and HCT-15 colorectal cancer cell viability in a concentration- and cell line-dependent manner with a significant reduction of viability following sole BV6 treatment. This effect was further enhanced by additional irradiation (2 or 8 Gy) at 4 h after BV6 treatment, most pronounced in SW480 and HT-29 cells (Fig. 1a)
Summary
We aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells. Tumor cells frequently develop strategies to escape cell death upon radio- and/or chemotherapeutic treatment which. In this context, members of the inhibitor of apoptosis (IAP) protein family recently gained attention as attractive target molecules for sensitizing tumor cells to radiation therapy [5, 6]. Overexpression of Survivin and a second well-studied member of this protein family, X-linked IAP (XIAP), is associated with a resistant phenotype in advanced rectal cancer after preoperative radiochemotherapy marked by increased local failure rates, distant metastasis and decreased overall survival [10, 11]
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