In the present study, the SMAC mimetic BV6 substantially increased 3D radiation response of three human colorectal cancer cell lines by degradation of cIAP1 and XIAP, thereby enhancing irradiation-induced apoptosis and hampering DNA DSB repair

In the present study, the SMAC mimetic BV6 substantially increased 3D radiation response of three human colorectal cancer cell lines by degradation of cIAP1 and XIAP, thereby enhancing irradiation-induced apoptosis and hampering DNA DSB repair. manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced H2AX/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 H2AX/53BP1-positive foci 24?h after 2?Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells. Conclusion The 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 material The online version of this article (doi:10.1186/s13014-015-0507-4) contains supplementary material, which is available to authorized users. Background Colorectal carcinoma is the third most prevalent cancer and constitutes the fourth most common cause of cancer-related death worldwide [1]. Since publication of the first results of the CAO/ARO/AIO-94 study, preoperative radiochemotherapy provides the standard treatment of locally advanced rectal cancer [2, 3]. However, tumor cells frequently develop strategies to escape cell death upon radio- and/or chemotherapeutic treatment which interferes with efficient treatment of the patients. To overcome therapeutic limitations, efforts have been made to identify factors resulting in a therapy resistance and to target those factors, which may improve clinical outcome [4]. 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]. Currently, eight different IAPs are known in mammals. Amongst them, Survivin has been extensively studied because of its multiple functions which comprise not only inhibition of Caspases and apoptosis but also regulation of cell division as part of the chromosomal passenger complex and radiation-induced damage repair [7C9]. Notably, 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]. A common structural feature of IAPs is their baculovirus IAP repeat (BIR) domain, present in different numbers in all IAPs and required for apoptosis inhibition [12]. This structural domain is responsible for multiple protein Mouse monoclonal to ATP2C1 interactions and regulation of IAP function. For Caspase inhibition, interaction of Survivin with XIAP by their BIR domains and with hepatitis B X-interacting protein (HBXIP) has been shown to be essential, while direct binding to Caspases 3, 7 and 9 is only mediated by XIAP [13, 14]. The carboxy-terminal Really Interesting New Gene (RING) domain, present for example in cellular IAP1 (cIAP1), cIAP2 and XIAP, functions as an E3 ubiquitin ligase and promotes ubiquitination and subsequent proteasomal degradation of the respective IAP and some of their binding partners Carbimazole [15, 16]. Amongst various IAP targeting approaches developed during the last years, substances mimicking the binding motif of the IAP antagonist Second Mitochondria-derived Activator of Caspase (SMAC) have gained growing attention. SMAC is released from mitochondria into the cytosol upon the induction of the intrinsic apoptosis pathway to negatively regulate IAP activity by binding to the BIR domains [17, 18]. The interaction between SMAC and XIAP, for example, prevents interaction of XIAP with Caspase 9 and subsequent activation of the apoptotic pathway [13]. Although the functions of cIAP1 and cIAP2 are less clear compared to XIAP Carbimazole Carbimazole and Survivin, it has been shown that both can function as E3 ubiquitin ligases and contribute to regulation of canonical and non-canonical nuclear factor kappa B (NF-B) signaling pathways and are involved Carbimazole in the upregulation of cytotoxic cytokines like tumor necrosis factor-alpha (TNF-) [15]. The latter renders human cancer cells susceptible to apoptosis induction in an autocrine/paracrine manner [19]. The bivalent SMAC mimetic BV6 binds to the BIR domains of IAP proteins, causing ubiquitination and proteasomal degradation of.