KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways
Highlights
► KNK437, a benzylidene lactam compound, is a novel radiosensitizer. ► KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1α under hypoxia. ► KNK437 abrogates hypoxia induced resistance to radiation.
Introduction
Stress ranging from heat stress to pathophysiological conditions induce HSPs [1]. These proteins function as molecular chaperones, highly conserved and cytoprotective [2]. Enhanced expression of HSPs in response to stress is mediated by the heat shock transcription factor 1 (HSF1) [2]. HSPs enhance DNA repair and inhibit cell death to activate an adaptive response to IR [3], [4], [5], [6]. Although, a number of HSPs have been implicated in cellular resistance to IR, only HSP90 has been effectively targeted. The most widely used HSP90 inhibitors such as geldenamycin and its clinically relevant analog 17AAG, sensitize cancer cells to radiation [7], [8]. However, these inhibitors activate HSF1 and subsequent induction of HSPs [9], [10]. Induction of HSPs by HSP90 inhibitors in turn limits their efficacy as radiosensitizers.
Hypoxia induces genetic instability and tumor invasion, resulting in a more malignant phenotype [11] and is a major limiting factor in the efficacy of IR against solid tumors. HIF-1 is an oxygen sensitive heterodimeric transcription factor responsible for cellular adaptation to hypoxic conditions. HIF-1 comprises α and β components where the α component is regulated by oxygen dependent protein degradation [12]. HIF-1 contributes to radioresistance by modulating the expression of genes involving angiogenesis and cell proliferation [13], [14]. Inhibition of HIF-1α by either genetic or pharmacological means sensitizes cancer cells to radiation [15], [16]. HIF-1α is mostly regulated at post-transcriptional levels of translation and protein stability [17], [18]. Various studies have reported that hypoxia induces HSPs which in turn stabilizes HIF-1α [19], [20], [21]. AKT signaling is an established upstream modulator of HIF-1α [18], [21], [22], [23]. Selective inhibition of AKT abrogates the accumulation of HIF-1α under hypoxia [18], [23].
KNK437 is a novel benzylidene lactam compound, isolated from an organic source library (Kaneka Corp., Osaka, Japan). KNK437 is characterized by its ability to inhibit stress induced synthesis of HSPs mediated by HSF1 [24]. The efficacy of KNK437 as a sensitizer to hyperthermia mediated cell killing has been validated in vivo [25]. When used along with the HSP90 inhibitor, 17-AAG, KNK437 abrogated the induction of HSP70 and exhibited synergy to reduce the survival of HL-60 cells [10]. In the present study, we examined whether KNK437 sensitizes cancer cells to IR and overrides hypoxia-induced radioresistance.
Section snippets
Chemicals and antibodies
All chemicals were purchased from Sigma unless indicated otherwise. KNK437 (heat shock protein inhibitor 1) was obtained from Calbiochem, Merck Chemicals Ltd., Nottingham, UK. Rabbit polyclonal antibodies against PARP, HIF-1α, HIF-1β, and Phospho-AKT (Ser473) and mouse monoclonal antibody against AKT were obtained from Cell Signaling Technology, Danvers, MA. Mouse monoclonal antibodies against HSP70, HSP27 were obtained Stressgen, Enzo Life Sciences, and Exeter, UK. Mouse monoclonal antibody
KNK437 sensitizes cancer cells to ionizing radiation
We hypothesized that KNK437 inhibits IR induced synthesis of HSPs, which in turn renders cells sensitive to radiotherapy. Two human tumor cell lines, MDA-MB-231 (breast) and T98G (glioma) were pre-treated with KNK437 (50 μM) as described in Section 2 before irradiation. Pre-treatment conditions were determined by dose response studies using the MTT assay (data not shown). As hypothesized, KNK437 reduced survival and clonogenicity of cells irradiated with single doses of 2 and 5 Gy (Fig. 1A).
Discussion
Cancer cells express high basal level of HSPs [33]. Extensive studies have been reported on the protective role of HSPs against radiation [3], [4], [5], [6]. A probable reason for the lack of induced synthesis of HSPs in irradiated cells under this study is the high basal level expression of both HSP70 and HSP27. Monomeric forms of HSF1 exist in complex with HSPs in the cytoplasm under normal conditions [32]. During stress, HSPs, which have a high affinity for denatured proteins dissociate from
Acknowledgments
The authors acknowledge funding to KMP from CR-UK (Grant No.: C1513/A7047).
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