U2OS cells were treated for the indicated period with LTX-315, staurosporine (STS) or 100 M carbonyl cyanide m-chlorophenyl hydrazine (CCCP) in the absence or presence of the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1 (Nec), followed by fixation and permeabilization of the cells, immunofluorescence staining for the detection of active caspase 3 (Casp3a) and counterstained with the chromatin dye Hoechst 33342. have been based on a sequence motif resembling the peptide KLAKLAK (K = lysine, L = leucine, A = alanine).1 Such peptides can be fused with plasma membrane transducing domains2 and targeted to specific tumor cell antigens3-6 the tumor-associated endothelium 7 or white adipose cells8 with the scope of generating agents that selectively ablate specific cell types in vivo, upon their systemic administration. Such peptides have been reported to induce apoptosis due to their capacity to induce mitochondrial membrane permeabilization, followed by the release of cytochrome and activation of caspases.3-11 Recently, an optimized antimicrobial peptide, LTX-315 has been designed based on the structure of bovine lactoferricin, which is one of the most studied antimicrobial peptides.12 LTX-315 has the particularity to cause the regression of B16 melanomas in vivo when it is administered into the tumor.12,13 This effect involves infiltration of the tumor by T lymphocytes Rabbit polyclonal to XPR1.The xenotropic and polytropic retrovirus receptor (XPR) is a cell surface receptor that mediatesinfection by polytropic and xenotropic murine leukemia viruses, designated P-MLV and X-MLVrespectively (1). In non-murine cells these receptors facilitate infection of both P-MLV and X-MLVretroviruses, while in mouse cells, XPR selectively permits infection by P-MLV only (2). XPR isclassified with other mammalian type C oncoretroviruses receptors, which include the chemokinereceptors that are required for HIV and simian immunodeficiency virus infection (3). XPR containsseveral hydrophobic domains indicating that it transverses the cell membrane multiple times, and itmay function as a phosphate transporter and participate in G protein-coupled signal transduction (4).Expression of XPR is detected in a wide variety of human tissues, including pancreas, kidney andheart, and it shares homology with proteins identified in nematode, fly, and plant, and with the yeastSYG1 (suppressor of yeast G alpha deletion) protein (5,6) and the stimulation of an anticancer immune response that protects immunocompetent mice cured from melanoma against subsequent rechallenge with B16 cells.12 Based on these observations, it has been suggested that LTX-315 may induce immunogenic cell death,12,13 a type of cell death that is able to improve the efficacy of anticancer therapies.14-24 Intrigued by these findings, we wondered which particular cell death modality would be induced by LTX-315, knowing that there is a constant debate on the question whether apoptosis or necrosis would constitute a more immunogenic type of cellular demise.15,25,26 Here, we report that LTX-315 fails to activate caspases and causes classical necrosis that is refractory to necroptosis inhibitors including necrostatin-1 and cyclosporine A. We also present ultrastructural 5-HT4 antagonist 1 evidence in favor of the hypothesis that LTX-315 induces a necrotic cell death phenotype. Results and Discussion Failure of LTX-315 to induce hallmarks of apoptosis The major morphological and biochemical hallmarks of apoptosis are nuclear condensation (pyknosis) with fragmentation (karyorhexis) and the activation of effector caspases, in particular caspase-3.27-29 Transmission electron microscopic observation of U2OS osteosarcoma cells treated with LTX-315 (6?h) did not reveal any morphological signs of nuclear apoptosis since nuclei appeared largely intact and major chromatin condensation was absent (Fig. 1). At low concentrations of LTX-315 (12.5 to 50 g/ml), which do not cause immediate cell death defined by plasma membrane permeabilization (see below), the only major morphological change consisted in the dilatation of mitochondria that often manifested a hollow appearance. At higher concentrations (100 g/ml), the vast majority of cells adopted a necrotic morphology with absent plasma membranes and vacuolated cytoplasms. Frequently, cellular remnants remained attached to the culture substrate while manifesting a ‘ghost’-like appearance (Fig. 1). Open in a separate window Figure 1. Ultrastructural characteristics of LTX-315-induced cell death. U2OS cells were either left untreated (control, Ctr) or treated with the indicated dose of LTX-315 for 6?hours followed by osmium tetroxide staining and transmission electron microscopy. Note the presence of dilated mitochondria in cells treated with 12.5 or 50 g/ml of LTX-315. We further analyzed the capacity of LTX-315 to induce chromatin condensation by means of 5-HT4 antagonist 1 fluorescence microscopy after Hoechst 33342 staining. This method was combined with the detection of activated, proteolytically mature caspase-3 (Casp3a) by immunofluorescence staining of fixed and permeabilized cells.30 The positive control, the pan-tyrosine kinase inhibitor staurosporine, 5-HT4 antagonist 1 induced a significant degree of caspase-3 activation (detectable as a positive immunofluorescence signal) and nuclear shrinkage (detectable by morphometric analysis of the surface area of the Hoechst 33342 staining). As an additional control, the pan-caspase inhibitor Z-VAD abolished the activation of caspase-3 and reduced chromatin condensation induced by staurosporine and the uncoupling agent CCCP while necrostatin-1, an inhibitor of the RIP1 kinase,31 did not interfere with these parameters (Fig. 2). In contrast, LTX-315 failed to induce both signs of apoptosis (Fig. 2). This result was obtained over a range of LTX-315 concentrations (from 12.5 to 200 g/ml) and at several time points (6?h, 24?h). Hence, LTX-315 is unable to induce the major morphological and biochemical signs of apoptosis. Open in a separate window Figure 2. Failure of LTX-315 to induce caspase-3 activation and nuclear shrinkage. U2OS cells were treated for the indicated period with LTX-315, staurosporine (STS) or 100 M carbonyl cyanide m-chlorophenyl hydrazine (CCCP) in the absence or presence of the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1 (Nec), followed by fixation and permeabilization of the cells, immunofluorescence staining for the detection of active caspase 3 (Casp3a) and counterstained with the chromatin dye Hoechst 33342. Representative images are shown in A (images obtained in the absence of Z-VAD and Nec). Quantitative results (means SD of triplicates) are shown in B and C. In B,.