Apoptosis necrosis or autophagy-it is the mode of cell demise that defines the response of surrounding cells and organs. autophagy signalling such as ER stress and phagosome formation is initiated. Importantly we also observed lysosomal membrane permeabilization. It is the integration of all signals that results in DNA degradation and a disruption of the plasma membrane. Our data thus suggest that Cd causes the activation of FCGR3A multiple death signals in parallel. The genotype (for example p53 positive or negative) as well as other factors may determine the initiation and rate of individual death signals. Differences in the signal mix and speed may explain the differing results recorded as to the Cd-induced mode of cell death thus far. In human endothelial cells it is the sum of most if not all of these signals that determine the mode of Cd-induced cell death: programmed necrosis. Electronic supplementary material The online version of this article (doi:10.1007/s00018-015-2094-9) contains supplementary material which is available to authorized users. Test or to one-sided ANOVA. Statistical analyses were performed Epiberberine using IBM SPSS Statistics 20.0 (SPSS Inc. USA). Results Chelation of Cd by EGTA prevents toxicity and Cd treatment induces DNA strand breaks in endothelial cells Pre-treatment of Cd incubated endothelial cells with the Ca2+ (Calcium) chelator EGTA (ethylene glycol tetra-acetic acid) significantly reduces the toxicity of this heavy metal. Quantification of flow cytometry-based Annexin V/Propidium Iodide (PI) staining revealed a significant inhibition of Cd-induced cell death by increasing EGTA concentrations after treatment with 15 or 30?μM Cd (Fig.?1a). To analyse the genotoxic effects of Cd on endothelial cells a Comet-Assay was performed. Figure?1c shows representative images of the Comet Assay from both control and Cd-treated cells after 12?h. The amount of Comet positive cells after Cd treatment was quantified and the results are displayed in Fig.?1b. Massive DNA strand breaks are observed after treatment with 15 or 30?μM Cd. However no influence of Cd on the cell cycle could be observed (Supplemental Material Figure S5). Fig.?1 Inhibition of Cd toxicity by EGTA and the effect of Cd on endothelial DNA. a Shows the quantification of Cd-induced cell death (Annexin V/PI staining) after pre-treatment of cells with increasing EGTA concentrations. (b) Quantification of Comet-tail positive … Cd treatment provokes an increase in intracellular Ca2+ concentration In addition to the rapid genotoxic effect Cd provokes a significant increase in intracellular Ca2+ concentration already detectable after 1?h of incubation (Supplemental Material Figure S1). Furthermore the involvement of the Ca2+ sensitive non-lysosomal cysteine protease calpain was analysed by the usage of the calpain I and II inhibitor (MDL 28170) showing a significant inhibition of Ca2+ flux in cells treated with 15?μM Cd but no effect in cells treated with 30?μM Cd after 24?h (Fig.?2b). To rule Epiberberine out the role of p53 in Cd-induced cell death as previously suggested  we generated p53 KD endothelial cells to address the question of whether Cd-induced DNA breaks result in a p53-dependent cell death response. Although p53 is involved in the Cd-induced cell death pathway a knock-down of p53 was not able to inhibit the intracellular Ca2+ flux induced by Cd after 24?h (Fig.?2a). Fig.?2 Cd induced increase in cytosolic Ca2+ concentration. a-d show flow cytometry-based quantifications of intracellular Ca2+ concentration after Cd treatment of endothelial cells for the indicated times. The Epiberberine effect of p53 KD (a) the incubation with … Involvement of BCL-XL in Cd-induced Ca2+ flux was analysed using endothelial cells overexpressing this anti-apoptotic protein and Fig.?2c shows that this overexpression is highly efficient in Epiberberine inhibiting Ca2+ flux. To test the hypothesis that Cd treatment induces a substantial release of Ca2+ from the endoplasmatic reticulum (ER) 2 (2-Aminoethoxydiphenyl borate) was used to inhibit InsP3 (Inositol 1 4 5 Ca2+ release. Flow cytometry-based analyses uncovered that 2APB incubation is not.