Cell-cell contacts prevent t-BuOOH-triggered ferroptosis and cellular damage in vitro by regulation of intracellular calcium.

Tert-butyl hydroperoxide (t-BuOOH) is an organic hydroperoxide widely used as a model compound to induce oxidative stress. It leads to a plethora of cellular damage, including lipid peroxidation, DNA double-strand breaks (DNA DSBs), and breakdown of the mitochondrial membrane potential (MMP). We could show in several cell lines that t-BuOOH induces ferroptosis, triggered by iron-dependent lipid peroxidation. We have further revealed that not only t-BuOOH-mediated ferroptosis, but also DNA DSBs and loss of MMP are prevented by cell-cell contacts. The underlying mechanisms are not known. Here, we show in murine fibroblasts and a human colon carcinoma cell line that t-BuOOH (50 or 100 µM, resp.) causes an increase in intracellular Ca2+, and that this increase is key to lipid peroxidation and ferroptosis, DNA DSB formation and dissipation of the MMP. We further demonstrate that cell-cell contacts prevent t-BuOOH-mediated raise in intracellular Ca2+. Hence, we provide novel insights into the mechanism of t-BuOOH-triggered cellular damage including ferroptosis and propose a model in which cell-cell contacts control intracellular Ca2+ levels to prevent lipid peroxidation, DNA DSB-formation and loss of MMP. Since Ca2+ is a central player of toxicity in response to oxidative stress and is involved in various cell death pathways, our observations suggest a broad protective function of cell-cell contacts against a variety of exogenous toxicants.

Cell-cell contacts protect against t-BuOOH-induced cellular damage and ferroptosis in vitro.

Ferroptosis is a recently discovered pathway of regulated necrosis dependent on iron and lipid peroxidation. It has gained broad attention since it is a promising approach to overcome resistance to apoptosis in cancer chemotherapy. We have recently identified tertiary-butyl hydroperoxide (t-BuOOH) as a novel inducer of ferroptosis. t-BuOOH is a widely used compound to induce oxidative stress in vitro. t-BuOOH induces lipid peroxidation and consequently ferroptosis in murine and human cell lines. t-BuOOH additionally results in a loss of mitochondrial membrane potential, formation of DNA double-strand breaks, and replication block. Here, we specifically address the question whether cell-cell contacts regulate t-BuOOH-induced ferroptosis and cellular damage. To this end, murine NIH3T3 or human HaCaT cells were seeded to confluence, but below their saturation density to allow the establishment of cell-cell contacts without inducing quiescence. Cells were then treated with t-BuOOH (50 or 200 µM, respectively). We revealed that cell-cell contacts reduce basal and t-BuOOH-triggered lipid peroxidation and consequently block ferroptosis. Similar results were obtained with the specific ferroptosis inducer erastin. Cell-cell contacts further protect against t-BuOOH-induced loss of mitochondrial membrane potential, and formation of DNA double-strand breaks. Interestingly, cell-cell contacts failed to prevent t-BuOOH-mediated replication block or formation of the oxidative base lesion 8-oxo-dG. Since evidence of protection against cell death was both (i) observed after treatment with hydrogen peroxide, methyl methanesulfonate or UV-C, and (ii) seen in several cell lines, we conclude that protection by cell-cell contacts is a widespread phenomenon. The impact of cell-cell contacts on toxicity might have important implications in cancer chemotherapy.

t-BuOOH induces ferroptosis in human and murine cell lines.

Reactive oxygen species (ROS)-induced apoptosis has been extensively studied. Increasing evidence suggests that ROS, for instance, induced by hydrogen peroxide (H2O2), might also trigger regulated necrotic cell death pathways. Almost nothing is known about the cell death pathways triggered by tertiary-butyl hydroperoxide (t-BuOOH), a widely used inducer of oxidative stress. The lipid peroxidation products induced by t-BuOOH are involved in the pathophysiology of many diseases, such as cancer, cardiovascular diseases, or diabetes. In this study, we exposed murine fibroblasts (NIH3T3) or human keratinocytes (HaCaT) to t-BuOOH (50 or 200 µM, respectively) which induced a rapid necrotic cell death. Well-established regulators of cell death, i.e., p53, poly(ADP)ribose polymerase-1 (PARP-1), the stress kinases p38 and c-Jun N-terminal-kinases 1/2 (JNK1/2), or receptor-interacting serine/threonine protein kinase 1 (RIPK1) and 3 (RIPK3), were not required for t-BuOOH-mediated cell death. Using the selective inhibitors ferrostatin-1 (1 µM) and liproxstatin-1 (1 µM), we identified ferroptosis, a recently discovered cell death mechanism dependent on iron and lipid peroxidation, as the main cell death pathway. Accordingly, t-BuOOH exposure resulted in a ferrostatin-1- and liproxstatin-1-sensitive increase in lipid peroxidation and cytosolic ROS. Ferroptosis was executed independently from other t-BuOOH-mediated cellular damages, i.e., loss of mitochondrial membrane potential, DNA double-strand breaks, or replication block. H2O2 did not cause ferroptosis at equitoxic concentrations (300 µM) and induced a (1) lower and (2) ferrostatin-1- or liproxstatin-1-insensitive increase in lipid peroxidation. We identify that t-BuOOH and H2O2 produce a different pattern of lipid peroxidation, thereby leading to different cell death pathways and present t-BuOOH as a novel inducer of ferroptosis.

Inhibition of ß-catenin signaling by phenobarbital in hepatoma cells in vitro.

The antiepileptic drug phenobarbital (PB) exerts hepatic effect based on indirect activation of the constitutive androstane receptor (CAR) via inhibition of the epidermal growth factor receptor (EGFR) and the kinase Src. It has furthermore been observed that in mice PB suppresses the growth of hepatocellular carcinoma with overactive signaling through the oncogenic Wnt/ß-catenin pathway, thus suggesting an interference of PB with ß-catenin signaling. The present work was aimed to characterize effects of PB on ß-catenin signaling at different cellular levels and to elucidate molecular details of the interaction of PB and ß-catenin in an in vitro system of mouse hepatoma cells. PB efficiently inhibited signaling through ß-catenin. This phenomenon was in-depth characterized at the levels of ß-catenin protein accumulation and transcriptional activity. Mechanistic analyses revealed that the effect of PB on ß-catenin signaling was independent of the activation of CAR and also independent of the cytosolic multi-protein complex responsible for physiological post-translation control of the ß-catenin pathway via initiation of ß-catenin degradation. Instead, evidence is provided that PB diminishes ß-catenin protein production by inhibition of protein synthesis via signal transduction through EGFR and Src. The proposed mechanism is well in agreement with previously published activities of PB at the EGFR and Src-mediated regulation of ß-catenin mRNA translation. Inhibition of ß-catenin signaling by PB through the proposed mechanism might explain the inhibitory effect of PB on the growth of specific sub-populations of mouse liver tumors. In conclusion, the present data comprehensively characterize the effect of PB on ß-catenin signaling in mouse hepatoma cells in vitro and provides mechanistic insight into the molecular processes underlying the observed effect.

Tumor promotion and inhibition by phenobarbital in livers of conditional Apc-deficient mice.

Activation of Wnt/ß-catenin signaling is important for human and rodent hepatocarcinogenesis. In mice, the tumor promoter phenobarbital (PB) selects for hepatocellular tumors with activating ß-catenin mutations via constitutive androstane receptor activation. PB-dependent tumor promotion was studied in mice with genetic inactivation of Apc, a negative regulator of ß-catenin, to circumvent the problem of randomly induced mutations by chemical initiators and to allow monitoring of PB- and Wnt/ß-catenin-dependent tumorigenesis in the absence of unknown genomic alterations. Moreover, the study was designed to investigate PB-induced proliferation of liver cells with activated ß-catenin. PB treatment provided Apc-deficient hepatocytes with only a minor proliferative advantage, and additional connexin 32 deficiency did not affect the proliferative response. PB significantly promoted the outgrowth of Apc-deficient hepatocellular adenoma (HCA), but simultaneously inhibited the formation of Apc-deficient hepatocellular carcinoma (HCC). The probability of tumor promotion by PB was calculated to be much lower for hepatocytes with loss of Apc, as compared to mutational ß-catenin activation. Comprehensive transcriptomic and phosphoproteomic characterization of HCA and HCC revealed molecular details of the two tumor types. HCC were characterized by a loss of differentiated hepatocellular gene expression, enhanced proliferative signaling, and massive over-activation of Wnt/ß-catenin signaling. In conclusion, PB exerts a dual role in liver tumor formation by promoting the growth of HCA but inhibiting the growth of HCC. Data demonstrate that one and the same compound can produce opposite effects on hepatocarcinogenesis, depending on context, highlighting the necessity to develop a more differentiated view on the tumorigenicity of this model compound.