Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO(2), CeO(2), SiO(2), and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO(2) and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO(2) and CeO(2). We also noted pronounced cytotoxicity for three out of four additional SiO(2) nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO(2) nanoparticles tested and for one of the supplementary SiO(2) nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO(2) nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn(2+) with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn(2+) could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO(2) nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum.

Amplification of the MycN oncogene characterizes a subset of highly aggressive neuroblastomas, the most common extracranial solid tumor of childhood. However, the significance of MycN amplification for tumor cell survival is controversial, since down-regulation of MycN was found to decrease markedly neuroblastoma sensitivity towards conventional anticancer drugs, cisplatin, and doxorubicin. Here, we show that a redox-silent analogue of vitamin E, α-tocopheryl succinate (α-TOS), which triggers apoptotic cell death via targeting mitochondria, can kill tumor cells irrespective of their MycN expression level. In cells overexpressing MycN, as well as cells in which MycN was switched off, α-TOS stimulated rapid entry of Ca(2+) into the cytosol, compromised Ca(2+) buffering capacity of the mitochondria and sensitized them towards mitochondrial permeability transition and subsequent apoptotic cell death. Prevention of mitochondrial Ca(2+) accumulation or chelation of cytosolic Ca(2+) rescued the cells. Thus, targeting mitochondria might be advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways.

BACKGROUND Prostaglandin E(2)(PGE(2)) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE(2) production, the expression pattern and localization of PGE(2) receptors and intracellular signal transduction pathways activated by PGE(2). PRINCIPAL FINDINGS A high expression of the PGE(2) receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE(2) and stimulation of serum-starved neuroblastoma cells with PGE(2) increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE(2)(dmPGE(2)) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE(2). Similarly, PGE(2) receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner. CONCLUSIONS These findings demonstrate that PGE(2) acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE(2) signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.

During the last years, there have been a number of reports that prove involvement of mitochondria in the pathogenesis of variety of disorders including cancer and neurodegenerative diseases. Alteration of vital mitochondrial functions - production of ATP, calcium buffering capacity, abnormal production of reactive oxygen species, can be potentially responsible for pathogenesis of cancer and neurodegenerative diseases. Involvement of mitochondria in various types of cell death makes them attractive targets for tumor cell elimination. This review describes the role of mitochondria in tumor cells and how targeting of mitochondria can be used as a tool in fighting cancer.

Cytochrome c is a well-known mitochondrial protein that fulfills life-supporting functions by transferring electrons to the respiratory chain to maintain ATP production. However, during the activation of apoptotic machinery, it is released from mitochondria and, being in the cytosol, it either triggers the activation of the caspase cascade in intrinsic apoptotic pathway, or it is involved in the amplification of extrinsic apoptotic signaling. Accumulating evidence suggests that only unmodified holocytochrome c is efficient in the stimulation of apoptosis. Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the complete or partial cytochrome c deficiency in vivo. Here, we discuss the importance of distinct amino acid residues for various functions of cytochrome c in cells and mice with targeted cytochrome c mutations.

PURPOSE: To investigate resveratrol (RSV) as a calorie restriction (CR) mimetic potentiator of platinum-based cancer drugs. METHODS: In ovarian carcinoma cell lines, the potentiating effects of RSV were assessed in sulforhodamine B-based growth assays and clonogenic assays. Flow cytometry was used to detect cell cycle effects, siRNA transfections for determining the involvement of SIRT1, and Western blotting for the assessment of altered protein expression and of autophagy. Intracellular ATP levels were detected with a commercial kit. RESULTS: Single-dose RSV co-treatment with cisplatin or carboplatin at inefficiently low doses had the clinically interesting effect of preventing regrowth of cancer cells after drug withdrawal. Of three cell lines tested, metastatic cells with low bioenergetic cellular index (i.e., more glycolytic) were particularly sensitive to combination treatment leading to PUMA induction, acute apoptosis, and autophagy. However, inhibition of regrowth and complete loss of clonogenicity was seen also without these events, in other cells. The underlying mechanism(s) was independent of effects reported to underlie the CR-mimetic cancer-preventive potential of RSV. Thus, SIRT1, estrogen receptors, AMPK activation or upregulation of mitobiogenesis, β-F(1)-ATPase or PTEN were not involved, and ATP levels did not decrease. CONCLUSIONS: RSV is an excellent candidate for potentiation of platinum treatment, rather than a cancer therapeutic drug in its own right. While SIRT1-dependent and lifespan-promoting effects of RSV are well-documented and may dominate in normal cells, the observed potentiation of platinum drugs does not require these mechanisms. We suggest that the responses of cancer cells to RSV differ greatly from those of normal cells.

The aim of this study was to investigate the involvement of membrane-bound microsomal glutathione transferase 1 (MGST1) in cellular resistance against oxidative stress as well as its mechanism of protection. MGST1 is ubiquitously expressed and predominantly located in the endoplasmic reticulum and outer mitochondrial membrane. Utilizing MCF7 cells overexpressing MGST1 we show significant protection against agents that are known to induce lipid peroxidation (e.g., cumene hydroperoxide and tert-butylhydroperoxide) and an end-product of lipid peroxidation (e.g., 4-hydroxy-2-nonenal). Furthermore, our results demonstrate that MGST1 protection can be enhanced by vitamin E when toxicity depends on oxidative stress, but not when direct alkylation is the dominant mechanism. Mitochondria in MGST1-overexpressing cells were shown to be protected from oxidative insult as measured by calcium loading capacity and respiration. MGST1 induces cellular resistance against cisplatin. Here we used vitamin E to elucidate whether oxidative stress caused by cisplatin is significant for cell toxicity. The results indicate that oxidative stress and induction of lipid peroxidation are not the most prominent toxic mechanism of cisplatin in our cell system. We thus conclude that MGST1 protects cells (and mitochondria) by both conjugation and glutathione peroxidase functions. A new protective mechanism against cisplatin is also indicated.

Summary Refractory anaemia with ring sideroblasts (RARS) is characterized by anaemia, erythroid apoptosis, cytochrome c release and mitochondrial ferritin accumulation. Granulocyte-colony-stimulating factor (G-CSF) inhibits the first three of these features in vitro and in vivo. To dissect the molecular mechanisms underlying the RARS phenotype and anti-apoptotic effects of G-CSF, erythroblasts generated from normal (NBM) and RARS marrow CD34(+) cells were cultured +/-G-CSF and subjected to gene expression analysis (GEP). Several erythropoiesis-associated genes that were deregulated in RARS CD34(+) cells showed normal expression in erythroblasts, underscoring the importance of differentiation-specific GEP. RARS erythroblasts showed a marked deregulation of several pathways including apoptosis, DNA damage repair, mitochondrial function and the JAK/Stat pathway. ABCB7, transporting iron from mitochondria to cytosol and associated with inherited ring sideroblast formation was severely suppressed and expression decreased with differentiation, while increasing in NBM cultures. The same pattern was observed for the mitochondrial integrity gene MFN2. Other downregulated key genes included STAT5B, HSPA5, FANCC and the negative apoptosis regulator MAP3K7. Methylation status of key downregulated genes was normal. The mitochondrial pathway including MFN2 was significantly modified by G-CSF, and several heat shock protein genes were upregulated, as evidence of anti-apoptotic protection of erythropoiesis. By contrast, G-CSF had no effect on iron-transport or erythropoiesis-associated genes.

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants commonly found as contaminants in the same food sources. Even though their neurotoxic effects are established, the mechanisms of action are not fully understood. In the present study, we have used the mouse hippocampal neuronal cell line HT22 to investigate the mechanisms of neuronal death induced by MeHg, PCB 153, and PCB 126, alone or in combination. All chemicals induced cell death with morphological changes compatible with either apoptosis or necrosis. Mitochondrial functions were impaired as shown by the significant decrease in mitochondrial Ca(2+) uptake capacity and ATP levels. MeHg, but not the PCBs, induced loss of mitochondrial membrane potential and release of cytochrome c into the cytosol. Also, pre-treatment with the antioxidant MnTBAP was protective only against cell death induced by MeHg. While caspase activation was absent, the Ca(2+)-dependent proteases calpains were activated after exposure to MeHg or the selected PCBs. Furthermore, lysosomal disruption was observed in the exposed cells. Accordingly, pre-treatment with the calpain specific inhibitor PD150606 and/or the cathepsin D inhibitor Pepstatin protected against the cytotoxicity of MeHg and PCBs, and the protection was significantly enhanced when the two inhibitors were combined. Simultaneous exposures to lower doses of MeHg and PCBs suggested mostly antagonistic interactions. Taken together, these data indicate that MeHg and PCBs induce caspase-independent cell death via parallel activation of calpains and lysosomal proteases, and that in this model oxidative stress does not play a major role in PCB toxicity.

Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo, etc, plays a crucial role in apoptosis induction. A redox-silent analogue of vitamin E, alpha-tocopheryl succinate (alpha-TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species and Bax-mediated permeabilization of the outer mitochondrial membrane. Here we show that alpha-TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells, and Jurkat T-lymphocytes. In particular, in addition to ROS production, alpha-TOS stimulates rapid Ca(2+)entry into the cells with subsequent accumulation of Ca(2+) in mitochondria - a prerequisite step for MPT induction. Alteration of mitochondrial Ca(2+) buffering capacity was observed as early as 8 h after incubation with alpha-TOS, when no activation of Bax was yet detected. Ca(2+) accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca(2+) uptake significantly mitigated the apoptotic response. Importantly, Ca(2+)-induced mitochondrial destabilization might co-operate with Bax-mediated OMM permeabilization to induce cytochrome c release from mitochondria.(c) 2010 UICC.