Combined effects of alternariols mixture on human colon carcinoma cells.

Mycotoxins are naturally occurring contaminants encountered at high levels in a wide variety of agricultural products intended for human and animal consumptions. Various Alternaria mycotoxins may occur simultaneously in small grain cereals. Considering the concomitant production of alternariol (AOH) and alternariol monomethyl ether (AME), it is expected that humans and animals are exposed to the mixture rather than to individual compounds. Therefore, we studied the interactive effects of binary mixture of alternariols (AOH and AME) on the human intestinal cell line, HCT116 cells. Exposure of HCT116 cells to low cytotoxic alternariols doses, resulted in a moderate cytotoxicity manifested by a loss in the cell viability mediated by an activation of the mitochondrial apoptotic process, associated with the opening of mitochondrial permeability transition pore (PTP) and the loss of the mitochondrial transmembrane potential (ΔΨm). However, when combined, they exert a significant increase in their toxic potential. Altogether, our study showed that AOH and AME combination is obviously additive.

In vitro investigation of toxicological interactions between the fusariotoxins deoxynivalenol and zearalenone.

It is expected that humans are exposed to combined mycotoxins, which occur simultaneously in the food items, than to individual compounds and that can increase their potential toxicity. Considering this coincident production, deoxynivalenol (DON) and zearalenone (ZEN) as they are produced by several Fusarium species, can interfere at a cellular level. Therefore, these two toxins were chosen to study their interactive effects on human colon carcinoma cells (HCT116), using the endpoints including cell viability, cell cycle analysis, mitochondrial transmembrane potential (ΔΨm) determination and permeability transition pore (PTP) opening. Our results showed that DON and ZEN caused a marked decrease of cell viability in a dose-dependent manner, mediated by an activation of the mitochondrial apoptotic process; characterized by PTP opening and the loss of ΔΨm. Nevertheless, combined DON and ZEN reduced all the toxicities observed with the mycotoxins separately. Therefore, the combination of the two mycotoxins appears as a sub-additive response.

Cell death induced by the Alternaria mycotoxin Alternariol.

Mycotoxins are unavoidable contaminants of most foods and feeds, and some are known to be detrimental to human health. It is thus worthwhile to understand how cells of the intestinal system, one of the primary targets of these toxins, respond to their toxic effects. In this study, human colon carcinoma cells were used to elucidate the cell death mode and the pathways triggered by Alternariol (AOH), the most important mycotoxin produced by Alternaria species, which are the most common mycoflora infecting small grain cereals worldwide. Treatment of cells with AOH resulted in a loss of cell viability by inducing apoptosis. AOH-induced apoptosis was mediated through a mitochondria-dependent pathway, characterized by a p53 activation, an opening of the mitochondrial permeability transition pore (PTP), a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O(2)(*-) and caspase 9 and 3 activation. Besides, deficiency of the pro-apoptotic protein Bax partially protected cells against AOH-induced mitochondrial alterations. In addition, experiments performed on purified mitochondria indicated that AOH does not directly target this organelle to induce cell death. Our results demonstrate for the first time that AOH-induced cytotoxicity is mediated by activation of the mitochondrial pathway of apoptosis in human colon carcinoma cells.

Involvement of mitochondria-mediated apoptosis in deoxynivalenol cytotoxicity.

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates cereal crops and harmfully affects the gastrointestinal tract. Since it is well known that mitochondria play a central role in apoptosis triggered by many stimuli, an effort was made to examine whether DON-induced cytotoxicity occurs through mitochondria-mediated apoptotic pathway. The intestinal system being one of the primary targets of mycotoxins, the human colon carcinoma cell line HCT116 was used in this study. Using flow cytometric analyses and immunofluorescence, we showed that DON at 100 µM induced a mitochondria-dependent apoptotic pathway associated with opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm), downstream generation of O2·â» and cytochrome c release. The DON-induced apoptosis was accompanied by an activation of caspase 9 and 3, as demonstrated by Western blot and caspase activity assay. In addition, by taking advantage of HCT116 cells invalidated for Bax, we showed that this pro-apoptotic protein favored mitochondrial alterations induced by the mycotoxin. Besides, incubation of purified mitochondria with DON indicated that this mycotoxin does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Altogether, our results indicate that mitochondria-related caspase-dependent apoptotic pathway is involved in this in vitro model of DON induced-cytotoxicity.

Increased expression of VDAC1 sensitizes carcinoma cells to apoptosis induced by DNA cross-linking agents.

A major clinical problem regarding antitumoral treatment with DNA cross-linking agents such as cisplatin (Cisp), mechlorethamine (HN2) or its derivative melphalan (MLP) is intrinsic or acquired resistance to therapy, which frequently results from a resistance to apoptosis induction. In this study, aimed to identify novel sensitizing targets to DNA cross-linker-induced cell death, we demonstrated that MLP, Cisp and HN2 induce mitochondrial permeability transition pore (PTP)-mediated apoptosis in cervical and colon carcinoma cells. This apoptotic pathway is characterized by dissipation of the mitochondrial membrane potential, production of ROS, mitochondrial translocation of Bax, release of apoptogenic factors, caspase activation and nuclear alterations. The opening of PTP and subsequent apoptosis was reduced in Bax deficient cells and in cells with elevated Bcl-2 level, but not in cells invalidated for Bak. We further showed that, among the pro-apoptotic PTP regulators tested (VDAC1, creatine kinase, ANT1 and ANT3), exogenous overexpression of VDAC1 was the most effective in enhancing Cisp- and MLP-induced apoptosis. In addition, pharmacologically induced up-regulation of VDAC1 by the chemotherapeutic agent arsenic trioxide (As(2)O(3)) greatly sensitized HeLa cells to Cisp and MLP treatment. These data indicate that increased expression of VDAC1 appears as a promising strategy to improve DNA cross-linker-induced chemotherapy.

The adenine nucleotide translocase 2, a mitochondrial target for anticancer biotherapy.

Apoptosis or programmed cell death is one of the most important signaling pathways, which controls the cell fate and is frequently impaired in cancer cells. The major consequences of apoptosis inhibition are the accumulation of mutated cells and their enhanced resistance to chemotherapeutic agents. More generally, intrinsic or acquired apoptosis resistance may favor tumor growth and dissemination of mutated cells, and this resistance can be responsible of treatment failure. Mitochondria are central organelles in the signaling pathway of apoptosis and have been proposed as favorite candidates for anticancer biotherapy because they accommodate potential biological targets. Indeed, although cancer cells are highly glycolytic and become energetically independent of oxidative phosphorylation. Mitochondrial proteins involved in the so-called mitochondrial membrane permeabilization (MMP), such as the adenine nucleotide translocase (ANT) can be instrumental to elicit cancer cell death. Thus, multiple pharmacological and molecular studies revealed ANT could be a promising therapeutic target for the following reasons: (i) ANT is a bi-functional protein, it mediates the vital exchange of cytosolic ADP and mitochondrial ATP and participates to MMP via its capacity to become a lethal pore in the mitochondrial inner membrane; (ii) both ANT functions are under the control of the (anti)-oncogenes from the Bax/Bcl-2 family, (iii) several chemotherapeutic agents directly modulate the pore-forming activity of ANT and (iv) ANT2 isoform, which is anti-apoptotic, can be overexpressed in human cancers and its invalidation sensitize cells to apoptosis. In this review, we will introduce the knowledge of the role of ANT in MMP, illustrate the modulation of ANT by several strategies and propose the possibility to target preferentially the ANT2 isoform for induction of cancer cell apoptosis.

Molecular events involved in ochratoxin A induced mitochondrial pathway of apoptosis, modulation by Bcl-2 family members.

In this study, we looked for the role of the mitochondrion in the cytotoxicity of ochratoxin A (OTA), which is one of the most abundant food-contaminating mycotoxins in the world. In different human carcinoma cell lines, OTA triggered a mitochondria-dependent apoptotic process, which is characterized by opening of the mitochondrial permeability transition pore (PTPC), loss of mitochondrial transmembrane potential (ΔΨ(m) ), increase in O(2) [chemp](-) production, mitochondrial relocalization of Bax, release of cytochrome c, and caspase activation. However, studies performed on purified organelles suggested that OTA does not directly target the mitochondrion. In addition, we showed that mitochondrial alterations induced by this mycotoxin are favored by the proapoptotic protein Bax, but not Bak. These alterations are prevented by the antiapoptotic proteins, Bcl-2 and to a lesser degree by Bcl-X(L). Taken together, these data indicate that although mitochondria, PTPC members and proteins of Bcl-2 family play a pivotal role in OTA-induced apoptosis, they do not constitute real targets to overcome its toxicity.

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells.

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.

Fusarial toxin-induced toxicity in cultured cells and in isolated mitochondria involves PTPC-dependent activation of the mitochondrial pathway of apoptosis.

Mycotoxins produced by the Fusarium molds can cause a variety of human diseases and economic losses in livestock. Fusaria produce predominantly two types of mycotoxins: the nonestrogenic trichothecenes including T-2 toxin and the mycoestrogens such as zearalenone (ZEN). In a previous report, we demonstrated that the hepatotoxicity of these mycotoxins involves the mitochondrial pathway of apoptosis. Here, we observed that both fusarotoxins induced cell death by a mitochondria-dependent apoptotic process which includes opening of the mitochondrial permeability transition pore complex (PTPC), loss of mitochondrial transmembrane potential, increase in O(2)(.-) production, mitochondrial relocalization of Bax, cytochrome c release, and caspase activation. Studies performed on isolated mouse liver mitochondria showed that both ZEN and T-2 toxin might act directly on mitochondria to induce a PTPC-dependent permeabilization of mitochondrial membranes. Moreover, they may target different members of PTPC. Indeed, although the inner membrane protein adenine nucleotide translocase could be the target of T-2 toxin, ZEN seems to target the outer membrane protein voltage-dependent anion channel. Cells pretreatment with the p53 inhibitor pifithrin-alpha suggested that ZEN but not T-2 toxin triggered a p53-dependent mitochondrial apoptotic pathway. Finally, mitochondrial alterations induced by ZEN and T-2 toxin are mediated by Bcl-2 family proteins, such as Bax, and prevented by Bcl-x(L) and to a lesser extent by Bcl-2. Taken together, these data indicate that mitochondria play a pivotal role in both ZEN- and T-2 toxin-induced apoptosis and that PTPC members and proteins of Bcl-2 family should be interesting targets to overcome fusarotoxin toxicity.

Role of the permeability transition pore complex in lethal inter-organelle crosstalk.

The ndoplasmic reticulum (ER) function is critical for multiple cellular activities. Hence, impairment of the physiological function of the ER, such as accumulation of unfolded proteins or disturbance of lumenal calcium homeostasis, leads to an evolutionarily conserved adaptive response known as the ER stress response. Activation of this self-protective pathway gives the cell a chance to restore normal ER function. In the case of prolonged or severe stress conditions, or if the ER dysfunctions cannot be compensated, apoptosis is ultimately activated to eliminate stressed cells. Although the molecular mechanisms involved in ER stress-mediated apoptosis are poorly understood, it is known that ER and mitochondria can cooperate to induce cell death. In this review, we discuss the commitment and development of the lethal crosstalk between ER and mitochondria and focus on the role of the mitochondrial permeability transition pore complex in these processes.

Different apoptotic pathways induced by zearalenone, T-2 toxin and ochratoxin A in human hepatoma cells.

Mycotoxins, secondary metabolites produced by moulds, have been shown to cause diverse toxic effects in animals and are also suspected of disease causation in humans. The present study compares the molecular mechanisms of the toxicity of zearalenone (ZEN), T-2 toxin and ochratoxin A (OTA) in human hepatoma cells HepG2. The three mycotoxins-induced a caspase-dependent mitochondrial apoptotic pathway. The mitochondrial alterations include: bax relocalisation into the mitochondrial outer membrane, loss of the mitochondrial transmembrane potential, PTPC opening, and cytochrome c (but not AIF) release. In the presence of ZEN and T-2 toxin, reactive oxygen species (ROS) level was highly increased at an early stage even before mitochondrial alterations were observed, whereas OTA-induced only O(2)(-) generation among total ROS. This ROS production appears as a consequence of mitochondrial alterations. HepG2 cell treatment with the p53 inhibitor pifithrin-alpha (PFT) and western blot analysis suggested that both ZEN and OTA, but not T-2 toxin, trigger a p53-dependent apoptotic pathway. These results clearly point to a central role of mitochondria in the apoptotic process induced by ZEN, T-2 toxin and OTA and provide new insights into the molecular mechanisms by which these mycotoxins might promote hepatotoxicty.

Binding of YY1 to the proximal region of the murine beta interferon promoter is essential to allow CBP recruitment and K8H4/K14H3 acetylation on the promoter region after virus infection.

Virus-induced activation of the beta interferon (IFN-beta) gene requires orderly recruitment of chromatin-remodeling complexes and time-regulated acetylation of histone residues K8H4 and K14H3 on the promoter region. We have previously shown that transcription factor Yin Yang 1 (YY1) binds the murine IFN-beta promoter at two sites (-122 and -90) regulating promoter transcriptional capacity with a dual activator/repressor role. In this work we demonstrate that both YY1 -122 and -90 sites are required for CBP recruitment and K8H4/K14H3 acetylation to take place on the IFN-beta promoter region after virus infection. A single point mutation introduced at either one of these two sites inhibiting YY1 binding completely disrupted CBP recruitment and K8H4/K14H3 acetylation independently of HMGI or IRF3 binding to the promoter. We have previously demonstrated that YY1 represses the transcriptional capacity of the IFN-beta promoter through its -90 site via histone deacetylation. Here we demonstrate that, in vivo, the binding of YY1 to the -90 site is constant all through virus infection whereas the binding of YY1 to the -122 site is activated after infection. We discuss here the capacity of YY1 to either repress (through histone deacetylase recruitment) or activate (through CBP recruitment) IFN-beta gene expression according to the occupancy of either only its -90 site or both its -122 and -90 sites.