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Dicitrinone B, a rare carbon-bridged citrinin dimer, was isolated from the marine-derived fungus, Penicillium citrinum. It was reported to have antitumor effects on tumor cells previously; however, the details of the mechanism remain unclear. In this study, we found that dicitrinone B inhibited the proliferation of multiple tumor types. Among them, the human malignant melanoma cell, A375, was confirmed to be the most sensitive. Morphologic evaluation, cell cycle arrest and apoptosis rate analysis results showed that dicitrinone B significantly induced A375 cell apoptosis. Subsequent observation of reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) reduction revealed that the apoptosis induced by dicitrinone B may be triggered by over-producing ROS. Further studies indicated that the apoptosis was associated with both intrinsic and extrinsic apoptosis pathways under the regulation of Bcl-2 family proteins. Caspase-9, caspase-8 and caspase-3 were activated during the process, leading to PARP cleavage. The pan-caspase inhibitor, Z-VAD-FMK, could reverse dicitrinone B-induced apoptosis, suggesting that it is a caspase-dependent pathway. Our data for the first time showed that dicitrinone B inhibits the proliferation of tumor cells by inducing cell apoptosis. Moreover, compared with the first-line chemotherapy drug, 5-fluorouracil (5-Fu), dicitrinone B showed much more potent anticancer efficacy, suggesting that it might serve as a potential antitumor agent.

Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8-24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Aims: With the identification of hypochlorous acid (HOCl) as a biomarker in diseased brains and endogenous detection of its modified proteins, HOCl might be implicated in the development of neurodegenerative disorders. However, its effect on neuronal cell death has not yet been investigated at gene expression level., Main Methods: Therefore, DNA microarray was performed for screening of HOCl-responsive genes in primary mouse cortical neurons. Neurotoxicity caused by physiological relevant HOCl (250μM) exhibited several biochemical markers of apoptosis., Key Findings: The biological processes affected during HOCl-mediated apoptosis included cell death, response to stress, cellular metabolism, and cell cycle. Among them, mRNAs level of cell death and stress response genes were up-regulated while expression of metabolism and cell cycle genes were down-regulated., Significance: Our results showed, for the first time, that HOCl induces apoptosis in cortical neurons by upregulating apoptotic genes and gene expression of stress response such as heat shock proteins and antioxidant proteins were enhanced to provide protection. These data form a foundation for the development of screening platforms and define targets for intervention in HOCl neuropathologies where HOCl-mediated injury is causative., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Nitric oxide (NO), ubiquitously expressed in the central nervous system, has been perceived to be a potential neuromodulator. Employing cultured murine primary cortical neurons, NO resulted in an inhibition of the ubiquitin-proteasome system (UPS) with a dose- and time-dependent decrease in cell viability. This is consistent with a previous study that reported a dysfunction of UPS with consequential apoptotic death in macrophage cell with NO treatment. However, it cannot be unclear if the drop in UPS efficiency is directly imposed on by NO. Therefore by using microarray analysis, our study revealed an early down-regulation or non-significant differential expression of genes encoding UPS proteins in NOC-18 (NO donor)-treated neurons as compared to an observed elevation of corresponding gene expression genes in lactacystin (classical proteasome inhibitor)-treated neurons (conducted earlier). Furthermore, time-course analysis of proteasome activity in NOC-18-treated neurons demonstrated a late onset of reduction. This is intriguing as it is well established that in an exclusive proteasome dysfunction-induced cell death, a compensatory feedback mechanism will be activated with an initial and concerted up-regulation of genes encoding proteins involved in UPS as seen when neurons were treated with lactacystin. Thus, it is highly suggestive that NO-triggered neuronal death takes on a different signaling cascade from that of a classical proteasome inhibitor, and that the late reduction of proteasome activity is a downstream event following the activation of apoptotic cellular signaling cascade. In intracellular condition, the proteasome is not NO preferred primary target responsible for the trigger of the cell death machinery. In conclusion, we presented novel findings that shed light into NO-induced cell death signaling cascade, which would be important in understanding the pathogenesis of neurodegenerative disorders such as Parkinson's disease.

Human thioredoxin reductase (TrxR) system is associated with cancer cell growth and anti-apoptosis process. Effects of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]ethane (BBSKE), a novel TrxR inhibitor, were investigated on human leukemia cell lines HL-60 and K562. BBSKE treatment induced cell growth inhibition and apoptosis in both cell lines. Apoptosis induced by BBSKE is through Bcl-2/Bax and caspase-3 pathways. Ehrlich's ascites carcinoma-bearing mice were used to investigate the anti-tumor effect of BBSKE in vivo. Tumor-bearing mice treated with BBSKE showed an increase of life span with a comparable effect to cyclophosphamide (CTX). These results suggest a potential usage of BBSKE as a therapeutic agent against non-solid tumors.

Studies have shown that the lipid peroxidation by-product, 4-hydroxynonenal (HNE), is involved in many pathological events in several neurodegenerative diseases. A number of signaling pathways mediating HNE-induced cell death in the brain have been proposed. However, the exact mechanism remains unknown. In the present study, we have examined the effects of HNE on cultured primary cortical neurons and found that HNE treatment leads to cell death via apoptosis. Both the caspase and calpain proteolytic systems were activated. There were also increased levels of phospho-p53 and cell cycle-related proteins. Gene transcription was further studied using microarray analysis. Results showed that majority of the genes associated with cell cycle regulation, response to stress, and signal transduction were differentially expressed. The various categories of differentially-expressed genes suggested that there are other parallel pathways regulating HNE-induced neuronal apoptosis. Collectively, these might help to elucidate similar molecular mechanisms involved during cell death in neurodegenerative diseases.

Studies suggest that cholesterol imbalance in the brain might be related to the development of neurological disorders. U18666A is a well-known amphiphile which inhibits intracellular cholesterol transport in treated cells. We have previously shown that U18666A leads to apoptosis and cholesterol accumulation in primary cortical neurons, which is associated with activation of caspases and calpains, hyperphosphorylation of tau, and increased oxidative stress markers. However, the mechanisms involved in U18666A-mediated apoptosis remain unknown. In this report, we sought to gain an insight into the molecular processes contributing to the neuronal apoptosis induced by U18666A. The microarray approach was used in conjunction with proteomics techniques to identify specific proteins which may serve as signature biomarkers during U18666A treatment. Eleven differentially expressed proteins were correlated at the gene expression level in a time-dependent manner. These proteins have been shown to play a role in lipid metabolism and transport, responses to cell death, protein folding and trafficking, and regulation of transcription. The identification of these differentially expressed proteins might provide a clue to decipher the intracellular biochemical changes during U18666A-mediated neuronal apoptosis. Our results provide, for the first time, a combined microarray and proteomics analysis of neuronal apoptosis mediated by inhibition of intracellular cholesterol transport. This new insight may greatly facilitate the study of neurodegenerative diseases., ((c) 2007 Wiley-Liss, Inc.)

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat-shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up-regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor-mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up-regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor-mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor-mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up-regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro-apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.

Objective: To examine the growth-inhibitory, apoptosis- and necrosis-inducing effects of realgar nano-particles (RNP) in human chronic myelogenous leukemia cell line K562 and acute myeloid leukemia cell line HL-60, and to find out the chemical species with efficacy., Method: A "solvent-relay" strategy was used for the preparation of RNP suspension. Cell viability was determined by MTT assay. Cell apoptosis and necrosis were characterized with Annexin V-PI double staining in association with flow cytometry and with morphological examination with Hoechst 33258 staining. Parallel experiments with arsenous acid (H3AsO3), the dominant form of arsenic trioxide in the solution, were conducted for comparison., Result: The mean diameter of RNP was 159.0 nm. RNP showed growth-inhibitory effect on both cell lines. The double staining test indicated that RNP induced both apoptosis and necrosis, and this was further confirmed by morphological examination., Conclusion: RNP induced both apoptosis and necrosis in leukemia cell lines K562 and HL-60. Thioarsenite species with both As-O and As-S bonds may be the active intermediates in the RNP.

YCT is a semipurified extract from Cratoxylum cochinchinense that has antioxidant properties and contains mostly mangiferin. We show here that YCT is selectively toxic to certain cell types and investigate the mechanisms of this toxicity in Jurkat T cells. By flow cytometric analyses, we show that YCT causes intense oxidative stress and a rise in cytosolic Ca(2+). This is followed by a rise in mitochondrial Ca(2+), release of cytochrome c, collapse of Deltapsi(m), a fall in ATP levels, and eventually cell death. The mechanism(s) of intense oxidative stress may involve a plasma membrane redox system, as cell death is inhibited by potassium ferricyanide. Cell death has some features of apoptosis (propidium iodide staining, externalization of phosphatidylserine, limited caspase-3 and -9 activities), but there was no internucleosomal DNA fragmentation.