The selectivity, as well as efficiency, of intercellular induction of apoptosis are based on reactive oxygen and nitrogen species (ROS and RNS). Extracellular superoxide anions generated specifically by transformed target cells represent the central element in this signaling system.

In a first signaling pathway, target cell-derived superoxide anions spontaneously dismutate tohydrogen peroxide, which fosters HOCl generation by a target cell-derived peroxidase. HOCl then interacts with target cell-derived superoxide anions and generates apoptosis-inducinghydroxyl radicals.

In a second signaling pathway, target cell-derived superoxide anions interact with effector cell-derived nitric oxide and form the apoptosis-inducer peroxynitrite.

In a third signaling pathway, HOCl and nitrite form toxic nitryl chloride. Our data show that nontransformed effector cells cause selective apoptosis induction in transformed cells through specific ROS and RNS interactions. Thereby, the efficiency and the selectivity of the reaction are determined by target cellderived superoxide anions.

In addition to intercellular induction of apoptosis, transformed cells are eliminated by TGF-beta-triggered, ROS-mediated autocrine/ paracrine self-destruction. In this process, the effector molecules, peroxidase and NO, are generated by the transformed target cells themselves and cooperate with their own extracellular ROS to induce apoptosis.

Tumor formation seems to require interference with or resistance to intercellular induction of apoptosis and to apoptotic self-destruction. Understanding and subsequent abrogation of tumor cell resistance may have a therapeutic potential.

Toxicol Appl Pharmacol. 2007 Sep 15;223(3):267-76. Epub 2007 Jun 7.

Kawiak A, Piosik J, Stasilojc G, Gwizdek-Wisniewska A, Marczak L, Stobiecki M, Bigda J, Lojkowska E.

Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.

http://www.ncbi.nlm.nih.gov/pubmed/17618663

Abstract:

* Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis.

* Plumbagin (found in many plants, including carnivora / venus flytrap), a naphthoquinone exhibiting anti-tumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex.

The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topo II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS.

In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation.

Results:

* These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II.

* Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA.

* The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis.

* Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.

Mol Pharmacol. 2005 Oct;68(4):983-94. Epub 2005 Jul 15.

Lu HR, Zhu H, Huang M, Chen Y, Cai YJ, Miao ZH, Zhang JS, Ding J.

Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Peoples Republic of China.

http://www.ncbi.nlm.nih.gov/pubmed/16024664

Abstract:

Reactive oxygen species (ROS) are produced by all aerobic cells and have been implicated in the regulation of diverse cellular functions, including intracellular signaling, transcription activation, proliferation, and apoptosis. Salvicine, a novel diterpenoid quinone compound, demonstrates a broad spectrum of antitumor activities.

Although salvicine is known to trap the DNA-topoisomerase II (Topo II) complex and induce DNA double-strand breaks (DSBs), its precise antitumor mechanisms remain to be clarified. In this study, we investigated whether salvicine altered the levels of ROS in breast cancer MCF-7 cells and whether these ROS contributed to the observed antitumoral activity.

* Our data revealed that salvicine stimulated intracellular ROS production and subsequently elicited notable DSBs.

* The addition of N-acetyl cysteine (NAC), an antioxidant, effectively attenuated the salvicine-induced ROS enhancement and subsequent DNA DSBs.

* Heat treatment reversed the accumulation of DNA DSBs, and the addition of NAC attenuated the Topo II-DNA cleavable complexes formation and the growth inhibition of salvicine-treated JN394top2-4 yeast cells, collectively indicating that Topo II is a target of the salvicine-induced ROS.

* On the other hand, when examining the impact of salvicine on DNA repair pathways, we unexpectedly observed that salvicine selectively down-regulated the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) protein levels and repressed DNA-PK kinase activity; both of these effects were attenuated by NAC pretreatment of MCF-7 cells.

* Finally and most importantly, NAC attenuated salvicine-induced apoptosis and cytotoxicity in MCF-7 cells.

Conclusion:

These results indicate that apart from its direct actions, salvicine generates ROS that modulate DNA damage and repair, contributing to the comprehensive biological consequences of salvicine treatment, such as DNA DSBs, apoptosis, and cytotoxicity in tumor cells.

The finding of salvicine-induced ROS provides new evidence for the molecular mechanisms of this compound.

Moreover, the effects of salvicine-induced ROS on Topo II and DNA-PK give new insights into the diverse biological activities of ROS.

Pharm Res. 2008 Sep;25(9):2171-80. Epub 2008 Jan 23.

Powolny AA, Singh SV.

Department of Pharmacology and Urology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

http://www.ncbi.nlm.nih.gov/pubmed/18213451

Abstract

PURPOSE: To investigate the mechanism of human prostate cancer cell growth inhibition by plumbagin, a constituent of the widely used medicinal herb Plumbago zeylanica L.

MATERIALS AND METHODS: Cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Cell cycle distribution and generation of reactive oxygen species (ROS) were determined by flow cytometry. The effect of plumbagin treatment on cellular redox status was determined by analysis of intracellular glutathione (GSH) levels and expression of genes involved in ROS metabolism.

RESULTS: Plumbagin treatment decreased viability of human prostate cancer cells (PC-3, LNCaP, and C4-2) irrespective of their androgen responsiveness or p53 status. Plumbagin-mediated decrease in cell viability correlated with apoptosis induction, which was accompanied by ROS generation and depletion of intracellular GSH levels. Pretreatment of cells with the antioxidant N-acetylcysteine inhibited plumbagin-mediated ROS generation and apoptosis. Plumbagin treatment also resulted in altered expression of genes responsible for ROS metabolism, including superoxide dismutase 2 (Mn-SOD).

CONCLUSION: The present study points towards an important role of ROS in plumbagin-induced apoptosis in human prostate cancer cells.

J Drug Target. 2007 Aug-Sep;15(7-8):475-86.

Fang J, Nakamura H, Iyer AK.

Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan. fangjun@ph.sojo-u.ac.jp

http://www.ncbi.nlm.nih.gov/pubmed/17671894

Abstract:

Reactive oxygen species (ROS), such as superoxide anion radicals (O.-2) and hydrogen peroxide (H2O2) are potentially harmful by-products of normal cellular metabolism that directly affect cellular functions. ROS is generated by all aerobic organisms and it seems to be indispensable for signal transduction pathways that regulate cell growth and reduction-oxidation (redox) status.

However, overproduction of these highly reactive oxygen metabolites can initiate lethal chain reactions, which involve oxidation and damage to structures that are crucial for cellular integrity and survival. In fact, many antitumor agents, such as vinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin, inostamycin, neocarzinostatin and many others exhibit antitumor activity via ROS-dependent activation of apoptotic cell death, suggesting potential use of ROS as an antitumor principle.

Thus, a unique anti-cancer strategy named "oxidation therapy" has been developed by inducing cytotoxic oxystress for cancer treatment.

This goal could be achieved mainly by two methods, namely:

(i) inducing the generation of ROS directly to solid tumors and

(ii) inhibiting the antioxidative enzyme (defense) system of tumor cells.

Since 1950s, many strategies have been employed based on the first method, namely, administration of ROS per se (e.g. H2O2) or ROS generating enzyme to tumor bearing animals. However no successful and practical results were obtained probably because of the lack of tumor selective ROS delivery and hence resulting in subsequent induction of severe side effects.

To overcome these obstacles, we developed polyethylene glycol (PEG) conjugated O.-2 or H2O2-generating enzymes, xanthine oxidase (XO) and D-amino acid oxidase (DAO) (PEG-DAO) respectively. More recently, a pegylated (PEG) zinc protoporphyrin (PEG-ZnPP) and a highly water soluble micellar formulation of ZnPP based on amphiphilic styrene maleic acid (SMA) copolymer, SMA-ZnPP, are prepared, which are potent inhibitors of heme oxygenase-1 (HO-1).

HO-1 is a major antioxidative enzyme of tumors, that is different in mechanism of catalase or superoxide dismutase (SOD). Consequently, both PEG-enzymes and PEG-ZnPP exhibited superior in vivo pharmacokinetics than their parental molecules, particularly in tumor delivery by taking advantage of the EPR effect of macromolecular nature, and thus showed remarkable antitumor effects suggesting the potentials of this anticancer therapeutic for clinical application.

Furthermore, it has been well known that many antioxidative enzymes such as catalase, SOD are down-regulated in most solid tumors in vivo. On the contrary, HO-1 is highly upregulated and it plays a very important role of antioxidation, because HO-1 generates biliverdin, which being converted to bilirubin exhibits a very potent antioxidative effect, and hence antiapoptosis in tumors.

Thus this oxidation therapy, by inhibiting this HO-1 dependent antioxidant (bilirubin) formation by ZnPP, and by enhancing ROS generation, is expected to offer a powerful therapeutic modality for future anticancer therapy.

Carnivora - Formic Acid Induces Yca1p-Independent Apoptosis-like Cell Death in the Yeast Saccharomyces Cerevisiae

FEMS Yeast Res. 2008 Jun;8(4):531-9. Epub 2008 Apr 29.

Du L, Su Y, Sun D, Zhu W, Wang J, Zhuang X, Zhou S, Lu Y.

Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Guangzhou, Republic of China.

http://www.ncbi.nlm.nih.gov/pubmed/18452540

Abstract

Formic acid disrupts mitochondrial electron transport and sequentially causes cell death in mammalian ocular cells by an unidentified molecular mechanism. Here, we show that a low concentration of formic acid induces apoptosis-like cell death in the budding yeast Saccharomyces cerevisiae, with several morphological and biochemical changes that are typical of apoptosis, including chromatin condensation, DNA fragmentation, externalization of phosphatidylserine, reactive oxygen species (ROS) production, loss of mitochondrial membrane potential and mitochondrion destruction. This process may not be dependent on the activation of Yca1p, the yeast caspase counterpart.

* In addition, the cell death induced by formic acid is associated with ROS burst,while intracellular ROS accumulate more rapidly and to a higher level in the YCA1 disruptant than in the wild-type strain during the progression of cell death.

* Our data indicate that formic acid induces yeast apoptosis via an Yca1p-independent pathway and it could be used as an extrinsic inducer for identifying the regulators downstream of ROS production in yeast.