Redox bases underlying the anti-tumor activity of garlic-contained organo-sulfur compounds: Implication in chemoprevention and chemotherapy

The beneficial effects of phytochemicals on human health have been extensively addressed. The majority of this outcome derives from their capability to function as antioxidants, thus the consumption of foods rich in these compounds is considered an advisable preventive therapy in slowing oxidative stress-mediated degenerative processes, such as those occurring during aging. Nevertheless, high concentrations of redox-active compounds could switch the antioxidant property to a pro-oxidant action leading to cell cycle arrest and death. This aspect place phytochemicals as promising therapeutics particularly for cancer prevention or treatment. Although their beneficial properties are known from ancient times, only during the recent years the molecular mechanisms underlying the anti-proliferative effects mediated by garlic-derived organo-sulfur compounds (OSC) are going to be clarified, with particular regard to what their pro-apoptotic features concerns. This chapter discusses the main findings that have contributed to the comprehension of OSC-mediated redox-dependent events governing growth arrest and apoptosis. Particularly, we report the mechanisms through which OSC have been suggested to generate reactive oxygen species and to modulate the redox state of specific reactive cysteines. Both processes will be argued as necessary events in inducing either irreversible damage to cellular macromolecules (e.g. DNA and cytoskeleton proteins), or waves of signaling finally resulting in the activation of the apoptotic program. In this perspective, the classes of proteins which have been indicated to represent the targets of OSC-mediated oxidative modifications, and to have a role in cellular redox response will be discussed

p38(MAPK)/p53 signalling axis mediates neuronal apoptosis in response to tetrahydrobiopterin-induced oxidative stress and glucose uptake inhibition: implication for neurodegeneration.

BH4 (tetrahydrobiopterin) induces neuronal demise via production of ROS (reactive oxygen species). In the present study we investigated the mechanisms of its toxicity and the redox signalling events responsible for the apoptotic commitment in SH-SY5Y neuroblastoma cells and in mouse primary cortical neurons. We identified in p38(MAPK)/p53 a BH4-responsive pro-apoptotic signalling axis, as demonstrated by the recovery of neuronal viability achieved by gene silencing or pharmacological inhibition of both p38(MAPK) and p53. BH4-induced oxidative stress was characterized by a decrease in the GSH/GSSG ratio, an increase in protein carbonylation and DNA damage. BH4 toxicity and the redox-activated apoptotic pathway were counteracted by the H2O2-scavengers catalase and N-acetylcysteine and enhanced by the GSH neo-synthesis inhibitor BSO (buthionine sulfoximine). We also demonstrated that BH4 impairs glucose uptake and utilization, which was prevented by catalase administration. This effect contributes to the neuronal demise, exacerbating BH4-induced nuclear damage and the activation of the pro-apoptotic p38(MAPK)/p53 axis. Inhibition of glucose uptake was also observed upon treatment with 6-hydroxydopamine, another redox-cycling molecule, suggesting a common mechanism of action for auto-oxidizable neurotoxins

Reactive oxygen species-dependent c-Jun NH2-terminal kinase/c-Jun signaling cascade mediates neuroblastoma cell death induced by diallyl disulfide

The pharmacological properties of garlic and its derivatives are long known, and their underling mechanisms are being extensively investigated. In this study we have addressed the effects of diallyl disulfide (DADS), an oil-soluble garlic molecule, on cell growth of neuroblastoma cell SH-SY5Y, focusing on the redox events associated with this compound. Treatment of SH-SY5Y cells with DADS resulted in arrest of cell cycle in G(2)/M phase and commitment to apoptosis through the activation of the mitochondrial pathway (Bcl-2 down-regulation, cytochrome c release into the cytosol, and activation of caspase-9 and caspase-3). The earliest oxidative event observed after DADS treatment was the increase of production of reactive oxygen species, which reached the maximum yield on 30 min of DADS treatment. The oxidative burst resulted in protein and lipid damage as demonstrated by protein carbonyl accumulation and lipid peroxidation. We demonstrated that apoptosis induction was highly dependent on the activation of the redox-sensitive c-Jun NH2-terminal kinase (JNK)/c-jun pathway. In particular, we established that DADS treatment induces JNK dissociation from glutathione S-transferase and its activation by phosphorylation. Moreover, treatment with JNK inhibitor I significantly reduced DADS-induced apoptosis and treatment with the spin trap 5,5'-dimethyl-1-pyrroline N-oxide or overexpression of the antioxidant enzyme copper, zinc superoxide dismutase, resulted in the inhibition of DADS-mediated toxicity through attenuation of JNK/c-jun pathway activation. Overall, the results suggest a pivotal role for oxidative stress in DADS-induced apoptosis and, taking into account that tumor cells are deficient in antioxidants, suggest a plausible utilization of this compound as an antiproliferative agent in cancer therapy

Under the ROS…thiol network is the principal suspect for autophagy commitment.

Low molecular weight and protein sulphydryls undergo reactive oxygen species (ROS)-mediated oxidation. However, in contrast to the irreversible damages that oxidative conditions yield on biomolecules, the oxidation of reactive cysteines frequently results in reversible modifications, which represent the prototype of the molecular mechanisms underlying redox signaling. Many proteins involved in a wide range of cellular processes have been classified as “redoxsensitive,” thereby modulating their function/activity dependent on the redox state of their critical thiols. Growing evidence from the past few years supports the idea that ROS production also correlates with the occurrence of autophagy. Nonetheless, the cysteine protease Atg4 remains the sole example of a protein whose redox regulation has been completely characterized and demonstrated to be necessary for the progression of autophagy. The principal aim of this commentary is to draw attention to the remarkable number of proteins that can fit the double role of: (i) being involved in autophagy, especially in autophagosome formation and (ii) sensing alterations of the cellular redox state by means of reactive cysteine residues. We will also attempt to provide a hypothetical model to explain the possible functional role of thiols in the occurrence of autophagy and outline a network of redox reactions likely concurring to allow the correct initiation and completion of autophagosomes

Biochemical mechanism of oxidative damage by redox-cycling drugs.

Biochemical mechanisms of production of redox intermediates of redox-cycling drugs include: photochemical events, either photoionization process or electron transfer from photoexcited states; electron exchange of reduced form of a drug with the oxy state of oxygen-binding hemoproteins; oxidation by catalytic metal centers (oxidases, peroxidases, oxygenases) of the reduced forms of drugs; or electron transfer to the oxidized form of a drug from activated intracellular electron transfer chain (mitochondria, microsomes, etc.). Further reaction of these drug free radicals can lead to oxidative damage by either direct attack of biological macromolecules or via oxygen reduction, giving O2-, H2O2, and OH. The reaction pathway depends on the presence of metal ions, natural scavengers, enzymes that control relative concentrations of reactive species, and availability of oxygen in the environment

Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment.

Kaempferol, a dietary cancer chemopreventive polyphenol, has been reported to trigger apoptosis in several tumor histotypes, but the mechanism underlying this phenomenon is not fully understood. Here, we demonstrate that in HeLa cells, kaempferol induces energetic failure due to inhibition of both glucose uptake and Complex I of the mitochondrial respiratory chain. As adaptive response, cells activate autophagy, the occurrence of which was established cytofluorometrically, upon acridine orange staining, and immunochemically, by following the increase of the autolysosome-associated form of the microtubule-associated protein light chain 3 (LC3-II). Autophagy is an early and reversible process occurring as survival mechanisms against apoptosis. Indeed, chemical inhibition of autophagy, by incubations with monensin, wortmannin, 3-methyladenine, or by silencing Atg5, significantly increases the extent of apoptosis, which takes place via the mitochondrial pathway, and shortens the time in which the apoptotic markers are detectable. We also demonstrate that autophagy depends on the early activation of the AMP-activated protein kinase (AMPK)/mTOR-mediated pathway. The overexpression of dominant negative AMPK results in a decrease of autophagic cells, a decrement of LC3-II levels, and a significant increase of apoptosis. Experiments performed with another carcinoma cell line yielded the same results, suggesting for kaempferol a unique mechanism of action

Kinetics of Electron Transfer between Azurin and Cytochrome 551 from Pseudomonas

Abstract The kinetics of electron transfer between the copper-containing protein azurin (Cu++/Cu+) and cytochrome 551 (Fe+++/Fe++) from Pseudomonas has been studied by rapid mixing methods. The reaction in both directions is fast; at low reagent concentrations (∼10-6 m) the apparent second order rate constant, at 20°, is about 3 x 106 m-1 sec-1 for the reaction Fe++ + Cu++ and 1.4 x 106 m-1 sec-1 for the reaction Fe+++ + Cu+. At high reagent concentrations the rates tend to reach a limiting value indicating that the reaction is not a simple second order process. The kinetics of the reactions of the reduced and oxidized forms of azurin and Pseudomonas cytochrome 551 with ferricyanide and dithionite has also been investigated. The rates of these reactions, at comparable reagent concentrations, are orders of magnitude lower than that between azurin and cytochrome 551

Glutamine deprivation enhances antitumor activity of 3-bromopyruvate through the stabilization of monocarboxylate transporter-1

Anticancer drug efficacy might be leveraged by strategies to target certain biochemical adaptations of tumors. Here we show how depriving cancer cells of glutamine can enhance the anticancer properties of 3-bromopyruvate, a halogenated analog of pyruvic acid. Glutamine deprival potentiated 3-bromopyruvate chemotherapy by increasing the stability of the monocarboxylate transporter-1, an effect that sensitized cells to metabolic oxidative stress and autophagic cell death. We further elucidated mechanisms through which resistance to chemopotentiation by glutamine deprival could be circumvented. Overall, our findings offer a preclinical proof-of-concept for how to employ 3-bromopyruvate or other monocarboxylic-based drugs to sensitize tumors to chemotherap

Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials

Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the 'sealed niche' of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD-DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD-DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co-cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle-specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD-DPSCs, as suggested by the consistent Ca(2+) release from these cells in response to endothelin-1 (ET-1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET-1 has been found to be superior in hD-DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD-DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD-DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therapy