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

N-Acetylaspartate Drives Oligodendroglial Differentiation via Histone Deacetylase Activation

An unmet clinical goal in demyelinating pathologies is to restore the myelin sheath prior to neural degeneration. N-acetylaspartate (NAA) is an acetylated derivative form of aspartate, abundant in the healthy brain but severely reduced during traumatic brain injury and in patients with neurodegenerative pathologies. How extracellular NAA variations impact the remyelination process and, thereby, the ability of oligodendrocytes to remyelinate axons remains unexplored. Here, we evaluated the remyelination properties of the oligodendroglial (OL) mouse cell line Oli-neuM under different concentrations of NAA using a combination of biochemical, qPCR, immunofluorescence assays, and in vitro engagement tests, at NAA doses compatible with those observed in healthy brains and during brain injury. We observed that oligodendroglia cells respond to decreasing levels of NAA by stimulating differentiation and promoting gene expression of myelin proteins in a temporally regulated manner. Low doses of NAA potently stimulate Oli-neuM to engage with synthetic axons. Furthermore, we show a concentration-dependent expression of specific histone deacetylases essential for MBP gene expression under NAA or Clobetasol treatment. These data are consistent with the idea that oligodendrocytes respond to lowering the NAA concentration by activating the remyelination process via deacetylase activation

Hindering NAT8L expression in hepatocellular carcinoma increases cytosolic aspartate delivery that fosters pentose phosphate pathway and purine biosynthesis promoting cell proliferation

N-acetylaspartate (NAA) is synthesized by the mitochondrial enzyme NAT8L, which uses acetyl-CoA and aspartate as substrates. These metabolites are fundamental for bioenergetics and anabolic requirements of highly proliferating cells, thus, NAT8L modulation may impinge on the metabolic reprogramming of cancer cells. Specifically, aspartate represents a limiting amino acid for nucleotide synthesis in cancer. Here, the expression of the NAT8L enzyme was modulated to verify how it impacts the metabolic adaptations and proliferative capacity of hepatocellular carcinoma. We demonstrated that NAT8L downregulation is asso-ciated with increased proliferation of hepatocellular carcinoma cells and immortalized hepatocytes. The over -expression of NAT8L instead decreased cell growth. The pro-tumoral effect of NAT8L silencing depended on glutamine oxidation and the rewiring of glucose metabolism. Mechanistically, NAT8L downregulation triggers aspartate outflow from mitochondria via the exporter SLC25A13 to promote glucose flux into the pentose phosphate pathway, boosting purine biosynthesis. These results were corroborated by the analyses of human and mouse hepatocellular carcinoma samples revealing a decrease in NAT8L expression compared to adjacent non -tumoral tissues. Overall, this work demonstrates that NAT8L expression in liver cells limits the cytosolic avail-ability of aspartate necessary for enhancing the pentose phosphate pathway and purine biosynthesis, counter-acting cell proliferation

A strong glutathione S-transferase inhibitor overcomes the P-glycoprotein-mediated resistance in tumor cells - 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) triggers a caspase-dependent apoptosis in MDR1-expressing leukemia cells

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol- 4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoproteinpositive tumors

GSH-C4 Acts as Anti-inflammatory Drug in Different Models of Canonical and Cell Autonomous Inflammation Through NFκB Inhibition

An imbalance in GSH/GSSG ratio represents a triggering event in pro-inflammatory cytokine production and inflammatory response. However, the molecular mechanism(s) through which GSH regulates macrophage and cell autonomous inflammation remains not deeply understood. Here, we investigated the effects of a derivative of GSH, the N-butanoyl glutathione (GSH-C4), a cell permeable compound, on lipopolisaccharide (LPS)-stimulated murine RAW 264.7 macrophages, and human macrophages. LPS alone induces a significant production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α and a significant decrement of GSH content. Such events were significantly abrogated by treatment with GSH-C4. Moreover, GSH-C4 was highly efficient in buffering cell autonomous inflammatory status of aged C2C12 myotubes and 3T3-L1 adipocytes by suppressing the production of pro-inflammatory cytokines. We found that inflammation was paralleled by a strong induction of the phosphorylated form of NFκB, which translocates into the nucleus; a process that was also efficiently inhibited by the treatment with GSH-C4. Overall, the evidence suggests that GSH decrement is required for efficient activation of an inflammatory condition and, at the same time, GSH-C4 can be envisaged as a good candidate to abrogate such process, expanding the anti-inflammatory role of this molecule in chronic inflammatory states

Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells

Sirtuin 6 (SIRT6) is a member of the NAD+-dependent class III deacetylase sirtuin family, which plays a key role in cancer by controlling transcription, genome stability, telomere integrity, DNA repair, and autophagy. Here we analyzed the molecular and biological effects of UBCS039, the first synthetic SIRT6 activator. Our data demonstrated that UBCS039 induced a time-dependent activation of autophagy in several human tumor cell lines, as evaluated by increased content of the lipidated form of LC3B by western blot and of autophagosomal puncta by microscopy analysis of GFP-LC3. UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity since the catalytic mutant H133Y failed to activate autophagy. At the molecular level, SIRT6-mediated autophagy was triggered by an increase of ROS levels, which, in turn, resulted in the activation of the AMPK-ULK1-mTOR signaling pathway. Interestingly, antioxidants were able to completely counteract UBCS039-induced autophagy, suggesting that ROS burst had a key role in upstream events leading to autophagy commitment. Finally, sustained activation of SIRT6 resulted in autophagy-related cell death, a process that was markedly attenuated using either a pan caspases inhibitor (zVAD-fmk) or an autophagy inhibitor (CQ). Overall, our results identified UBCS039 as an efficient SIRT6 activator, thereby providing a proof of principle that modulation of the enzyme can influence therapeutic strategy by enhancing autophagy-dependent cell death

PYRROLO[1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) induce apoptosis in K562 cells

BACKGROUND: The objective of this study was to gain insight into the molecular mechanism of induced cell death (apoptosis) by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model. METHODS: We focused our attention on some members of the PBTDs family to test their potential apoptotic activity in K562 cells. Important apoptotic activity was demonstrated, as evidenced by the concentration and percentage of cell death quantified by measuring PI-uptake by flow cytometry, and DNA fragmentation analyzed by agarose gel electrophoresis, generating a characteristic ladder pattern of discontinuous DNA fragments. The expression of Bcl-2 family was tested using western blotting and transfection method. RESULTS: PBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADP-ribose)polymerase (PARP) cleavage. PBTD-1 and -3 treatment resulted in caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we used K562/vector and K562/bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with K562/vector, K562/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 10 muM PBTD-1 and -3 for 24 h produced morphological features of apoptosis and DNA fragmentation in K562/vector cells, respectively. In contrast, PBTD-1 and -3-induced caspase-3 activation and apoptosis were inhibited in K562/Bcl-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis. CONCLUSION: These results indicate that PBTDs effectively induce apoptosis of K562 leukemia cells through the activation of caspase cascades. In addition, these findings indicate that Bcl-2 inhibits PBTD-1 and -3 induced-apoptosis via a mechanism that interferes with cytocrome c release, and the activity of caspase-3, which is involved in the execution of apoptosis

Therapeutic targeting of replicative immortality

One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed “senescence,” can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells’ heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy

Cancer prevention and therapy through the modulation of the tumor microenvironment

Cancer arises in the context of an in vivo tumor microenvironment. This microenvironment is both a cause and consequence of tumorigenesis. Tumor and host cells co-evolve dynamically through indirect and direct cellular interactions, eliciting multiscale effects on many biological programs, including cellular proliferation, growth, and metabolism, as well as angiogenesis and hypoxia and innate and adaptive immunity. Here we highlight specific biological processes that could be exploited as targets for the prevention and therapy of cancer. Specifically, we describe how inhibition of targets such as cholesterol synthesis and metabolites, reactive oxygen species and hypoxia, macrophage activation and conversion, indoleamine 2,3-dioxygenase regulation of dendritic cells, vascular endothelial growth factor regulation of angiogenesis, fibrosis inhibition, endoglin, and Janus kinase signaling emerge as examples of important potential nexuses in the regulation of tumorigenesis and the tumor microenvironment that can be targeted. We have also identified therapeutic agents as approaches, in particular natural products such as berberine, resveratrol, onionin A, epigallocatechin gallate, genistein, curcumin, naringenin, desoxyrhapontigenin, piperine, and zerumbone, that may warrant further investigation to target the tumor microenvironment for the treatment and/or prevention of cancer