nitroarginine and Neoplasms

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Humans; Lipogenesis; Neoplasms; Oxidative Stress; Pentose Phosphate Pathway; Phosphogluconate Dehydrogenase; Protein Serine-Threonine Kinases; Ribulosephosphates; Signal Transduction

Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cytotoxins; Female; HCT116 Cells; HT29 Cells; Humans; Hypoxia; Immunohistochemistry; Mice; Mice, Inbred C3H; Mice, Inbred NOD; Mice, SCID; Neoplasms; Neovascularization, Pathologic; Nitric Oxide Synthase; Nitroarginine; Tirapazamine; Treatment Outcome; Triazines; Tumor Burden; Xenograft Model Antitumor Assays

This study was undertaken to develop a method to quantitatively monitor the effect of inhibition of nitric oxide synthase (NOS) on tumour vascular activity using dynamic contrast-enhanced computed tomography (DCE-CT). The DCE-CT studies were performed in 13 anaesthetized rats bearing tumours. To investigate the effect of NOS inhibition, N-nitro-L-arginine (L-NNA) was intravenously administered in eight rats, while only the vehicle was administered in five rats. The contrast enhancement (CE) images were generated by subtracting the CT images before and after the administration of contrast agent. The tumour blood volume (TBV) images were also generated. The CE significantly decreased after L-NNA administration, while there were no significant changes when only the vehicle was administered. There was a good correlation between CE and TBV, suggesting that CE mainly reflects TBV. In conclusion, the present method appears to be useful for monitoring the effect of NOS inhibition on tumour vascular activity.

Topics: Animals; Blood Volume; Enzyme Inhibitors; Male; Neoplasms; Nitric Oxide Synthase; Nitroarginine; Radiographic Image Enhancement; Rats; Rats, Inbred F344; Tomography, X-Ray Computed

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the 'ground state', remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation, we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.

Topics: Animals; Cell Survival; Cells, Cultured; Mice; Molecular Structure; Neoplasms; Neurons; Pharmaceutical Preparations; Sensitivity and Specificity; Stem Cells

Topics: Blood Volume; Enzyme Inhibitors; Humans; Neoplasms; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Regional Blood Flow

The role of nitric oxide (NO) in the response to Photofrin-based photodynamic therapy (PDT) was investigated using mouse tumour models characterized by either relatively high or low endogenous NO production (RIF and SCCVII vs EMT6 and FsaR, respectively). The NO synthase inhibitors Nomega-nitro-L-arginine (L-NNA) or Nomega-nitro-L-arginine methyl ester (L-NAME), administered to mice immediately after PDT light treatment of subcutaneously growing tumours, markedly enhanced the cure rate of RIF and SCCVII models, but produced no obvious benefit with the EMT6 and FsaR models. Laser Doppler flowmetry measurement revealed that both L-NNA and L-NAME strongly inhibit blood flow in RIF and SCCVII tumours, but not in EMT6 and FsaR tumours. When injected intravenously immediately after PDT light treatment, L-NAME dramatically augmented the decrease in blood flow in SCCVII tumours induced by PDT. The pattern of blood flow alterations in tumours following PDT indicates that, even with curative doses, regular circulation may be restored in some vessels after episodes of partial or complete obstruction. Such conditions are conducive to the induction of ischaemia-reperfusion injury, which is instigated by the formation of superoxide radical. The administration of superoxide dismutase immediately after PDT resulted in a decrease in tumour cure rates, thus confirming the involvement of superoxide in the anti-tumour effect. The results of this study demonstrate that NO participates in the events associated with PDT-mediated tumour destruction, particularly in the vascular response that is of critical importance for the curative outcome of this therapy. The level of endogenous production of NO in tumours appears to be one of the determinants of sensitivity to PDT.

Topics: Animals; Enzyme Inhibitors; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Neoplasm Transplantation; Neoplasms; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Photochemotherapy

It may be therapeutically advantageous to alter tumour blood supply specifically. Nitric oxide is a potent vasodilator which is produced in many tissues by the enzyme nitric oxide synthase (NOS). We have transfected cDNA for the inducible isoform of this enzyme (iNOS), under the control of the radiation-inducible promoter WAF1. The activity of the promoter was initially assessed using green fluorescent protein (GFP) in both endothelial cells and rat tail artery segments. Induction of protein expression by 9.5- and 4.5-fold respectively, was observed after a radiation dose of 4 Gy. Artery sections were then transfected with the WAF1/iNOS construct; this gave five-fold induction of iNOS protein after a dose of 4 Gy. The transfected artery was also tested functionally for relaxation, indicative of NO production. One hour after exposure to 4 Gy there was a significant (65%) relaxation of artery segments that had been preconstricted with phenylephrine. This could be partially reversed by the NOS inhibitor nitro-L-arginine. This study demonstrates that we can regulate vascular tone using an X-ray inducible promoter.

Topics: Animals; Blotting, Western; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Endothelium, Vascular; Gene Expression; Genetic Therapy; Male; Neoplasms; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Phenylephrine; Promoter Regions, Genetic; Rats; Rats, Wistar; Vasoconstrictor Agents; Vasodilation; X-Rays

The roles of tumor necrosis factor (TNF alpha) and reactive nitrogen intermediates (RNI) as effectors of macrophage-mediated tumor cell killing were investigated in a variety of tumor cell lines. Three TNF alpha-sensitive tumor targets were also susceptible to resting bone-marrow-derived mononuclear phagocytes (BMMP). This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha, indicating that TNF alpha is the major effector of macrophage-mediated killing of these targets. The other 21 tumor cell lines examined were resistant to TNF alpha but, in their large majority, were more or less susceptible to killing by interferon gamma (IFN gamma)- and Corynebacterium parvum (CP)-activated BMMP. Among the various analogues of L-arginine used to assess the role of L-arginine-derived RNI as mediators of macrophage tumoricidal activity, NG-monomethyl-L-arginine (NMMA) was most efficient in suppressing RNI secretion by activated macrophages. In some macrophage tumor-cell combinations, NMMA inhibited both the generation of RNI and the expression of tumoricidal activity in a dose-dependent manner, suggesting a central role for RNI as effectors. In other combinations, NMMA in concentrations that abolished secretion of RNI either affected tumor-cell killing only after its induction by IFN gamma, or not at all. The findings not only support the thesis that macrophages posses various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophage activation and the properties of the tumor-cell type.

Topics: Animals; Arginine; Argininosuccinic Acid; Cell Line; Cell Survival; Humans; Immunity, Cellular; Interferon-gamma; Macrophage Activation; Macrophages; Male; Mice; Neoplasms; Nitroarginine; omega-N-Methylarginine; Propionibacterium acnes; Rats; Tumor Necrosis Factor-alpha