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Non-muscle invasive bladder cancer is a common tumour in men and women. In case of resistance to the standard therapeutic agents, gemcitabine can be used as off-label instillation therapy into the bladder. To reduce potential side effects, continuous efforts are made to optimise the therapeutic potential of drugs, thereby reducing the effective dose and consequently the pharmacological burden of the medication. We recently demonstrated that it is possible to significantly increase the therapeutic efficacy of mitomycin C against a bladder carcinoma cell line by exposure to non-toxic doses of blue light (453 nm). In the present study, we investigated whether the therapeutically supportive effect of blue light can be further enhanced by the additional use of the wavelength-specific photosensitiser riboflavin. We found that the gemcitabine-induced cytotoxicity of bladder cancer cell lines (BFTC-905, SW-1710, RT-112) was significantly enhanced by non-toxic doses of blue light in the presence of riboflavin. Enhanced cytotoxicity correlated with decreased levels of mitochondrial ATP synthesis and increased lipid peroxidation was most likely the result of increased oxidative stress. Due to these properties, blue light in combination with riboflavin could represent an effective therapy option with few side effects and increase the success of local treatment of bladder cancer, whereby the dose of the chemotherapeutic agent used and thus the chemical load could be significantly reduced with similar or improved therapeutic success. [ABSTRACT FROM AUTHOR]

Oxidative stress may cause endothelial dysfunction and vascular disease. It has been shown that NO protects endothelial cells (EC) against [H.sub.2][O.sub.2]-induced toxicity. In addition, it is known that NO within cells induces a zinc release from proteins containing zinc-sulfur complexes. The aim of this study was to investigate whether zinc released intracellularly by NO plays a signaling role in the NO-mediated protection against [H.sub.2][O.sub.2] in rat aortic EC. Our results show that the NO-mediated protection toward [H.sub.2][O.sub.2] depends on the activities of glutathione peroxidase and glutamate cysteine ligase (GCL), the rate-limiting enzyme of glutathione (GSH) de novo biosynthesis. Moreover, NO increases the synthesis of the antioxidant GSH by inducing the expression of the catalytic subunit of GCL (GCLC). Chelating intracellular 'free' zinc abrogates the NO-mediated increase of GCLC and of cellular GSH levels. As a consequence, the NO-mediated protection against [H.sub.2][O.sub.2]-induced toxicity is impaired. We also show that under proinflammatory conditions, both cellular NO synthesis and intracellular 'free' zinc are required to maintain the cellular GSH levels. Using RNA interference and laser scanning microscopy, we found that the NO-induced expression of GCLC depends on the activation of the transcription factor Nrf2 but not on the activity of the 'zinc-sensing' transcription factor MTF-1. These findings show that intracellular 'free' zinc plays a signaling role in the protective activity of NO and could explain why maintenance of an adequate zinc status in the endothelium is important to protect from oxidative stress and the development of vascular disease. inducible nitric oxide synthase; zinc signaling; oxidative stress; glutamate cysteine ligase; Nrf2

[Zn.sup.2+] is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic [Zn.sup.2+] release from the zinc storing protein metallothionein (MT) and probably other proteins that complex [Zn.sup.2+] via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of [Zn.sup.2+]. This nuclear [Zn.sup.2+] release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated [Zn.sup.2+] trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular [Zn.sup.2+] release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular [Zn.sup.2+] trafficking and homeostasis.

The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions. endothelium; inflammation; nitric oxide; inducible nitric oxide synthase