NADPH oxidase inhibitor VAS2870 prevents staurosporine-induced cell death in rat astrocytes.

Background Astrocytes maintain central nerve system homeostasis and are relatively resistant to cell death. Dysfunction of cell death mechanisms may underlie glioblastoma genesis and resistance to cancer therapy; therefore more detailed understanding of astrocytic death modalities is needed in order to design effective therapy. The purpose of this study was to determine the effect of VAS2870, a pan-NADPH oxidase inhibitor, on staurosporine-induced cell death in astrocytes. Materials and methods Cultured rat astrocytes were treated with staurosporine as activator of cell death. Cell viability, production of reactive oxygen species (ROS), and mitochondrial potential were examined using flow cytometric analysis, while chemiluminescence analysis was performed to assess caspase 3/7 activity and cellular ATP. Results We show here for the first time, that VAS2870 is able to prevent staurosporine-induced cell death. Staurosporine exerts its toxic effect through increased generation of ROS, while VAS2870 reduces the level of ROS. Further, VAS2870 partially restores mitochondrial inner membrane potential and level of ATP in staurosporine treated cells. Conclusions Staurosporine induces cell death in cultured rat astrocytes through oxidative stress. Generation of ROS, mitochondrial membrane potential and energy level are sensitive to VAS2870, which suggests NADPH oxidases as an important effector of cell death. Consequently, NADPH oxidases activation pathway could be an important target to modulate astrocytic death.

Staurosporine induces apoptosis and necroptosis in cultured rat astrocytes.

Apoptosis and necroptosis are highly regulated, interconnected forms of a cell death. The distinction between them is critical, because necroptosis may cause significant cell loss and local inflammation, whereas apoptosis is essential for tissue homeostasis. The same stimulus can induce both apoptosis and necroptosis. Both forms of a cell death were detected in various pathologies, including pathologies in the central nervous system. Astrocytes are a large, heterogeneous cell population in the central nervous system, with many supportive, developmental functions. Although their demise may seriously impair normal functions of the central nervous system, it is still poorly understood. In this study, apoptosis and necroptosis were induced in cultured rat astrocytes by staurosporine. When a low concentration (10(-7) M) of staurosporine was applied, a significantly increased proportion of early apoptotic cells was detected after regeneration in a staurosporine free medium. The proportion of necroptotic cells was already increased without regeneration after 3 hours of exposure to staurosporine. When a higher (10(-6) M) concentration of staurosporine was applied, further significantly increased necroptosis was detected after regeneration in a staurosporine free medium. Necroptosis was significantly reduced when RIP1 kinase was inhibited by necrostatin-1, whereas inhibition of caspases with z-vad-fmk, an irreversible pan-caspase inhibitor, did not prevent necroptosis. This report of necroptosis induced by staurosporine represents a simple approach for the in vitro induction and detection of apoptosis and necroptosis.

Staurosporine induces different cell death forms in cultured rat astrocytes.

BACKGROUND: Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. MATERIALS AND METHODS: Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. RESULTS: We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10(-7) M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10(-6) M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. CONCLUSION: Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis.

Differentiation of Borrelia burgdorferi sensu lato species by temperature gradient gel electrophoresis.

Borrelia burgdorferi sensu lato is a multispecies complex of pathogenic spirochetes, causing Lyme borreliosis. Due to clinical, epidemiological, and taxonomical implications, there is a need for identification of isolated Borrelia strains. In the present study, we have optimized TGGE for B. burgdorferi sensu lato species differentiation and the results were compared with two reference methods, namely PFGE and restriction of 5S-23S intergenic space region PCR product. A differentiation of B. garinii, B. afzelii, and B. burgdorferi senso stricto species with TGGE was possible and intraspecies variation was detected. Results compared between TGGE, PFGE, and restriction of 5S-23S intergenic space region PCR product showed no difference in specificity of species identification.

Comparison of pulsed-field gel electrophoresis (PFGE) and two different polymerase chain reactions (PCRs) for species identification of Borrelia burgdorferi sensu lato strains.

The purpose of the present study was to compare the findings of three different molecular-biological methods for Borrelia burgdorferi sensu lato species identification: (i) large DNA fragments pattern obtained with Mlul restriction endonuclease and separated with pulsed-field gel electrophoresis (PFGE); (ii) polymerase chain reaction (PCR), the region inside the 16S rRNA gene multiplied with species-specific primers; and (iii) PCR, the interspace region between the 5S and 23S rRNA genes amplified, the PCR product restricted with Msel restriction endonuclease and fragments separated in polyacrylamide gel. Forty-eight Borrelia strains isolated from diverse clinical materials and two tick strains were analyzed. Each of the 50 isolates analyzed by PFGE was found to be a single species: 30 B. afzelii, 14 B. garinii, and 6 B. burgdorferi sensu stricto. PCR amplification of 16S rRNA with species-specific primers revealed a single species in 41/50 samples and in nine samples two species were detected. PCR of the 5S-23S interspace region restricted with MseI restriction endonuclease detected a single species in 48/50 samples and a mixture of two species was found in 2/50 samples. In all cases where a single species was identified using PCR the species was in accordance with the PFGE result, and in all cases where a mixture of two species was identified by PCR one of the species was the same as that detected by PFGE. Using a criterion of complete concordance of the results a significant difference in species identification was found when PFGE and the 16S rRNA PCR were compared (p = 0.0026), but not between 5S-23S interspace PCR and PFGE (p = 0.4949) or between 16S rRNA and 5S-23S interspace PCRs (p = 0.0552). PCR assays were faster and easier to perform than PFGE for Borrelia species identification, however PFGE remains a standard procedure for analyzing isolates and demonstrating heterogeneity within species.