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20 H.J. Jang et al. / Neuroscience Letters 443 (2008) 17–22nuclear fragmentation (Fig. 1B). To examine whether oxidativestress is specific for TIND or not, we investigated the effects ofz-VAD-fmk and antioxidants on the vinblastine-induced neuronaldeaths. Treatment with z-VAD-fmk significantly attenuated thevinblastine-induced neuronal death, but antioxidants such as troloxand ascorbic acid or NADPH oxidase inhibitors such as apocynin andDPI failed to inhibit the vinblastine-induced death (Fig. 1D). Thesefindings indicate that oxidative stress is uniquely involved in TINDbut not in vinblastine-induced deaths.To delineate whether the oxidative stress is involved in upstreamor downstream of microtubule inhibition by taxol, we observedthe effects of taxol on microtubule network using fluorescenceimmunocytochemistry for tubulin. After treating neurons withtaxol for 80 min, neurite beadings were identified (Fig. 2B). So weinvestigated the effect of trolox and apocynin on the formation ofneurite beadings by taxol. Treatment with either trolox or apocynindid not affect formation of the neurite beadings (Fig. 2C andD). These results suggest that oxidative stress is downstream eventof microtubule inhibition by taxol.Based on our findings and reports that all subunits of NADPHoxidase are expressed in cultured cortical cells [10,15] and thatactivation of this enzyme is involved in ROS-induced oxidative celldeath in neurodegenerative diseases [9,18], we hypothesize thatactivation of NADPH oxidase may also be involved in TIND in mixedcortical cultures. First, we examined p47 phox and gp91 phox expressionat the mRNA level by RT-PCR analysis and at the protein levelby western blot. Whereas cortical cultures expressed low levels ofp47 phox and gp91 phox mRNA, 2-h exposure to taxol substantiallyincreased the mRNA levels for p47 phox and gp91 phox in cortical cultures(Fig. 3A). Western blot assays confirmed that taxol exposureincreased the expressions of both p47 phox and gp91 phox after 3 h(Fig. 3B) and induced translocation of the p47 phox to the membranein cortical cultures, a signature event for the activation of NADPHoxidase (Fig. 3C).Next, we examined whether the enhanced expression of NADPHoxidase contributed to ROS generation after exposure to taxol.Exposure of mixed cortical cultures to taxol for 3 h markedlyincreased cellular DCF fluorescence, an indicator for superoxide,compared with sham-washed controls. Treatment with eitherapocynin, an NADPH oxidase inhibitor [19], or trolox, a vitamin Eanalog, markedly reduced the DCF fluorescence (Fig. 4A). We monitoredthe changes in DCF fluorescence for 8 h after exposure to taxol.As shown in Fig. 4B, taxol markedly increased the fluorescence forup to 2 h and then gradually increased the fluorescence for up to6 h. Treatment with either apocynin or trolox significantly reducedthe increase of DCF fluorescence.The next question was whether ROS generated by NADPH oxidasecontributed to TIND. Treatment with known NADPH oxidaseFig. 3. Induction and activation of p47 phox and gp91 phox in mixed cortical cultures exposed to taxol. (A) RT-PCR for p47 phox and gp91 phox in sham-washed control cultures(S) and in sister cultures after 2-h exposure to 300 nM taxol (T). The panels are representative of three independent experiments. HPRT was used as a house-keeping gene.Bars denote densitometer readings of RT-PCR signals expressed as folds of respective control values (mean ± S.E.M.; n = 3). *P < 0.05, compared with respective controls. (B)Western blots for p47 phox and gp91 phox in sham-washed control cultures (S) and in sister cultures after 3-h exposure to 300 nM taxol (T). The panels are representative ofthree independent experiments. (C) Western blot for p47 phox in the cytosolic fraction and the membrane fraction of cortical cultures, 3 h after sham wash (S) and 300 nMtaxol exposure (T).
H.J. Jang et al. / Neuroscience Letters 443 (2008) 17–22 21Fig. 4. Reduction of taxol-induced ROS generation by apocynin or trolox. (A) DCF fluorescencein cortical cultures 3 h after sham wash (Sham) or 3 h exposure to 300 nMtaxol without (Tax) or with addition of 500 M apocynin (+Apo) or 100 M trolox(+Tro). (B) Relative ROS generation was analyzed by measuring DCF fluorescence incortical cultures exposed to 300 nM taxol without () or with 500 M apocynin (),100 Mtrolox(○) for the indicated times. Each point and bar is mean ± S.E.M. from6 to 12 wells. *P < 0.05, compared with taxol-treated group.inhibitors, such as apocynin, AEBSF [6] or DPI [12], significantlyreduced the neuronal cell death (Fig. 5A). Furthermore, suppressingthe expression of gp91 phox with siRNA also significantly attenuatedTIND (Fig. 5B).Taxol mainly causes peripheral neuropathy in humans and itsCNS toxicity is rare and transient [20]. In the present study, wedemonstrated that TIND is apoptotic based on morphological features,such as chromatin condensation and nuclear fragmentation,and intervention with anti-apoptotic drugs in mouse cortical cultures.Although taxol is known to induce apoptotic cell death inproliferating cells, taxol also induces apoptotic cell death in nonproliferatingcells like cortical neurons and cardiomyocytes [7,17].Interestingly, it has been suggested that taxol may be useful fortreating Alzheimer’s disease due to its stabilization of microtubules[13].The attenuation of TIND by treatment with antioxidants indicatesthat oxidative stress is involved in this process. Increasedoxidative stress is an important underlying factor for the pathogenesisof a number of neurodegenerative diseases, includingAlzheimer disease (AD), multiple sclerosis (MS) and stroke. Thepresent study has demonstrated that NADPH oxidase is expressedin mixed cortical cultures and exposure of the cortical cultures totaxol induces the expression of this enzyme. Several lines of evi-Fig. 5. Attenuation of taxol-induced neuronal death by NADPH oxidase inhibitorsand knockdown of gp91 phox by siRNA. (A) Effect of 500 M apocynin, 50 M AEBSFand 100 nM DPI on the 300 nM taxol-induced neuronal death in mixed corticalcultures. Each column and bar is the mean ± S.E.M. from 8 to 12 wells. *P < 0.05,compared with taxol-treated group. (B) Effect of knockdown of gp91 phox with siRNAon the 300 nM taxol-induced neuronal death in mixed cortical cultures. Each columnand bar is the mean ± S.E.M. from 8 to 12 wells. *P < 0.05, compared withtaxol-treated control group.dence indicate that NADPH oxidase participates in the process ofneuronal death in some pathologic conditions. For example, activationof NADPH oxidase was also observed in Alzheimer’s diseasebrain [16], and NADPH oxidase participates in the neuronal deathinduced by beta-amyloid peptide [9], in zinc-induced neuronaldeath [15] and in BDNF-induced neuronal death in cultured neurons[10].In the present study of mixed cortical cultures, the contributionby NADPH oxidase to TIND was directly supported by findingsthat antioxidants and inhibitors of NADPH oxidase attenuate bothROS generation and TIND. Furthermore, suppression of NADPH oxidasewith siRNA for gp91 phox also significantly reduced TIND. Thisagain supports the hypothesis that oxidative stress through activationof NADPH oxidase is a major mechanism in TIND. Consistentwith these findings, a recent study showed that taxol killed cancercells by generating ROS through enhancing the activity of NADPHoxidase [3].Vinblastine and taxol are involved in microtubule-disruptinganticancer drug, but they have opposite action on microtubule polymerization[8]. In present study, vinblastine also induced apoptotic
Page 2 and 3: 56MethodsCell culture, reagents and
Page 4 and 5: 1314DiscussionThe prognosis of brea
Page 6 and 7: 21with antibodies against cyclin B1
Page 8 and 9: ARTICLE IN PRESSBiochemical and Bio
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Page 14 and 15: 2present on the cell surface:LPA 1
Page 16 and 17: 4Table 1Taxol(nM)Effect of LPA on T
Page 18 and 19: 6Lysophosphatidate inhibits Taxol-i
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