On Nietzsche’s Concept of ‘European Nihilism’

<div><p>Aplog-1 is a simplified analog of the tumor-promoting aplysiatoxin with anti-proliferative and cytotoxic activities against several cancer cell lines. Our recent findings have suggested that protein kinase Cδ (PKCδ) could be one of the target proteins of aplog-1. In this study, we synthesized amide-aplog-1 (<b>3</b>), in which the C-1 ester group was replaced with an amide group, to improve chemical stability <i>in vivo</i>. Unfortunately, <b>3</b> exhibited seventy-fold weaker binding affinity to the C1B domain of PKCδ than that of aplog-1, and negligible anti-proliferative and cytotoxic activities even at 10⁻⁴ M. A conformational analysis and density functional theory calculations indicated that the stable conformation of <b>3</b> differed from that of aplog-1. Since 27-methyl and 27-methoxy derivatives (<b>1</b>, <b>2</b>) without the ability to bind to PKC isozymes exhibited marked anti-proliferative and cytotoxic activities at 10⁻⁴ M, <b>3</b> may be an inactive control to identify the target proteins of aplogs.</p></div

Possible Contribution of Zerumbone-Induced Proteo-Stress to Its Anti-Inflammatory Functions via the Activation of Heat Shock Factor 1

<div><p>Zerumbone is a sesquiterpene present in <i>Zinger zerumbet</i>. Many studies have demonstrated its marked anti-inflammatory and anti-carcinogenesis activities. Recently, we showed that zerumbone binds to numerous proteins with scant selectivity and induces the expression of heat shock proteins (HSPs) in hepatocytes. To dampen proteo-toxic stress, organisms have a stress-responsive molecular machinery, known as heat shock response. Heat shock factor 1 (HSF1) plays a key role in this protein quality control system by promoting activation of HSPs. In this study, we investigated whether zerumbone-induced HSF1 activation contributes to its anti-inflammatory functions in stimulated macrophages. Our findings showed that zerumbone increased cellular protein aggregates and promoted nuclear translocation of HSF1 for HSP expression. Interestingly, HSF1 down-regulation attenuated the suppressive effects of zerumbone on mRNA and protein expressions of pro-inflammatory genes, including inducible nitric oxide synthase and interlukin-1β. These results suggest that proteo-stress induced by zerumbone activates HSF1 for exhibiting its anti-inflammatory functions.</p></div

Zerumbone and HS induced proteo-stress.

<p>(A) RAW264.7 cells were treated with zerumbone (0–100 μM) for 6 hr, then lysed for western blot analysis using anti-zerumbone-adducts Ab. (B) Cells were treated with zerumbone (0–50 μM) for 6 or 12 hr, then lysed for filter trap assay using anti-zerumbone-adducts Ab. (C) Cells were treated with HS (incubation at 43°C in water bath) for 0–60 min or zerumbone (0–50 μM for 12 hr, then lysed for filter trap assay using anti-ubiquitin Ab.</p

Proposed mechanisms of biological functions by zerumbone.

<p>Zerumbone may specifically bind to Keap1 and activates Nrf2, whose activation plays an important role in its detoxification and anti-oxidation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161282#pone.0161282.ref008" target="_blank">8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161282#pone.0161282.ref002" target="_blank">2</a>]. In addition to this mode of action, zerumbone is bound to various cellular proteins with low selectivity. Such non-specific interaction can induce proteo-stress and subsequently activate HSF1, which was found to partially contribute to anti-inflammatory functions of zerumbone.</p

NAC and PBA attenuated both proteo-stress and anti-inflammatory functions of zerumbone.

<p>(A, B) RAW264.7 cells were pretreated with NAC (0–10 mM) for 1 hr. After incubation of the cells with zerumbone (0, 50 μM) for another 12 hr, cells were lysed for filter trap assay using anti-ubiquitin and anti-zerumbone-adducts Abs. (C) Cells were pretreated with NAC (0–10 mM) for 1 hr. After incubation of the cells with zerumbone (0–10 μM) for another 1 hr, cells were exposed to LPS (100 ng/mL) for 12 hr. Then, cells were subjected to western blot analysis using anti-COX-2, anti-iNOS, and anti-α-tubulin Abs. Also, after LPS stimulation for 24 hr, the supernatants were subjected to Griess assay. *<i>P</i> < 0.001. (D) Cells were pretreated with PBA (0, 1 mM) for 1 hr. After incubation of the cells with zerumbone (0, 50 μM) for another 12 hr, filter trap assay was done using anti-ubiquitin and anti-zerumbone-adducts Abs. (E) Cells were pretreated with PBA (0, 1 mM) for 1 hr. After incubation of the cells with zerumbone (0–10 μM) for another 1 hr, cells were exposed to LPS (100 ng/mL) for 12 hr. Then, cells were subjected to western blot analysis using anti-COX-2, anti-iNOS, and anti-α-tubulin Abs. Also, after LPS stimulation for 24 hr, the supernatants were subjected to Griess assay. *<i>P</i> < 0.01.</p

Primers used for real-time RT-PCR

<p>Primers used for real-time RT-PCR</p

HS treatment and geldanamycin had anti-inflammatory functions.

<p>(A) RAW264.7 cells were treated with HS (incubation at 43°C in water bath) for 30 min. After recovery at 37°C for another 0–6 hr, cells were lysed for western blot analysis using anti-COX-2, anti-iNOS, and anti-α-tubulin Abs. (B) Cells were pretreated with geldanamycin (0–1 μM) for 6hr, then exposed to LPS (100 ng/mL) for 12 hr. Then, cells were lysed for western blot analysis using anti-COX-2, anti-iNOS, and anti-α-tubulin Abs.</p

Zerumbone and HS promoted nuclear translocation of HSF1 and induced HSP70 expressions.

<p>(A, B) RAW264.7 cells were treated with the vehicle (DMSO), zerumbone (50 μM), or geldanamycin (1 μM) for 0–3 hr, then subjected to Nuclear-cytoplasmic fractionation. Cells were lysed for western blot analysis using anti-HSF1, anti-laminB (as a nucleus fraction marker), and anti-α-tubulin (cytoplasm) Abs. As a positive control, cells were treated with HS (incubation at 43°C in water bath) for 60 min. (C) Cells were treated with or without HS (incubation at 43°C in water bath) for 30 min. After recovery at 37°C for another 0–12 hr, cells were lysed for western blot analysis using anti-HSP70 and anti-α-tubulin Abs. (D) Cells were treated with vehicle (DMSO), zerumbone (50 μM), or geldanamycin (0.2, 1 μM) for 6–24 hr, then lysed for western blot analysis using anti-HSP70 and anti-α-tubulin Abs.</p

Structural optimization of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin, as an anticancer lead

<div><p>Aplog-1 is a simplified analog of debromoaplysiatoxin (DAT) with potent tumor-promoting and proinflammatory activities. Aplog-1 and DAT exhibited anti-proliferative activities against several human cancer cell lines, whereas aplog-1 did not have tumor-promoting nor proinflammatory activities. We have recently found 10-methyl-aplog-1 (<b>1</b>) to have strong anti-proliferative activity compared with aplog-1. To further investigate the structural factors involved in the tumor-promoting, proinflammatory, and anti-proliferative activities, two dimethyl derivatives of aplog-1 (<b>2</b>, <b>3</b>) were synthesized, where two methyl groups were installed at positions 4 and 10 or 10 and 12. 10,12-Dimethyl-aplog-1 (<b>2</b>) had stronger inhibitory effects on the growth of several human cancer cell lines than <b>1</b> and DAT, but exhibited no tumor-promoting and proinflammatory activities. In contrast, 4,10-dimethyl-aplog-1 (<b>3)</b> displayed weak tumor-promoting and proinflammatory activities along with anti-proliferative activity similar to that of <b>1</b> and DAT. Compound <b>2</b> would be the optimized seed for anticancer drugs among the simplified analogs of DAT.</p></div

Toxicity in Rat Primary Neurons through the Cellular Oxidative Stress Induced by the Turn Formation at Positions 22 and 23 of Aβ42

The 42-mer amyloid β-protein (Aβ42) aggregates to form soluble oligomers that cause memory loss and synaptotoxicity in Alzheimer’s disease (AD). Oxidative stress is closely related to the pathogenesis of AD. We previously identified the toxic conformer of Aβ42 with a turn at positions 22 and 23 (“toxic turn”) by solid-state NMR and demonstrated that a monoclonal antibody (11A1) against the toxic turn in Aβ42 mainly detected the oligomer in the brains of AD patients. Our recent study suggested that oxidative stress is a key factor of the oligomerization and cognitive impairment induced by Aβ overproduction in vivo. However, the involvement of the toxic conformer in Aβ42-induced oxidative damage remains unclear. To investigate this mechanism, we examined the levels of intracellular reactive oxygen species (ROS) and neurotoxicity in rat primary neurons using E22P-Aβ42, a mutant that induces a turn at positions 22 and 23, and E22V-Aβ42, a turn-preventing mutant. E22P-Aβ42, but not E22V-Aβ42, induced greater ROS production than Wt-Aβ42 in addition to potent neurotoxicity. Interestingly, the formation of the toxic conformer in both E22P-Aβ42 and Wt-Aβ42 probed by the 11A1 antibody preceded Aβ42-induced neurotoxicity. Trolox (a radical scavenger) and Congo red (an aggregation inhibitor) significantly prevented the neurotoxicity and intracellular ROS induced by E22P-Aβ42 and Wt-Aβ42, respectively. These results suggest that Aβ42-mediated toxicity is caused by the turn that favors toxic oligomers, which increase generation of ROS