CAM includes clinicohistopathologically heterogeneous disease entities. Among CAM entities, anti-TIF1-γ-Ab(+) CAM has characteristically shown a close temporal association with cancer detection and the histopathologic findings of dC5b-9 and VFs, and CAM with NAM is a subset of anti-TIF1-γ-Ab(-) CAM.

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine "phenylahistin" (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.

Oxidative stress is associated with beta-amyloid peptide (A beta) accumulation in the brains of Alzheimer's disease patients. A beta is generated upon the sequential proteolytic cleavage of transmembrane amyloid precursor protein (APP) by two membrane-bound proteases, beta-secretase (BACE1) and the gamma-secretase complex comprising presenilin 1 (PS1), nicastrin, APH-1 and PEN-2. Recent evidence suggests that significant amounts of BACE1 and gamma-secretase components localize in the cholesterol-rich region of membranes known as lipid rafts, where A beta production occurs preferentially. In this study, we investigated the effects of oxidative stress on the BACE1 and gamma-secretase components in lipid rafts using human neuroblastoma SH-SY5Y cells exposed to ethacrynic acid (EA), a compound that induces cellular glutathione depletion. Following exposure of cells to EA, heme oxygenase-1, a marker protein of oxidative stress, was strongly induced. Moreover, treatment with EA resulted in a significant increase in PS1 protein levels, but not those of nicastrin, APH-1, PEN-2 or BACE1, in both cell lysates and the lipid raft fraction. This increase in PS1 protein expression was prevented by co-treatment with an antioxidant, N-acetylcysteine (NAC). EA additionally induced a significant increase in PS1 mRNA expression, which was inhibited by NAC. Finally, EA treatment was found to promote A beta secretion from cells expressing Swedish mutant APP. It appears that in our cell culture model, oxidative stress enhances PS1 protein levels in lipid rafts via up-regulation of PS1 transcription, which may constitute the mechanism underlying the oxidative stress-associated promotion of A beta production.

β-Secretase, BACE1 is a neuron-specific membrane-associated protease that cleaves amyloid precursor protein (APP) to generate β-amyloid protein (Aβ). BACE1 is partially localized in lipid rafts. We investigated whether lipid raft localization of BACE1 affects Aβ production in neurons using a palmitoylation-deficient mutant and further analyzed the relationship between palmitoylation of BACE1 and its shedding and dimerization. We initially confirmed that BACE1 is mainly palmitoylated at four C-terminal cysteine residues in stably transfected neuroblastoma cells. We found that raft localization of mutant BACE1 lacking the palmitoylation modification was markedly reduced in comparison to wild-type BACE1 in neuroblastoma cells as well as rat primary cortical neurons expressing BACE1 via recombinant adenoviruses. In primary neurons, expression of wild-type and mutant BACE1 enhanced production of Aβ from endogenous or overexpressed APP to similar extents with the β-C-terminal fragment (β-CTF) of APP mainly distributed in nonraft fractions. Similarly, β-CTF was recovered mainly in nonraft fractions of neurons expressing Swedish mutant APP only. These results show that raft association of BACE1 does not influence β-cleavage of APP and Aβ production in neurons, and support the view that BACE1 cleaves APP mainly in nonraft domains. Thus, we propose a model of neuronal Aβ generation involving mobilization of β-CTF from nonraft to raft domains. Additionally, we obtained data indicating that palmitoylation plays a role in BACE1 shedding but not dimerization.