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6-O-Palmitoyl ascorbic acid (PAA) has recently been used as a substitute for ascorbic acid because of its greater potency as an antioxidant. In detailed concentration response studies distinct cytotoxic effects of PAA at concentrations exceeding 100 μM were reported. Here we examined and further characterized this cytotoxicity. While ascorbic acid was tolerated well up to millimolar concentrations, PAA revealed an lc50 between 125 and 150 μM in rat GH3 tumor cells. Morphological and biochemical observations suggested the induction of apoptosis at concentrations exceeding 125 μM with a prominent activation of caspase 3 at 250 μM after 4 hr. A subsequent pronounced fragmentation of DNA (DNA-ladder) was detected after 6 hr and was further enhanced after 12 hr. The activation of caspases and the cleavage of its substrate PARP was preceded by a distinct increase in the phosphorylation of stress activated JNK-kinases. This observation suggested that the agent affected signal transduction mechanisms regulating protein phosphorylation at serine/threonine residues in the cell. No effect of PAA on protein phosphatase 2A (PP2A)-like activity was observed while magnesium-dependent protein phosphatase activity, presumably PP2C, was inhibited concentration-dependently up to 75% at the respective concentrations. Thus, the cytotoxic, pro-apoptotic effect of PAA might be related to the inhibition of PP2C and the activation of JNK. [Copyright &y& Elsevier]

In the present study, we outlined the part of the molecule mediating the prominent pro-apoptotic effect of the Michael adduct of ascorbic acid with p-chloro-nitrostyrene, a new synthetic phosphatase inhibitor. The nitrostyrene (NS) moiety was identified as the structure essential for apoptosis induction. NS and its ascorbic acid adducts displayed LC(50) values of 10-25 microM with no significant reduction of potency in okadaic acid resistant cells overexpressing the MDR1 P-glycoprotein. Induction of apoptosis by NS derivatives and the protein phosphatase 2A inhibitor cantharidic acid was proven by the analysis of caspase-3 activation and subsequent fragmentation of DNA. Further structure activity analysis revealed the necessity of the nitro group at the beta-position of the side chain. The pro-apoptotic potential of adducts of NS with pyrimidine- or pyridine-derivatives varied between NS and a progressive reduction in potency up to a nearly complete loss of cytotoxicity. Substitutions at the benzene core of NS suggested a prominent enhancement of toxicity only by substitutions at the 2- or 3-position. Heterocyclic aromatics can substitute for the benzene ring of NS albeit with a 2-3-fold reduced potency. In conclusion, nitrostyrene was identified as the core structure mediating the pro-apoptotic effect of a new synthetic phosphatase inhibitor. Further studies defined a nitrovinyl side chain attached to an aromatic ring as the pharmacophore structure of a new group of pro-apoptotic agents. These observations present the basis for the development of a new group of anticancer drugs.

Until now guinea-pigs have been rarely used to investigate formation and deposition of Alzheimer's disease-associated amyloid beta peptides despite the sequence identity of human and guinea-pig amyloid beta peptides being known, and the overall similarity of human and guinea-pig amyloid precursor protein. We now describe a primary cell culture system of mixed fetal guinea-pig brain cells, which we have applied to characterize endogenous amyloid precursor protein processing and amyloid beta formation. These cell cultures were established at embryonic day 24 of guinea-pigs after comparison of selected stages of guinea-pig ontogenetic development with the known ontogeny of rats, and were characterized by immunocytochemical detection of neuronal and glial marker proteins. Amyloid precursor protein expression, processing and amyloid beta formation increased in parallel with cellular maturation during cultivation and reached a stable phase after approximately 14 days in vitro therefore providing a suitable time for analysis. Aged cultures display strong neuronal amyloid precursor protein immunoreactivity and an altered profile of amyloid precursor protein isoform messenger RNA expression due to glial proliferation as single neurons were shown to retain their typical pattern of amyloid precursor protein expression. We show that amyloid precursor protein in guinea-pig cells is processed by different protease activities which most likely represent alpha- and beta-secretase, leading to the generation of soluble amyloid precursor protein derivatives. Furthermore, endogenous amyloid precursor protein processing leads to production of substantial amounts of amyloid beta-peptides which accumulate in conditioned culture medium. Amyloid beta was readily detectable by western blot analysis and was shown to consist of approximately 80-90% amyloid beta(1-40). We suggest that primary guinea-pig cell cultures provide a valuable tool in amyloid research that resembles amyloid precursor protein processing under physiological concentrations and, therefore, the situation in humans more closely than current rodent models. It should be especially useful in screening experiments for secretase inhibiting compounds.