PubMed · 14215532
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A P KLOTZ. 1964-11-16. IRRITABLE COLON.. https://pubmed.ncbi.nlm.nih.gov/14215532/
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The marine alkaloid ascididemin (ASC) was shown to exert cytotoxicity even against multidrug-resistant cancer cells. Here, we address the signaling pathways utilized by ASC to trigger apoptosis in Jurkat leukemia T cells. We show that ASC (0.5-20 microM) induces a mitochondrial pathway that requires the activation of the initiator caspase-2 upstream of mitochondria. ASC-triggered apoptosis occurred independent of CD95, but required mitochondrial dysfunction. The activation of caspase-2 was shown to precede the processing of caspase-8, -9 and -3. The specific caspase-2 inhibitor zVDVADfmk abrogated ASC-induced DNA fragmentation almost completely. Overexpression of Bcl-x(L) blocked caspase-8 but not caspase-2 processing. Conversely, caspase-2 inhibition strongly reduced caspase-9 activation. As a possible link between caspase-2 and mitochondrial dysfunction, Bid was found to be cleaved by ASC. In addition, JNK was activated by ASC upstream of mitochondria via reactive oxygen species. The specific JNK inhibitor SP600125 partially inhibited caspase-2 and -9 processing as well as cytochrome c release and DNA fragmentation indicating that JNK contributes to, but is not necessary for ASC-mediated apoptosis. Thus, ASC triggers a pathway in which early activation of caspase-2 provides a possible link between its DNA-damaging activity and the induction of mitochondrial dysfunction. The activation of JNK contributes to this signaling upstream of mitochondria.
API-ionspray MS and MS/MS techniques have been utilized to elucidate the structures of 20 bisbenzylisoquinoline alkaloids, consisting of 17 diether and three monoether links of two benzyltetrahydroisoquinoline units, which were isolated and identified previously from a variety of Thalictrum sp. (Ranunculaceae family). Apparent protonated molecular ions ([M+H](+)) and very intense doubly-protonated molecular ion ([M+2H](++), 100% of relative abundance) in Q1 Scan MS spectra and prominent as well as diagnostic product ions for the structural information in MS/MS spectra were observed in nanogram quantities for all investigated alkaloids.
Sanguinarine is a quaternary benzo[c]phenanthridine alkaloid, extracted from the argemone oil, which produced severe human intoxications. To investigate the sanguinarine biotransformation, we develop a simple extraction process and a high performance liquid chromatographic separation coupled to a sensitive fluorometric detection of sanguinarine in cell culture medium, as well as in rat urine and plasma. After extraction with an acidified organic solvent, sanguinarine elution is performed within 15 min on a Nucleosil C18 column with a gradient using 0.2% formic acid/water/acetonitrile as mobile phase. Extracted and standard sanguinarine are characterized by mass spectrometry. The extraction recovery of sanguinarine is about 80% in cell culture medium and in rat urine, but lower in plasma. This convenient high performance liquid chromatography (HPLC) method allows to quantify sanguinarine over concentrations ranged 10-2000 ng ml(-1). The limit of fluorometric detection is 0.5 ng. Under these conditions, the lower limit of quantification of sanguinarine is 50 ng ml(-1) in cell culture medium and in rat urine and 100 ng ml(-1) in rat plasma. This analytical HPLC method is specific, linear and reproducible in all media and is suitable for quantitative determination of sanguinarine in biological fluids.