Molecular Pharmacology Of Marine Bioactive Alkaloid Fascaplysin

Fascaplysin, which was originally isolated from marine sponge Fascaplysinopsis sp. in 1988 withβ-carboline structure, is a promising candidate for cancer therapeutics owing to its multiple antitumor activities including CDK4 selective inhibition, DNA intercalation, and angiogenesis. Although the cytotoxic activity of fascaplysin against cancer cell lines was obvious, the exact mode of action and molecular mechanism for this marine natural product remain unclear. Previous researches of our laboratory indicated that apoptosis induced by fascaplysin might be important for its cytotoxic activity. In this study, the route for synthesis of fascaplysin was investigated; gene expression profiling, western blot, flow cytometric analysis, and metabonomics technique were used to explore the molecular mechanism of fascaplysin, especially the mechanism of apoptosis.Fascaplysin was synthesized according to the route reported by Radchenko (1997) with improvements. O-bromophenylacetic acid was converted into o-bromophenylacetyl chloride before reacting with tryptamine to give amid 3.γ-MnO2, the most effective oxidant comparing to other forms of MnO2, was used to obtain intermediate 6. Structures of the products had been established by MS, IR and NMR spectra. MTT assay and Hoechst staining were used to assess the antitumor activity of fascaplysin following the synthesis on hepatocellular carcinoma cell line Bel-7402. Fascaplysin had been synthesized in seven steps from O-bromophenylacetic acid in about 15% overall yield. MTT assay indicated that fascaplysin inhibited human hepatocarcinoma cells BeL-7402 proliferation in a time- and dose-dependent manner. Hoechst staining showed that BeL-7402 displayed apoptotic morphology characters in a dose- and time-dependent manner. These results suggested that the route used for the synthesis of fascaplysin was more practical. The genome-wide oligonucleotide microarray for human, contains 21522 known and predicted genes, was used to investigate the transcriptome of human umbilical vein endothelial cells (HUVEC) and BeL-7402 in response to fascaplysin treatment. Analysis of the regulated genes showed that a number of them involved in apoptosis in both cell lines. There were 49 apoptosis-related genes in HUVEC, and 55 in BeL-7402, including tumor necrosis factor (TNF) superfamily, TNF receptor superfamily, death domain protein, Bcl-2 family, and genes relating to inflammation, DNA damage and apoptosis. These results suggested that TRAIL apoptotic pathway and mitochondrial apoptotic pathway might be activated.We demonstrated by western blot and flow cytometry that, in vitro, fascaplysin sensitized HUVEC and BeL-7402 cells to TRAIL-induced apoptosis by up-regulating the expression of DR5. Fascaplysin increased DR5, cleaved caspase-9 and active caspase-3 and decreased procaspase-8 and Bid at protein level. The apoptosis induced by fascaplysin in HUVEC was markedly blocked by a dominat negative form of DR5 (TRAIL R2/Fc chimera) or caspase inhibitors such as Z-VAD-FMK, Z-DEVD-FMK, Z-IETD-FMK and Z-LEHD-FMK. The effects of TRAIL R2/Fc chimera and Z-IETD-FMK were more significant. The early stage apoptotic rates in HUVEC cells were decreased from 19.79% to 8.09%, 8.95%, respectively. The late apoptotic rates were decreased from 11.54% to 3.2%, 4.06%, respectively. Futhermore, MTT assay indicated that subtoxic dose of fascaplysin enhanced TRAIL-induced apoptosis in HUVEC and BeL-7402. The apoptosis in HUVEC induced by co-treatment with fascaplysin and TRAIL was significantly blocked by TRAIL R2/Fc chimera or caspase inhibitors including Z-IETD-FMK and Z-LEHD-FMK. The early stage apoptotic rates in HUVEC cells were decreased from 22.97% to 5.28%, 11.3%, respectively. The late apoptotic rates were decreased from 14.02% to 4.38%, 2.66%, respectively. We also found that the Bax/Bcl-2 ratio was increased by fascaplysin in HUVEC indicating the activation of mitochondria pathway.Cell metabolites from BeL-7402 treated with 0.61μg/ml fascaplysin for several time points were studied by a metabonomic method based on ultra performance liquid chromatography coupled to mass spectrometry. Principle component analysis (PCA) and orthogonal-projection to latent structures discriminant analysis (O-PLS-DA) were built to separate the control treatment and drug treatment and to find compounds that are expressed in significantly different amounts between the two populations. Results showed that the fascaplysin-treated group and the control-treated group could be seperated after 12 h, and obviously separated after 24 h. 26 metabolite ions were considered as potential biomarkers according to the Variable important in the Project (VIP) value and S-plot. Down-regulation of sphingomyeline and up-regulation of phosphatidylcholine, sphinganine, N,N-dimethyl-safingol and phytosphingosine indicated that fascaplysin treatment might activate the sphingolipid signaling pathway.