Pharmacodynamic And Mechanism Studies Of (+)-13a-(S)-Deoxytylophorinine, A Novel Anti-Cancer Phenanthroindolizidine Alkaloid

Phenanthroindolizidine alkaloids are a family of plant-derived compounds with various pharmacological activities. Its chemical structure is different from any known anticancer drugs. Phenanthroindolizidine alkaloid exhibits many biological activities, including anticancer, antiinfiamatory, anti-ameobicidal and anti-viral activities. Specific biomolecular targets of this kind of alkaloids have not yet been clearly identified. (+)-13a-(S)-Deoxytylophorinine (short for CAT) is a new phenanthroindolizidine alkaloid, originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata, which are from family of Asclepiadaceae. Its content from plant is very low. The total synthesis of this compound resolved the major problem of its low natural availability.At first, we tested the in vitro and in vivo anticancer activities of this compound and some of its derivatives. They exhibited favorable anticancer activities in many cancer cells from different tissue origins, with IC50 in the range of 10-7-10-8mol/L. In mouse xenograft model, CAT and its salts, in several dosage regimen, can well inhibit the growth of transplanted tumors.CAT's solubility in water is rather poor. In order to improve its water solubility, several salts of CAT were made, including malate, citrate and tartrate. In vitro and in vivo evaluations proved that the salts of CAT are equivalent to CAT in its anticancer activities. CAT can be administrated p.o. in CMC suspensions, while the salts can also be administrated i.v. However, due to the severe vescular irritation, the tolerance to animals is rather poor, the effects of intravenous administration were not better.The distributions of CAT in blood and brain tissues are also tested. CAT and its salts can come into brain through brain blood barrier.5min after oral administration, CAT can be detected in brain and it comes to its peak concentration after 15min.2 hours later, the concentration of CAT can still be maintained in certain concentration level. So, this proves that CAT can penetrate blood brain barrier and may be used to treat brain tumor by oral administrations.Several compounds from phenanthroindolizidine alkaloid family were reported to exhibit severe neurotoxicity. We also preliminarily evaluated the neurotoxicity of CAT by rat pheochromocytoma PC 12 cell neurite-outgrowth assay which is a dependable model for predicting neurotoxicity in vitro. Compared to vincristinine, an anticancer drug with severe neurotoxicity, CAT exerts no neurotoxicity even at relatively high concentrations.Interactions between this compound and DNA sequences were studied in details with circular dichroic spectroscopy and Fluorescence spectroscopy. Concentration-dependent interactions were observed and this compound seemed to interact sequence-specifically with AT-repeated sequence of double-helical DNA. Such interactions were proved to be intercalating rather than groove binding by viscosity measurements. Interactions between CAT and RNA were also tested, and concentration-dependent interactions were observed.The enantiomer of CAT,-CAT, has only one different chiral carbon atom with CAT. However,-CAT's in vitro and in vivo anticancer activities were far inferior to its enantiomer. We further userd gene chip and proteomic technology to detect the discrepancy of gene expression profile and protein expression profile of human glioma U251 cells, treated by CAT and its enantiomer. Several genes related to apoptosis, cell cycle regulation, signaling transduction and growth regulation as well as genes related to DNA replication, reparation, transcription regulation and ribosome translation were found upregulated or downregulated after the treatment of CAT, rather than its enantiomer. We also find several proteins by proteomic technology that expressed differentially after the treatment of CAT and its enantiomer.In short, CAT was a novel plant-derived anticancer alkaloid of distinct mechanisms, with broad perspect for further development. CAT can be orally administrated and can penetrate the blood brain barrier without obvious neurotoxicity. CAT or its derivatives can well be developed into a new anticancer drug for glioma or other brain cancers.