ABSTRACT
A high-throughput screening assay was developed and applied to a large library of natural product extract samples, in order to identify compounds which preferentially inhibited the in vitro 2D growth of a highly metastatic osteosarcoma cell line (MG63.3) compared to a cognate parental cell line (MG63) with low metastatic potential. Evaluation of differentially active natural product extracts with bioassay-guided fractionation led to the identification of lovastatin (IC50 = 11 µm) and the limonoid toosendanin (IC50 = 26 nm). Other statins and limonoids were then tested, and cerivastatin was identified as a particularly potent (IC50 < 0.1 µm) and selective agent. These compounds potently and selectively induced apoptosis in MG63.3 cells, but not MG63. Assays with other cell pairs were used to examine the generality of these results. Statins and limonoids may represent unexplored opportunities for development of modulators of osteosarcoma metastasis. As cerivastatin was previously approved for clinical use, it could be considered for repurposing in osteosarcoma, pending validation in further models.
Subject(s)
Biological Products/pharmacology , Cell Proliferation/drug effects , High-Throughput Screening Assays/methods , Biological Products/chemistry , Biological Products/isolation & purification , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Line , Cell Movement/drug effects , Cell Survival/drug effects , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/isolation & purification , Drugs, Chinese Herbal/pharmacology , Humans , Lovastatin/chemistry , Lovastatin/isolation & purification , Lovastatin/pharmacology , Melia/chemistry , Melia/metabolism , Monascus/chemistry , Monascus/metabolism , Osteosarcoma/metabolism , Osteosarcoma/pathology , Plant Extracts/chemistry , Pyridines/chemistry , Pyridines/isolation & purification , Pyridines/pharmacology , Seeds/chemistry , Seeds/metabolismABSTRACT
The fruits of Melia toosendan Sieb. et Zucc. (Meliaceae) are a source of bioactive limonoids that can be used as effective pesticides. In this study, two novel limonoids, 6-acetylsendanal and 6-ketocinamodiol, were isolated together with fourteen known compounds, namely four protolimonoids, six trichilin-class limonoids, and four C-seco limonoids. The structures of the new compounds were determined by extensive spectroscopic analyses (HR-ESI-MS, UV, IR, 1D and 2D NMR). The bioassay results revealed that eleven of the extracted limonoids exhibited interesting antifeedant activities against the larvae of Pieris rapae with AFC50 values in the range of 0.11-1.79â mm. Particularly, mesendanin H, with an AFC50 value of 0.11â mm, exhibited a higher activity than the positive control toosendanin. Information on new bioactive limonoids may provide further insight into M. toosendan as a source of bioactive components.
Subject(s)
Limonins/chemistry , Melia/chemistry , Animals , Butterflies/drug effects , Butterflies/growth & development , Drugs, Chinese Herbal/pharmacology , Fruit/chemistry , Fruit/metabolism , Larva/drug effects , Larva/growth & development , Limonins/isolation & purification , Limonins/pharmacology , Magnetic Resonance Spectroscopy , Melia/metabolism , Molecular Conformation , Spectrometry, Mass, Electrospray IonizationABSTRACT
Toosendanin (TSN) is a hepatotoxic triterpenoid extracted from Melia toosendan Sieb et Zucc. Considering that TSN contains the structural alert of the furan ring, it is believed that bioactivation of TSN may be responsible for its toxicity. Herein, the bioactivation potential and metabolism profiles of TSN were investigated. After an oral administration of 10 mg/kg TSN to rats, esterolysis and conjugation with amino acids were identified as the main metabolic pathways. The same types of conjugates were detected in liver microsomes in an NADPH-dependent manner. According to the remaining amount of the parent drug, the reactivity of trapping reagents with TSN reactive metabolites was sorted in a decreasing order of N(α)-(tert-butoxycarbonyl)-l-lysine (Boc-Lys) > alanine, lysine, taurine, phenylalanine, serine, glutamic acid, glycine, and glutathione (GSH) > cysteine. No conjugates were observed in NADPH and N-acetyl cysteine (NAC)-supplemented human liver microsomal incubations. Further phenotyping studies and the chemical synthesis of the major conjugated standards proved that TSN was bioactivated by CYP3A4 and yielded a cis-butene-1,4-dial intermediate, which was prone to undergo 1,2-addition with the amino group of amino acids and GSH to form 3-pyrroline-2-one adducts. The sulfydryl group of GSH also attacked the intermediate and yielded S-conjugates by 1,4- or 1,2-addition, which would form pyrrole conjugates by further reacting with the amino group. Compared to the well-recognized S-conjugation of the furan ring, N-conjugation with multiple amino acids and GSH played a more important part in the elimination of reactive metabolites of TSN. The significance of these conjugates requires further investigation.
Subject(s)
Amino Acids/chemistry , Cytochrome P-450 CYP3A/metabolism , Drugs, Chinese Herbal/analysis , Furans/metabolism , Glutathione/chemistry , Animals , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP3A/genetics , Drugs, Chinese Herbal/metabolism , Furans/chemistry , Humans , Mass Spectrometry , Melia/chemistry , Melia/metabolism , Microsomes, Liver/metabolism , Nitrogen/chemistry , Rats , Rats, Wistar , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Taurine/chemistryABSTRACT
Meliacine (MA), an antiviral principle present in partially purified leaf extracts of Melia azedarach L., reduces viral load and abolishes the inflammatory reaction and neovascularization during the development of herpetic stromal keratitis in mice. 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), obtained from MA, displays anti-herpetic and immunomodulatory activities in vitro. We investigated whether CDM interferes with the angiogenic process. CDM impeded VEGF transcription in LPS-stimulated and HSV-1-infected cells. It proved to have neither cytotoxic nor antiproliferative effect in HUVEC and to restrain HUVEC migration and formation of capillary-like tubes. Moreover, MA inhibits LMM3 tumor-induced neovascularization in vivo. We postulate that the antiangiogenic activity of CDM displayed in vitro as a consequence of their immunomodulatory properties is responsible for the antiangiogenic activity of MA in vivo, which would be associated with the lack of neovascularization in murine HSV-1-induced ocular disease.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antiviral Agents/pharmacology , Immunologic Factors/pharmacology , Animals , Cell Proliferation , Chemistry, Pharmaceutical/methods , Chemotaxis , Drug Design , Female , Gene Expression Regulation , Herpesvirus 1, Human/metabolism , Humans , Lipopolysaccharides/metabolism , Melia/metabolism , Mice , Mice, Inbred BALB C , Neoplasms/metabolism , Neoplasms/pathology , Neovascularization, Pathologic , Peptides/pharmacology , Plant Extracts/pharmacology , Plant Proteins/pharmacology , Vascular Endothelial Growth Factor A/biosynthesisABSTRACT
To elucidate the effects of redox balance regulation on epidermal pigmentation, we used an antioxidant-rich extract of the herb Melia toosendan (dried mature fruits) to assess its effect on endothelin-1 (EDN1)-stimulated pigmentation in human epidermal equivalents and analyzed its biological mechanism of action. Addition of the Melia toosendan extract elicited a marked depigmenting effect on EDN1-stimulated pigmentation after 14 days of treatment, which was accompanied by a significant decrease in eumelanin content. Real-time RT-PCR and Western blotting revealed that the EDN1-stimulated expression of melanocyte-specific proteins (including tyrosinase) was significantly suppressed at the gene and protein levels by the extract. Signaling analysis with specific inhibitors and immunoblots revealed that in melanoma cells treated with the extract, there was a marked deficiency in the EDN1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB. Since all those proteins are downstream phosphorylation targets of PKC activity, these findings indicate that the Melia toosendan extract attenuates the EDN1-stimulated pigmentation by preferentially inhibiting PKC activity within melanocytes.