Embryotoxicity evaluation of atractylodin and ß-eudesmol using the zebrafish model.

Atractylodin and ß-eudesmol are the major active ingredients of Atractylodes lancea (Thunb) DC. (AL). Both compounds exhibit various pharmacological activities, including anticancer activity against cholangiocarcinoma. Despite the widespread use of this plant in traditional medicine in China, Japan, Korea, and Thailand, studies of their toxicological profiles are limited. The present study aimed to evaluate the embryotoxicity of atractylodin and ß-eudesmol using the zebrafish model. Zebrafish embryos were exposed to a series of concentrations (6.3, 12.5, 25, 50, and 100 µM) of each compound up to 72 h post-fertilization (hpf). The results showed that atractylodin and ß-eudesmol induced mortality of zebrafish embryos with the 50% lethal concentration (LC50) of 36.8 and 53.0 µM, respectively. Both compounds also caused embryonic deformities, including pericardial edema, malformed head, yolk sac edema, and truncated body. Only ß-eudesmol decreased the hatching rates, while atractylodin reduced the heart rates of the zebrafish embryos. Additionally, both compounds increased reactive oxygen species (ROS) production and altered the transcriptional expression levels of superoxide dismutase 1 (sod1), catalase (cat), and glutathione S-transferase pi 2 (gstp2) genes. In conclusion, atractylodin and ß-eudesmol induce mortality, developmental toxicity, and oxidative stress in zebrafish embryos. These findings may imply similar toxicity of both compounds in humans.

The Hepatotoxicity of Alantolactone and Germacrone: Their Influence on Cholesterol and Lipid Metabolism in Differentiated HepaRG Cells.

The sesquiterpenes alantolactone (ATL) and germacrone (GER) are potential anticancer agents of natural origin. Their toxicity and biological activity have been evaluated using the differentiated HepaRG (dHepaRG) cells, a hepatocyte-like model. The half-maximal inhibitory concentrations of cell viability after 24-h treatment of dHepaRG cells are approximately 60 µM for ATL and 250 µM for GER. However, both sesquiterpenes induce reactive oxygen species (ROS) formation in non-toxic concentrations and significantly dysregulate the mRNA expression of several functional markers of mature hepatocytes. They similarly decrease the protein level of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and their transcription target, intercellular adhesion molecule 1 (ICAM-1). Based on the results of a BATMAN-TCM analysis, the effects of sesquiterpenes on cholesterol and lipid metabolism were studied. Sesquiterpene-mediated dysregulation of both cholesterol and lipid metabolism was observed, during which these compounds influenced the protein expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol regulatory element-binding protein 2 (SREBP-2), as well as the mRNA expression of HMGCR, CYP19A1, PLIN2, FASN, SCD, ACACB, and GPAM genes. In conclusion, the two sesquiterpenes caused ROS induction at non-toxic concentrations and alterations in cholesterol and lipid metabolism at slightly toxic and toxic concentrations, suggesting a risk of liver damage if administered to humans.

Eudesmacarbonate, a Eudesmane-Type Sesquiterpene from a Marine Filamentous Cyanobacterial Mat (Oscillatoriales) in the Florida Keys.

A new, cyclic carbonate eudesmane-type sesquiterpene, eudesmacarbonate (1), was isolated from marine filamentous cyanobacterial mats associated with apparent ingestion-related intoxications of captive bottlenose dolphins in the Florida Keys. Sequencing of 16S rDNA revealed that mats were composed of closely related Oscillatoriacean species including a previously undocumented species of Neolyngbya. The structure of 1 was elucidated by (+)-HRESIMS, 1D and 2D NMR, single-crystal X-ray diffraction, and vibrational circular dichroism data. Toxicity of 1 was assessed in the zebrafish embryo/larval model, and 1 was found to exhibit effects qualitatively similar to those observed for the known neurotoxin brevetoxin-2 and consistent with neurobehavioral impairment.

Anticancer activity using positron emission tomography-computed tomography and pharmacokinetics of ß-eudesmol in human cholangiocarcinoma xenografted nude mouse model.

Cholangiocarcinoma (CCA) is an important public health problem in several parts of South East Asia, particularly in Thailand. The limited availability of effective diagnostic tools for early stage CCA, including chemotherapeutic options, constitutes a major problem for treatment and control of CCA. The aim of the present study was to assess the anti-CCA activity and pharmacokinetics of ß-eudesmol in CCA-xenografted nude mouse model and healthy mice. Positron emission tomography-computed tomography (PET-CT) with (18)F-fluorodeoxyglucose was used for detecting and monitoring tumour development, and PET-CT with technetium-99m was used to investigate its pharmacokinetics property. Results support the role of PET-CT as a potential tool for detecting and monitoring the progress of lung metastasis. Tumour size and lung metastasis were significantly inhibited by 91.6% (of baseline) and 95% (of total lung mass), respectively, following treatment with high-dose ß-eudesmol (100 mg/kg body weight for 30 days). Survival time was prolonged by 64.4% compared with untreated controls. Systemic clearance of the compound was rapid, particularly during the first 60 min. The compound was distributed to the vital organs at maximum levels 2 h after oral administration and 15 min after intravenous injection. Results from the present study suggest the potential of ß-eudesmol as a promising candidate for further development as an anti-CCA drug with respect to its pharmacodynamics and pharmacokinetic properties. PET-CT, with radiotracers (18)F-fluorodeoxyglucose and technetium-99m, was shown to be a reliable tool in the investigation of anti-CCA and pharmacokinetic properties of ß-eudesmol in CCA-xenografted and healthy mice.

Triggering of programmed erythrocyte death by alantolactone.

The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 µM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 µM), ceramide abundance (60 µM) and DCFDA fluorescence (≥40 µM). The effect of alantolactone (60 µM) on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca2+]i, ceramide abundance and oxidative stress.

Alantolactone induces cell apoptosis partially through down-regulation of testes-specific protease 50 expression.

Testes-specific protease 50 (TSP50) is aberrantly expressed in many cancer biopsies and plays a crucial role in tumorigenesis, which make it a potential cancer therapeutic target for drug discovery. Here, we constructed a firefly luciferase reporter driven by the TSP50 gene promoter to screen natural compounds capable of inhibiting the expression of TSP50. Then we identified alantolactone, a sesquiterpene lactone, could efficiently inhibit the promoter activity of TSP50 gene, further results revealed that alantolactone also efficiently inhibited the expression of TSP50 in both mRNA and protein levels. Moreover, we found alantolactone could increase the ratio of Bax/Bcl-2, and activate caspase-9 and caspase-3 in the cancer cells with high expression of TSP50, surprisingly, the same effects can also be observed in the same cells just by knockdown of TSP50 gene expression. Furthermore, our results suggested that overexpression of TSP50 decreased the cell sensitivity to alantolactone-induced apoptosis in those cancer cells. Taken together, these results suggest that alantolactone induces mitochondrial-dependent apoptosis at least partially via down-regulation of TSP50 expression.

Japonicone A suppresses growth of Burkitt lymphoma cells through its effect on NF-κB.

PURPOSE: NF-κB, a transcriptional regulator of diverse genes involved in cell survival, proliferation, adhesion, and apoptosis, has been implicated in various malignancies. We discovered a potent natural NF-κB inhibitor, Japonicone A, from the traditional herb Inula japonica Thunb, evaluated its preclinical pharmacology and therapeutic activity, and investigated the underlying mechanisms of action for its antitumor activity. EXPERIMENTAL DESIGN: Various types of cancer and normal cells were exposed to Japonicone A for cytotoxicity screening, followed by determination of cell apoptosis and cell-cycle arrest. Western blotting, immunostaining, and gene reporter assay were used to analyze NF-κB activity. Two xenograft models were used for therapeutic efficacy evaluation. RESULTS: Japonicone A killed cancer cells but had low cytotoxicity to normal cells. Burkitt lymphoma cells were particularly sensitive. Japonicone A inhibited the growth and proliferation of Raji, BJAB, and NAMALWA lymphoma cells and resulted in G2-M phase arrest and apoptosis. Furthermore, exposure of cells to Japonicone A caused inactivation of the TNF-α-TAK1-IKK-NF-κB axis and inhibition of TNF-α-stimulated NF-κB activity and nuclear translocation, followed by downregulation of NF-κB target genes involved in cell apoptosis (Bcl-2, Bcl-xL, XIAP, TRAF2) and in the cell cycle and growth (cyclin D, c-Myc). Moreover, Japonicone A inhibited local growth and dissemination of cancer cells to multiple organs in vivo. CONCLUSION: Japonicone A exerts significant anticancer effects on Burkitt lymphoma cells in vitro and in vivo through targeting of the NF-κB signaling cascade. These results highlight the potential of Japonicone A as a chemotherapeutic agent and warrant its development as a therapy for lymphomas.