Preliminary investigation of the safe dose of oleandrin when administered orally to Beagles.

OBJECTIVE: The purpose of the study reported herein was to determine the dose of oleander extract and oleandrin (the key pharmacologically active constituent) that could be safely administered PO to dogs. ANIMALS: 42 purebred Beagle dogs were used to study an extract of Nerium oleander. METHODS: 3 studies were performed in 42 purebred young adult (ages 12 months or older) Beagle dogs using a supercritical fluid extract of N oleander leaves. The first study was an 8-day initial dose-ranging study in 2 dogs, a second 7-day repeat-dosing study was performed in 4 dogs, and the final study was performed in 32 dogs where test subjects were given extract or placebo once daily for 28 consecutive days via oral (gavage) administration followed by a 14-day recovery period. RESULTS: At 2.3 µg/kg of oleandrin, there were no observable adverse effects during the duration of the study. Adverse effects were not seen until doses exceeded 6.9 µg/kg of oleandrin, at which time mild, reversible clinical signs were noted. However, a dose > 460 µg of oleandrin/kg was fatal in 1 of 2 dogs in this study. CLINICAL RELEVANCE: The studies reported here, taken in totality, suggest that doses exceeding 6.9 µg/kg of oleandrin may be associated with cardiac abnormalities. An estimated no treatment effective adverse event oral dose of oleandrin appears to be 4.6 µg of oleandrin/kg. Higher doses may be tolerable but should be used with appropriate monitoring.

Antitumour effect of odoroside A and its derivative on human leukaemia cells through the ROS/JNK pathway.

Oleandrigenin-3-O-ß-D-diginoside (a derivative of odoroside A), isolated and purified by our group, has seldom been explored for its pharmacological activity. This study aimed at clarifying the mechanisms towards the leukaemia-suppressive role of odoroside A (compound #1) and its derivative, oleandrigenin-3-O-ß-D-diginoside (compound #2) isolated from Nerium oleander. Viability and nuclear morphology change were assessed by CCK-8 assay and fluorescence microscope, respectively. Then, the cell apoptosis and autophagy induced by the compounds were detected by flow cytometry and Western blot. Xenograft model of nude mice was also applied to measure the leukaemia-suppressive effects of compound #2 in vivo. The result displayed that compound #1 and compound #2 inhibited the proliferation of HL60 and K562 cells and stronger effects were found in HL60 than K562 cells. Both of the compounds induced a dose-dependent apoptosis and autophagy in HL60 cells, where compound #2 was more potent than compound #1. Compound #2 also demonstrated a time-dependent apoptosis and autophagy in HL60 cells. Furthermore, ROS generation and JNK phosphorylation occurred in a dose-dependent manner in the cells treated with compound #2. Mitochondria also played critical role, proved by the decrease of Bcl-2, the release of cyto c to cytosol and the activation of caspase-3 and caspase-9. Moreover, the antitumour effects of compound #2 were validated in the nude mouse xenograft model in vivo. Odoroside A and its derivative inhibited the growth of leukaemia by inducing apoptosis and autophagy through the activation of ROS/JNK pathway. These results suggest that the compounds can serve as potential antitumour agents against leukaemia, especially acute myeloid leukaemia (AML).

Authentication of tejocote (Crataegus mexicana) dietary supplements based on DNA barcoding and chemical profiling.

Tejocote (Crataegus mexicana, Mexican hawthorn), known as a weight-loss supplement, has been marketed online and is easily available for overseas direct purchase. Alipotec (brand name) is known as one of the most popular products containing tejocote in Mexico and other countries. However, adverse effects have been reported by users of these supplements. Therefore it is necessary to find the reason for the side effect. Dietary supplement samples labelled as containing tejocote were analysed using mass spectrometry and DNA barcoding analysis. Our results demonstrate that Alipotec samples contained ingredients from different species, yellow oleander instead of tejocote. The rpoB barcode region was able to differentiate between tejocote and yellow oleander species. Moreover, it was also observed that three compounds, including thevetin B, neriifolin, and digitoxigenin, clearly distinguish between tejocote and yellow oleander samples. This is the first and preliminary investigation to use an integrated approach of both chemical and genomic profiling for the authentication of dietary supplement containing tejocote.

Neuroprotective effects of mitoquinone and oleandrin on Parkinson's disease model in zebrafish.

Aim: The aim of this study is to investigate the possible protective effects of mitoquinone and oleandrin on rotenone induced Parkinson's disease in zebrafish. Materials and methods: Adult zebrafish were exposed to rotenone and mitoquinone for 30 days. Biochemical parameters were determined by spectrophotometric method and Parkinson's disease-related gene expressions were determined by reverse transcription polymerase chain reaction method. Measurement of neurotransmitters was performed by liquid chromatography tandem-mass spectrometry instrument. The accumulation of synuclein was demonstrated by immunohistochemical staining. In vitro thiazolyl blue tetrazolium bromide method was applied to determine the mitochondrial function of synaptosomal brain fractions using rotenone as a neurotoxic agent and mitoquinone and oleandrin as neuroprotective agents. Results: Mitoquinone improved the oxidant-antioxidant balance and neurotransmitter levels that were disrupted by rotenone. Mitoquinone also ameliorated the expressions of Parkinson's disease-related gene expressions that were disrupted by rotenone. According to thiazolyl blue tetrazolium bromide assay results, mitoquinone and oleandrin increased mitochondrial function which was decreased due to rotenone exposure. Conclusion: Based on the results of our study, positive effects of mitoquinone were observed in Parkinson's disease model induced by rotenone in zebrafish.

Oleandrin synergizes with cisplatin in human osteosarcoma cells by enhancing cell apoptosis through activation of the p38 MAPK signaling pathway.

PURPOSE: Our previous studies have reported the antitumor effect of oleandrin on osteosarcoma; however, its chemosensitizing effect in osteosarcoma treatment is still unknown. Therefore, we explored the sensitizing effects of oleandrin to cisplatin in osteosarcoma and investigated the potential mechanisms. METHODS: After exposure to oleandrin and/or cisplatin, CCK-8 and colony formation assays, DAPI staining and flow cytometry were performed to detect cell proliferation and apoptosis in 143B, U-2OS and MG-63 osteosarcoma cells. The median-effect analysis was applied to evaluate the combined effect. Western blot was used to determine the expression of related proteins. Osteosarcoma xenografts and histological observations were applied to confirm the combined effect in vivo. RESULTS: Compared with cisplatin or oleandrin alone, the combined treatment significantly inhibited cell proliferation and induced cell apoptosis. The median-effect analysis indicated a synergistic cytotoxic effect. The combined treatment downregulated Bcl-2 and upregulated Bax and cleaved caspase-3, -8 and -9. And the suppression of caspases reduced cell death. Furthermore, oleandrin alone or with cisplatin, activated the p38 MAPK/Elk-1 pathway. The inhibition of the p38 MAPK pathway increased cell viability and reduced apoptosis. In vivo, the combined treatment was also verified to significantly inhibit tumor growth, induce apoptosis and activate the p38 MAPK pathway. CONCLUSIONS: The combination of oleandrin with cisplatin exerts a synergistic antitumor effect in osteosarcoma, which relates to the activation of the p38 MAPK pathway.

3'-epi-12ß-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells.

Rationale: Cardenolides have potential as anticancer drugs. 3'-epi-12ß-hydroxyfroside (HyFS) is a new cardenolide structure isolated by our research group, but its molecular mechanisms remain poorly understood. This study investigates the relationship between its antitumor activities and autophagy in lung cancer cells. Methods: Cell growth and proliferation were detected by MTT, lactate dehydrogenase (LDH) release, 5-ethynyl-20-deoxyuridine (EDU) and colony formation assays. Cell apoptosis was detected by flow cytometry. Autophagic and signal proteins were detected by Western blotting. Markers of autophagy and autophagy flux were also detected by immunofluorescence, transmission electron microscopy and acridine orange staining. Real time RT-PCR was used to analyze the gene expression of Hsp90. Hsp90 ubiquitination was detected by coimmunoprecipitation. The antitumore activities of HyFS were observed in nude mice. Results: HyFS treatment inhibited cell proliferation and induced autophagy in A549 and H460 lung cancer cells, but stronger inhibition of cell proliferation and induction of cell apoptosis were shown when HyFS-mediated autophagy was blocked. The Hsp90/Akt/mTOR axis was found to be involved in the activation of HyFS-mediated autophagy. Evidence of direct interaction between Hsp90 and Akt was observed. HyFS treatment resulted in decreased levels of heat shock protein 90 (Hsp90) and phosphorylated Akt, overexpression of Hsp90 increased activation of autophagy, and inhibition of Hsp90 expression decreased autophagy. In addition, ubiquitin-mediated degradation of Hsp90 and subsequent dephosphorylation of its client protein Akt were also found in HyFS-treated lung cancer cells. Moreover, combination treatment with HyFS and chloroquine showed remarkably increased tumor inhibition in both A549- and H460-bearing mice. Conclusion: Our results demonstrate that HyFS induced cytoprotective autophagy through ubiquitin-mediated degradation of Hsp90, which further blocked the Akt/mTOR pathway in lung cancer cells. Thus, a combination of a HyFS-like cardenolide and an autophagic inhibitor is a potential alternative approach for the treatment of lung cancer.

The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway.

PURPOSE: Evidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer. However, the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown. The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells. METHODS: The cytotoxicity and IC50 of five small molecule compounds (oleandrin, neriifolin, strophanthidin, gitoxigenin, and convallatoxin) in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays, respectively. Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide, followed by flow cytometry. Intracellular Ca2+ was determined using Fluo-3 AM,glutathione (GSH) levels were measured using a GSH detection kit,and the activity of caspase-3, -9 was measured using a peptide substrate. BAX, pro-caspase-3, -9, cytochrome C and BCL-2 expression were determined by Western blotting. RESULTS: Oleandrin significantly decreased cell viabilities in SW480, HCT116 and RKO cells. The IC50 for SW480 cells was 0.02 µM, whereas for NCM460 cells 0.56 µM. More interestingly, the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells. Oleandrin downregulated protein expression of pro-caspase-3, -9, but enhanced caspase-3, -9 activities. These effects were accompanied by upregulation of protein expression of cytochrome C and BAX, and downregulation of BCL-2 protein expression in a concentration-dependent manner. Furthermore, oleandrin increased intracellular Ca2+ concentration, but decreased GSH concentration in the cells. CONCLUSIONS: The present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway. Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.

Strophalloside induces apoptosis of SGC-7901 cells through the mitochondrion-dependent caspase-3 pathway.

Cardenolides with special chemical structures have been considered as effective anti-cancer drugs in clinic trials. Strophalloside is a cardenolide we recently isolated from Antiaris toxicaria obtained from Hainan, China. The aim of this study was to investigate the possible anticancer effects induced by strophalloside and the underlying molecular mechanism. Gastric carcinoma SGC-7901 cells were treated with strophalloside at various concentrations for different times, and resulting cell viability was determined by the MTT assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Apoptosis were measured by Annexin V-FITC/PI and Hoechst staining. The changes of mitochondrial transmembrane potential were examined by a JC-1 kit. The expressions of pro-apoptotic protein cytochrome c, caspase-3 and caspase-9 were detected by western blotting analysis. The results showed that strophalloside was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion in a time- and dose-dependent manner. Mitochondrial membrane potential declined and the concentration of cytochrome c increased in cytoplasm and caspase-3 and caspase-9 were cleaved into activated states, suggesting that cytochrome c was released from the mitochondrion to cytoplasm and finally activated the caspase-dependent apoptosis pathway. Our results indicate that strophalloside is a potential anticancer drug.

Kaempferol glycosides and cardenolide glycosides, cytotoxic constituents from the seeds of Draba nemorosa (Brassicaceae).

Bioassay-directed fractionation of a methanolic extract from the seeds of Draba nemorosa (Brassicaceae) led to isolation of a new flavonol glycoside, drabanemoroside (5, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranose) along with four known flavonoid derivatives (1-4), four cardenolide glycosides (6-9). Kaempferol glycosides 2 and 5 showed strong cytotoxicity against human small lung cancer cell line A549 and melanoma SK-Mel-2 with an IC(50) of 0.5 microg/mL and 1.9 microg/mL, respectively. Cardenolide glycosides 6-9 showed potent cytotoxicity (A549) in the range of 0.01-0.032 microg/mL. Their structures were characterized based on spectroscopic data (2D NMR, HRTOFMS, IR, and UV) and comparison of literature values. The carbohydrate units were also confirmed by comparing the hydrolysate of 5 with authentic monosaccharides.

Ouabain, a circulating hormone secreted by the adrenals, is pivotal in cardiovascular disease. Fact or fantasy?

Substantial evidence points to the presence in human plasma of an inhibitor of the sodium/potassium pump which plays a central role in the pathophysiology of circulatory disorders, including essential hypertension. Studies from the 1980/90s claimed that this inhibitor was identical or very similar in structure to plant-derived ouabain and was synthesized by the adrenal cortex. However, the physical evidence in studies reporting isolation and identification of ouabain in human plasma appears insecure on closer examination. Additionally, reported circulating levels of immunoreactive ouabain in humans vary greatly, the ability of the human adrenal glands to secrete ouabain is questionable and the original commercial assay for measuring immunoreactive ouabain is no longer available. We submit that the position of ouabain as an endogenous, adrenally produced regulator of the sodium pump is of such importance that the current evidence needs either to put on a more secure footing or to lose its current status.

Mimicking human bipolar ion dysregulation models mania in rats.

Psychiatric diseases in general, and bipolar illness in particular, are difficult to model in animals since the subjective nature of the core symptoms appears to preclude objective observation of behavioral changes. An adequate animal model of a psychiatric condition must fulfill three core criteria: share pathophysiological characteristics of the human condition (face validity), have similar behavioral manifestations as the human disease (construct validity), and improve with medications that improve the symptoms seen in afflicted humans (predictive validity). The ouabain model for bipolar illness mimics a widely reproduced biologic abnormality in mania: reduced sodium pump activity. An intracerebroventricular (ICV) administration of 5microL 10(-3)M ouabain induces motoric hyperactivity preventable by lithium, carbamazepine, and haloperidol. ICV ouabain may also produce environmentally dependent hypoactivity. The model, however, has not yet been examined for other potential manic behavior in rats such as reduced need for sleep, increased sexual activity, or increased irritability. While additional characterization of the model is required, the ouabain model for bipolar illness is the only available animal model that fulfills the three criteria for an adequate animal model for bipolar illness.

[Effect of griffithin on anticancer activity and apoptosis of cancer cells in vitro].

To study the anticancer activity of griffithin from Streptocaulon griffithii Hook. f. and its effect on apoptosis of cancer cells in vitro, the inhibitory effect of griffithin on cell proliferation was studied by MTT assay, the cell apoptosis was observed by AO/EB double decoration assay and flow cytometry. Griffithin exhibited high anticancer activity on four human cancer cell lines, with IC50 ranged from 0.17 - 0.43 microg x mL(-1). Griffithin also induced apoptosis of PC-3 cells. Griffithin had anticancer activity and induced apoptosis of cancer cells.

Antiproliferative cardenolides from Periploca graeca.

Nine cardiotonic steroids, six 17beta-cardenolides (2, 4, 6-9) and three 17alpha-cardenolides (1, 3, 5) have been identified from the chloroform and chloroform-methanol extracts of Periploca graeca L. (Asclepiadaceae) stems. Among these, compound 5, the 17alpha-isomer of periplocin 6, was identified as a new compound. The antiproliferative effects of these compounds were tested in vitro in the hormone-independent prostate cancer cell line PC-3. Five of these compounds, all 17beta-isomers with a 14beta-OH group and at least one sugar molecule (4, 6-9), showed a very strong antiproliferative effect, with IC50 values between 18 and 50 nM. Compound 2, the only 17beta-cardenolide aglycone, had an IC50 value of 0.6 microM, which is 13 to 16 times higher than the values found for the corresponding cardenolides with one or two sugars. The IC50 values found for the three 17alpha-isomers were significantly higher (5.4 and 7.3 microM), with IC50 ratios (17alpha-cardenolide/17beta-cardenolide) of up to 192. In the 17alpha-cardenolide series, the presence of a sugar unit does not seem to have a significant effect on the IC50 values. This is the first report showing a markedly different cytotoxicities between the 17alpha- and 17beta-isomers in the cardenolide series.

Novel digitalis-like factor, marinobufotoxin, isolated from cultured Y-1 cells, and its hypertensive effect in rats.

Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 microg/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufagenin-like immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135+/-3.0 versus 126+/-2.0 mm Hg and 1.41+/-0.286 versus 0.34+/-0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.

5alpha-cardenolides from Kanahia laniflora inhibit ionotropic acetylcholine receptors.

5alpha-cardenolides isolated from Kanahia laniflora are inhibitors of muscle-type nicotinic acetylcholine receptors expressed in TE671 cells with IC (50) values in the range of 27 - 60 microM, as determined by whole-cell patch-clamp electrophysiological experiments.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

A rare cause of complete heart block after transdermal botanical treatment for psoriasis.

We report the case of a 59-year-old man with a new 3 degrees AV block with a history of psoriasis. After implantation of a definitive DDDR pacemaker, the patient reported a transdermal self-medication with an extract of Nerium oleander for the treatment of his psoriasis. The pharmacological, epidemiological, and clinical features are discussed in brief.

Anti-tumour activity of Digitalis purpurea L. subsp. heywoodii.

Recent research has shown the anticancer effects of digitalis compounds suggesting their possible use in medical oncology. Four extracts obtained from the leaves of Digitalis purpurea subsp. heywoodii have been assessed for cytotoxic activity against three human cancer cell lines, using the SRB assay. All of them showed high cytotoxicity, producing IC50 values in the 0.78 - 15 microg/mL range with the methanolic extract being the most active, in non toxic concentrations. Steroid glycosides (gitoxigenin derivatives) were detected in this methanolic extract. Gitoxigenin and gitoxin were evaluated in the SRB assay using the three human cancer cell lines, showing IC50 values in the 0.13 - 2.8 microM range, with the renal adenocarcinoma cancer cell line (TK-10) being the most sensitive one. Morphological apoptosis evaluation of the methanolic extract and both compounds on the TK-10 cell line showed that their cytotoxicity was mediated by an apoptotic effect. Finally, possible mechanisms involved in apoptosis induction by digitalis compounds are discussed.