Mundulea Sericea Research Articles

PI3K/AKT Activity and Functional Relevance in Deguelin Induced Apoptosis in MDS Cell Line.

Abstract 4977 IntroductionPI3K/AKT signalling pathway is involved in cell growth, proliferation and apoptosis. PI3K/AKT constitutive activation is observed in several solid tumors and leukemic cells. Inhibition of PI3K/AKT activity using specific inhibitor, LY294002, results in apoptosis. Deguelin is a natural product isolated from the leguminous, Mundulea sericea, with antitumorigenic effect in vitro and in vivo. The inhibition effect of Deguelin in PI3K/AKT signalling pathway in leukaemia cells was also observed in vitro. AimsWe evaluated PI3K/AKT activation using p-AKT expression by flow cytometry in P39 cell line and CD34+ hematopoietic progenitors. PI3K/AKT activity inhibition mediated by deguelin and its proapoptotic effect was tested in P39 cell line. Material and MethodsThe high-risk MDS cell line P39 and leukaemia cell lines, Jurkat and HL60, were maintained in RPMI1640 and FBS 10%. Jurkat and HL60 were used as positive and negative control for p-AKT expression, respectively. Cell lines and CD34+ cells from bone marrow healthy donors (n=5) were assessed for p-AKT expression. Flow cytometry detection of intracellular Ser 473 p-AKT was performed after permeabilization and fixation with a saponin based solution with Tween20 and FACSlysing solution (Becton Dickinson-BD). An alexa-fluor 488-conjugated rabbit antibody to Ser 473 p-AKT (Cell Signalling Technology) was employed. A double immunostaining procedure using CD45-PerCP and CD34+PE was performed to analyse p-AKT expression in CD34+ cells from bone marrow healthy donors. After incubation, cells were analysed on a FACSCalibur (BD). The p-AKT activity was determined using Kolmogorov-Smirnov test (D). Difference between groups was analyzed using the Mann Whitney test. For treatment and determination of apoptosis, cells were exposed to deguelin (at concentrations of 10-300nM) and LY294002 (15-50uM) for 24-48h. To determine apoptotic changes, cells were stained withy Annexin-V/FITC and propidium iodide and examined on a FACScalibur (BD). ResultsJurkat cells showed constitutive PI3K/AKT activation with a mean D value for p-AKT expression of D=0,84 ± 0,02. P39 cells also showed a constitutive PI3K/AKT activation with p-AKT expression as high as in Jurkat cells (mean D= 0,76±0,07). P-AKT expression was significant lower in CD34+cells from health donors (D=0,38±0,06) than in Jurkat and P39, respectively (p=0,0043 and p=0,015). Inhibition of p-AKT was observed using different concentrations of LY294002 (15-50uM) in Jurkat cells and apoptosis was observed after 24 and 48h when cells were treated with 50uM. For P39, although apoptosis was observed, no p-AKT inhibition was detected after LY294002 treatment. Treatment with deguelin induced apoptosis in Jurkat via p-AKT inhibition. No effect on apoptosis or p-AKT expression was observed in P39 cell line after deguelin treatment. ConclusionsPI3/AKT constitutive activation was observed in P39 cell line and suggests that PI3/AKT can play a role in MDS progression. Deguelin effect in PI3K/AKT pathway can be cell specific, as observed in Jurkat but not in P39 cell line. Thus, deguelin in combination with other agents should be tested as a potential therapeutic option for MDS via PI3K/AKT inhibition. DisclosuresNo relevant conflicts of interest to declare.

Deguelin Induces Cell Growth Arrest and Cell Death Via the Suppression of BCR-ABL and Survivin Expression in BCR-ABL-T315I Mutant Cells.

Abstract 4255Development of kinase domain mutation is a major drug-resistance mechanism for tyrosine kinase inhibitor (TKI) in cancer therapy. A particularly challenging example is found in Philadelphia chromosome-positive chronic myelogenous leukemia (CML) where TKI STI571 is ineffective against the BCR-ABL mutant, E255K or T315I. Therefore, new agents that effectively kill these cells with kinase domain mutation are absolutely necessary. Deguelin is a rotenoid from African plant Mundulea sericea, which has been recently shown to suppress the lung tumorigenesis via inhibiting the activation of PI3kinase-Akt signaling. More recently, deguelin has been shown to bind to ATP-binding pocket of heat shock protein (Hsp) 90 and inhibit functions of Hsp90 client proteins. However, the effects of deguelin on BCR-ABL mutant transformed cells are poorly understood. We investigated the effects of deguelin on the cell growth and cell death of BaF/3 pre-B-cells transformed with BCR-ABL-wild type (WT), BCR-ABL-E255K and BCR-ABL-T315I (kindly provided by Dr. Kimura, Saga University, Saga, Japan), by trypan-blue exclusion and Annexin V stain. Deguelin inhibited the proliferation of these transformed cells in a time-and a dose-dependent manner (IC50=1.0mM). In addition, flow cytometric analysis revealed a higher frequency of Annexin V(+) cells in deguelin-treated cells (mean ± SD : WT 80.1±15.3%, E255K 82.3±3.5%, T315I 94.0±0.4%, P< 0.05, respectively), compared to that in untreated cells (WT 11.5±1.6%, E255K 11.9±2.0%, T315I 15.3±4.8%, respectively). These results indicate that deguelin induces the cell-growth arrest and cell death in these cells. To better understand the mechanism of these effects, we examined the intracellular signaling and the expression of apoptosis associated proteins by immunoblot analysis. We found that deguelin suppressed BCR-ABL protein expression and prevented CrkL phosphorylation in these cells. In addition, the inhibitor of apoptosis protein, survivin expression was decreased at 24 hours after deguelin treatment. These results suggested that deguelin suppresses BCR-ABL and survivin expression, and inhibits CrkL phosphorylation in BCR-ABL mutant and WT cells. Since deguelin has been shown to inhibit the function of Hsp90 client proteins, deguelin may destabilize BCR-ABL and survivin by inhibiting Hsp90 function. Taken together, deguelin appears to induce cell growth arrest and apoptosis of BCR-ABL mutant and WT transformed cells via the suppression of BCR-ABL and survivin expression. Thus, deguelin merits further investigation as a potential therapeutic agent for CML with T315I ABL mutation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Deguelin Induces Cell Growth Arrest and Cell Death by Destabilizing Phosphorylated STAT3 and Survivin in Adult T-Cell Leukemia Cells.

Abstract 4784Adult T-cell leukemia (ATL) cells are remarkably resistant to conventional chemotherapy. Therefore, new agents that effectively kill these cells are needed. Deguelin is a rotenoid from African plant Mundulea sericea which has been recently shown to suppress the lung tumorigenesis via inhibiting the activation of PI3kinase-Akt signaling. More recently, deguelin has been shown to bind to ATP-binding pocket of heat shock protein (Hsp) 90 and inhibit functions of Hsp90 client proteins. However, the effects of deguelin on ATL cells are poorly understood. To investigate the effects, we examined the cell growth and cell death of human T-cell leukemia virus type 1 (HTLV-1)- transformed cells, KUT-1 and MT-2 (kindly provided by Dr. Hanada, Kagoshima Medical Center, Kagoshima and Dr. Harashima, Hayashibara Institute Inc., Okayama, Japan, respectively) by trypan-blue exclusion and Annexin V/PI stain. Deguelin inhibited the proliferation of KUT-1 and MT-2 cells in a time- and dose-dependent manner (IC50=0.5μM). In addition, flow cytometric analysis revealed a higher frequency of Annexin V(+) cells in deguelin-treated cells compared to that in untreated cells (KUT-1 P< 0.01, MT-2 P< 0.01, respectively). These results indicate that deguelin induces the cell growth arrest and cell death of ATL cells. To better understand the mechanism of these effects, we examined the intracellular signaling and the expression of apoptosis associated proteins by immunoblot analysis. We found that deguelin prevented the tyrosine phosphorylation of the transcription factor signal transducer and activator of transcription (STAT) 3, which is constitutively activated in these cells. In addition, the inhibitor of apoptosis protein, survivin expression was decreased 24 hours after deguelin treatment, indicating that deguelin induces the cell growth arrest and apoptosis via at least in part the inhibition of STAT3 phosphorylation and the down-regulation of survivin expression. To determine the mechanism of inhibition of STAT3 phosphorylation and survivin expression, MT-2 cells were treated with proteasome inhibitor MG132 and/or deguelin. MG132 restored STAT3 dephosphorylation and down-regulation of survivin expression by deguelin. In addition, quantitative RT-PCR assay showed that deguelin treatment did not alter survivin gene expression. These results suggested that deguelin induces the degradation of phosphorylated STAT3 and survivin via the ubiquitination/proteasome pathway in ATL cells. Since deguelin has been shown to inhibit the function of Hsp90 client proteins, deguelin may destabilize phosphorylated STAT3 and survivin by inhibiting Hsp90 function. Taken together, deguelin appears to induce cell growth arrest and apoptosis of ATL cells via the suppression of the tyrosine phosphorylation of STAT3 and down-regulation of survivin. Thus, deguelin merits further investigation as a potential therapeutic agent for this incurable disease. Disclosures:No relevant conflicts of interest to declare.

Deguelin, a Novel Hsp90 Inhibitor, Suppresses STAT3 and STAT5 Activity and Induces Apoptosis in the Human T-Cell Leukemia Virus Type 1-Transformed Cells.

Adult T-cell leukemia (ATL) cells are remarkably resistant to conventional chemotherapy. Therefore, new agents that effectively kill these cells are needed. Deguelin is a rotenoid from African plant Mundulea sericea which has been recently shown to suppress the lung tumorigenesis via inhibiting the activation of PI3kinase-Akt signaling. More recently, deguelin has been shown to bind to ATP-binding pocket of heat shock protein (Hsp) 90 and inhibit functions of Hsp90 client proteins. However, the effect of deguelin on ATL cells is not known. We investigated the effects of deguelin on the cell growth and cell death of human T-cell leukemia virus type 1 (HTLV-1)- transformed cells, KUT-1 and MT-2 (kindly provided by Dr. Hanada, Kagoshima Medical Center, Kagoshima and Dr. Kibata, Hayashibara Biochemical Laboratory Inc., Okayama, Japan, respectively) by trypan-blue exclusion and MTT assays, and annexin V/PI staining. Deguelin inhibited the proliferation of KUT-1 and MT-2 cells in a time-and a dose-dependent manner and had much less cytotoxic effects on normal human peripheral blood mononuclear cells. In addition, flow cytometric analysis revealed a higher frequency of annexin V-positive cells in deguelin treated cells compared to that in untreated cells. These results indicate that deguelin induces the cell-growth arrest and cell death in ATL cells. To better understand the mechanism of these effects, we examined the intracellular signaling and the expression of apoptosis associated proteins by immunoblot analysis. We found that deguelin prevented the tyrosine phosphorylation of the transcription factor signal transducer and activator of transcription (STAT) 3 and STAT5, both of which are constitutively activated in these cells. In addition, the inhibitor of apoptosis protein, XIAP expression decreased at 24 hours after deguelin treatment, indicating that deguelin induces the cell growth arrest and apoptosis via at least in part the inhibition of STAT3 and STAT5 phosphorylation and the downregulation of XIAP expression. We next examined whether deguelin suppresses janus kinase (JAK) 3 phosphorylation in KUT-1 cells using immunoprecipitation assay. However, deguelin treatment did not inhibit JAK3 phosphorylation, suggesting that the inhibition of STAT5 phosphorylation by deguelin is independent of JAK3 in KUT-1 cells. Since deguelin has been shown to inhibit the function of Hsp90 client proteins, we examined the protein expression of cyclin dependent kinase (Cdk) 4, which is known as one of the Hsp90 client proteins. Deguelin treatment reduced Cdk4 expression in both cells, suggesting that deguelin may destabilize Cdk4 via inhibiting the Hsp90 function. Taken together, deguelin appears to inhibit the proliferation and induce apoptosis of ATL cells via preventing the tyrosine phosphorylation of STAT proteins and destabilizing Cdk4. Thus, deguelin merits further investigation as a potential therapeutic agent for this incurable disease.

Antiangiogenic Effect of Deguelin on Choroidal Neovascularization

Age-related macular degeneration is the leading cause of blindness in the elderly. Choroidal neovascularization (CNV) leads to severe vision loss in patients of age-related macular degeneration. Previously, we have demonstrated that deguelin, isolated from plants in the Mundulea sericea family, is a chemopreventive agent. This study evaluates the antiangiogenic effect of deguelin on CNV. The toxicity of deguelin was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human umbilical vein endothelial cells (HUVECs) as well as histological examination and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining in the deguelin-injected retina. Antiangiogenic activity of deguelin was evaluated by in vitro tube formation assay of HUVECs and in vivo angiogenesis of chick chorioallantoic membrane (CAM). In C57BL/6 mice with laser-induced CNV, deguelin or phosphate-buffered saline was injected intravitreously. CNV lesions were examined by fluorescence angiography and vessel counting in cross-sections. Deguelin showed no effect on cell viability of HUVECs and no retinal toxicity in a concentration range of 0.01 to 1 microM. Deguelin effectively inhibited in vitro tube formation of HUVECs and in vivo angiogenesis of CAM. Interestingly, deguelin significantly reduced CNV and its leakage in mouse model of laser photocoagulation-induced CNV. Our data suggests that deguelin is a potent inhibitor of CNV and may be applied in the treatment of other vasoproliferative retinopathies such as retinopathy of prematurity and diabetic retinopathy.

Deguelin, an Akt Inhibitor, Suppresses IκBα Kinase Activation Leading to Suppression of NF-κB-Regulated Gene Expression, Potentiation of Apoptosis, and Inhibition of Cellular Invasion

Deguelin, a constituent of the bark of the African plant Mundulea sericea (Leguminosae), exhibits antiproliferative and anticarcinogenic activities through a mechanism that is not well understood. Because various steps in carcinogenesis are regulated by NF-kappaB, we postulated that the activity of deguelin is mediated through this transcription factor. We found that deguelin suppressed NF-kappaB activation induced by carcinogens, tumor promoters, growth factors, and inflammatory stimuli. This suppression was not cell-type specific, because NF-kappaB activation was suppressed in both lymphoid and epithelial cells. Moreover, constitutive NF-kappaB activation was also blocked by deguelin. The suppression of TNF-induced NF-kappaB activation by deguelin occurred through the inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Deguelin also suppressed the NF-kappaB reporter activity induced by TNFR1, TNFR-associated death domain, TNFR-associated factor 2, and IkappaBalpha kinase, but not that induced by p65. The inhibition of NF-kappaB activation thereby led to the down-regulation of gene products involved in cell survival, proliferation, and invasion. Suppression of these gene products by deguelin enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results demonstrate that deguelin inhibits the NF-kappaB activation pathway, which may explain its role in the suppression of carcinogenesis and cellular proliferation.

Molecular mechanisms of deguelin-induced apoptosis in transformed human bronchial epithelial cells

Increasing evidence has demonstrated that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway plays an important role in cell proliferation, apoptosis, angiogenesis, adhesion, invasion, and migration, functions that are critical to cancer cell survival and metastasis. Increased expression of activated Akt has been observed in the early stages of tobacco-induced lung carcinogenesis. Moreover, blocking the PI3K/Akt pathway specifically inhibits the proliferation of non-small cell lung cancer (NSCLC) cells, indicating that the PI3K/Akt pathway is a potential target for chemoprevention and therapy in lung cancer. The aim of this work is to study the lung cancer chemopreventive potential of PI3K/Akt inhibitors using an in vitro lung carcinogenesis model. We found that genetic or pharmacologic approaches targeting the PI3K/Akt pathway inhibited the proliferation of premalignant and malignant human bronchial epithelial (HBE) cells. After screening several natural products to identify a potential lung cancer chemopreventive agent, we have found that deguelin, a rotenoid isolated from Mundulea sericea (Leguminosae), specifically inhibits the growth of transformed HBE and NSCLC cells by inducing cell-cycle arrest in the G2/M phase and apoptosis, with no detectable toxic effects on normal HBE cells, most likely due to the agent’s ability to inhibit PI3K/Akt-mediated signaling pathways. The specific sensitivity of premalignant and malignant HBE and NSCLC cells to deguelin suggests that this drug could be clinically useful for chemoprevention in early-stage lung carcinogenesis and for therapy in confirmed lung cancer.

Plants used for poison fishing in tropical Africa

Fishing with the aid of poisonous plants was formerly very common in Africa. Today this easy and simple method of fishing is forbidden but still practised in remote areas. The poisonous ingredients are pounded and thrown into a pool or dammed sections of a small river. After a time which varies according to conditions the fish begin to rise to the surface of the water and can readily be taken by hand. In general, the fish can be eaten without problems. 325 fish-poisoning plants, spread among 71 plant families with 183 genera, are presented. The closely related groups of Caesalpiniaceae, Mimosaceae and Papilionaceae clearly dominate. It is also remarkable that a great proportion are Euphorbiaceae. The plants most used are Tephrosia vogelii, Mundulea sericea, Euphorbia tirucalli, Gnidia kraussiana, Adenia lobat, Balanites aegyptiaca, Swartzia madagascariensis, Neoratanenia mitis, Tetrapleura tetraptera and Strychnos aculeata. Many fishing poisons play an important part in the preparation of arrow poisons and in traditional medicine.

Inhibitory effect of munetone, an isoflavonoid, on 12- O-tetradecanoylphorbol 13-acetate-induced ornithine decarboxylase activity

Starting with an extract derived from the bark of Mundulea sericea Willd. (Leguminosae) that was active in the process of inhibiting 12- O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase activity (ODC) in cultured mouse epidermal ME 308 cells, the isoflavonoid munetone was isolated and identified as an active principle (IC 50 = 46 ng/ml). Topical application of munetone (0.04–5 μmol) to the skin of CD-1 mice 2 h prior to treatment with TPA (10 nmol) resulted in dose-dependent inhibition of epidermal ODC activity. In addition, munetone inhibited TPA-independent c-Myc-induced ODC activity with cultured BALB/c c-MycER cells, as well as 7,12-dimethylbenz[ a]anthracene (DMBA)-induced preneoplastic lesion formation in a mouse mammary gland organ culture (MMOC) system. These data suggest the potential of munetone to serve as a cancer chemopreventive agent by virtue of blocking the process of tumor promotion.

Rotenoids mediate potent cancer chemopreventive activity through transcriptional regulation of ornithine decarboxylase.

For the discovery of new cancer chemopreventive agents, we have studied the potential of plant extracts to inhibit phorbol ester-induced ornithine decarboxylase (ODC) activity in cell culture. Four active rotenoids were obtained from the African plant Mundulea sericea (Leguminosae). These isolates were highly potent when evaluated for inhibition of chemically induced preneoplastic lesions in mammary organ culture and inhibition of papillomas in the two-stage mouse skin model, and they appear to function by a unique mechanism at the level of ODC messenger RNA expression. Based on our findings, rotenoids can be regarded as promising new chemopreventive or anticancer agents.

Rotenoids and chalcones from Mundulea sericea that inhibit phorbol ester-induced ornithine decarboxylase activity

Two novel rotenoids, (−)-13α-hydroxydeguelin and (−)-13α-hydroxytephrosin, and a new chalcone, munsericin, were isolated from the bark of Mundulea sericea, and their structures were elucidated by spectroscopic methods. Also obtained were the parent rotenoids, deguelin and tephrosin, and the known chalcone, 4-hydroxy-lonchocarpin. The rotenoids and chalcones exhibited potent inhibitory activity against phorbol ester-induced ornithine decarboxylase activity in cultured mouse 308 epidermal cells.

2-Aminoimidazole in seeds of Mundulea sericea

An amine present as ca 0.9 % of the fr. wt of the seeds of the legume Mundulea sericea has been isolated and characterised as 2-aminoimidazole