Triterpenoids from Desfontainia spinosa

The leaves and stem of Desfontainia spinosa were found to contain eleven urs-12-ene triterpene compounds. The major compound present was ursolic acid. Four other known compounds were isolated, i.e. ursolic acid acetate, pomolic acid, 23-hydroxytormentic acid and nigaichigoside Fl, its ester glucoside. The other six compounds are novel and have been identified as 24-hydroxytormentic acid, 7α-hydroxytormentic acid, 7α,23-dihydroxytormentic acid and their corresponding ester glucosides. It is unlikely that these compounds contribute to the reputed narcotic properties of this plant.     

Fluorescent labeling of ursolic acid with FITC for investigation of its cytotoxic activity using confocal microscopy

Fluorescent labeling is a widely-used approach in the study of intracellular processes. This method is becoming increasingly popular for studying small bioactive molecules of natural origin; it allows us to estimate the vital intracellular changes which occur under their influence. We propose a new approach for visualization of the intracellular distribution of triterpene acids, based on fluorescent labeling by fluoresceine isothiocyanate. As a model compound we took the most widely-used and best-studied acid in the ursane series – ursolic acid, as this enabled us to compare the results obtained during our research with the available data, in order to evaluate the validity of the proposed method. Experimental tracing of the dynamics of penetration and distribution of the labeled ursolic acid has shown that when the acid enters the cell, it initially localizes on the inner membranes where the predicted target Akt1/protein kinase B – a protein that inhibits apoptosis – is located.     

Antibacterial activity of triterpene acids and semi-synthetic derivatives against oral pathogens

Triterpene acids (ursolic, oleanoic, gypsogenic, and sumaresinolic acids) isolated from Miconia species, along with a mixture of ursolic and oleanolic acids and a mixture of maslinic and 2-a-hydroxyursolic acids, as well as ursolic acid derivatives were evaluated against the following microorganisms: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus sobrinus, and Enterococcus faecalis, which are potentially responsible for the formation of dental caries in humans. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) during the evaluation of the antibacterial activity. All the isolated compounds, mixtures, and semi-synthetic derivatives displayed activity against all the tested bacteria, showing that they are promising antiplaque and anticaries agents. Ursolic and oleanolic acids displayed the most intense antibacterial effect, with MIC values ranging from 30 microg/mL to 80 microg/mL. The MIC values of ursolic acid derivatives, as well as those obtained for the mixture of ursolic and oleanolic acids showed that these compounds do not have higher antibacterial activity when compared with the activity observed with either ursolic acid or oleanolic acid alone. With regard to the structure-activity relationship of triterpene acids and derivatives, it is suggested that both hydroxy and carboxy groups present in the triterpenes are important for their antibacterial activity against oral pathogens.     

Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/Akt signaling pathways

Celastrol, a plant triterpene has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. In the present report, we investigated the effect of celastrol on proliferation of various cancer cell lines. The mechanism, by which this triterpene exerts its apoptotic effects, was also examined in detail. We found that celastrol inhibited the proliferation of wide variety of human tumor cell types including multiple myeloma, hepatocellular carcinoma, gastric cancer, prostate cancer, renal cell carcinoma, head and neck carcinoma, non-small cell lung carcinoma, melanoma, glioma, and breast cancer with concentrations as low as 1 μM. Growth inhibitory effects of celastrol correlated with a decrease in the levels of cyclin D1 and cyclin E, but concomitant increase in the levels of p21 and p27. The apoptosis induced by celastrol was indicated by the activation of caspase-8, bid cleavage, caspase-9 activation, caspase-3 activation, PARP cleavage and through the down regulation of anti-apoptototic proteins. The apoptotic effects of celastrol were preceded by activation of JNK and down-regulation of Akt activation. JNK was needed for celastrol-induced apoptosis, and inhibition of JNK by pharmacological inhibitor abolished the apoptotic effects. Overall, our results indicate that celastrol can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and down-regulation of anti-apoptotic protein expression.     

Isolation of pomolic acid from Chamaenerion angustifolium and the evaluation of its potential genotoxicity in bacterial test systems

The lipophilic components of the medicinal plant, the fireweed (Chamaenerion angustifolium), have been examined using the raw materials of a three-year harvest. Twenty eight aliphatic and six triterpene acids have been detected for the first time by chromatography—mass spectrometry analysis. It has been shown using the Ames test and the SOS chromotest that pomolic acid possesses no genotoxic and mutagenic properties.     

The inhibition of BDNF/TrkB/PI3K/Akt signal mediated by AG1601 promotes apoptosis in malignant glioma

Malignant glioma is the most aggressive primary brain tumor and has a poor survival rate. Even if extensive methods are preformed to treat glioma, the mortality rate is still very high. It is necessary for discovering and developing new drugs for malignant glioma treatment. AG1601 is one of AG-series drugs, including AG1031 and AG1503, and it has been optimized on the original basis. In our study, we found that AG1601 markedly inhibited proliferation and promoted C6 glioma cell apoptosis in vitro. AG1601 also reduced the size and weight of glioma in vivo. The growth ability of glioma was significantly inhibited after treatment with AG1601. It also showed that the expression levels of BDNF/TrkB/PI3K/Akt signal related proteins were obviously decreased in C6 glioma cells after treatment with AG1601 in vivo and in vitro. We also found that BDNF, as the activator of BDNF/TrkB/PI3K/Akt signal, reversed the anti-proliferation and pro-apoptosis of C6 glioma cells caused by AG1601. K252a, a specific inhibitor of TrkB, and AG1601 in combination aggravated C6 glioma cell apoptosis. These results indicate that AG1601 has good effects on the anti-proliferation and pro-apoptosis of malignant glioma via BDNF/TrkB/PI3K/Akt signal and could be considered as a potential drug in treating malignant glioma.     

Triterpene acids from the leaves of Perilla frutescens and their anti-inflammatory and antitumor-promoting effects

Nine triterpene acids, viz., six of the ursane type, ursolic acid (1), corosolic acid (2), 3-epicorosolic acid (3), pomolic acid (4), tormentic acid (5) and hyptadienic acid (6), and three of the oleanane type, oleanolic acid (7), augustic acid (8) and 3-epimaslinic acid (9), among which 1 constituted the most predominant triterpene acid, were isolated and identified from ethanol extracts of the leaves of red perilla [Perilla frutescens (L.) Britton var. acuta Kudo] and green perilla [P. frutescens (L.) Britton var. acuta Kudo forma viridis Makino]. These eight compounds, 1, 2, 4-9, were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg/ear) in mice. All the compounds tested showed a marked anti-inflammatory effect, with a 50% inhibitory dose (ID50) of 0.09-0.3 mg per ear. In addition, an evaluation against the Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA showed five compounds, 1-3, 5 and 9, with a potent inhibitory effect on EBV-EA induction (91-93% inhibition at 1x10(3) mol ratio/TPA). Furthermore, compound 5 exhibited strong antitumor-promoting activity in an in vivo two-stage carcinogenesis test of mouse tumor by using 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator and TPA as a promoter.     

Lipids,Mitochondria and Cell Death: Implications in Neuro-oncology

Polyunsaturated fatty acids (PUFAs) are known to inhibit cell proliferation of many tumour types both in vitro and in vivo. Their capacity to interfere with cell proliferation has been linked to their induction of reactive oxygen species (ROS) production in tumour tissues leading to cell death through apoptosis. However, the exact mechanisms of action of PUFAs are far from clear, particularly in brain tumours. The loss of bound hexokinase from the mitochondrial voltage-dependent anion channel has been directly related to loss of protection from apoptosis, and PUFAs can induce this loss of bound hexokinase in tumour cells. Tumour cells overexpressing Akt activity, including gliomas, are sensitised to ROS damage by the Akt protein and may be good targets for chemotherapeutic agents, which produce ROS, such as PUFAs. Cardiolipin peroxidation may be an initial event in the release of cytochrome c from the mitochondria, and enriching cardiolipin with PUFA acyl chains may lead to increased peroxidation and therefore an increase in apoptosis. A better understanding of the metabolism of fatty acids and eicosanoids in primary brain tumours such as gliomas and their influence on energy balance will be fundamental to the possible targeting of mitochondria in tumour treatment.     

Pentacyclic triterpenoids from the aerial parts of Lantana camara and their nematicidal activity

Two new olean-12-ene triterpenoids, camarolic acid (1) and lantrigloylic acid (2), have been isolated from the aerial parts of Lantana camara, along with ten known triterpenes, namely, camaric acid, lantanolic acid, lantanilic acid, pomolic acid, camarinic acid, lantoic acid, camarin, lantacin, camarinin, and ursolic acid. The new compounds have been characterized as 3,25-epoxy-3alpha-hydroxy-22beta-{[(S)-3-hydroxy-2-methylidenebutanoyl]oxy}olean-12-en-28-oic acid (1) and 3,25-epoxy-3alpha-hydroxy-22beta-[(3-methylbut-2-enoyl)oxy]olea-9(11),12-dien-28-oic acid (2) through spectroscopic studies and a chemical transformation. Seven of the constituents, namely pomolic acid, lantanolic acid, lantoic acid, camarin, lantacin, camarinin, and ursolic acid, were tested for nematicidal activity against root-knot nematode Meloidogyne incognita. Pomolic acid, lantanolic acid, and lantoic acid showed 100% mortality at 1 mg/ml concentration after 24 h, while camarin, lantacin, camarinin, and ursolic acid exhibited 100% mortality at this concentration after 48 h. These results are comparable to those obtained with the conventional nematicide furadan (100% mortality at 1 mg/ml concentration after 24 h).     

PI3K通路抑制剂LY294002对人脑胶质瘤细胞株U87细胞衰老和凋亡的影响

目的:PI3K/Akt信号通路是与胶质瘤发生发展密切相关的核心通路之一,LY294002是该通路的特异性抑制剂。本研究通过探讨PI3K通路抑制剂LY294002对U87胶质瘤细胞系细胞衰老及凋亡的影响,从而为胶质瘤患者治疗的新策略奠定理论基础。方法:将体外培养的人脑胶质瘤U87细胞株分为DMSO处理的对照组和LY294002(100μM)处理的实验组,采用β-半乳糖苷酶染色和流式细胞术的方法,分别检测并比较两组肿瘤细胞衰老和凋亡的情况。结果:LY294002处理组U87胶质瘤细胞的衰老指数(32.20±4.46%)显著高于DMSO对照组(3.40±1.61%,t=6.254,P0.001)。另外,与DMSO对照组相比,凋亡蛋白caspase-3mRNA的表达在LY294002处理组胶质瘤细胞中显著上调(t=8.923,P0.05)。LY294002处理组肿瘤细胞的凋亡指数(80.10±4.832%)明显高于DMSO对照组(4.260±1.073%,t=8.923,P0.05)。结论:LY294002既能够诱导肿瘤细胞衰老,又能够诱导肿瘤细胞凋亡,然而其诱导胶质瘤细胞凋亡的能力占据主导地位,为其发挥抗胶质瘤效应的主要途径。另外,在LY294002的持续作用下,部分衰老的肿瘤细胞或许会发生凋亡。这些结论为为临床增强胶质瘤患者的联合化疗奠定了理论基础。     

Underlying Mechanism of Quercetin-induced Cell Death in Human Glioma Cells

There has been considerable interest in recent years in the anti-tumor activities of flavonoids. Quercetin, a ubiquitous bioactive flavonoid, can inhibit proliferation and induce apoptosis in a variety of cancer cells. However, the precise molecular mechanism by which quercetin induces apoptosis in cancer cells is poorly understood. The present study was undertaken to examine the effect of quercetin on cell viability and to determine its underlying mechanism in human glioma cells. Quercetin resulted in loss of cell viability in a dose- and time-dependent manner and the decrease in cell viability was mainly attributed to cell death. Quercetin did not increase reactive oxygen species (ROS) generation and the quercetin-induced cell death was also not affected by antioxidants, suggesting that ROS generation is not involved in loss of cell viability. Western blot analysis showed that quercetin treatment caused rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Transient transfection with constitutively active forms of MEK and Akt protected against the quercetin-induced loss of cell viability. Quercetin-induced depolarization of mitochondrial membrane potential. Caspase activity was stimulated by quercetin and caspase inhibitors prevented the quercetin-induced loss of cell viability. Quercetin resulted in a decrease in expression of survivin, antiapoptotic proteins. Taken together, these findings suggest that quercetin results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of ERK, Akt, and survivin.     

Regulation of the phosphatidylinositol 3-kinase-Akt and the mitogen-activated protein kinase pathways by ursolic acid in human endometrial cancer cells

The phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the mitogen activated protein kinase (MAPK) pathway are important in the development and proliferation of various human cancers. It has been found recently that ursolic acid treatment affects growth and apoptosis in cancer cells. We sought to determine whether prominent signaling pathways, including the PI3K-Akt pathway and the MAPK (JNK, P38, and P44/42) pathway mediate these effects. Endometrial cancer cells often have high levels of phosphorylated Akt seen in conjunction with a PTEN mutation or deletion. Elevation in Akt protects the cancer cell from apoptosis. Ursolic acid treatment moderately decreased PI3K levels in SNG-II cells. Treatment also decreased phospho-Akt and phospho-P44/42 in a dose- and time-dependent fashion, dramatically in SNG-II cells and moderately in HEC108 cells. This effect was most pronounced following treatment with 50 mum ursolic acid for 72 h. Our study found inhibition of both the PI3K-Akt pathway and the MAPK pathway in two endometrial cancer cell lines, SNG-II and the poorly differentiated HEC108 cell line.     

Sodium vanadate inhibits apoptosis in malignant glioma cells: A role for Akt/PKB

The dual signal hypothesis of apoptosis holds that a common signal can activate both apoptotic and proliferative pathways. The fate of a cell is dependent on which of these two pathways predominates. In the MAPK family of kinases, ERK and JNK have been proposed to mediate apoptosis whereas the PI3K-stimulated kinase, Akt/PKB, has been shown to inhibit apoptosis. The object of this study was to determine the role of these kinases in a glioma model of apoptosis. We have previously shown that K252a induces apoptosis and inhibits kinase activity. In this study we confirm these results and shown that the protein tyrosine phosphatase inhibitor sodium vanadate activates ERK, JNK and Akt/PKB, but does not stimulate proliferation. Vanadate did protect T98G cells from K252a-induced apoptosis, an effect that was abolished by addition of the PI3K inhibitor wortmannin. This suggests that PI3K and Akt/PKB may be responsible for mediating vanadate's protective effect on glioma cells. We conclude that the intracellular balance between protein phosphorylation pathways is a critical determinant of both cell proliferation and cell death.     

EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth

Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G(1). Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27(kip1) levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion.     

The Adenosine A3 Receptor Agonist Cl-IB-MECA Induces Cell Death Through Ca2+/ROS-Dependent Down Regulation of ERK and Akt in A172 Human Glioma Cells

Adenosine A₃ receptor (A3AR) is coupled to G proteins that are involved in a variety of intracellular signaling pathways and physiological functions. 2-Chloro-N ⁶-(3-iodobenzyl) adenosine-5??-N-methylcarboxamide (Cl-IB-MECA), an agonist of A3AR, has been reported to induce cell death in various cancer cells. However, the effect of CI-IB-MECA on glioma cell growth is not clear. This study was undertaken to examine the effect of CI-IB-MECA on glioma cell viability and to determine its molecular mechanism. CI-IB-MECA inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Treatment of CI-IB-MECA resulted in an increase in intracellular Ca²⁺ followed by enhanced reactive oxygen species (ROS) generation. EGTA and N-acetylcysteine (NAC) blocked the cell death induced by CI-IB-MECA, suggesting that Ca²⁺ and ROS are involved in the Cl-IB-MECA-induced cell death. Western blot analysis showed that CI-IB-MECA induced the down-regulation of extracellular signal-regulated kinases (ERK) and Akt, which was prevented by EGTA, NAC, and the A3AR antagonist MRS1191. Transfection of constitutively active forms of MEK, the upstream kinase of ERK, and Akt prevented the cell death. CI-IB-MECA induced caspase-3 activation and the CI-IB-MECA-induced cell death was blocked by the caspase inhibitors DEVD-CHO and z-VAD-FMK. In addition, expression of XIAP and Survivin were decreased in cells treated with Cl-IB-MECA. Collectively, these findings demonstrate that CI-IB-MECA induce a caspase-dependent cell death through suppression of ERK and Akt mediated by an increase in intracellular Ca²⁺ and ROS generation in human glioma cells. These suggest that A3AR agonists may be a potential therapeutic agent for induction of apoptosis in human glioma cells.     

Ursolic acid derivatives from Bangladeshi medicinal plant, Saurauja roxburghii: Isolation and cytotoxic activity against A431 and C6 glioma cell lines

Ursene-type pentacyclic triterpenes, including the ursolic acid, corosolic acid, and a new ursene-type pentacyclic triterpene, 7,24-dihydroxy ursolic acid, were isolated from the methanolic extract of the leaves of the Bangladeshi medicinal plant, Saurauja roxburghii, a higher plant indigenous to South East Asia and some part of North America. They were tested for the cytotoxicity against C6 rat glioma and A431 human epidermoid carcinoma cell lines. Although they have the same ursene-type pentacyclic triterpene core structure, the position and numbers of hydroxyl groups on the terpene structure significantly affected the activity and the selectivity to the cell lines.     

Quantitative analysis of anti-apoptotic function of Akt in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells under normal and stressed conditions

The serine/threonine kinases Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma have been implicated in preventing cells from undergoing apoptosis. Although several small molecule inhibitors of Akt have been reported to induce apoptosis in cancer cells, these inhibitors may have additional targets. In the current study, we used an Akt3 small interfering RNA (Akt3 siRNA) to analyze apoptosis induction in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells (MEF-Akt1,2-DKO). Our data indicated that Akt3 siRNA inhibited Akt3 protein expression in a dose-dependent manner. As a result, phosphorylation of Akt and its downstream targets, including FKHRL1 and GSK3alpha/beta, were reduced accordingly. The treatment also induced apoptosis in MEF-Akt1,2-DKO cells. However, apoptosis induction is significant only when more than 80% of Akt3 protein was depleted. Reintroducing Akt3 totally rescued Akt3-siRNA-induced apoptosis in MEF-Akt1,2-DKO cells. In addition, reintroducing Akt1 also inhibited apoptosis induced by Akt3 siRNA. Moreover, Akt3 siRNA potentiated different stress-induced apoptosis in MEF-Akt1,2-DKO cells at a lower dose when compared with what is required for apoptosis induction by itself. Our study suggests that only a small portion of Akt is active in wild-type MEF cells and a threshold of Akt inhibition is required to induce apoptosis by pure Akt inhibitors. In addition, our data indicate that cells under stress require more Akt for its survival.     

RLIP76 Depletion Enhances Autophagic Flux in U251 Cells

Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.