EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells

Aims

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro.

Main methods

Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes.

Key findings

We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation.

Significance

Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer.     

Cytotoxic and Apoptosis‐Inducing Activities,and Anti‐Tumor‐Promoting Effects of Cyanogenated and Oxygenated Triterpenes

Two of each semisynthetic lanostane‐ and cycloartane‐type triterpenes with a cyano‐enone functionality, i.e., 13 and 18 , and 23 and 28 , respectively, sixteen of their synthetic intermediates, 9 – 12, 14 – 17, 19 – 22 , and 24 – 27 , along with seven semisynthetic oxygenated triterpene acetates, 29 – 35 , and eight natural hydroxy triterpenes, 1 – 8 , were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines. One natural triterpene, 8 , and ten semisynthetic triterpenes, 9, 13, 15, 18, 23, 25, 28, 29, 32 , and 33 , exhibited potent cytotoxicities against one or more cell lines with IC₅₀ values in the range of 1.4–9.9 μM . Two lanostane‐type triterpenes with a cyano‐enone functionality, 3‐oxolanosta‐1,8,24‐triene‐2‐carbonitrile ( 13 ) and 3‐oxolanosta‐1,8‐diene‐2‐carbonitrile ( 18 ), induced apoptosis in HL60 cells, as observed by membrane phospholipid exposure in flow cytometry. Western blot analysis showed that 13 and 18 significantly reduced procaspases‐3, ‐8, and ‐9, and increased cleaved caspases‐3, ‐8, and ‐9. These findings indicated that compounds 13 and 18 induced apoptosis in HL60 cells via both the mitochondrial and the death receptor‐mediated pathways. In addition, upon evaluation of the inhibitory effects on Epstein? Barr virus early antigen (EBV‐EA) activation induced with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells, seven natural triterpenes, 1 – 6 and 8 , and ten semisynthetic triterpenes, 9, 10, 14, 15, 19, 20, 24, 25, 29 , and 30 , exhibited inhibitory effects which were higher than that of β‐carotene, a vitamin A precursor studied widely in cancer‐chemoprevention animal models.     

Triterpenes from Ferula linkii

Two new natural triterpenes, 3β-acetoxyolean-9,12-diene and 3β-acetoxy-6β-hydroxyolean-9,12-diene, were isolated from Ferula linkii.     

Effects of sesquiterpenes and triterpenes from the rhizome of Alisma orientale on nitric oxide production in lipopolysaccharide-activated macrophages: absolute stereostructures of alismaketones-B 23-acetate and -C 23-acetate

The methanolic extract from a Chinese herbal medicine, the rhizome of Alisma orientale, was found to exhibit inhibitory activity of nitric oxide (NO) production in lipopolysaccharide (LPS)activated macrophages. Novel triterpenes, alismaketones-B 23-acetate and -C 23-acetate, were isolated from the active extract together with eight sesquiterpenes and eighteen protostane-type triterpenes. The absolute stereostructures of new triterpenes were characterized on the basis of chemical and physicochemical evidence, which included the chemical correlations with known triterpenes. The guaiane-type sesquiterpenes (alismol, orientalols A and C) and protostane- and seco-protostane-types triterpenes (alisols C monoacetate, E-23-acetate, F, H, I, L-23-acetate, and M-23-acetate, alismaketones-B 23-acetate and -C 23-acetate, alismalactone 23-acetate, and 3-methylalismalactone 23-acetate) inhibited LPS-induced NO production (IC50 = 8.4-68 microM). Other triterpenes (alisols A, A monoacetate, B, B monoacetate, E, G, K-23-acetate, and N-23-acetate and 11-deoxyalisol B) also showed the potent inhibitory activity, but they showed cytotoxic effects more than 30 microM (MTT assay). In addition, alismol and alisol F were found to suppress iNOS induction.     

Induction of apoptosis in HeLa cells by 3beta-hydroxyurs-12-en-27-oic acid

3Beta-hydroxyurs-12-en-27-oic acid (1), a pentacyclic triterpenoid isolated from the rhizomes of Astilbe chinensis, was structurally very similar to ursolic acid, with the only difference being the interchange of the COOH and Me group at C(14) and C(17). Ursane-type triterpene with a COOH group at C(14) is present in a limited number of natural resources. Compound 1 was found to exhibit more distinctive cytotoxicity toward human cervical squamous carcinoma (HeLa) cells than ursolic acid, suggesting that the position of the COOH group significantly affects the cytotoxicity of ursane-type pentacyclic triterpenes with a COOH group. To elucidate the underlying biological mechanism responsible for the cytotoxicity of 1, we investigated its growth-inhibitory and apoptosis-inducing effect on HeLa cells. Compound 1 induced a marked concentration- and time-dependent inhibition of cell proliferation with an IC50 value of 6.80+/-0.88 microg/ml following 48 h incubation. The drug-treated HeLa cells displayed typical morphological apoptotic characteristics and formation of DNA ladders in agarose-gel electrophoresis. Flow cytometric analysis showed that the cell cycle was arrested in G0/G1 phase by 1, and the apoptotic rate of HeLa cells treated for 48 h with 20 microg/ml of 1 was 21.08+/-2.14%. Also, 1 increased and decreased the expression of Bax and Bcl-2 proteins, respectively, and lowered the mitochondrial transmembrane potential (delta psi(m)). The peptidic caspase-3 inhibitor DEVD-CHO (NH2-Asp-Glu-Val-Asp-CHO, at 2 microM) could increase the viability of HeLa cells previously treated with 1. These results indicate that 1 induces efficient cell apoptosis through down-regulating Bcl-2 expression, up-regulating Bax expression, lowering delta psi(m), and by activating the caspase-3 pathway.     

Cytotoxic triterpenes from the aerial roots of Ficus microcarpa

Six triterpenes, 3beta-acetoxy-12,19-dioxo-13(18)-oleanene (1), 3beta-acetoxy-19(29)-taraxasten-20alpha-ol (2), 3beta-acetoxy-21alpha,22alpha-epoxytaraxastan-20alpha-ol (3), 3,22-dioxo-20-taraxastene (4), 3beta-acetoxy-11alpha,12alpha-epoxy-16-oxo-14-taraxerene (5), 3beta-acetoxy-25-methoxylanosta-8,23-diene (6) along with nine known triterpenes, 3beta-acetoxy-11alpha,12alpha-epoxy-14-taraxerene (7), 3beta-acetoxy-25-hydroxylanosta-8,23-diene (8), oleanonic acid (9), acetylbetulinic acid (10), betulonic acid (11), acetylursolic acid (12), ursonic acid (13), ursolic acid (14), and 3-oxofriedelan-28-oic acid (15) were isolated from the aerial roots of Ficus microcarpa, and their structures elucidated by spectroscopic methods. The in vitro cytotoxic efficacy of these triterpenes was investigated using three human cancer cell lines, namely, HONE-1 nasopharyngeal carcinoma, KB oral epidermoid carcinoma, and HT29 colorectal carcinoma cells. Compound 8 and pentacyclic triterpenes 9-15 possessing a carboxylic acid functionality at C-28 showed significant cytotoxic activities against the aforementioned cell lines and gave IC50 values in the range 4.0-9.4 microM.     

Actinidic Acid,a New Triterpene Phytoalexin from Unripe Kiwi Fruit

Seven phytoalexins (1-7), including a new compound, were isolated from the peel of unripe kiwi fruit (Actinidia deliciosa cv. Golden King) that had been wounded and inoculated with Colletotrichum musae. The new phytoalexin (1) was identified as 2α,3β,23-trihydroxy-12,20(30)-ursadien-28-oic acid, and named actinidic acid. Phytoalexins 2-6 are known triterpenes but have not previously been described as phytoalexins. Phytoalexin 7 is the same triterpene as the phytoalexin of nectarine fruit.     

茯苓中三萜类和多糖类成分的研究进展

主要对中药茯苓中的三萜类和多糖类成分进行了综述。到目前为止已从茯苓的菌核和菌丝中分离到三萜类物质39个,其中羊毛甾-8-烯型三萜12个,羊毛甾-7,9(11)-二烯型三萜16个,3,4-开环-羊毛甾-7,9(11)-二烯型三萜7个,3,4-开环-羊毛甾-8-烯型三萜2个,三环二萜类1个,齐墩果烷型三萜1个;分离到多糖类物质23个。     

Tirucallane-type triterpenes from Juliania adstringens

Five tirucallane-type triterpenes were isolated along with nine known triterpenes from the bark of Juliania adstringens. The structures of the five triterpenes were determined by analysis of their 1H and 13C NMR and mass spectral data, and each compound exhibited growth inhibitory activity against leukemia cells (L-1210).     

A novel Ganoderma lucidum G0119 fermentation strategy for enhanced triterpenes production by statistical process optimization and addition of oleic acid

A novel enhanced triterpenes fermentation production process by Ganoderma lucidum G0119 with the addition of oleic acid in the medium has been developed and optimized. All of the six exogenous additives tested were found to exhibit stimulatory effect on mycelial growth and triterpenes biosynthesis by G. lucidum. The results show that oleic acid addition had significant role in promoting triterpenes production. The optimal concentration and time of oleic acid addition were determined to be 30 mL/L and 0 h, respectively. Furthermore, three significant factors influencing triterpenes production were identified as glucose, magnesium sulfate and temperature using the Plackett–Burman design. The optimized conditions by central composite design were 27.83 g/L glucose, 1.32 g/L magnesium sulfate, 26.2°C temperature. The triterpenes fermentation yield with the optimized medium based on actual confirmatory experimental data in 6 L fermentor was 1.076 g/L versus the statistical model predicted value of 1.080 g/L. Our innovatively developed triterpenes fermentation production technology and process has been proven to produce high triterpenes productivity and yield conceivably useful for industrial production.     

3β-hydroxy-21β-e-cinnamoyloxyolean-12-en-28-oic acid,a triterpenoid from enterolobium contorstisiliquum

The triterpenes 3β-hydroxy-21β-E-cinnamoyloxyolean-12-en-20-oic acid, 3β,21β-dihydroxyolean-12-en-28-oic acid (machaerinic acid) and its lactone (3β-hydroxyolean-12-en-21β→28-lactone) were isolated from the fruits of Enterolobium contorstisiliquum. Methyl and ethyl esters of 3β,21β-dihydroxyolean-12-en-oic acid were isolated and characterized as artifacts. The structures of these triterpenes have been established by a study of their chemical and spectroscopic (IR, MS and NMR) data.     

Ganoderma lucidum total triterpenes prevent radiation-induced DNA damage and apoptosis in splenic lymphocytes in vitro

The development of radioprotective agents has been the subject of intense research, especially in the field of radiotherapy. In this study, we examined the radioprotective activity of the total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst in mouse splenic lymphocytes in vitro. Using the MTT assay, Ganoderma triterpenes were found to have no effect on cell viability, indicating that they are non-toxic to splenic lymphocytes. The effect of the total triterpenes on DNA damage and apoptosis induced by radiation was analyzed using the comet assay, DNA ladder assay and flow cytometric analysis. Total triterpenes were found to be highly effective in preventing DNA laddering, even at low concentrations (25μg/ml). The comet assay demonstrated that the G. triterpenes effectively prevented DNA damage, and flow cytometry revealed a reduction in apoptotic cells. The effect of the total triterpenes on intracellular reactive oxygen species (ROS) level and endogenous antioxidant enzyme activity in splenic lymphocytes were determined to elucidate possible radioprotective mechanisms. Total triterpenes successfully reduced the formation of intracellular ROS and enhanced endogenous antioxidant enzyme activity in splenic lymphocytes following irradiation. Thus, these findings indicate that the total triterpenes isolated from G. lucidum have a remarkable ability to protect normal cells from radiation-induced damage, which suggests therapeutic potential.     

Selective ultrasound-assisted extractions of lipophilic constituents from Betula alleghaniensis and B. papyrifera wood at low temperatures

Betula alleghaniensis and B. papyrifera are widely distributed in the province of Québec (Canada) and, since these trees are valuable exports for the local lumber industry, large amounts of their residual ligneous biomass are available for further exploitation. Betula species are well known for their significant concentrations of triterpenes, some of which were recently discovered to present promising bioactivity. The secondary transformation of birch biomass could therefore become important for many industries, particularly the pharmaceutical industry. In the present study, extracts from birch sawdust were obtained using an optimised ultrasound-assisted extraction in which the careful choice of temperature permitted a selective extraction of the targeted triterpenes. Moreover, compared with the classical Soxhlet method, higher extraction yields were obtained in a shorter time. The lipophilic extracts obtained using dichloromethane as a solvent were analysed by GC-MS and the major compounds identified as lupane-type cyclic triterpenoids accompanied by the non-cyclic triterpene squalene. Numerous aliphatic long-chain fatty acids were also found in the extracts together with phytosterols. Betulonic acid and squalene, the major extract constituents for both B. alleghaniensis and B. papyrifera, are both bioactive molecules.     

Triterpenes from Euonymus revolutus

Fourteen pentacyclic triterpenes, which included ten D:A-friedooleananes, three lup-20(29)-enes and 2α,3α-dihydroxy-olean-12-en-28-oic acid, were isolated from the stem bark extract of Euonymus revolutus.     

分光光度法测定灵芝三萜含量的干扰因素探讨

根据药典方法,以齐墩果酸为对照品,对灵芝中含有的三萜、甾醇和脂肪酸3种类型化合物进行分光光度法测定,并对影响三萜含量测定的因素进行分析。结果表明灵芝中的甾醇和脂肪酸类化合物会干扰所有的测定结果,尤其影响灵芝孢子中三萜含量的测定。灵芝子实体中三萜化合物的结构特征,造成了其测定值远远低于真实值。因此,分光光度法不适用于测定灵芝子实体、菌丝体和孢子及其相关产品中的三萜含量。     

Triterpenoids from enkianthus campanulatus

From the leaves of Enkianthus campanulatus were isolated three new triterpenes, 3-oxo-19,23,24-trihydroxyurs-12-en-28-oic acid, 3β,6β, 19,23-tetrahydroxyurs-12-en-28-oic acid and 3β,6β,23-trihydroxyurs-12-en-28-oic acid.     

牛樟芝固态发酵菌丝体三萜及多糖提取工艺优化研究

为研究牛樟芝固态发酵菌丝体中三萜和多糖的最佳提取工艺，选取浸提时间、料液比和提取温度3个因素，分别设置3个水平，以三萜和多糖得率为指标，并采用正交试验法进行分析。结果表明，三萜的最佳提取工艺条件为：浸提时间3 h、料液比1∶30 (g·mL-1)、温度70 ℃，在此条件下，得率为3.43%；多糖的最佳提取工艺为：浸提时间2 h、料液比1∶20 (g·mL-1)、温度95 ℃，在此条件下得率为4.71%。研究结果为牛樟芝固态发酵菌丝体中三萜和多糖的提取工艺提供了参考。     

Terpenoids of Rhododendron japonicum

From Rhododendron japonicum were isolated two diterpene glucosides, pieroside B and grayanoside B, and two new triterpenes, 2α,3α,24-trihydroxy     

灵芝羊毛甾烷型三萜抗肿瘤构效关系初探

多篇文献报道灵芝三萜具有良好的抗肿瘤活性,但其构效关系和作用靶点尚未系统比较。本研究以45种灵芝三萜为研究对象,通过对小鼠白血病细胞L1210的增殖抑制测定,来评价化合物抗肿瘤的能力。结果表明,灵芝菌丝体三萜和子实体中性三萜的活性较强。进一步采用Discovery Studio分子对接技术探讨了12种活性较强的灵芝三萜与5种抗肿瘤作用相关蛋白p53、Bcl-xl、EGFR、IL-2和VEGFR2之间可能的作用靶点,推测三萜化合物抗肿瘤能力与其结构上的乙酰氧基和母环上的共轭双键有关。其中,具有这一特征的含有3个乙酰氧基的ganoderic acid T,其抗肿瘤活性最强。本研究为灵芝三萜活性位点的寻找和结构改造提供了参考。