Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.     

中国红豆杉细胞经重复诱导和蔗糖饲喂后云南紫杉烷C生产的相应基因表达变化

红豆杉悬浮培养细胞具有可持续生产抗癌药物紫杉醇及其他紫杉烷的潜力。在中国红豆杉悬浮培养细胞中,云南紫杉烷 C(Tc) 是主要的次生代谢产物。为促使代谢前体由生成其他紫杉烷的代谢支路转到生产紫杉醇,实验采用实时定量PCR技术 (RQ-PCR) 揭示细胞培养过程中紫杉醇及紫杉烷合成关键基因的动态变化。在细胞培养的第7天和第12天,以100 μmol/L 2,3-二羟丙基茉莉酸 (DHPJA) 进行诱导,同时在第7天饲喂20 g/L的蔗糖,在此过程中考察6个关键基因 (TASY,TDAT,T5αH,TαH,T10βH和T14βH) 的表达变化。上述联合调控手段使得Tc产量在第1次诱导8 d后达 (554.46±21.28) mg/L,第2次诱导9 d后高达 (997.72±1.51) mg/L。代谢早期基因TASY和TDAT在第1次诱导后表达量分别提高了182和98倍,在第2次诱导后表达量分别提高了208和131倍。在每次诱导后基因表达量提高约持续24 h,之后下降。其他4个基因 (T5αH、TαH、T10βH和T14βH) 的情况有所不同。基因TαH在2次诱导后表达量分别提高了3 061和1 016倍。其他3个基因T5αH、T10βH、T14βH在第1次诱导后表达量分别提高13、38、20倍,在第2次诱导后分别提高7、16、6倍。RQ-PCR结果表明基因表达和Tc积累之间存在紧密相关性：基因表达的变化与Tc产量的变化相一致,诱导可提高6个基因的表达量。基因的高表达随着培养过程逐渐衰减,再次诱导可再次促使基因的高表达。     

中国红豆杉中一个新的紫杉烷二萜

从中国红豆杉(Taxus chinensis)枝叶中分离得到4个紫杉烷二萜,通过波谱分析分别确定为：1β-羟基-2α,7β-二去乙酰基巴卡亭I(1),1β-羟基巴卡亭I(2),2α,5α,7β,9α,10β,13α-六乙酰氧基紫杉-4(20),11-二烯(3)和2-去乙酰氧基-5-去肉桂酰taxinine J(4)其中化合物1为新化合物。     

Regioselectivity of taxoid-O-acetyltransferases: heterologous expression and characterization of a new taxadien-5alpha-ol-O-acetyltransferase

In addition to the anticancer drug Taxol, yew (Taxus) species produce a large variety of other taxane diterpenoids which differ mainly in the type of acyl and aroyl groups appended to the many hydroxyl functions on the taxane core; acetate esters are particularly common. Taxol bears an acetate at C10 and another at C4 thought to originate by intramolecular migration of a C5 acetate function in the process of oxetane ring formation, but many other naturally occurring taxoids bear acetate groups at C1, C2, C7, C9, and C13, in addition to C5 and C10. cDNAs encoding a taxoid 5alpha-O-acetyltransferase (taxadien-5alpha-ol as substrate) and a taxoid 10beta-O-acetyltransferase (10-deacetylbaccatin III as substrate) have been acquired from a recently isolated family of Taxus acyl/aroyltransferase clones. To explore the origins of other acetylated taxoids, the group of recombinant Taxus acyltransferases was investigated with a range of polyhydroxylated taxoids as substrates. From this survey, a new acetyltransferase clone (denoted TAX19) was identified that was capable of acetylating taxadien-5alpha-ol with activity comparable to that of the previously identified 5alpha-O-acetyltransferase (clone TAX1). However, when these two recombinant enzymes were presented with taxadien-triol and tetraol substrates, they exhibited different regiospecificities. The TAX1 enzyme preferentially acetylates the "northern" hemisphere hydroxyls at C9 and C10, whereas the TAX19 enzyme preferentially acetylates the "east-west" pole positions at C5 and C13. The TAX1 enzyme possesses the lowest KM value with taxadien-5alpha-ol (an early pathway metabolite) as substrate, with much higher KM values for the polyhydroxylated taxoid substrates, whereas the TAX19 enzyme possesses lower KM values (than the TAX1 transferase) for all taxoid substrates tested. These results suggest that both TAX1 and TAX19 acyltransferases may function at the early C5 acetylation step of taxoid metabolism, and that the TAX19 acyltransferase, because of its broader specificity for polyhydroxylated taxoids, may also function later in metabolism and be responsible for the production of many other acetylated taxoids.     

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.     

利用RNAi抑制曼地亚红豆杉细胞紫杉烷14β-羟基化酶基因的表达

目的：利用RNA干扰技术抑制紫杉烷14β-羟基化酶(简称14OH)基因的表达。方法：以曼地亚红豆杉(Taxus×media)为材料,通过农杆菌介导的双元载体转化系统将针对14OH基因构建的RNA干扰载体导入该植物细胞中。应用PCR-Southern技术对转基因细胞进行了鉴定。另外,利用RT-PCR技术检测14OH基因mRNA表达水平,并采用HPLC技术对转基因细胞系中三种C-14氧取代的紫杉烷成分进行测定。结果：Southern检测证实转基因实验获得成功,RT-PCR结果表明转基因细胞系中14OH基因mRNA表达水平与对照组相比显著下降,HPLC分析显示C-14氧取代的紫杉烷含量也有明显降低。结论：该RNA干扰技术有效地抑制了曼地亚红豆杉细胞内14OH基因的表达,有望为提高紫杉醇的生物合成产量提供一条新途径。     

中国红豆杉枝叶中的紫杉烷二萜

从中国红豆杉(Taxus chinensis)枝叶的乙醇提取物中分离得到8个紫杉烷二萜,通过波谱分析分别确定为：14p羟基巴卡亭Ⅵ(1),巴卡亭Ⅵ(2),巴卡亭Ⅳ(3),1β-去羟基巴卡亭Ⅳ(4),云南红豆杉酯甲(5),2α-去乙酰-2α-苯甲酰基-13α-乙酰基云南紫杉亭(6),5α-羟基-2α,7βp,9α,10β,13α-五乙酰氧基紫杉-4(20),11-二烯(7)和Taxaein(8)。其中化合物1为新化合物,并报道八个化合物在丙酮中测得的核磁共振信号,化合物8的数据属首次报道。     

Structure-activity relationship study of taxoids for their ability to activate murine macrophages as well as inhibit the growth of macrophage-like cells

A series of new taxoids modified at the C-3', C-3'N, C-10, C-2 and C-7 positions has been designed, synthesized and evaluated for their potency to induce NO and TNF production by peritoneal murine macrophages (Mphi) from LPS-responsive C3H/HeN and LPS-hyporesponsive C3H/HeJ strains and human blood cells, and for their ability to inhibit the growth of Mphi-like cell lines J774.1 and J7.DEF3. The SAR-study has shown that the nature of the substituents at these positions have critical effect on the induction of TNF and NO production by Mphi. Positions C-3' and C-10 are the most flexible and an intriguing effect of the length of the substituents at the C-10 position is observed for taxoids bearing a straight chain alkanoyl moiety. An aromatic group at the C-3'N and C-2 positions is required for the activity, while only hydroxyl or acetyl substituents seem to be tolerated at the C-7 position. The natural stereochemistry in the C-13 isoserine side chain of the taxoids is an absolute requirement for macrophage activation. It has also been clearly shown that there is no correlation between the ability of the taxoids to induce TNF/NO production in C3H/HeN Mphi and the cytotoxicity against Mphi-like cells.     

The 13C-NMR Features of Natural Taxane Diterpenoid Compounds

The paper reviewed the 13C-NMR features of natural taxane diterpenoids according to their carbon-skeleton types. In the 13C-NMR it is easy to distinguish the 6/8/6 and 5/7/6 membered rings by observation of the 13C-NMR data of C-1 and C-15. The remarkable differences of the resonance of C -13 and C-12 were found. In addition, based on various chemical environments man y obscured chemical shifts of carbons can be distinguished, such as the oxygenat ed tertiary carbons of C-2, C-5, C-7, C-9 and C-13, and sometimes between C-9 an d C-10, as well as between aliphatic quaternary carbons of C-8 and C-15, and sec ondary carbons of C-6 and C-14. All the above-mentioned characteristics are helpful for structural elucidation and assignments of the carbon signals of taxoids.     

The influence of ultraviolet radiation on the content of pharmacologically active taxoids in yew tissues

The aim of the work was the investigation of the influence of UV radiation on the taxoids contents in yew tissues. The experiment was performed using Taxus baccata var. Aurea Corr. twigs irradiated with UV C (lambda = 254 nm) and UV A (lambda = 366 nm) in comparison to control samples. Multistep procedure of sample preparation was applied before the analysis of paclitaxel and 10-DAB III: SPE using alumina - for purification from the chlorophylles, waxes and polar balasts as well as zonal micropreparative TLC on silica - for isolation of partially separated fractions. The quantitation of some taxoids in the isolated fractions was performed using RP-HPLC procedure in system C18/acetonitrile + water. The experiments with UV-A and especially UV-C radiation showed changes in concentrations of paclitaxel and its precursor-10-deacetylbaccatin III. The results can be utilized to increase the yield of the taxoids isolated for medicinal and practical purposes.     

Simultaneous identification and determination of major taxoids from extracts of Taxus chinensis cell cultures

Liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/ MS) and high-performance liquid chromatography (HPLC) methods have been used to identify and quantify the major taxoids from extracts of Taxus chinensis cell cultures. Chromatography was carried out on a reverse phase C18 column with isocratic-mode elution. By analytically comparing LC/ESI-MS/MS of the extracts with that of the available reference substances and literature, six taxoids were identified as taxuyunnanine C (Tc, 1), yunnanxane (2), 2alpha,5alpha10beta-triacetoxy-14beta-propionyloxytaxa-4(20),11-diene (3), 2alpha,5alpha, 10beta-triacetoxy-14beta-(2-methyl)butyryloxytaxa-4(20),11-diene (4), taxol (5), and baccatin III (B III, 6), respectively. Among them, 2, 3 and 4 were assigned in the absence of the corresponding reference substances, and 3 and 4 were detected in this cell line for the first time. The identification was validated by NMR spectra. The precise quantification of 1 and 5 was made using HPLC. The limit of detection (LOD), 0.5 microg/ml for 5, 1.5 microg/ml for 1, and the linearity and accuracy of the quantitative method were evaluated, indicating a wide linear range and satisfactory accuracy. The amounts of other identified taxoids were calculated on the basis of comparison of the absolute response factors of similar structural substances. The proposed method provides a rapid, conventional and reliable tool to characterize and study cell lines for elucidating the taxane biosynthesis.     

Chemical Studies on Taxus canadensis

A series of new taxanes, 1 – 93 , have been isolated, together with 37 known taxoids including Taxol^® (paclitaxel) and cephalomannine, from the Canadian yew, Taxus canadensis (Taxaceae) in the past 30 years. These new taxoids possess various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, and 6/12 ring systems and six new taxanes with four novel skeletons, i.e., a taxane with a 6/6/8/6 ring system, a taxane with a [3.3.3] propellane skeleton, three taxanes with [3.3.3] [3.4.5] dipropellane sytems, as well as a novel taxane with a unique 5/5/4/6/6/6 hexacyclic skeleton, containing a unique [3.3.2] propellane, were isolated for the first time from natural sources. It should be emphasized that 13‐acetyl‐9‐dihydrobaccatin III, a very useful starting material for the semisynthesis of Taxol^® and Taxotere^®, represents the most abundant taxane in the needles of this yew tree. These findings establish the above mentioned yew tree as significantly different from the remaining species. On the other hand, some chemical modifications on the taxanes isolated from this plant were carried out.     

Catabolism of [4-14C]testosterone by subcellular fractions of human prostate

1. Incubation conditions were established in experiments with human-prostate homogenates for almost complete conversion of [4-(14)C]testosterone into at least ten transformation products. 2. Whole homogenates of tissue with benign hypertrophy were shown to contain 3alpha-, 3beta- and 17beta-hydroxy steroid dehydrogenases, Delta(4)-3-oxo steroid 5alpha- and 5beta- reductases and unidentified hydroxylases. 3. Most of the 17beta-hydroxy steroid-dehydrogenase activity was located in the mitochondria, which showed little other activity. 4. The 3alpha- and 3beta-hydroxy steroid dehydrogenases and the 5beta-reductase were located in the high-speed supernatant and required supplementation with NADPH for activity. 5. The 5alpha-reductase was located in both microsomal and high-speed supernatant fractions and also required supplementation with NADPH.     

Synthesis and structure-activity relationships of taxuyunnanine C derivatives as multidrug resistance modulator in MDR cancer cells

A series of new generation taxoids bearing a bulky group on different positions such as C-2, C-5, C-7, C-9, C-10 or C-14 were obtained by chemical modifications and biotransformation of taxuyunnanine C (1) and its analogs, 4, 5, and 10. Compounds 3, 5, 6, 8, and 9a showed significant activity toward calcein accumulation in MDR 2780AD cells. The most effective compound 9a with a cinnamoyloxy group at C-14 and a hydroxyl group at C-10 was actually efficient for the cellular accumulation of the anticancer agent, vincristine, in MDR 2780AD cells. The enhancing effects of 6 and 9a for taxol, adriamycin, and vincristine were at the same levels as those of verapamil toward MDR 2780AD cells. Thus, compounds 6 and 9a can modulate the multidrug resistance of cancer cells. The cytotoxicity (IC(50)) of the compounds was examined against human normal cell line, WI-38, and cancer model cell lines, VA-13 and HepG2. Since compounds 6 and 8 had no cytotoxicity, they were expected to be lead compounds of MDR cancer reversal agents. On the contrary, compounds 3, 5, and 9a showed cell growth inhibitory activity toward VA-13 and/or HepG2 as well as accumulation activity of calcein and/or vincristine in MDR 2780AD and they were expected to be lead compounds of new-type anticancer agents.     

Age and sex differences in the metabolism of C19-delta5-steroids in human amniotic epithelium

In vitro transformation of [4-(14)C]-dehydroepiandrosterone and [4-(14)C]-5-androstenediol in human amniotic epithelium from the first and the second trimester was studied. Total conversion of both substrates used and formation of their "more polar" metabolites increased with advancing gestational age. The reduction of 17-oxo or the oxidation of 17 beta-hydroxy groups reached a maximum between weeks 17 and 20. A significant decrease in the conversion of delta 5-3 beta-hydroxy group from week 11 was evident. No significant sex differences were observed.     

Analysis of biosynthetic fluctuations of cultured Taxus seedlings using a metabolomic approach

Fluctuations in the biosynthesis of taxoids in 1–5 year old cultured seedlings of Taxus chinensis var. mairei were investigated using LC–IT-TOF-MS and a metabolomics approach. In the total ion chromatogram (TIC) of the extracts, 16 prominent peaks were observed. Ten compounds were identified by comparison of retention times and MS/MS spectra with those of reference compounds. An additional 6 taxoids were isolated by preparative HPLC and identified by comparison of their spectroscopic data with those reported in the literature. It was clarified that the relative concentrations of taxoids with 4(20) double bonds are high at early stages of cultivation. On the other hand, relatively higher amounts of 5-acetoxy taxoids oxidized at the 4- and 10- positions and taxoids having 5(20)-oxetane rings were found at later stages of cultivation. This approach provides practical information on the biosynthetic flow of taxoids in cultured yew seedlings.     

东北红豆杉的化学成分和药理作用研究进展

本文综述了东北红豆杉(Taxus cuspidata)在化学成分和药理学方面的研究进展,着重强调了近年来紫杉烷类化合物的化学研究和生理活性。     

天然紫杉烷类二萜化合物的核磁共振碳谱规律的探讨

紫杉醇（ｔａｘｏｌ）［1］是一种结构新颖、抗肿瘤作用机制独特的新型抗肿瘤药,现已成为全世界研究的热点［2］。紫杉醇属于天然紫杉烷类二萜化合物,因此,许多学者试图从自然界中寻找抗肿瘤作用更强的紫杉烷类二萜成分［3］。迄今已发现这类化合物30 0多个,并发现了一些新的骨架类型［4 - 8］。紫杉烷类化合物骨架类型多,取代位置和取代基种类更多,且几乎都为含氧取代基,因而结构解析较为复杂。无疑,核磁共振技术的应用对解析结构起到至关重要的作用。氢谱、碳谱、ＮＯＥ、ＤＥＰＴ及各种二维技术（1Ｈ＿1ＨＣＯＳＹ、ＮＯＥＳＹ、…     

beta-Hydroxy acyl-CoA inhibition of mitochondrial ATP production

beta-Hydroxypalmitoyl-CoA and beta-hydroxystearoyl-CoA were synthesized, purified and quantitated. beta-Hydroxypalmitoyl-CoA and beta-hydroxystearoyl-CoA instantly and reversibly inhibited oxidative phosphorylation by rabbit heart mitochondria oxidizing pyruvate. [8-14C]ADP uptake studies showed that the beta-hydroxy acyl-CoA species linearly inhibited the adenine nucleotide translocase system. Free beta-hydroxy fatty acids at comparable concentrations (0.005 mM) did not affect ADP uptake or state III respiration.     

Syntheses and biological activity of C-3'-difluoromethyl-taxoids

A series of new taxoids bearing difluoromethyl group at the C-3' position and modifications at the C-10 and C-14 positions has been synthesized and their biological activities studied. The in vitro cytotoxicity assay results indicate that these newly developed taxoids exhibit comparable to several times better activity against drug-sensitive cell line LCC6-WT, and 40-70 times better activity against the corresponding drug-resistant cancer cell line LCC6-MDR as compared to that of paclitaxel. Apoptosis analysis has revealed the exceptional activity of SB-T-12843 (1e) in inducing apoptosis in both MDR-bearing and MDR-negative cancer cells.