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Availability of isotope-labelled metabolites often proves to be an essential prerequisite for the successful identification of a biosynthetic pathway. Also for the metabolic engineering isotope-labelled, either radioactive or non-radioactive, biosynthetic intermediates represent valuable tools for the assessment of metabolic flux, identification of unexpected biosynthetic side reactions, or for the confirmation of the functionality of the engineered reaction step. Most often the required compounds are neither available commercially nor prepared by chemical means within an acceptable time span and effort. Biocatalytic synthesis of early pathway intermediates may offer an attractive solution for this problem. For the metabolic engineering of taxol biosynthesis in a heterologous host, like yeast, isotope-labelled taxanes represent useful tools for the establishment and functional assessment of the introduced biosynthetic steps. Using Taxus chinensis taxadiene synthase expressed in the heterologous organism Pichia pastoris, we describe a method for the biocatalytic synthesis of tritium ((3)H)-labelled taxa-4(5),11(12)-diene, which represents the pathway committing biosynthetic precursor for all taxoid diterpenoids.

Oral administration of the taxanes docetaxel and paclitaxel is hampered by their affinity for drug transporters, especially ABCB1 (P-glycoprotein, Pgp); extensive first-pass metabolism by cytochrome P450 3A (CYP3A); and poor drug solubility. Preclinical studies in Pgp-deficient and wild-type mice demonstrated that modulation of either Pgp or CYP3A resulted in high systemic exposure to docetaxel or paclitaxel. This concept could successfully be translated to clinical trials.

Purpose: To evaluate HER2 status in residual tumor identified at the time of surgery in patients not achieving a pathologic complete response (pCR) and to determine the effect of alterations in HER2 status on recurrence-free survival (RFS)., Experimental Design: Clinicopathologic data for patients with HER2-overexpressing breast cancer receiving neoadjuvant therapy with a taxane, anthracycline, and concomitant trastuzumab between 2004 and 2007 were reviewed. Surgical specimens for patients achieving less than a pCR were assessed to determine if there was enough residual tissue to evaluate posttreatment HER2 status. RFS was determined using the Kaplan-Meier method and compared by the log-rank statistic., Results: A pCR was achieved in 72 of the 142 (50.7%) patients. Residual tumor was sufficient to assess posttreatment HER2 status in 25 patients. Fluorescence in situ hybridization done on pretreatment specimens confirmed HER2 amplification before beginning therapy. Eight (32.0%) posttreatment tumors were found to be HER2-negative by fluorescence in situ hybridization. At a median follow-up of 37 months (range, 8-56 months), the RFS was significantly better for patients with tumors that retained HER2 amplification (87.5% versus 50%, P = 0.04)., Conclusion: High pCR rates are achieved in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab in combination with anthracyclines and taxanes. One third of patients with significant residual disease loses HER2 amplification, and this change is associated with poor RFS. Residual tumor identified at the time of surgery should be reassessed for HER2 status, and novel adjuvant therapy strategies need to be studied in this population.

Taxanes exhibit a high tendency to epimerize at C-7 under physiological conditions. This study aimed to investigate the composite effect of C-7 configuration and other substructural elements on the metabolic properties of taxanes. Cephalomannine, 7-epi-cephalomannine, 10-deacetyl-paclitaxel, and 7-epi-10-deacetyl-paclitaxel were chosen as model compounds. In human liver microsomes, 7-epi-cephalomannine was subject to C-13 lateral chain (M-1) and diterpenoid core monohydroxylation (M-2), mediated by cytochrome P450 (CYP) 3A4 and CYP2C8, respectively. However, only one 7-epi-10-deacetyl-paclitaxel metabolite (M), monohydroxylated at taxane ring by CYP2C8, was detected. In comparison with cephalomannine, the catalytic efficiency of CYP2C8 for 7-epi-cephalomannine was about five-fold higher due to the decreased K(m). Although CYP2C8 showed a high capacity for metabolizing 7-epi-10-deacetyl-paclitaxel, 10-deacetyl-paclitaxel was hardly metabolized under the identical incubation conditions. In conclusion, C-7 configuration represents one of the most important structural determinants in taxanes metabolism.

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1microM of l-phenylalanine (6.2gL(-1)). Addition of 1microM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7microg L(-1)), which was not detected in the untreated control culture. Supplementation with 100microM of l-phenylalanine together with 100microM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3microg L(-1) (control untreated culture) to 568.2microg L(-1), the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8microg L(-1)) was observed when the medium was supplemented with 100microM of the precursor in combination with methyl jasmonate. Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.

We have isolated endophytic fungi from Indian yew tree, Taxus baccata and then screened for taxol production. Out of the forty fungal cultures screened, one fungus Gliocladium sp. was found to produce taxol and 10DAB III (10 Deacetyl baccatin III). These compounds were purified by TLC, HPLC and characterized using UV-Spectroscopy, ESI-MS, MS/MS and proton NMR. One liter of Gliocladium sp. culture yielded 10 microg of taxol and 65 microg of 10 DAB III. The purified taxol from the fungus showed cytotoxicity towards cancer lines HL-60 (leukemia), A431 (epidermal carcinoma) and MCF-7 (breast cancer).

Seven pairs of epimers and one pair of isomeric metabolites of taxanes, each pair of which have similar structures but different retention behaviors, together with additional 13 taxanes with different substitutions were chosen to investigate the quantitative structure-retention relationship (QSRR) of taxanes in ultra fast liquid chromatography (UFLC). Monte Carlo variable selection (MCVS) method was adopted to choose descriptors. The selected four descriptors were used to build QSRR model with multi-linear regression (MLR) and artificial neural network (ANN) modeling techniques. Both linear and nonlinear models show good predictive ability, of which ANN model was better with the determination coefficient R(2) for training, validation and test set being 0.9892, 0.9747 and 0.9840, respectively. The results of 100 times' leave-12-out cross validation showed the robustness of this model. All the isomers can be correctly differentiated by this model. According to the selected descriptors, the three dimensional structural information was critical for recognition of epimers. Hydrophobic interaction was the uppermost factor for retention in UFLC. Molecules' polarizability and polarity properties were also closely correlated with retention behaviors. This QSRR model will be useful for separation and identification of taxanes including epimers and metabolites from botanical or biological samples.

Purpose: The purpose of the present article was to evaluate indications, regimens, treatment modalities, and predictive factors of response to treatment in locally advanced squamous cell carcinoma of the head and neck (SCCHN)., Methods: An expert panel including otolaryngology and head and neck surgery specialists, oncologists, radiotherapists and biologists analyzed the literature providing a synthesis and giving some recommendations., Synthesis: Findings from the main randomized phase III trials highlight that the TPF regimen (docetaxel, cisplatin, fluorouracil) represent a preferential option when induction chemotherapy is indicated in either operable or non-operable patients. Given the potential fragility of patients presenting with SCCHN, treatment modalities in routine use require applying preventive measures and tailored follow-up according to each patient's profile. As regards predictive factors of response to TPF regimen, no factor is currently validated, but ongoing trials should provide better knowledge., Conclusion: Progresses in induction chemotherapy have allowed improving the prognosis of patients with locally advanced SCCHN. The TPF regimen represents a major improvement in this indication, and ongoing strategic clinical trials should refine its indications.

Docetaxel (DTX) is one of the most active chemotherapeutic agents for treating metastatic breast cancer. Its aqueous solubility is very low, hence the available formulation of DTX for clinical use consists of high concentrations of tween80, which has been associated with several hypersensitivity reactions. To reduce the systemic toxicity of DTX as well as to avoid the use of tween80, in this study DTX was chemically conjugated with human serum albumin via a succinic spacer. A high-performance liquid chromatography method was developed for the determination of DTX-albumin conjugate. T47D and SKOV3 cells were used for the evaluation of the in vitro cytotoxicity of the conjugate by MTT assay. Studies were then done on balb/c mice to elucidate the tissue distribution of conjugates after intravenous administration. The albumin-conjugated formulation of DTX with the particle size of 90-110 nm showed enhanced solubility and in vivo characteristics and significantly higher cytotoxicity against tumor cells, for example, IC50 of 6.30 +/- 0.73 nM for T47D cell line compared to free DTX with IC50 of 39.4 +/- 1.75 nM. Conjugation also maintained DTX plasma level at 16.19% up to 2 h after injection compared with 2.51% for Taxotere, hence increasing the chance of nanoparticles uptake by tumor cells., (Copyright 2009 Wiley-Liss, Inc.)

In order to find the minimal structural requirements to maintain microtubule binding, 12 taxol analogues have been docked to the taxol binding site of tubulin. By comparing the interactions of each analogue with beta-tubulin, the structure-activity relationships are summarized as follow: C-2 benzoyl and taxane ring systems are the essential groups for microtubule binding, the improvements of bioactivity and bioavailability are dependent on the substituents at positions C-1, C-4, C-7, C-9, C-10, and C-14, whereas the C-13 side chain mainly provides a specific binding.

Four systems of nanoparticles of biodegradable polymers were developed in this research for oral delivery of anticancer drugs with Docetaxel used as a model drug, which include the poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), the poly(lactide)-vitamin E TPGS nanoparticles (PLA-TPGS NPs), the poly(lactic-co-glycolic acid)-montmorillonite nanoparticles (PLGA/MMT NPs) and the poly(lactide)-vitamin E TPGS/montmorillonite nanoparticles (PLA-TPGS/MMT NPs). Vitamin E TPGS stands for d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), which is a water-soluble derivative of natural vitamin E formed by esterification of vitamin E succinate with polyethylene glycol (PEG) 1000. The design was made to take advantages of TPGS in nanoparticle technology such as high emulsification effects and high drug encapsulation efficiency, and those in drug formulation such as high cellular adhesion and adsorption. MMT of similar effects is also a detoxifier, which may cure some side effects caused by the formulated drug. The drug-loaded NPs were prepared by a modified solvent extraction/evaporation method and then characterized for their MMT content, size and size distribution, surface charge and morphology, physical status and encapsulation efficiency of the drug in the NPs, and in vitro drug release profile. Cellular uptake of the coumarin 6-loaded NPs was investigated. In vitro cancer cell viability experiment showed that judged by IC(50), the PLA-TPGS/MMT NP formulation was found 2.89, 3.98, 2.12-fold more effective and the PLA-TPGS NP formulation could be 1.774, 2.58, 1.58-fold more effective than the Taxotere((R)) after 24, 48, 72h treatment, respectively. In vivo PK experiment with SD rats showed that oral administration of the PLA-TPGS/MMT NP formulation and the PLA-TPGS NP formulation could achieve 26.4 and 20.6 times longer half-life respectively than i.v. administration of Taxotere((R)) at the same 10mg/kg dose. One dose oral administration of the NP formulations could realize almost 3 week sustained chemotherapy in comparison of 22h of i.v. administration of Taxotere((R)). The oral bioavailability can be enhanced from 3.59% for Taxotere((R)) to 78% for the PLA-TPGS/MMT NP formulation and 91% for the PLA-TPGS NP formulation respectively. Oral chemotherapy by nanoparticles of biodegradable polymers is feasible.

MicroRNAs (miRNAs) are important regulators of gene expression that are increasing being implicated in controlling plant development and its interaction with the environment. The advent of new high-throughput sequencing technologies has enabled both the discovery and quantification of miRNAs from a diverse range of species. In this study, we employed high throughput Illumina sequencing to identify miRNAs from Taxus chinensis (T. chinensis) cells to investigate the effect of the taxoid elicitor methyl jasmonate (MJ) on miRNA expression. In a dataset of approximately 6.6 million sequences, a total of 58 miRNAs, belonging to 25 families were identified. A majority of them are conserved between angiosperms and gymnosperms. However, two miRNAs (miR1310 and miR1314) appear gymnosperm-specific, with miR1314 likely to exist as a cluster. MJ treatment significantly affected the expression of specific miRNAs; 14 miRNAs from 7 different families (miR156, miR168, miR169, miR172, miR396, miR480 and mir1310) were down regulated whereas 3 miRNAs from 2 families (miR164 and miR390) were up regulated.

The native N-debenzoyl-2'-deoxypaclitaxel:N-benzoyltransferase (NDTBT), from Taxus plants, transfers a benzoyl group from the corresponding CoA thioester to the amino group of the beta-phenylalanine side chain of N-debenzoyl-2'-deoxypaclitaxel, which is purportedly on the paclitaxel (Taxol) biosynthetic pathway. To elucidate the substrate specificity of NDTBT overexpressed in Escherichia coli, the purified enzyme was incubated with semisynthetically derived N-debenzoyltaxoid substrates and aroyl CoA donors (benzoyl; ortho-, meta-, and para-substituted benzoyls; various heterole carbonyls; alkanoyls; and butenoyl), which were obtained from commercial sources or synthesized via a mixed anhydride method. Several unnatural N-aroyl-N-debenzoyl-2'-deoxypaclitaxel analogues were biocatalytically assembled with catalytic efficiencies (V(max)/K(M)) ranging between 0.15 and 1.74 nmol.min(-1).mM(-1). In addition, several N-acyl-N-debenzoylpaclitaxel variants were biosynthesized when N-debenzoylpaclitaxel and N-de(tert-butoxycarbonyl)docetaxel (i.e., 10-deacetyl-N-debenzoylpaclitaxel) were used as substrates. The relative velocity (v(rel)) for NDTBT with the latter two N-debenzoyl taxane substrates ranged between approximately 1% and 200% for the array of aroyl CoAs compared to benzoyl CoA. Interestingly, NDTBT transferred hexanoyl, acetyl, and butyryl more rapidly than butenoyl or benzoyl from the CoA donor to taxanes with isoserinoyl side chains, whereas N-debenzoyl-2'-deoxypaclitaxel was more rapidly converted to its N-benzoyl derivative than to its N-alkanoyl or N-butenoyl congeners. Biocatalytic N-acyl transfer of novel acyl groups to the amino functional group of N-debenzoylpaclitaxel and its 2'-deoxy precursor reveal the surprisingly indiscriminate specificity of this transferase. This feature of NDTBT potentially provides a tool for alternative biocatalytic N-aroylation/alkanoylation to construct next generation taxanes or other novel bioactive diterpene compounds.

To achieve a better understanding of taxol metabolism and accumulation in Taxus cell cultures, a T. baccata cell line growing for 20 days in a selected growth medium was treated at the beginning of the experiment with several concentrations of taxol (25, 50, 100 and 200mgL(-1)). Compared with an untreated control, all these taxol concentrations stimulated cell-associated taxol content (up to 32.7 times in the presence of 200mgL(-1) exogenous taxol), although higher concentrations significantly depressed cell viability. DNA laddering analysis revealed that the viability reduction was not related to apoptosis, suggesting that taxol itself was the primary responsible factor. On the basis of RT-PCR expression analysis of genes encoding taxadiene synthase (ts) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) from treated and nontreated T. baccata cell line cultures, it was observed that exogenous taxol clearly induced the mRNA levels of both taxane-related enzymes. Additionally, we found that exogenous taxol caused a considerable increase in taxadiene synthase activity, although in no case did this coincide with the highest levels of taxol observed at the end of the culture. The effect of exogenous taxol on the content of other related taxanes was also considered.

The C-7 chiral centre in paclitaxel is subject to epimerization under physiological conditions, thus making 7-epi-paclitaxel as the principal degradant. This study was designed to characterize the cytochrome P450 (CYP) enzymes involved in 7-epi-paclitaxel metabolism, and to examine possible metabolic interactions that this C-7 epimer may have with paclitaxel. In human liver microsomes, 7-epi-paclitaxel was oxidized to two monohydroxylated metabolites while the metabolic sites occurred at the C-13 side-chain for M-1 and taxane core ring for M-2. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that M-1 was generated predominantly by CYP3A4 and M-2 by CYP2C8. Co-incubation of 7-epi-paclitaxel with paclitaxel in human liver microsomes resulted in potent inhibition of 6alpha-hydroxypaclitaxel formation (IC((50)) = 2.1 +/- 0.2 muM), thus decreasing the metabolic elimination of paclitaxel. In conclusion, both CYP3A4 and CYP2C8 play a major role in biotransformation of 7-epi-paclitaxel in human liver microsomes. The existence of epimeric interactions between paclitaxel and its degradant might be a noteworthy factor resulting in the complex pharmacokinetic profile of paclitaxel.

The purpose of this study was to evaluate the affinity of docetaxel for 14 transporter proteins and assess the functional significance of 17 variants in five genes involved in drug elimination. Among the transfected models investigated, OATP1B3 (SLCO1B3) was identified as the most efficient influx transporter for docetaxel. None of the observed genotypes (SLCO1B3, ABCB1, and ABCC2) was related with docetaxel clearance in 92 white patients (P > 0.17). However, the simultaneous presence of the CYP3A4*1B and CYP3A5*1A alleles was associated with a 64% increase in docetaxel clearance (P = 0.0015), independent of both sex and CYP3A activity (as determined using the erythromycin breath test). This haplotype was also associated with increased midazolam clearance in another population (P = 0.0198). An analysis of the CYP3A locus among CEPH-HapMap samples revealed that CYP3A4*1B is present exclusively among a subset of CYP3A5 expressors. Therefore, future studies should first stratify the population on the basis of CYP3A5 genotype and then compare CYP3A activity between individuals with and without the CYP3A4*1B allele.

Over 200 derivatives have been obtained through chemo-enzymatic transformation of the taxanes derived from cell cultures of Taxus chinensis. The reversal activity towards MDR tumor cells and cyto-toxicity of most these compounds were evaluated, and several derivatives exhibited powerful MDR reversal activity. The substrate-specificities of the two most important biotransformation reactions-C-7beta and 9alpha hydroxylations, were investigated and preliminarily concluded. In addition, some key intermediates in hypothetically biosynthetic pathway of taxoid were obtained. These results indicate that biotransformation combined with chemical transformation is a powerful approach to structural diversification of natural products, lead compound discovery and the study on the structure activity relationships of the derivatives. Moreover, biotransformation may mimic some steps in biosynthetic pathway of natural products and provide useful hints as to their biogenetic study.

More than 50 kinds of endophytic fungi associated with Taxus chinensis, were isolated and examined as potential source of the imposing anticancer drug taxol. Of these, 4 isolates show ability to produce taxane when measured with the competitive inhibition enzyme immunoassay method. The most promising clone, DA10, identified as Mucor rouxianus sp., is the first rouxianus reported as taxol-production fungus. The presence of taxol and its important precursors, such as 10-diacetyl baccatinIII (10-DAB) and baccatinIII, in the culture of this fungus was confirmed by reactivity with taxane-specific monoclonal antibody, comparative chromatographic and mass spectrometric behavior, cytotoxity to liver carcinoma 7402, and molecular cloning of kernel fragment of taxadiene synthase gene.

The 10-deacetylbaccatin III:10beta-O-acetyltransferase isolated from Taxus cuspidata regiospecifically transfers short-chain alkanoyl groups from their corresponding CoA thioesters to the C10 hydroxyl of 10-deacetylbaccatin III. This 10-O-acetyltransferase along with five other Taxus acyltransferases on the paclitaxel (Taxol) biosynthetic pathway and one additional Taxus-derived acyltransferases of unknown function were screened for 4-O-acetyltransferase activity against 4-deacetylbaccatin III, 7-acetyl-, 13-acetyl-, and 7,13-diacetyl-4-deacetylbaccatin III. These 4-deacyl derivatives were semisynthesized from the natural product baccatin III via silyl protecting group manipulation, regioselective reductive ester cleavage with sodium bis(2-methoxyethoxy)aluminum hydride, and regioselective acetylation with acetic anhydride. Assays with the 4-deacetylated diterpene substrates and acetyl CoA revealed the taxane 10beta-O-acetyltransferase was able to catalyze the 4-O-acetylation of 4-deacetylbaccatin III to baccatin III and 13-acetyl-4-deacetylbacatin III to 13-acetylbaccatin III, although each was converted at lesser efficiency than with the natural substrate. In contrast, this enzyme was unable to acetylate 7-acetyl-4-deacetylbaccatin III and 7,13-diacetyl-4-deacetylbaccatin III substrates at C4, suggesting that the C7 hydroxyl of baccatin III must remain deacylated for enzyme function. The biocatalytic transfer of an acyl group to the tertiary hydroxyl on the oxetane moiety at C4 of the taxane ring demonstrates that the regiochemistry of the 10beta-acetyltransferase is mutable.

A novel single-walled carbon nanotube (SWNT)-based tumor-targeted drug delivery system (DDS) has been developed, which consists of a functionalized SWNT linked to tumor-targeting modules as well as prodrug modules. There are three key features of this nanoscale DDS: (a) use of functionalized SWNTs as a biocompatible platform for the delivery of therapeutic drugs or diagnostics, (b) conjugation of prodrug modules of an anticancer agent (taxoid with a cleavable linker) that is activated to its cytotoxic form inside the tumor cells upon internalization and in situ drug release, and (c) attachment of tumor-recognition modules (biotin and a spacer) to the nanotube surface. To prove the efficacy of this DDS, three fluorescent and fluorogenic molecular probes were designed, synthesized, characterized, and subjected to the analysis of the receptor-mediated endocytosis and drug release inside the cancer cells (L1210FR leukemia cell line) by means of confocal fluorescence microscopy. The specificity and cytotoxicity of the conjugate have also been assessed and compared with L1210 and human noncancerous cell lines. Then, it has unambiguously been proven that this tumor-targeting DDS works exactly as designed and shows high potency toward specific cancer cell lines, thereby forming a solid foundation for further development.

LPS is the primary ligand of Toll-like receptor 4, activating it through binding to its accessory protein MD-2. Murine but not human cells expressing MD-2/TLR4 are also activated by paclitaxel. Paclitaxel binds to human MD-2. The binding site of paclitaxel overlaps with the binding site of bis-ANS and LPS, which results in the ability of taxanes to inhibit LPS signaling in the system with human receptors. Circular dichroic spectra of human MD-2 indicated differences in the chemical environment in the presence of paclitaxel and docetaxel. Molecular docking identified the interacting residues of MD-2 and suggests that hydrophobic interactions govern the binding, while the C-3'N group where the paclitaxel and docetaxel differ is exposed on the surface of MD-2.

Background: Anthracyclines and taxanes are commonly used in the treatment of breast cancer. However, tumor resistance to these drugs often develops, possibly due to overexpression of drug transporters. It remains unclear whether drug resistance in vitro occurs at clinically relevant doses of chemotherapy drugs and whether both the onset and magnitude of drug resistance can be temporally and causally correlated with the enhanced expression and activity of specific drug transporters. To address these issues, MCF-7 cells were selected for survival in increasing concentrations of doxorubicin (MCF-7DOX-2), epirubicin (MCF-7EPI), paclitaxel (MCF-7TAX-2), or docetaxel (MCF-7TXT). During selection cells were assessed for drug sensitivity, drug uptake, and the expression of various drug transporters., Results: In all cases, resistance was only achieved when selection reached a specific threshold dose, which was well within the clinical range. A reduction in drug uptake was temporally correlated with the acquisition of drug resistance for all cell lines, but further increases in drug resistance at doses above threshold were unrelated to changes in cellular drug uptake. Elevated expression of one or more drug transporters was seen at or above the threshold dose, but the identity, number, and temporal pattern of drug transporter induction varied with the drug used as selection agent. The pan drug transporter inhibitor cyclosporin A was able to partially or completely restore drug accumulation in the drug-resistant cell lines, but had only partial to no effect on drug sensitivity. The inability of cyclosporin A to restore drug sensitivity suggests the presence of additional mechanisms of drug resistance., Conclusion: This study indicates that drug resistance is achieved in breast tumour cells only upon exposure to concentrations of drug at or above a specific selection dose. While changes in drug accumulation and the expression of drug transporters does occur at the threshold dose, the magnitude of resistance cannot be attributed solely to changes in drug accumulation or the activity of drug transporters. The identities of these additional drug-transporter-independent mechanisms are discussed, including their likely clinical relevance.

Supramolecular assemblies have attracted a great attention, due to their intriguing topologies and their application in various fields such as nanodevices, sensors, molecular switches, and drug delivery systems. In this study, we prepared the monosubstituted insulin with poly(ethylene glycol) (PEG, MW about 2200) and its cyclodextrin (CyD) polypseudorotaxanes. The pegylated insulin formed polypseudorotaxanes with alpha- and gamma-CyDs, by inserting one PEG chain in the alpha-CyD cavity and two PEG chains in the gamma-CyD cavity. The pegylated insulin/alpha- and gamma-CyD polypseudorotaxanes were less soluble in water and the release rate of the drug decreased in the order of drug alone>the gamma-CyD polypseudorotaxane>the alpha-CyD polypseudorotaxane. The plasma levels of the pegylated insulin after subcutaneous administration of the gamma-CyD polypseudorotaxane to rats were significantly prolonged, accompanying an increase in the area under plasma concentration-time curve, which was clearly reflected in the prolonged hypoglycemic effect. The results indicated that the pegylated insulin/CyD polypseudorotaxanes can work as a sustained drug release system, and the polypseudorotaxane formation with CyDs may be useful as a sustained drug delivery technique for other pegylated proteins and peptides.

Basing on the synthesis of pH-sensitive amphiphilic block copolymer poly (2-ethyl-2-oxazoline)-poly (D, L-lactide)(PEOz-PDLLA), this paper presents the preparation of docetaxel-loaded pH-sensitive block copolymer micelles using film dispersion method. The critical micelle concentration (CMC) was measured by pyrene fluorescent probe technique. The entrapment efficiency and drug-loaded amount were determined by HPLC. The morphology, diameter and surface potential of the micelles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential analyzer, respectively. The in vitro release behavior of DTX from polymeric micelles was investigated using dialysis method. The results indicated that the CMC, drug-loaded amount and entrapment efficiency of the micelles was 1.0 x 10(-3) g x L(-1), 15.0% and 91.1%, respectively. The micelles had a narrow size distribution, with a mean diameter of 28.7 nm. The micelle was globular-shaped and its zeta potential was (1.19 +/- 0.12) mV. In pH 7.4 PBS, docetaxel was released in a sustained manner from the micelles; while in PBS at pH 5.0, drug was released more rapidly, which suggested the pH-sensitive drug release behavior of the PEOz-PDLLA micelles. According to all the studies above, it can be concluded that the PEOz-PDLLA block copolymer micelles may be applied as promising drug delivery system for hydrophobic anti-tumor drugs.

The time-course of expression of dbat and dbtnbt genes involved in the later steps of paclitaxel biosynthesis and the intracellular taxane accumulation were investigated through a 64-day subculture interval of VI/M1 and VI/M2 Taxus baccata callus cultures. HPLC proved traces of baccatin III and an intracellular content of paclitaxel up to 90 microg/g DW. The steady-state of the respective gene transcripts was measured by quantitative real-time RT-PCR. The expression profile of dbat and dbtnbt genes was slightly different and varied within the subculture. The highest level of dbat expression was detected 24 h after inoculation followed by a decrease in both cultures. In contrast with dbat no substantially high expression of the dbtnbt gene after inoculation was observed. The impact of the conditions during inoculation on gene expression is discussed. Although the increase in transcriptional activity of both genes positively correlated with callus growth, the intracellular accumulation of paclitaxel varied during subculture with the maximum in the stationary (VI/M1) or at the end of the linear (VI/M2) phase. The increase of the steady-state mRNA level of the dbtnbt gene was followed by paclitaxel accumulation with a delay of approx. 28 (VI/M1) and 14 days (VI/M2).

A combination method of using microneedle pretreatment and elastic liposomes was developed to increase skin permeation of drugs with high molecular weight and poor water solubility. Docetaxel (DTX, MW=807.9) was chosen as a model drug. DTX liposomal systems with and without elastic properties were prepared and characterized. The effect of the developed formulations on the permeation of DTX across both rat and porcine skin was investigated in vitro. The combination effect of microneedle pretreatment and elastic liposomes on the permeability of DTX was evaluated using porcine skin in vitro. The following results were obtained: (1) Elastic liposomes loaded with DTX can enhance transdermal delivery of DTX without microneedle treatment. (2) An enhanced transdermal flux (1.3-1.4 microg/cm(2)/h) for DTX from all liposomal formulations was observed after microneedle treatment. Importantly, the lag time obtained following the application of elastic liposomes through microneedle-treated skin was decreased by nearly 70% compared with that obtained from conventional liposomes. These results suggest that the combination of elastic liposomes with microneedle pretreatment can be a useful method to increase skin permeation of drugs with high molecular weight and poor water solubility.

The regional variability of Taxus rhizosphere bacterial community composition and diversity was studied by comparative analysis of three large 16S rRNA gene clone libraries from the Taxus rhizosphere in different regions of China (subtropical and temperate regions). One hundred and forty-six clones were screened for three libraries. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the abundance of sequences affiliated with Gammaproteobacteria, Betaproteobacteria, and Actinobacteria was higher in the library from the T. xmedia rhizosphere of the temperate region compared with the subtropical Taxus mairei rhizosphere. On the other hand, Acidobacteria was more abundant in libraries from the subtropical Taxus mairei rhizosphere. Richness estimates and diversity indices of three libraries revealed major differences, indicating a higher richness in the Taxus rhizosphere bacterial communities of the subtropical region and considerable variability in the bacterial community composition within this region. By enrichment culture, a novel Actinobacteria strain DICP16 was isolated from the T. xmedia rhizosphere of the temperate region and was identified as Leifsonia shinshuensis sp. via 16S rRNA gene and gyrase B sequence analyses. DICP16 was able to remove the xylosyl group from 7-xylosyl-10-deacetylbaccatin III and 7-xylosyl-10-deacetylpaclitaxel, thereby making the xylosyltaxanes available as sources of 10-deacetylbaccatin III and the anticancer drug paclitaxel. Taken together, the present studies provide, for the first time, the knowledge of the biodiversity of microorganisms populating Taxus rhizospheres.

The microtubule binding affinities of a series of synthetic taxanes have been measured with the aims of dissecting individual group contributions and obtaining a rationale for the design of novel compounds with the ability to overcome drug resistance. As previously observed for epothilones, the positive and negative contributions of the different substituents to the binding free energies are cumulative. By combining the most favorable substitutions we increased the binding affinity of paclitaxel 500-fold. Insight into the structural basis for this improvement was gained with molecular modeling and NMR data obtained for microtubule-bound docetaxel. Taxanes with affinities for microtubules well above their affinities for P-glycoprotein are shown not to be affected by multidrug resistance. This finding strongly indicates that optimization of the ligand-target interaction is a good strategy to overcome multidrug resistance mediated by efflux pumps.

Trichomonas gallinae and Tritrichomonas foetus are flagellated parasitic protozoa of the upper digestive tract of birds and the urogenital tract of cattle, respectively. Both of these species are important in the veterinary field, due to the fact that they cause significant economic losses. Therefore, we investigated the morphology of these parasites by studying microtubule cytoskeleton organization. FLUTAX-2, an active fluorescent derivative of Taxol, was used in this study. This fluorescent taxoid binds to polymerized alphabeta-tubulin dimers. Our results showed that FLUTAX-2 was able to bind to and stabilize microtubules of intact T. gallinae and T. foetus trophozoites, allowing the microtubular cytoskeleton to be easily observed by fluorescence microscopy. T. foetus and T. gallinae had no differences in their FLUTAX-2 binding profiles. Further studies may allow this technique to be improved, and it may possibly be used as a routine laboratory method for the diagnosis of avian and bovine trichomonosis.

Taxa-4(5),11(12)-diene is the first committed precursor of functionalized taxanes such as paclitaxel, a successful anticancer drug. Biosynthesis of taxanes in yew involves several oxidations, a number of which have been shown to be catalyzed by cytochrome P-450 oxygenases. Hydroxylation of the C-5alpha of taxa-4(5),11(12)-diene is believed to be the first of these oxidations, and a gene encoding a taxa-4(5),11(12)-diene 5alpha-hydroxylase (CYP725A4) was recently described (Jennewein, S., Long, R. M., Williams, R. M., and Croteau, R. (2004) Chem. Biol. 11, 379-387). In an attempt to produce the early components of the paclitaxel pathway by a metabolic engineering approach, cDNAs encoding taxa-4(5),11(12)-diene synthase and CYP725A4 were introduced in Nicotiana sylvestris for specific expression in trichome cells. Their co-expression did not lead to the production of the expected 5alpha-hydroxytaxa-4(20),11(12)-diene. Instead, taxa-4(5),11(12)-diene was quantitatively converted to a novel taxane that was purified and characterized. Its structure was determined by NMR analysis and found to be that of 5(12)-oxa-3(11)-cyclotaxane (OCT) in which the eight-carbon B-ring from taxa-4(5),11(12)-diene is divided into two fused five-carbon rings. In addition, OCT contains an ether bridge linking C-5 and C-12 from opposite sides of the molecule. OCT was also the sole major product obtained after incubation of taxa-4(5),11(12)-diene with NADPH and microsomes prepared from recombinant yeast expressing CYP725A4. The rearrangement of the taxa-4(5),11(12)-diene ring system is thus mediated by CYP725A4 only and does not rely on additional enzymes or factors present in the plant. The complex structure of OCT led us to propose a reaction mechanism involving a sequence of events so far unknown in P-450 catalysis.

The natural product cyclostreptin reacts covalently and stoichiometrically with microtubules, at either of two amino acid residues of beta-tubulin, Thr-218 or Asn-226, but much less extensively and only at Thr-218 in unpolymerized tubulin. It was found that 8-acetylcyclostreptin (8AcCS) induces tubulin assembly in a manner almost identical with that of cyclostreptin. We therefore synthesized [ (14)C-acetyl]8AcCS and studied the kinetics of its interaction with glutaraldehyde-stabilized microtubules and with unassembled tubulin. With the microtubules, we found that 8AcCS bound rapidly, with a minimal (unmeasurable with the radiolabeled analogue) lag prior to the occurrence of the covalent reaction. Apparent reaction rate constants for the overall reaction ranged from 6.2 x 10 (2) M (-1) s (-1) at 0 degrees C to 5.6 x 10 (3) M (-1) s (-1) at 20 degrees C. The rate constants obtained at 0 and 10 degrees C indicate an activation energy for the reaction of about 27 kcal/mol, while those obtained at 10 and 20 degrees C indicate an activation energy of about 7.7 kcal/mol. With the unpolymerized tubulin, we did find a minimal covalent reaction occurred without apparent microtubule assembly, but a substantial reaction only occurred following assembly. In conclusion, the radiolabeled 8AcCS shows that an extensive covalent interaction of ligand with tubulin requires microtubule assembly and that the covalent reaction occurs rapidly after the initial binding interaction.

To investigate how taxane's substituents at C3' affect its metabolism, we compared the metabolism of cephalomannine and paclitaxel, a pair of analogs that differ slightly at the C3' position. After cephalomannine was incubated with human liver microsomes in an NADPH-generating system, two monohydroxylated metabolites (M1 and M2) were detected by liquid chromatography/tandem mass spectrometry. C4'' (M1) and C6alpha (M2) were proposed as the possible hydroxylation sites, and the structure of M1 was confirmed by (1)H NMR. Chemical inhibition studies and assays with recombinant human cytochromes P450 (P450s) indicated that 4''-hydroxycephalomannine was generated predominantly by CYP3A4 and 6alpha-hydroxycephalomannine by CYP2C8. The overall biotransformation rate between paclitaxel and cephalomannine differed slightly (184 vs. 145 pmol/min/mg), but the average ratio of metabolites hydroxylated at the C13 side chain to C6alpha for paclitaxel and cephalomannine varied significantly (15:85 vs. 64:36) in five human liver samples. Compared with paclitaxel, the major hydroxylation site transferred from C6alpha to C4'', and the main metabolizing P450 changed from CYP2C8 to CYP3A4 for cephalomannine. In the incubation system with rat or minipig liver microsomes, only 4''-hydroxycephalomannine was detected, and its formation was inhibited by CYP3A inhibitors. Molecular docking by AutoDock suggested that cephalomannine adopted an orientation in favor of 4''-hydroxylation, whereas paclitaxel adopted an orientation favoring 3'-p-hydroxylation. Kinetic studies showed that CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel due to an increased V(m). Our results demonstrate that relatively minor modification of taxane at C3' has major consequence on the metabolism.

For years, the microtubule-stabilizing agents paclitaxel and docetaxel (progenitors of the family of taxanes) have been the most successful anticancer drugs currently used in clinics. However, both drugs are associated with notorious side effects, drug resistance, and cross resistance with other chemotherapeutic agents. These limitations have led to the search for new drugs with improved biological activity. In the present paper, we discuss the interaction of taxanes with the tubulin/microtubule system by the formulation of 6 QSARs. Hydrophobicity of the substituents (pi) is found to be one of the most important determinants of the activity followed by steric parameters. Parabolic correlations (eqs 3 and 7) with B5 and pi are the most encouraging examples, where the optimum values of these parameters are well defined. We believe that these two QSARs may prove to be adequate predictive models that can help to provide guidance in design/synthesis and subsequently yield very specific compounds (IV and VIII) that may have high biological activities. On the basis of these two QSARs 3 and 7, 18 compounds (IV-12- IV-22 and VIII-16- VIII-22) are suggested as potential synthetic targets. Cross-validation, quality factor (Q), Fischer statistics (F), and Y-randomization tests have validated all the QSAR models.

Paclitaxel (Taxol((R))) binding to the conformation of human serum albumin assumed in the presence of long-chain fatty acids was studied by automated docking. Reduced binding affinities at both the primary and secondary sites were predicted, compared to those characterizing the interaction with the fatty acid-free protein. The baccatin core of paclitaxel was found to play a more important role than its C13 side chain in determining the ligand binding mode as well as in contributing to the overall binding energy at the primary site.

Luteibacter sp., a new bacterium isolated from the soil around a Taxus cuspidata Sieb. et Zucc plant, was studied for its capability to metabolize cephalomannine (1). After preparative fermentation, eight metabolites were obtained and characterized as baccatin III (2), baccatin V (3), 10-deacetylbaccatin III (4), 10-deacetyl-10-oxobaccatin V (5), 7-epicephalomannine (6), 10-deacetylcephalomannine (7), 10-deacetyl-7-epicephalomannine (8), and 3'-N-debenzoyl-3'-N-(2-methylbutyryl)-7-epitaxol (9). Among these metabolites, 9 is a new compound. Epimerization of the 7beta-OH group and hydrolysis of the C-13 side-chain were the two major reactions in this bioprocess. However, the biotransformation of 7beta-D-xylosyl-10-deacetyltaxol (10) with the same strain yielded a C-13 side-chain eliminated product without epimerization at C-7 (11). Metabolites 5-9 and 11, together with 1 and paclitaxel, were evaluated for their inhibitory activities against five human cancer cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780). All these compounds showed less potent activities than paclitaxel, which is currently used in clinical chemotherapy.

The effect of common organic solvents on the activities of various human cytochromes P450 has been reported. However, very little is known about their influence on CYP2B6 and CYP2C8 enzymes. The purpose of this study was to investigate the effect of solvents on the kinetics of representative CYP2B6 (bupropion hydroxylase) and CYP2C8 (paclitaxel hydroxylase) reactions in human liver microsomes. Methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied at increasing volumes (v/v). Acetonitrile, DMSO, and ethanol were shown to increase the Km and decrease the intrinsic clearance (CLint) of CYP2B6-mediated bupropion hydroxylation in a concentration-dependent manner. These solvents did not noticeably alter the Vmax at concentrations of < or =1% (v/v). Unlike the other solvents studied, the effect of methanol (< or =0.5%, v/v) on CYP2B6 kinetics was negligible. Both DMSO and ethanol increased the Km and decreased the CL(int) of CYP2C8-mediated paclitaxel hydroxylation in a concentration-dependent manner. Acetonitrile had minimal influence on CYP2C8 enzyme kinetics at concentrations of < or =1% (v/v). Methanol decreased the Km of paclitaxel at low concentrations followed by an increase at concentrations of > or =2% (v/v). This differential influence on Km resulted in an increased CLint at low concentrations followed by a decrease at high concentrations. The studied solvents had minimal influence on Vmax of paclitaxel. Collectively, DMSO and ethanol were not suitable for characterizing CYP2B6- and CYP2C8-mediated reactions because they showed concentration-dependent inhibition. Methanol and acetonitrile at concentrations of < or =0.5% and < or =1% (v/v) appeared to be suitable for the measurement of CYP2B6- and CYP2C8-mediated activities, respectively.

First-pass metabolism is a common cause of incomplete and variable absolute bioavailability for an orally dosed drug. The drug-metabolizing enzyme CYP3A4 is often implicated in this process, resulting, in some cases, in systemic exposures of less than 15% of the administered dose. By creating an elegant CYP3A4-transgenic mouse model, van Herwaarden et al. show in this issue of the JCI that first-pass metabolism of the anticancer agent docetaxel by the gut wall, and not the liver, is likely to be the major cause of its low oral bioavailability in humans (see the related article beginning on page 3583). This study helps explain interpatient differences in efficacy and safety following oral therapy with approved CYP3A4 substrates and provides a powerful new tool for preclinical predictions of first-pass metabolism for new drugs in development.

Objective: The anticancer drug docetaxel is extensively metabolized by cytochrome P450 (CYP) 3A isozymes. Furthermore, docetaxel is also a substrate for the transmembrane ATP-binding cassette efflux transporter protein ABCB1. CYP3A-inhibition significantly reduces docetaxel total systemic clearance, on average by 50%. However, data on the effect of CYP3A-inhibition on the fecal and urinary excretion of docetaxel are lacking. To further elucidate the role of CYP3A- and ABCB1-mediated elimination pathways for docetaxel we investigated the effect of the potent CYP3A-inhibitor, and also ABCB1-inhibitor, ketoconazole on the fecal and urinary disposition of docetaxel in cancer patients., Methods: Fifteen patients were treated with docetaxel (100 mg/m2), followed 3 weeks later by a reduced dose in combination with orally administered ketoconazole, or vice versa. Six patients were also administered [3H]-radiolabeled docetaxel. Fecal and urinary specimens, collected up to 72-h post-infusion, were analyzed for cumulative parent drug and radioactivity excretion., Results: Ketoconazole coadministration increased fecal parent drug excretion twofold from 2.6 +/- 2.8 to 5.2 +/- 5.4% (mean +/- SD, P = 0.03) but did not affect urinary parent drug excretion (P = 0.69). The sum of fecal and urinary parent drug excretion was 5.3 +/- 3.0% for docetaxel alone and 7.8 +/- 5.6% in the presence of ketoconazole, respectively (P = 0.04). Total recovered radioactivity values were 45.8 +/- 19.1 and 32.4 +/- 19.7%, respectively (P = 0.23)., Conclusion: CYP3A-inhibition by ketoconazole increases fecal parent drug excretion twofold in cancer patients. A more pronounced increase was not achieved, most likely due to concomitant intestinal ABCB1-inhibition.

Response rates of tumours to docetaxel (DOCT) are 45-60% in advanced breast cancer but problems associated with side effects, drug resistance and high costs occur. Conjugated linoleic acids (CLAs) also have anti-tumorigenic activity that elicits similar changes in oncogene expression to DOCT and could augment DOCT efficacy. CLA isomers appear to differ in cytotoxicity toward cancer cells. Effects of two CLA isomers on cytotoxicity of DOCT in breast cancer cells (MCF-7; MDA-MB-231) in vitro were assessed. Cells were incubated up to 72 h with 40 microM each of LA or CLA isomers (cis-9, trans-10 CLA, or trans-10, cis-12 CLA) or a 50:50 isomer mix, alone or with DOCT (0-64 microM); a pilot study determined IC(50) and IC(70) concentrations. Treatments were concurrent (CLA and DOCT together) or sequential (CLA then DOCT). MTT assay determined cell viability. Trans-10, cis-12 CLA was the most effective fatty acid (P<0.001) and increased with treatment time. IC(50) and IC(70) concentrations of DOCT were determined, concurrently or sequentially, with and without fatty acids, in the two cell types. Concurrent treatment with trans-10, cis-12 CLA and DOCT augmented inhibition of cell growth in one or both cell lines (decreased IC(50) and IC(70) in MCF-7; P<0.05 but only IC(50) in MDA-MB-231; P<0.05). CLA mix reduced IC(50) and IC(70) in MDA-MB-231 (P<0.001) but not in MCF-7. Cis-9, trans-11 CLA and LA had no effect. Sequential treatment with CLAs then DOCT reduced IC(50) and IC(70) in MCF-7 but not in MDA-MB-231. The latter had increased IC(50) and IC(70) with LA treatment (P<0.05) and increased IC(70) with cis-9, trans-11 CLA (P<0.05) with sequential but not concurrent treatment. Longer pre-incubation times with trans-10, cis-12 CLA (24-72 h) elicited greater reductions in IC(50) and IC(70) in MCF-7 cells. Results show that CLA isomers augment anti-tumour effects of docetaxel in breast cancer cells and suggest possible dual treatment regimens.

A full-length cDNA encoding 10-deacetylbaccatin III-10-O-acetyl transferase (designated as TmDBAT), which catalyzes the acetylation of the C-10 hydroxyl group of the advanced metabolite 10-deacetylbaccatin III (10-DAB) to yield baccatin III, the immediate diterpenoid precursor of Taxol, was isolated from Taxus x media. Heterologous expression of TmDBAT in E. coli demonstrated that TmDBAT was a functional gene. Tissue expression pattern analysis revealed that TmDBAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, implying that TmDBAT was tissue-specific. Expression profiling analysis of TmDBAT under different elicitor treatments including silver nitrate, ammonium ceric sulphate and methyl jasmonate indicated that TmDBAT was an elicitor-responsive gene. Southern blot analysis suggested that TmDBAT belonged to a small multigene family.

This review covers advances in the discovery, preclinical and clinical development of potential anticancer agents based upon the diterpenoid taxane skeleton. The anticancer properties of approved clinical agents of this class are not discussed, but the review documents how, 13 years post-approval of paclitaxel (Taxol), the base taxane structure is still utilized as the starting skeleton for molecules with improved pharmacological and toxicological properties. Paclitaxel may in fact be the first example of a 'tunable' anticancer agent. In addition, paclitaxel, and perhaps other taxanes in due course, has activities beyond the known antitumor indications, with an example being the use of paclitaxel-coated stents in cardiovascular therapies.

Purpose: We previously reported that the expression of Aurora-A was frequently up-regulated in human esophageal squamous cell carcinoma (ESCC) tissues as well as cell lines and the up-regulation contributed to a poor prognosis. In this study, we assessed the possibility of Aurora-A suppression as a therapeutic target for ESCC using ESCC cell lines., Experimental Design: We established subclones using vector-based short hairpin RNA (shRNA). Then, we investigated the effect of Aurora-A suppression on proliferation and cell cycle changes in vitro. Next, chemosensitivity against docetaxel was investigated by tetrazolium salt-based proliferation assay (WST assay) and cell number determinations, and furthermore, the type of cell death induced by docetaxel was analyzed by flow cytometry. Finally, to examine the effect of Aurora-A shRNA on proliferation and chemosensitivity against docetaxel in vivo, a s.c. tumor formation assay in nude mice was done., Results: We established two genetically different stable cell lines (510 A and 1440 A) in which levels of Aurora-A were reduced. Cell growth was inhibited by 38.7% in 510 A and by 24.3% in 1440 A in vitro compared with empty vector-transfected controls (510 m and 1440 m), and this growth inhibition was mediated through G(2)-M arrest as confirmed by flow cytometry. Next, in a WST assay, the IC(50) for Aurora-A shRNA-transfected cells was lower than that of empty vector-transfected cells (510 A, 2.7 x 10(-7) mol/L; 510 m, 4.8 x 10(-7) mol/L; 1440 A, 2.6 x 10(-7) mol/L; 1440 m, 4.9 x 10(-7) mol/L). In addition, 0.3 nmol/L docetaxel induced a notable level of apoptosis in Aurora-A shRNA-transfected cells compared with empty vector-transfected cells. In the assay of s.c. tumors in nude mice, tumor growth in 510 A was inhibited by 36.1% compared with that in 510 m, and in tumors treated with docetaxel, the suppression of Aurora-A resulted in 44.0% tumor growth suppression in vivo., Conclusions: These results indicated that Aurora-A might play an important role in chemosensitivity to docetaxel, and the suppression of its expression might be a potential therapeutic target for ESCC.

Breast cancer is the second most common cause of cancer-related death in women in the US and the UK, accounting for 15-17% of all female cancer deaths. Current treatment strategies include hormone therapy, such as anti-estrogens (tamoxifen) and aromatase inhibitors (exemastane, anastrozole, letrozole), as well as cytotoxics, such as the taxanes (paclitaxel, docetaxel). With multiple therapy choices, a method to prospectively screen patients prior to therapy selection is now needed. Pharmacogenetics seeks to develop screening mechanisms to optimise drug therapy. DNA variations in metabolism, transport and drug target genes may contribute to chemotherapy efficacy and toxicities. The status of the identification of genetic markers for breast cancer therapy selection is highlighted in this review.

Previous in vitro studies revealed that the 10-deacetylbaccatin III 10beta-O-acetyltransferase (DBAT) from Taxus can catalyze the transfer of acetyl, propionyl or n-butyryl from CoA to the C10-hydroxyl of 10-deacetylbaccatin III. Accordingly, Escherichia coli JM109 were transformed to recombinantly express dbat, and this enzyme function was coupled to that of acetyl-CoA synthase (acs, EC 6.2.1.1) expressed from and regulated by genes encoded on the bacterial chromosome. Incubation of the bacteria with 10-deacetylbaccatin III and increasing concentrations of acetic acid revealed an in vivo conversion ( approximately 10%) of substrate to natural product baccatin III (C10-acetylated), which was remarkably similar to the relative conversion without acid supplementation. Incubation of the modified E. coli with 5 mM propionic acid, revealed a fivefold increase in the conversion ( approximately 13%) of 10-deacetylbaccatin III to 10-deacetyl-10-propionylbaccatin III, compared to approximately 2% conversion in the absence of exogenous propionate. To produce the butyrylbaccatin III analog in vivo, bacteria were engineered to co-express the dbat and atoAD (EC 2.8.3.8) genes; the latter encodes an acetoacetate: acetyl-CoA CoA-transferase that activates butyrate to butyryl CoA. The bacteria were incubated with 10-deacetylbaccatin III and 25-100 mM butyrate, and a maximum of approximately 2.6% conversion to 10-butyrylbaccatin III was observed compared to approximately 0.6% conversion when no exogenous butyrate was supplied.

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.

Administering Taxus suspension cells with labeled 5alpha-hydroxytaxadiene and 5alpha,10beta-dihydroxytaxadiene, and the corresponding 5alpha-acetate esters, demonstrated that acetylation at C5 of the monool precursor promotes the formation of 14beta-hydroxy taxoids, such as taxuyunnanine C, at the expense of 13alpha-hydroxy taxoids, including Taxol and its congeners, but that the major bifurcation in taxoid biosynthesis, toward 13alpha- or 14beta-hydroxy taxoids, occurs after 10beta-hydroxylation of the taxane core.

Doxorubicin is known to cause cardiomyopathy and congestive heart failure (CHF) upon chronic administration. A major obstacle to doxorubicin-containing multiagent therapies pertains to the possible development of cardiomyopathy and CHF at lower than expected cumulative doses of doxorubicin. For example, the cardiac toxicity of doxorubicin is aggravated by the anti-HER2 antibody Trastuzumab or by the tubulin-active taxane paclitaxel; however, the mechanisms by which Trastuzumab and paclitaxel aggravate doxorubicin-induced cardiotoxicity are mechanistically distinct: Trastuzumab interferes with cardiac-specific survival factors that help the heart to withstand stressor agents like anthracyclines, while paclitaxel acts by stimulating the formation of anthracycline metabolites that play a key role in the mechanism of cardiac failure. Here, we briefly review the molecular mechanisms of the cardiotoxic synergism of Trastuzumab or paclitaxel with doxorubicin, and we attempt to briefly outline how the mechanistic know-how translates into the clinical strategies for improving the safety of anthracycline-based multiagent therapies.

In the microenvironment of solid growing tumors, pronounced hypoxia or extracellular acidosis is commonly found. The aim of this study was the analysis of the cytotoxic effect of different chemotherapeutic agents (cisplatin, daunorubicin, docetaxel) under these conditions in vitro. Prostate carcinoma cells (R3327-AT1) were exposed to hypoxia (pO2 < 0.5 mmHg) or extracellular acidosis (pH = 6.6) for 6h. After 3h, cytotoxic drugs were added. The cytotoxic effect was assessed by measuring caspase 3-activity (apoptosis), LDH release (necrosis) and repopulation of the cells after chemotherapy (cell death). Compared to aerobic control conditions, severe hypoxia over 6 h per se led to a slight increase in apoptosis, necrosis and cell death. With all three chemotherapeutic agents, hypoxia led to a reduced (by approx. 25%) caspase 3-activity and a marked increase in necrosis. However, the overall cytotoxicity of the drug was not affected by O2-deficiency. By contrast, during extracellular acidosis, the cytotoxic effect of daunorubicin was reduced by 40%, preferentially due to a marked reduction in apoptosis. With cisplatin and docetaxel no change in overall cell death was detected. However, for daunorubicin the tumor-pH seems to have a strong impact on cytotoxicity. With this chemotherapeutic drug the therapeutic efficacy is markedly reduced in an acidotic environment.