Presence Of Paclitaxel Research Articles

Cytoskeleton-interacting activity of geiparvarin, diethylstilbestrol and conjugates

Geiparvarin, a natural compound isolated from the leaves of Geijera parviflora, inhibits the growth of various tumor cell lines with a mode of action which may be attributed to its anti-microtubular activity. Our previous findings indicated that geiparvarin is able to inhibit the in vitro polymerization of tubulin and to derange the microtubular network in fibroblasts more effectively in the presence of paclitaxel. To further explore its biological activity here we have studied the effects exerted on the other components of the cytoskeleton by geiparvarin and two derivatives obtained by conjugating the 3(2H)-furanone ring of geiparvarin with diethylstilbestrol (DES). Firstly, observations by electron microscopy confirmed anti-microtubular properties, a near-total absence of microtubules is detected when tubulin is incubated with drugs in the presence of paclitaxel, whereas microtubule formation is not inhibited by drugs when assembly is induced by guanosine 5′-triphosphate (GTP). Immunofluorescence assays demonstrated that geiparvarin and DES act in a vinblastine-like fashion, causing a marked depletion of intermediate filaments while the network of microfilaments is not affected. Both the conjugates alter the ‘stress fibers’ organization of actin and disrupt the vimentin pattern; generally they derange cytoskeleton more markedly than the parent compounds. The cell growth inhibiting effects of geiparvarin and derivatives are dose-dependent; they vary according to the cell line used, when compounds were administered either alone or simultaneously with paclitaxel. Unlike other anti-microtubule agents, they do not exhibit cell-cycle compartment specificity and do not influence thymidine uptake in the cell.

A phase I and pharmacologic study of capecitabine and paclitaxel in breast cancer patients

Based on preclinical studies demonstrating that treatment with paclitaxel upregulates intratumoral thymidine phosphorylase (dTHdPase), which catalyzes the final step in the conversion of the oral fluoropyrimidine capecitabine to 5-fluorouracil (5-FU), as well as the overlapping spectra of activity for these agents, particularly in metastatic breast cancer, this phase I study evaluated the feasibility of administering capecitabine on an intermittent schedule in combination with paclitaxel in previously-treated patients with locally advanced or metastatic breast cancer. The study also sought to recommend doses for subsequent disease-specific studies, identify clinically significant pharmacokinetic interactions, and detect preliminary antitumor activity. Nineteen previously treated women with metastatic breast cancer whose prior treatment included neither paclitaxel or capecitabine received one hundred one courses of capecitabine and paclitaxel. Paclitaxel was administered as a three-hour intravenous (i.v.) infusion at a fixed dose of 175 mg/m2 and capecitabine was administered as 2 divided daily doses for 14 days followed by a seven-day rest period every 3 weeks. The dose of capecitabine was increased from a starting dose of 1650 mg/m2/d. The plasma sampling scheme in the first course permitted characterization of the pharmacokinetics of each agent given alone and concurrently to detect major pharmacokinetic interactions. Palmar plantar erythrodysesthesia (hand foot syndrome) and neutropenia were the principal dose-limiting toxicities (DLT). Other toxicities included diarrhea and transient hyperbilirubinemia. Three of eight new patients treated with capecitabine 2000 mg/m2/d and paclitaxel 175 mg/m2 experienced DLT in the first course, whereas none of eleven new patients treated with capecitabine 1650 mg/m2/d and paclitaxel 175 mg/m2 developed DLT. Pharmacokinetic studies indicated that capecitabine did not grossly affect the pharmacokinetics of paclitaxel, and there were no major effects of paclitaxel on the pharmacokinetics of capecitabine and capecitabine metabolites. However, AUC values for the major 5-FU catabolite, fluorobeta-alanine (FBAL), were significantly lower in the presence of paclitaxel. Two complete and seven partial responses (56% response rate) were observed in sixteen patients with measurable disease; four of six patients whose disease was previously treated with high-dose chemotherapy and hematopoietic stem-cell support had major responses. Seven of nineteen patients had stable disease as their best response. Recommended combination doses of capecitabine on an intermittent schedule and paclitaxel are capecitabine 1650 mg/m2/d orally for 14 days and paclitaxel 175 mg/m2 i.v. every 3 weeks. The favorable preclinical interactions between capecitabine and paclitaxel, as well as the acceptable toxicity profile and antitumor activity in patients with metastatic breast cancer, support further clinical evaluations to determine an optimal role for the combination of capecitabine and paclitaxel in breast cancer and other relevant malignancies.

MEK Inhibition Enhances Paclitaxel-induced Tumor Apoptosis

The anti-cancer drug paclitaxel (Taxol) alters microtubule assembly and activates pro-apoptotic signaling pathways. Previously, we and others found that paclitaxel activates endogenous JNK in tumor cells, and the activation of JNK contributes to tumor cell apoptosis. Here we find that paclitaxel activates the prosurvival MEK/ERK pathway, which conversely may compromise the efficacy of paclitaxel. Hence, a combination treatment of paclitaxel and MEK inhibitors was pursued to determine whether this treatment could lead to enhanced apoptosis. The inhibition of MEK/ERK with a pharmacologic inhibitor, U0126, together with paclitaxel resulted in a dramatic enhancement of apoptosis that is four times more than the additive value of the two drugs alone. Enhanced apoptosis was verified by the terminal transferase-mediated dUTP nick end labeling assay, by an enzyme-linked immunosorbent assay for histone-associated DNA fragments, and by flow cytometric analysis for DNA content. Specificity of the pharmacologic inhibitor was confirmed by the use of (a) a second MEK/ERK inhibitor and (b) a transdominant-negative MEK. Enhanced apoptosis was verified in breast, ovarian, and lung tumor cell lines, suggesting this effect is not cell type-specific. This is the first report of enhanced apoptosis detected in the presence of paclitaxel and MEK inhibition and suggests a new anticancer strategy.

Paclitaxel-induced apoptosis in non–small cell lung cancer cell lines is associated with increased caspase-3 activity

Objective: Our objective was to determine whether paclitaxel-induced apoptosis in human lung cancer cells is Fas dependent. Methods: Human lung cancer cell lines were evaluated for morphologic evidence of apoptosis, DNA fragmentation (TUNEL positivity), and caspase-3 activation after paclitaxel treatment. Human lung adenocarcinoma, squamous cell carcinoma, undifferentiated lung carcinoma, and bronchoalveolar carcinoma cell lines were each cultured in 10 μmol/L paclitaxel. Results: After 24 hours of culture in paclitaxel, a 22% to 69% increase in the number of apoptotic cells was evident by means of methylene blue-azure A-eosin staining with characteristic blebbing and nuclear condensation. TUNEL assay also confirmed an increase of 19.9% to 73.0% of cells with nuclear fragmentation. Caspase-3 activity, assayed by Z-DEVD cleavage, increased from 20% to 215% ( P < .05). ZB4, an antagonistic anti-Fas antibody, did not block paclitaxel induction of caspase-3 activity (155.8 vs 165.8 U, not significant). Apoptotic morphologic changes were inhibited in cells cultured in the presence of paclitaxel and Ac-DEVD-CHO, a caspase-3 inhibitor. Conclusions: Paclitaxel induces apoptosis in lung cancer cell lines, as assessed by a consistent increase in caspase-3 activity, DNA laddering, and characteristic morphologic changes. Paclitaxel-induced apoptosis in human lung cancer cells is associated with caspase-3 activation but is not Fas dependent. (J Thorac Cardiovasc Surg 2000;119:795-803)

Equilibrium studies of a fluorescent paclitaxel derivative binding to microtubules.

A fluorescent derivative of paclitaxel, 3'-N-m-aminobenzamido-3'-N-debenzamidopaclitaxel (N-AB-PT), has been prepared in order to probe paclitaxel-microtubule interactions. Fluorescence spectroscopy was used to quantitatively assess the association of N-AB-PT with microtubules. N-AB-PT was found equipotent with paclitaxel in promoting microtubule polymerization. Paclitaxel and N-AB-PT underwent rapid exchange with each other on microtubules assembled from GTP-, GDP-, and GMPCPP-tubulin. The equilibrium binding parameters for N-AB-PT to microtubules assembled from GTP-tubulin were derived through fluorescence titration. N-AB-PT bound to two types of sites on microtubules (K(d1) = 61 +/- 7.0 nM and K(d2) = 3.3 +/- 0.54 microM). The stoichiometry of each site was less than one ligand per tubulin dimer in the microtubule (n(1) = 0.81 +/- 0.03 and n(2) = 0.44 +/- 0.02). The binding experiments were repeated after exchanging the GTP for GDP or for GMPCPP. It was found that N-AB-PT bound to a single site on microtubules assembled from GDP-tubulin with a dissociation constant of 2.5 +/- 0.29 microM, and that N-AB-PT bound to a single site on microtubules assembled from GMPCPP-tubulin with a dissociation constant of 15 +/- 4.0 nM. It therefore appears that microtubules contain two types of binding sites for paclitaxel and that the binding site affinity for paclitaxel depends on the nucleotide content of tubulin. It has been established that paclitaxel binding does not inhibit GTP hydrolysis and microtubules assembled from GTP-tubulin in the presence of paclitaxel contain almost exclusively GDP at the E-site. We propose that although all the subunits of the microtubule at steady state are the same "GDP-tubulin-paclitaxel", they are formed through two paths: paclitaxel binding to a tubulin subunit before its E-site GTP hydrolysis is of high affinity, and paclitaxel binding to a tubulin subunit containing hydrolyzed GDP at its E-site is of low affinity.

A radioimmunoassay for the measurement of paclitaxel and taxanes in biological specimens using commercially available reagents.

We have developed a rapid and sensitive radioimmunoassay for the measurement of paclitaxel and related taxanes in biological specimens using commercially available reagents. The presence of paclitaxel competitively inhibited the binding of radioactive paclitaxel to polyclonal anti-taxane antibody or anti-taxane monoclonal antibody. When using polyclonal antibody, this method detected paclitaxel in concentrations as low as 0.87 nM and was also effective in 20% methanol. This radioimmunoassay is useful for the measurement of paclitaxel and taxanes in biological specimens like culture medium of paclitaxel-producing microorganisms and may be useful for the measurement of paclitaxel and related compounds in other potential natural sources.

Isolation of a novel microorganism, pestalotia heterocornis, producing paclitaxel

Pestalotia heterocornis was isolated from soil collected in yew forest and was shown to produce paclitaxel in semisynthetic liquid media. The presence of paclitaxel in the fungal extract was confirmed by FAB mass spectrometry and NMR spectroscopy. The maximum yield of paclitaxel was 31 &mgr;g per liter. Optimal paclitaxel production occurred after 5-7 days in a 20-liter scale fermentation at 23 degrees C. These results indicate that P. heterocornis is an excellent candidate for consideration in fermentation technology. Copyright 1999 John Wiley & Sons, Inc.

Determination of taxane concentrations in Taxus canadensis clippings using high performance liquid chromatographic analysis with an internal standard

Clippings of Taxus canadensis were collected and divided into needles, twigs and whole clippings samples. The biomass was extracted and analysed for taxanes using high performance liquid chromatography. The addition of n-octylbenzamide as an internal standard was found to reduce the standard deviation of calculated yields by a third. Paclitaxel was found to be highest in needles (0.0434% dry wt). The presence of paclitaxel was confirmed by photodiode array spectra. Impurities were found to co-elute with other taxanes. A compound was tentatively identified as 9-dihydro-13-acetylbaccatin III based on residence time and UV spectra. The effect of storage on paclitaxel content was investigated and it was found that paclitaxel was stable in dried needles stored at room temperature for at least 1 year. Copyright © 1999 John Wiley & Sons, Ltd.

Differential Assembly Kinetics of α-Tubulin Isoforms in the Presence of Paclitaxel

The antitumor drug paclitaxel (PTX) inhibits cell growth by binding to microtubules, the eukaryotic structures consisting of α- and β-tubulin. PTX also promotes the assembly of tubulin in the absence of microtubule-associated proteins. Although recent studies have implicated β-tubulin as the site of PTX binding, no information is available that relates α-tubulin to the binding site. In an effort to understand whether the α-tubulin is involved in the drug binding, we have studied the assembly of α-tubulin isoforms in the presence of PTX. The assembly results in the presence of 10 μM paclitaxel (PTX) show that the isoforms assemble at differential rates. The rate of assembly for tyrosinated Mα1/2 is about three-fold higher than that of the nontyrosinated Mα1/2 isoform. Such a strikingly different assembly behavior of the α-tubulin isoforms indicates that α-tubulin may be involved in the interaction of PTX with microtubules.

Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues.

To determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent analogues in which a (dimethylamino)benzoyl group has been introduced into the 7- and 10-positions, and the benzoyl groups at the 2- and N- as well as the 3'-phenyl ring have been modified with dimethylamino functions. In a tubulin assembly assay, the N-m- and N-p-(dimethylamino)benzoyl derivatives had activities comparable to the activity of paclitaxel. The 2-, 3'-, and 10-analogues had slightly reduced activity, and the 7-derivative was about 5% as active as paclitaxel. On the basis of the results of studies of the effect of solvents on the fluorescence emission spectra, it is proposed that the unbound analogues form hydrogen bonds with protic solvents. But the 7- and 10-substituted analogues appear to be more affected by protic solvents than the other analogues. Previously, we studied the binding of the N-meta derivative to tubulin and microtubules [Sengupta, S., et al. (1995) Biochemistry 34, 11889-11894]. In this study, we extended the studies to include the 2-, 7-, and 10-derivatives. Similar to the N-substituted analogue, binding of the 2-derivative to tubulin was accompanied by a large blue shift, whereas a very small shift occurred when the 7- and 10-substituted derivatives bound. The 2- and N-substituted analogues bind to microtubules with an increase in fluorescence intensity over that which was observed with tubulin, whereas binding of the 7- and 10-substituted analogues was accompanied by a large quenching in fluorescence. This quenching may be due to the presence of charged residues in the protein near the 7- and 10-(dimethylamino)benzoyl groups or to pi stacking of the groups with an aromatic side chain. The presence of paclitaxel with microtubules prevented the fluorescence increase of the 2- and N-derivatives and quenching of the 7- and 10-derivatives. The difference in behavior of the fluorescent analogues upon binding to polymerized tubulin, coupled with the solvent studies on the free drugs, suggests that the 2- and N-benzoyl groups of paclitaxel bind in a hydrophobic pocket of tubulin but could participate in hydrogen bonding, and the 7- and 10-positions are in a more hydrophilic environment.

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography.

Vinorelbine tartrate and paclitaxel combinations: enhanced activity against in vivo P388 murine leukemia cells.

Many critical cellular functions such as mitosis, cell movement, and maintenance of cell structure are performed by microtubules. Antimicrotubule agents (which disrupt or block the formation of microtubules) are among the most widely used anticancer drugs and have contributed to the curative therapy of many neoplasms. Recently, two new antimicrotubule agents, vinorelbine tartrate (Navelbine) and paclitaxel (Taxol) have demonstrated clinical activity against ovarian, breast, and non-small-cell lung carcinomas. These agents target microtubules at different sites, and they both interfere with mitotic spindle function. Since vinorelbine tartrate and paclitaxel have shown a similar antitumor profile in clinical trials thus far, it is reasonable to expect that they may be used interchangeably in some combination therapies or perhaps with each other in the same treatment regimen. On the basis of their similar activity profile in clinical trials, we decided to investigate the therapeutic outcome of a vinorelbine tartrate and paclitaxel binary drug combination, even though they appeared to have overlapping toxic effects. We wanted to ascertain the effect of this binary drug combination, in an in vivo setting, as it related to host toxicity and antitumor activity. CDF-1 female mice that were implanted intraperitoneally with one million P388 murine leukemia cells were treated intraperitoneally with vinorelbine tartrate, paclitaxel, or a combination of the two drugs on a day-1, -5, and -9 dosing schedule. Experimental groups had between five and eight mice per group. Vinorelbine tartrate was administered at either 8, 12, 16, 20, or 24 mg/kg and paclitaxel at either 4.5, 18, or 36 mg/kg. As single agents, neither vinorelbine tartrate nor paclitaxel generated meaningful numbers of 60-day cures (i.e., tumor free at day 60). In contrast, optimal combination regimens produced 60-day cures in more than 80% of the mice. The LD10 (dose lethal to 10% of the mice) of vinorelbine tartrate increased approximately 2.5-fold in the presence of paclitaxel and allowed otherwise lethal vinorelbine tartrate doses to be administered safely, which may have contributed to the antitumor efficacy of the combinations. The effect of the time delay between vinorelbine tartrate and paclitaxel administration on toxicity and cures appeared to be contingent on the vinorelbine tartrate dose. Results suggest that the overlapping toxic effects of vinorelbine tartrate and paclitaxel might not be a deterrent to their use in combination drug therapy. When used appropriately, rather than having enhanced toxic effects, otherwise toxic doses were better tolerated and survival improved over single-agent therapy.

Cycloheximide inhibits the cytotoxicity of paclitaxel (Taxol®)

Treatment of human breast (MCF-7) and lung (A549) adenocarcinoma cell lines with 10 micrograms/ml cycloheximide provided substantial protection from paclitaxel-induced cytotoxicity. Addition of cycloheximide to cells at 0, 6, 12 or 18 h into a 24 h exposure to paclitaxel resulted in cytotoxicity similar to that found in cells treated with paclitaxel alone for only 0, 6, 12 or 18 h, respectively. DNA flow cytometry showed that paclitaxel blocked cells in G2/M. Mitotic index studies demonstrated that paclitaxel arrested cells in mitosis and that prolonged exposure to paclitaxel resulted in the development of multiple micronuclei. Concurrent incubation of cells in cycloheximide prevented the development of a G2/M block, mitotic arrest and micronuclei formation. The addition of cycloheximide to cells at 6 or 12 h into a 24 h exposure to paclitaxel reduced the degree of G2/M block to that produced by incubation of cells in paclitaxel alone for only 6 or 12 h. Mitotic index studies confirmed that cells treated with cycloheximide during paclitaxel exposure had a marked reduction in the percentage of cells in mitosis. However, the percentage of paclitaxel-treated cells which had multiple micronuclei was increased in cells treated with cycloheximide. These results indicate that entry into mitosis is a prerequisite for paclitaxel-induced cytotoxicity and that cycloheximide reduces cytotoxicity due to paclitaxel by preventing cells from entering mitosis. However, once cells have entered mitosis in the presence of paclitaxel, protein synthesis is not required for the development of multiple micronuclei and cytotoxicity.