Taxane Concentrations Research Articles

Long-term taxane exposure and transdifferentiation of prostate cancer in vitro.

254 Background: Development of aggressive variants of metastatic castration-resistant prostate cancer (AVPC) is a major challenge in the course of therapy but the underlying mechanisms of aggressive transdifferentiation are not completely understood and appropriate tumor models are missing. Here, we investigated the consequences of long-term taxane exposure on hormone-independent, BRCA2-mutated, AR-V7-positive 22Rv1 cells. Methods: 22Rv1 cells were treated with stepwise increased taxane concentrations for 10 months. Individual clones were picked and further cultured in media containing either docetaxel (Doce) or cabazitaxel (Caba). Passage-matched cells were maintained in culture without treatment. Further characterization was carried out using proliferation, migration, metabolic, and colony formation assays as well as proteomics, RNAseq analyses and xenotransplantation in immunodeficient mice. Results: In total, three single cell 22Rv1-DR clones (50-100-fold resistance to Doce) and three 22Rv1-CR clones (80-150-fold resistance to Caba) were successfully established. All clones showed cross-resistance to either drug. Expectedly, treatment-induced overexpression of ABCB1 was detected and validated. Moreover, alteration of drug resistance related SLC7A5, SLC3A2, and SLC25A24 genes was observed. Additionally, an enrichment analyses identified, among others, neuroendocrine transdifferentiation (GO-term “Neuroendocrine tumors”, p=4.46e-5) to be stimulated in prostate 22Rv1 cells under long-term treatment with Doce or Caba. In line with this, the neuroendocrine features were validated in vitro as well as in xenotransplanted tumors in vivo with upregulation of synaptophysin, chromogranin and neuron specific enolase accompanied by downregulation of the androgen receptor (AR) and upregulation of AR spice variants. Additionally, neuritic morphology, shift to higher nuclear-plasma ratio, partial loss of adherent properties and growth slowdown, along with higher migratory activity were detected. Conclusions: Long-term taxane exposure of 22Rv1 cells resulted in the development of neuroendocrine traits in individual cell clones that have successfully been translated into stable cell lines. Thus, we provide a new cell line model for secondary therapy-induced neuroendocrine transdifferentiation. Further in-depth analysis to identify individual alterations in the course of therapy is currently ongoing.

Paclitaxel use in pregnancy: neonatal follow-up of infants with positive detection of intact paclitaxel and metabolites in meconium at birth.

Up to 3years of age, follow-up of neonates exposed to Paclitaxel in utero is reassuring. Continued observation of neonatal development is essential. • Chemotherapy during the second and third trimester of pregnancy does not result in an increase in congenital malformations or developmental delay. • In non-human primate studies by Van Calsteren et al., variable plasma and/or tissue concentrations of taxanes, carboplatin, and trastuzumab were encountered in the fetal compartment. • Pilot data reported by the current investigators proved that paclitaxel crosses the human placenta. • This current article provides medical and developmental follow up on the newborns from this exposure for 3 years after birth.

Mechanisms of inhibition of endothelial cell migration by taxanes

Several antiangiogenic mechanisms have been proposed for the widely-used cancer chemotherapeutic drugs taxotere (docetaxel) and taxol (paclitaxel), but none has been definitively identified. We analyzed their effects at a range of concentrations on migration and mitosis of human umbilical vein endothelial cells (HUVECs) on microtubule and focal adhesion morphology and microtubule dynamic instability during migration. Both taxanes inhibited migration by inhibiting both maintenance of the forward direction of the cell and by slowing migration over the entire contorted path length. At low (but not all) taxane concentrations that inhibit HUVEC migration, the shortening rates and shortening lengths of microtubules at the leading edge were strongly inhibited; peripheral microtubules were reduced in number and fewer targeted focal adhesions; focal adhesions doubled in length and became ring-shaped, elongate, and reduced in number; and an increase in stabilized microtubules occurred centrally. HUVEC migration was 13-19-fold more sensitive to taxanes than was mitosis confirming that taxanes exhibit significant effects in addition to mitotic arrest that may contribute to their overall antitumor effects. No relationship was detected between centrosome position and migration characteristics. The data suggest that taxanes inhibit migration, at least in part, by inhibiting the dynamic instability of microtubules that possibly interact with guanine nucleotide exchange factors and thus with the focal adhesion-associated G-proteins that then lead to disruption of the regulated formation and turnover of focal adhesions. A mechanism is presented by which subcytotoxic concentrations of taxanes might stabilize dynamic instability of a few microtubules and thereby inhibit migration and angiogenesis.

Abstract A99: Docetaxel-resistance can be overcome by enhancing intratumoral taxane concentrations to a threshold level for efficacy.

Abstract Introduction / objectives: Metastatic castration resistant prostate cancer (mCRPC) patients are treated with docetaxel (DOC) as a first line of treatment. Although DOC improves survival of mCRPC patients, resistance to DOC is commonly developed, hampering clinical efficacy. We hypothesized that there is a minimum exposure level for antitumor activity and therefore investigated the relation between intratumoral concentrations and antitumor activity for DOC and second-line treatment cabazitaxel (CABA) in DOC-naive and DOC-resistant patient-derived prostate cancer (PC) xenograft models. Materials and methods: DOC-resistant PC xenografts were generated previously by treatment of tumor-bearing mice with a single dose of DOC (33 mg/kg) followed by additional dosing at growth recurrence and upon subsequent passaging. Intratumoral concentrations of DOC and CABA were measured in the following xenografts: PC374 (DOC-sensitive), PC339 (poorly sensitive) and in the PC339-DOC resistant xenograft. Naïve PC339 and PC374 bearing mice were treated with a single injection of DOC or CABA of 8.3; 16.5 or 33 mg/kg. PC339-DOC -bearing mice, resistant to DOC at 33 mg/kg, were treated with a single injection of DOC 16.5; 33 or 50 mg/kg and sacrificed at 7 days post treatment. Intratumoral concentrations of DOC and CABA were correlated to the log ratio of tumor volume (TV) at start of treatment (day 0) divided by TV at sacrifice (day 7). Results: In mice dosed with DOC 33 mg /kg, intratumoral concentrations were higher in PC374 compared to PC339: 1.6 ± 05ng/mg tissue and 0.28 ± 0.13 ng/mg tissue, respectively (P =0.001). Moreover, CABA levels were higher than those of DOC in both PC374 and PC339 tumors: 5.3 ± 1.3 ng/mg tissue and 4.1 ± 1.8 ng/mg tissue, respectively (P= 0.0005, P= 0.005). In PC339 tumors, CABA antitumor activity and intratumoral concentrations were strongly correlated (r = -0.87, P <0.0001). Although DOC at 33 mg/kg was inactive in PC339-DOC bearing mice, increased dosing to 50 mg/kg may overcame drug resistance, resulting in a significant increase in intratumoral concentrations from 0.29 ± 0.09 ng/mg after dosing with 33 mg/kg to 1.1 ± 0.25 ng/mg after treatment with 50 mg/kg (P=0.005). In this xenograft antitumor response and intratumoral concentrations were strongly correlated (r=-0.84, P=0.004). For both DOC and CABA, a minimum exposure level (or ‘threshold’ concentration) for anti-tumor efficacy of 1.5 ng taxane/mg tumor tissue was found for naïve and DOC-resistant PC xenografts. Conclusions: These preclinical data pinpoint a sustained relationship between intratumoral concentrations and antitumor activity of taxanes in DOC resistant PC models. A minimum exposure level for effective intratumoral concentrations for both taxanes was defined that still applies in DOC-resistance. These data indicate that efforts to improve taxane uptake in DOC-resistant tumors may still be a relevant strategy to improve treatment outcome of mCRPC patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A99. Citation Format: Ellen S. de Morree, Corrina M. de Ridder, Peter de Bruijn, Herman Burger, Erik A.C. Wiemer, Ron H.J. Mathijssen, Ronald de Wit, Wytske van Weerden. Docetaxel-resistance can be overcome by enhancing intratumoral taxane concentrations to a threshold level for efficacy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A99.

Transplacental transfer of paclitaxel, docetaxel, carboplatin, and trastuzumab in a baboon model.

The paucity of data on fetal effects of prenatal exposure to chemotherapy prompted us to study the transplacental transport of commonly used anticancer agents in a pregnant baboon model. Single or combination chemotherapy with paclitaxel, docetaxel, carboplatin, and trastuzumab was administered to 9 baboons at a mean (SD) gestational age of 117 (26) days (paclitaxel, 100 mg/m2 [n = 2]; docetaxel, 100 mg/m2 [n = 2]; paclitaxel, 175 mg/m2 with carboplatin, area under the curve of 6 at standard dosage [n = 2] and 50% dosage [n = 1]; docetaxel, 75 mg/m2 with carboplatin, area under the curve 6 [n = 1]; and docetaxel, 75 mg/m2 with trastuzumab, 8 mg/kg [n = 1]). Serial fetal and maternal blood samples, amniotic fluid, maternal urine, and fetal and maternal tissue samples were collected for the first 76 hours after drug infusion. Levels of carboplatin were determined by atomic absorption spectrometry, docetaxel and paclitaxel by high-performance liquid chromatography, and trastuzumab by enzyme-linked immunosorbent assay. Fetal plasma concentrations of carboplatin averaged 57.5% (14.2%) of maternal concentrations (n = 7). Fetal plasma concentrations were 1.5% (0.8%) of maternal concentrations (n = 7). Immediately after ending the infusion, paclitaxel was not detectable in fetal tissues, whereas, after 3 hours, fetal tissues contained 15% of maternal tissue concentrations.Docetaxel could not be detected in fetal blood samples (n = 9). In the first 3 hours after docetaxel infusion, fetal tissues contained 5.0% to 50.0% of maternal tissue concentrations, whereas equal fetal and maternal tissue concentrations were found after 26 and 76 hours.The transplacental passages of trastuzumab were 85.0% and 3.0%, 2 and 26 hours after trastuzumab infusion, respectively. After 26 hours, amniotic fluid contained 36.4% of the fetal plasma concentration. Fetal tissue concentrations varied between 5.0% and 14.0% of the maternal concentration. Variable plasma and/or tissue concentrations of taxanes, carboplatin, and trastuzumab were encountered in the fetal compartment. These data are important when cancer treatment is considered during pregnancy and underline the need for long-term follow-up of children after prenatal exposure to these cytotoxic agents.

Seasons Study of Four Important Taxanes and Purification of 10-Deacetylbaccatin III from the Needles of Taxus baccata L. by Two-Dimensional Liquid Chromatography

Simultaneous seasonal variation of four analogous taxanes, namely taxol (paclitaxel), cephalomannine, 10-deacetyl baccatin III (10-DAB), and baccatin III, from the needle of Iranian yew (Taxus baccata L.) has been monitored. Powdered samples of needles have been subjected to microwave-assisted extraction (MAE) and the extracted sample has been purified by liquid-liquid extraction (LLE) and analysis by reverse phase high performance liquid chromatography (RP-HPLC). These samples were identified by the LC-MS/MS system. The results indicated that taxane concentrations depend on seasonal and location. Analyses of these samples showed that 10-DAB has higher concentration than other taxanes and its highest concentration has been obtained in August. Thus, a heart cutting two-dimensional liquid chromatography was used for obtaining better resolution of taxanes. The first column in the analytical scale chromatography was a Eurospher-100 C8 and the second was Nucleosil-100 C18 which has been eluted at a flow rate of 1 mL min−1 with 70:30 v/v and 50:50 v/v water/acetonitrile, respectively. Optimized conditions in analytical chromatography have been extended to semipreparative scale in which the first column was a Eurospher-100 C8 and the second was a Eurospher-100 C18, both eluted with 70:30 v/v water/acetonitrile at a flow rate of 10 mL min−1. Detection has been carried out at a wavelength of 227 nm throughout the analysis in both scales. Final purity of the fraction containing 10-DAB, collected from the semipreparative column, is 82% after the first semipreparative run. Results have been confirmed by spiking of the standard 10-DAB sample and analysis by nuclear magnetic resonance (NMR).

Proteomic investigation of taxol and taxotere resistance and invasiveness in a squamous lung carcinoma cell line

Pulse selections on a chemotherapy naive squamous lung carcinoma cell line, SKMES-1, with clinically relevant concentrations of taxanes (taxol or taxotere) resulted in the development of a stable taxotere-resistant variant, SKMES-1-Taxotere and an unstable taxol-resistant variant, SKMES-1-Taxol. Both variants exhibited increased invasiveness in vitro. The unstable nature of SKMES-1-Taxol facilitated looking at factors involved in loss of taxol resistance and increased invasion. The taxotere and taxol-resistant cell lines were 5.9-fold and 12.5-fold resistant to taxotere and taxol respectively. Alterations in expression of/or point mutations in tubulin, the main target of taxanes, is a major mechanism or resistance. However, alterations in expression of beta tubulin were not consistent in the taxane-selected variants. Cross-resistance to adriamycin, vincristine and etoposide (VP-16) was consistent with overexpression of P-glycoprotein (P-gp). However, P-gp alone is not sufficient to confer the complete multiple drug resistance phenotype as all cell lines exhibited cross-resistance to 5-Fluorouracil (5-FU) and more than one mechanism has been linked to taxane resistance. There was no correlation between the fall of taxol resistance in SKMES-1-Taxol and P-gp expression indicating the loss in resistance to be independent of P-gp expression. Furthermore, resistance to the other drugs was not unstable in SKMES-1-Taxol suggesting some parallel mechanisms of resistance. Two-dimensional electrophoresis coupled with matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify alterations in expression of specific proteins associated with taxane resistance. A large number of differentially regulated proteins were identified in the resistant and invasive variants affecting cellular processes including stress response, protein turnover and cytoskeleton proteins.

Seasonal Changes in the Concentration of Major Taxanes in the Biomass of Wild Canada Yew (Taxus canadensis. Marsh.)

Seasonal variations in taxane concentrations in Taxus canadensis. Marsh. (Canada yew) were measured at 15 sampling dates from September 2004 to September 2005 by clipping 3-year-old shoots from the same 15 plants. Four age classes of needles and stems were separated and individually weighed and analyzed for the three major taxanes in T. canadensis.: 10-deacetylbaccatin III (10-DAB III), 9-dihydro-13-acetylbaccatin III (9-DHAB III), and paclitaxel (PAC). The dry weight fraction of each foliage age class decreased with increasing age; that is, proportionately more young than old foliage (current > current + 1 year > current + 2 year) was present in a 3-year-old stem. The ratio of needle:stem dry weight also decreased with increasing foliage age. Taxane concentrations varied widely, depending on the tissue, its age, and time of year. Peak concentrations were observed for a short period at the end of the growing season (August–September). The lowest taxane levels were found during the period of active growth: April through July. PAC and 9-DHAB III concentrations were higher in needles, but young stems contained the highest amounts of 10-DAB III. Levels of all three taxanes were higher in young than in old stems, likely due to increasing amounts of taxane-poor stem wood. Composite taxane concentrations were calculated for 3-year-old foliage from the component needle and stem weights and taxane analyses. It is suggested that summer harvesting between April and July (the low point in annual taxane levels) yields the poorest quality biomass, and harvesting from August onward is preferable.

Paclitaxel and Docetaxel Stimulation of Doxorubicinol Formation in the Human Heart: Implications for Cardiotoxicity of Doxorubicin-Taxane Chemotherapies

Antitumor therapy with the anthracycline doxorubicin is limited by a dose-related cardiotoxicity that is aggravated by a concomitant administration of the taxane paclitaxel. Previous limited studies with isolated human heart cytosol showed that paclitaxel was able to stimulate an NADPH-dependent reduction of doxorubicin to its toxic secondary alcohol metabolite doxorubicinol. Here we characterized that 0.25 to 2.5 microM paclitaxel caused allosteric effects that increased doxorubicinol formation in human heart cytosol, whereas 5 to 10 microM paclitaxel decreased doxorubicinol formation. The closely related taxane docetaxel caused similar effects. Basal or taxane-stimulated doxorubicinol formation was blunted by 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione (AL1576), a specific inhibitor of aldehyde reductases. Doxorubicinol was measured also in the cytosol of human myocardial strips incubated in plasma and exposed to doxorubicin in the absence or presence of paclitaxel or docetaxel and their clinical vehicles Cremophor EL or polysorbate 80. Low concentrations of taxanes stimulated doxorubicinol formation, whereas high concentrations decreased it. Doxorubicinol formation reached its maximum on adding plasma with 6 microM paclitaxel or docetaxel; this corresponded to the partitioning of 1.5 to 2.5 microM taxanes in the cytosol of the strips. Taxane-stimulated doxorubicinol formation was not mediated by vehicles, nor was it caused by increased doxorubicin uptake or de novo protein synthesis; however, doxorubicinol formation was blunted by AL1576. These results show that allosteric interactions with cytoplasmic aldehyde reductases enable paclitaxel or docetaxel to stimulate doxorubicinol formation in human heart. This information serves metabolic insights into the risk of cardiotoxicity induced by doxorubicin-taxane therapies.

Taxanes Synergize with the Bispecific Antibody MDXH447 to Enhance Antibody-Dependent Cell-Mediated Cytotoxicity

The interaction between antibody based therapy and cytotoxic chemotherapy is complex. To explore these interactions we investigated, in vitro, the effects of IC20 growth inhibitory concentrations of taxanes on bispecific antibody-mediated tumor cell cytotoxicity. MDXH447 is a bispecific antibody with specificity for the high affinity IgG receptor (CD64) and the type I epidermal growth factor receptor type (EGF-R). A431 cells, an epidermoid carcinoma cell line that over expresses EGF-R, were exposed to a range of IC20 growth inhibitory concentrations of paclitaxel or docetaxel. Interferon gamma activated monocytes were armed with MDXH447 and a standard chromium release antibody-dependent cell-mediated cytotoxicity (ADCC) assay was performed. Using the Chou and Talalay median effect analysis, we found that MDXH447-mediated ADCC was enhanced when A431 target cells were pretreated with paclitaxel or docetaxel. Median effect analysis of these interactions supported a synergistic interaction (CI < 1). Pretreatment of A431 cells with taxanes did not increase EGF-R expression compared to untreated controls. A431 epidermoid carcinoma cells pretreated with IC20 growth inhibitory concentrations of taxanes enhanced interferon gamma activated monocyte mediated ADCC killing through MDXH447.

Seasonal variations in the content of taxanes in the bark of Taxus baccata L.

Analytical determination by HPLC of five taxanes—paclitaxel, cephalomannine, 10-deacetylpaclitaxel, 10-deacetylcephalomannine and taxcultine—in extracts from the bark of Taxus baccata indicates that taxane concentrations vary seasonally. The highest concentration was found in October and the lowest in January for all taxanes examined. Copyright © 1999 John Wiley & Sons, Ltd.

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.

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

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

Genetic variation and parameter estimates in Taxusbrevifolia (Pacific yew)

Pacific yew (Taxusbrevifolia Nutt.) is a shade-tolerant gymnosperm native to the western United States and Canada. It recently gained attention as the source of Taxol® (paclitaxel), a promising new anticancer drug. Large-scale harvest of mature Pacific yew trees for the extraction of paclitaxel has resulted in the need for improved forest management practices and an increased understanding of the amount and distribution of genetic variation in the species. We partitioned estimates of genetic variance for allozyme, metric, and taxane traits into region, population, family, and within family components in seedling common-garden tests. Genetic diversity, genetic distance, and Nei's Gst values were estimated based on gene frequencies for 22 isozyme loci. Concentrations of taxanes were determined for needles and roots using HPLC. Populations of Pacific yew are more distinct from one another than is typical of long-lived, wind-pollinated conifers in western North America, but there is little regional differentiation. Yew populations have notably less allozyme diversity than most other gymnosperms with similar life-history characteristics. Most genetic variation in all traits occurs within the population, and much of that is within family. Heritabilities for growth and taxane traits ranged from low to moderately high. Gene conservation or management strategies should include broad sampling among and within populations of Pacific yew. Opportunities for genetic selection to develop improved lines or cultivars for the production of paclitaxel exist, but use of currently domesticated yew species is more time and cost efficient.

Seasonal and tissue variation in taxane concentrations of Taxus brevifolia

Analysis of seven taxanes: taxol, baccatin III, 10-deacetyltaxol, 10-deacetylbaccatin III, 7-xylosyl-10-deacetyltaxol, cephalomannine and brevifoliol in extracts from bark and foliage of pacific yew ( Taxus brevifolia) showed a gradient of decreasing concentration from stem base to branch tip. This decrease is attributed to the generally higher concentration of taxanes in the phloem tissue and the decrease in inner bark thickness from base to branch tip. Analysis of taxanes extracted from stem bark and needles sampled over a growing season showed that most taxane concentrations were significantly lower in the needles than in the bark. Typically, taxane concentrations in bark increased from May through August; whereas, in needles, concentrations changed little during that period. Two exceptions were baccatin III, which in the summer reached levels equivalent to bark, and brevifoliol which increased from March to August, reaching levels in needles nine times greater than bark.