Novel fluoro- and hydroxyl-containing jasmonate derivatives as highly efficient elicitors in suspension cultures of Taxus chinensis

To develop more effective abiotic elicitors for cell suspension cultures of T. chinensis to meet the needs for paclitaxel as anti-tumor drug, some fluoro- or hydroxyl-containing groups are introduced to the ester moiety of jasmonic acid by the esterification or acylation with bis(trichloromethyl) carbonate and corresponding alcohol. Some of them are found to be novel and effective elicitors, which can enhance the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate (MJA) in T. chinensis cell cultures.     

Association of Drosophila cysteine string proteins with membranes

Cysteine string proteins are putative synaptic vesicle proteins that lack a transmembrane domain. Our analysis shows that Drosophila cysteine string proteins are extensively modified by hydroxylamine-sensitive fatty acylation. This modification could be responsible for association of csp's with membranes. Extensive deacylation of Dcsp's by a 20 h incubation in 1 M hydroxylamine, pH 7.0, or methanolic KOH produces a protein of 6–7 kDa lower mass than untreated Dcsp's. Surprisingly, the hydroxylamine treatment does not cause release of Dcsp's from membranes. On the other hand, alkaline stripping of membranes isolated from Drosophila brain by 0.1 M sodium carbonate, pH 11.5, causes a significant release of Dcsp's from membranes into the cytosol. These results indicate that fatty acylation may not form the main anchor of Dcsp's in membranes. Taking advantage of the endocytotic block in the Drosophila mutant shibire^ts1, we analyzed the acylation states of Dcsp's in two stages during synaptic vesicle recycling and found no evidence for an acylation/ deacylation cycle of Dcsp's in the brain nerve terminals.     

New Taxol (paclitaxel) prodrugs designed for ADEPT and PMT strategies in cancer chemotherapy

Two new glucuronide paclitaxel prodrugs have been synthesized. Linked to the 2'-OH of the drug by a carbonate function, they include a self-immolative spacer bearing an arylnitro or arylamino group between the drug and the glucuronic acid residue. Both prodrugs were well detoxified and easily cleaved in the presence of beta-D-glucuronidase with fast removal of the spacer, releasing paclitaxel. The arylamino spacer-containing prodrug, more stable than the corresponding nitro analogue, was selected for further studies.     

Synergistic anti-tumor activity of isochaihulactone and paclitaxel on human lung cancer cells

Drug resistance frequently develops in tumors during chemotherapy. Therefore, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Here, we show that isochaihulactone (K8) enhanced paclitaxel-induced apoptotic death in human lung cancer cells, and the enhancing effect was related to increased NSAID-activated gene-1 (NAG-1) expression. CalcuSyn software was used to evaluate the synergistic interaction of K8 and paclitaxel on human lung cancer cells; the synergistic effect of K8 in combination with paclitaxel was increased more than either of these drugs alone. Furthermore, the activity of ERK1/2 was enhanced by the combination of K8 and paclitaxel, and an ERK1/2 inhibitor dramatically inhibited NAG-1 expression in human lung cancer cells. Therefore, this synergistic apoptotic effect in human lung cancer cells may be directly associated with K8-induced NAG-1 expression through ERK1/2 activation. Moreover, over-expression of NAG-1 enhanced K8/paclitaxel-induced apoptosis in human lung cancer cells. In addition, treatment of nude mice with K8 combined with paclitaxel induced phospho-ERK1/2 and NAG-1 expression in vivo. Targeting of NAG-1 signaling could enhance therapeutic efficacy in lung cancer. Our results reveal that activation of NAG-1 by K8 enhanced the therapeutic efficacy of paclitaxel in human lung cancer cells via the ERK1/2 signaling pathway.     

Modulating paclitaxel bioavailability for targeting prostate cancer

Four novel water-soluble peptide-paclitaxel conjugates were designed and synthesized as prostate-specific antigen (PSA)-activated prodrugs for prostate cancer therapy. These prodrugs were composed of a peptide, HSSKLQ or SSKYQ, each of which is selectively cleavable by PSA; a self-immolative linker, either para-aminobenzyl alcohol (PABS) or ethylene diamine (EDA); and the parent drug, paclitaxel. Introduction of a PABA or EDA linker between the peptide and paclitaxel in prodrugs 2-5 resulted in products with an increased rate of hydrolysis by PSA. The stability of prodrugs 2 and 3, with the PABA linker, was poor in the serum-containing medium because of the weak carbonate bond between the PABA and paclitaxel; however, this disadvantage was overcome by introducing a carbamate bond using an EDA linker in prodrugs 4 and 5. Thus, the incorporation of an EDA linker increased both the stability and PSA-mediated activation of these prodrugs. The cytotoxicity of each prodrug, as compared to paclitaxel, was determined against a variety of cell lines, including the PSA-secreting CWR22Rv1 prostate cancer cell line. The EDA-derived prodrug of paclitaxel 5 was stable and capable of being efficiently converted to an active drug that killed cells specifically in the presence of PSA, suggesting that this prodrug and similarly designed PSA-cleavable prodrugs may have potential as prostate cancer-specific therapeutic agents.     

Synthesis and characterization of novel biodegradable poly(carbonate ester)s with photolabile protecting groups

Novel biodegradable poly(carbonate ester)s with photolabile protecting groups were synthesized by ring-opening copolymerization of L-lactide (LA) with 5-methyl-5-(2-nitro-benzoxycarbonyl)-1,3-dioxan-2-one (MNC) with diethyl zinc (Et2Zn) as catalyst. The poly(L-lactide-co-5-methyl-5-carboxyl-1,3-dioxan-2-one) (P(LA-co-MCC)) was obtained by UV irradiation of poly(L-lactide acid-co-5-methyl-5-(2-nitro-benzoxycarbonyl)-1,3-dioxan-2-one) (P(LA-co-MNC)) to remove the protective 2-nitrobenzyl group. The free carboxyl groups on the copolymers P(LA-co-MCC) were reacted with paclitaxel, a common antitumor drug. Gel permeation chromatography and NMR studies confirmed the copolymer structures and successful attachment of paclitaxel to the copolymer.     

Overexpression of IL-6 but not IL-8 increases paclitaxel resistance of U-2OS human osteosarcoma cells

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OS(IL-6) are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OS(IL-8) transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.     

Paclitaxel reduces regulatory T cell numbers and inhibitory function and enhances the anti-tumor effects of the TLR9 agonist PF-3512676 in the mouse

The anti-tumor properties of Toll-like receptor (TLR) 9 agonist CpG oligodeoxynucleotides (ODN) are enhanced by combinations with several cytotoxic chemotherapy regimens. The mechanisms of this added benefit, however, remain unclear. We now report that, similar to the depletion of regulatory T cells (Treg) using anti-CD25, paclitaxel increased the anti-tumor effect of the TLR9 agonist PF-3512676 in a CD8⁺ T cell-dependent fashion. Paclitaxel treatment decreased Treg numbers in a TLR4-independent fashion, and preferentially affected cycling Treg expressing high levels of FoxP3. The paclitaxel-induced reduction in Treg FoxP3 expression was associated with reduced inhibitory function. Adoptively transferred tumor-antigen specific CD8⁺ T cells proliferated better in mice treated with paclitaxel and their recruitment in the tumor was increased. However, the systemic frequency of PF-3512676-induced tumor-antigen specific effector CD8⁺ T cells decreased with paclitaxel, suggesting opposite effects of paclitaxel on the anti-tumor response. Finally, gene expression profiling and studies of tumor-associated immune cells revealed a complex modulation of the PF-3512676-induced immune response by paclitaxel, including a decrease of IL-10 expression and an increase in IL-17-secreting CD4⁺ T cells. Collectively, these data suggest that paclitaxel combined with PF-3512676 may not only promote a better anti-tumor CD8⁺ response though increased recruitment in the tumor, possibly through Treg depletion and suppression, but also exerts more complex immune modulatory effects.     

Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity

Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.     

Changes in plasma levels of inflammatory cytokines in response to paclitaxel chemotherapy

BACKGROUND: Flu-like symptoms are common, early transient side effects of paclitaxel chemotherapy. We hypothesized that these symptoms may be due to release of inflammatory cytokines in response to treatment. The objective of this study was to assess changes in plasma levels of interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha during chemotherapy and to correlate these changes with musculoskeletal symptoms. METHODS: Ninety patients with breast cancer were included; 70 patients received single agent paclitaxel either weekly or every 3 weeks and 20 received FAC (5-FU, doxorubicin, cyclophosphamide) chemotherapy. Fifteen healthy volunteers were included as controls. Cytokines and symptoms were measured before starting therapy, on day 3 and on the last day of one treatment cycle. RESULTS: At baseline, all subjects had measurable levels of IL-8 but only 49% had IL-12, 45% had IL-10, 32% had IL-6, and 21% had IL-1beta or TNF-alpha in their plasma. There was no difference in baseline cytokine levels between cancer patients and the healthy volunteers. Schedule-dependent transient changes in the levels of 3 cytokines were observed in the paclitaxel treated patients. In the every 3-week paclitaxel group, IL-6 and IL-8 increased whereas in the weekly paclitaxel group IL-10 increased significantly compared to baseline. Fatigue and flu-like symptoms were also worse on day 3. In the weekly paclitaxel group, increase in IL-10 level correlated positively with joint pain (p=0.003). In the every 3-week paclitaxel group, increase in IL-8 level correlated positively with flu-like symptom (p=0.008). In the FAC-treated group and among the healthy volunteers none of these cytokines increased significantly. CONCLUSIONS: Weekly paclitaxel induces transient increase in IL-10 levels whereas every 3-week higher dose treatments induce IL-8 and IL-6 in the plasma. These changes correlate with joint pain and flu-like symptoms.     

LRP16影响宫颈癌Siha细胞对化疗药物的敏感性

目的:探讨抑制LRP16的表达对宫颈癌Siha细胞的化疗药物敏感性的影响。方法:将抑制LRP16表达的小干扰RNA:negativecontrol-si RNA(NC)、si RNA-374(si374)转染入Siha宫颈鳞癌细胞系中,通过顺铂(DDP)和紫杉醇(TAX)的处理后,采用CCK-8检测不同浓度紫杉醇、顺铂作用宫颈癌细胞系Siha48 h后,计算出细胞被抑制一半时顺铂、紫杉醇的药物浓度(IC50);使用Hoechst33342染色观察细胞凋亡,采用流式细胞仪检测顺铂IC50作用Siha细胞48小时后的细胞凋亡情况,紫杉醇IC50作用Siha细胞之后的细胞周期分布情况。结果:CCK-8检测转染的Siha细胞增殖活性受到抑制,Hoechst33342染色观察转染的Siha细胞凋亡明显增加,流式细胞仪检测凋亡显示,si374+顺铂的早期凋亡率22.15±2.24,NC+顺铂12.45±2.72,流式细胞仪检测周期显示G2/M(%),si374+紫杉醇29.94±1.87,NC+紫杉醇17.66±2.32。结论:LRP16基因表达下调之后,抑制Siha细胞的增殖、促进其凋亡,使细胞周期滞留于G2/M期,从而提高Siha细胞的化疗敏感性。     

Acylation of L-asparaginase with total retention of enzymatic activity

Acylation of L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) with complete retention of catalytic activity was achieved. Several parameters of the acylation method, based on the binding of palmitoyl residues to epsilon-NH2 groups of protein, were optimized. The correlation between the acylation degree of L-asparaginase and the retention of catalytic activity was established. For a palmitoyl chloride/protein molar ratio ranging from 50 to 900, a degree of modification of 10 to 30% and a retention of catalytic activity of 98 to 60% respectively, was observed. Hydrophobicity of 30% acylated protein was correlated with turbidity in water and octanol and was compared with the native protein. Acylated protein incorporated into liposomes, showed an increase in catalytic activity in intact form as compared to the native enzyme. By the introduction of a sequential acylation cycle, an improvement of the degree of modification with a maximal value at 50% was obtained. Total retention of catalytic activity was achieved by acylation in the presence of 8 mM L-asparagine in a reactional medium.     

Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model

INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.     

ATP-independent control of Vac8 palmitoylation by a SNARE subcomplex on yeast vacuoles

Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation.     

Kinetic studies of the acylation of pig muscle–d-glyceraldehyde 3-phosphate dehydrogenase by 1,3-diphosphoglycerate and of proton uptake and release in the overall enzyme mechanism

In the presence of NAD(+) the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD(+) and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H(+) indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5-8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results.     

Low-dose paclitaxel enhances the anti-tumor efficacy of GM-CSF surface-modified whole-tumor-cell vaccine in mouse model of prostate cancer

Chemotherapy combined with a tumor vaccine is an attractive approach in cancer therapy. This study was designed to investigate the optimal schedule and mechanisms of action of a novel GM-CSF (granulocyte?Cmacrophage colony-stimulating factor) surface-modified tumor-cell vaccine in combination with paclitaxel in the treatment of mouse RM-1 prostate cancer. First, the anti-tumor efficiencies of various dosage of paclitaxel (4, 20, 40?mg/kg) in combination with the vaccine in different administration sequences were examined in the mouse RM-1 prostate cancer model. Then, the in vivo and in vitro effects of various dosage of paclitaxel on RM-1 cells, T cells, and DCs (dendritic cells) were evaluated. The results showed that: (a) the GM-CSF-surface-modified tumor-cell vaccine was more potent at inducing the uptake of tumor antigens by DCs than irradiated tumor cells plus free GM-CSF; (b) 4?mg/kg paclitaxel combined with the GM-CSF-surface-modified tumor-cell vaccine was the most effective at enhancing tumor regression in RM-1 prostate cancer mice when the vaccine was administrated 2?days after paclitaxel; and (c) administration of 4?mg/kg paclitaxel followed by the vaccine induced the highest degree of CD8⁺ T-cell infiltration in tumor tissue, suggesting that the induction of tumor-specific immune response had occurred. These findings suggested that the GM-CSF-surface-modified tumor-cell vaccine may have potential clinical benefit for patients with prostate cancer when it is combined with paclitaxel. Furthermore, the effect of immunochemotherapy depends on careful selection of paclitaxel dosage and the sequence of paclitaxel/vaccine administration.     

Mathematical deconvolution uncovers the genetic regulatory signal of cancer cellular heterogeneity on resistance to paclitaxel

Drug resistance remains a major problem in combating malignancies, resulting critical the resistance to paclitaxel used in the treatment of many different cancers. Elucidating the cellular heterogeneity composition of tumours may be relevant to designing more effective treatment strategies on drug resistance. In particular, such heterogeneity correlates with the measurement of gene expression below the population level. However, experimental assays capturing differential response are limited and cannot discern the variation in gene expression specific to different cellular types in tumour populations. These limitations led us to consider a mathematical modelling approach, in which the gene expression of cellular subpopulations is recovered by deconvolution. Mathematically, the deconvolution is a multi-linear regression-based problem. We combined herein data on cellular subpopulation frequency composition with gene expression values from 16 tumour lines (8 resistant and 8 sensitive to paclitaxel treatment) to find genes that are differentially expressed between paclitaxel resistant and paclitaxel sensitive tumour lines in different cellular subpopulations. The results indicate that many genes differentially expressed between paclitaxel resistant and sensitive cancer lines are only detected when considering their heterogeneous cellular composition. Overall, our methodology is thought to keep in mind phenotypic heterogeneity improving our resolution in the identification of biomarkers on resistance to chemo-therapeutic agents.     

Kinetics and Mechanism of Ultrasound-assisted Extraction of Paclitaxel from <Emphasis Type="Italic">Taxus chinensis</Emphasis>

Batch experimental studies were carried out for the ultrasound-assisted extraction of paclitaxel from Taxus chinensis while varying parameters such as ultrasound power, extraction temperature and contact time. The extraction of the majority of the paclitaxel (~99%) was achieved from the biomass by a single extraction at 380W of ultrasound power for a period of 10 min. The kinetics data obtained for the paclitaxel extractions, and the dominant role played by intraparticle diffusion, were found to be in concordance with the pseudo-second-order model, and the intraparticle diffusion model respectively. The effective diffusion coefficient of paclitaxel (4.1882 × 10^-13 ~ 5.7093 × 10^-13 m²/s) and the mass transfer coefficient (4.705 × 10^-8 ~ 14.1160 × 10^-8 m/s) increased when the extraction temperature and ultrasound power were raised.