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Multidrug resistance (MDR) is the main cause of failure in the chemotherapy of cancer patients. The present study aimed to evaluate the effects of sesquiterpene coumarins of Ferula gummosafruits on P-glycoprotein (P-gp)–mediated MDR. Drimane-type sesquiterpene coumarins from the fruits of F. gummosawere extracted with dichloromethane and subjected to column chromatography. The effects of the isolated compounds on P-gp–mediated MDR were evaluated in the breast cancer cell line MCF-7 which shows high resistance to doxoribicin (MCF-7/Dox). Phytochemical investigation of dichloromethane extract of F. gummosafruits resulted in three sesquiterpene coumarins including conferone (1), mogoltacin (2), and feselol (3). The structures of these compounds were confirmed by 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy. Exposure of cells to conferone, mogoltacin, feselol, and verapamil (positive control) enhanced doxorubicin uptake by MCF-7/Dox cells. This effect was dose dependent, but varied with the structure of the chemical. At 25μM, all the tested sesquiterpene coumarins restored at least 50% of the reference uptake (uptake by sensitive cells); but at 10μM, their potency varied where conferone showed the highest potency and feselol showed the lowest potency. Conferone, mogoltacin, and feselol from F. gummosasuppress P-gp–mediated drug efflux in highly resistant human breast cancer cells.

Background. Since the improvement of chemotherapy with safe molecules is needed for better efficacy without supplementary toxicity, we investigated the feasibility and tolerability of the combination of docetaxel and curcumin, a polyphenolic derivative extracted from Curcuma longa Patients and methods. Patients with advanced or metastatic breast cancer were eligible. Docetaxel (100 mg/m2) was administered as a 1-hour i.v. infusion every 3 weeks on d1 for 6 cycles. Curcumin was orally given from 500 mg/d for 7 consecutive days by cycle (from d-4 to d+2) and escalated until a dose-limiting toxicity should occur. The primary endpoint of this study was to determine the maximal tolerated dose of the combination of dose-escalating curcumin and standard dose of docetaxel chemotherapy in advanced and metastatic breast cancer patients. Secondary objectives included toxicity, safety, vascular endothelial growth factor and tumor markers measurements, and assessment of objective and clinical responses to the combination therapy. Results. Fourteen patients were accrued in this open-label phase I trial. At the last dose level of curcumin, 3 dose-limiting toxicities were observed and 2 out of 3 patients at this dose level refused to continue treatment, leading us to define the maximal tolerated dose of curcumin at 8000 mg/d. Eight patients out of 14 had measurable lesions according to RECIST criteria, with 5 PR and 3 SD. Some improvements as biological and clinical responses were observed in most patients. Conclusion. The recommended dose of curcumin is 6000 mg/d for 7 consecutive days every 3 weeks in combination with a standard dose of docetaxel. From the encouraging efficacy results, a comparative phase II trial of this regimen plus docetaxel versus docetaxel alone is ongoing in advanced and metastatic breast cancer patients.

The treatment of chemoresistant tumors represents an important challenge in the field of oncology. Primary or acquired overexpression of ATP-dependent transporters, in particular P-glycoprotein (Pgp, MDR1 protein), is a major cause of multidrug resistance and reduced patient survival. Sustained efforts have thereby been undertaken to find agents overcoming this resistance. This review provides a chemical and biological overview on bioactive metabolites from the marine field (natural molecules and analogues) that can overcome or circumvent resistance to ATP-dependent efflux pumps, their mechanisms of action and their structure-activity relationships. Their clinical relevance and status are presented. Active compounds (often microtubule-interacting agents) have been isolated from sponges and ascidians and, in lesser extent from cnidarians, and molluscs. The toxicity and the reversal activity can be uncoupled but, marine metabolites usually maintain high toxicity in multiresistant cancer cells. Certain display synergistic effects with clinically important anticancer drugs. The marine drug recently approved for cancer therapy [Trabectedin (Yondelis®)] and those entered into clinical trials act on multiple targets and, circumvent or overcome chemoresistance through very unusual mechanisms of action. Pharmacological and clinical data suggest that metabolites from the marine field could provide new therapeutic options for patients with tumors resistant to conventional therapy.

Formation of a furan derivative was observed in the course of a synthetic approach towards a new carotenoid metabolite, starting from a propargylic ketone, in the presence of mercuric triflate-tetramethylurea complex.

Overexpression of the protein transporter P-glycoprotein (Pgp, MDR1) at the cell surface is a major cause of multidrug resistance (MDR) and poor response to treatment in cancer chemotherapy and therapy for leishmaniasis. The present study shows that conferone, a sesquiterpene coumarin ether isolated for the first time from FERULA SCHTSCHUROWSKIANA, endemic in Uzbekistan, enhances the cell toxicity of vinblastine (VBL) in MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) cells. Conferone presents the advantage to mediate this effect at safe concentrations. At 10 ?M, it efficiently competes with the photoactivatable cyclosporin A analogue (SDZ 212 - 122) for the binding to Pgp and accumulates [ 3H]-VBL to a higher extent than cyclosporin A or cnidiadin. [ 3H]-VBL accumulation is dose-dependent and correlates with the inhibition of Pgp photolabeling affinity, supporting the hypothesis that conferone sensitizes MDCK-MDR1 cells to VBL by competitively inhibiting drug efflux. In MDCK-MDR1 cells, [ 3H]-VBL accumulation appears to be almost completely dependent on inhibition of Pgp transport. However, the strict specificity of conferone to this efflux pump has to be demonstrated in cell lines expressing other protein transporters. Collectively, our findings identify conferone as a powerful modulator of Pgp transport and a promising molecule for the treatment of MDR malignancies and leishmaniasis. Complementary IN VITRO and IN VIVO studies are, however, needed to assess the value of conferone as a reversal drug in human therapy. Considering its high affinity for Pgp, conferone may have an additional usefulness as a tool for the design or the (hemi)synthesis of agents probing Pgp. To our knowledge, this is the first report identifying sesquiterpene coumarins from FERULA as possible drug candidates for the reversion of MDR encoded by the MDR1 gene or the synthesis of agents probing Pgp.

The plasminogen activator/plasmin system represents a key component of the proteolytic machinery underlying angiogenesis. In this work, we investigated the effect of Neovastat (Æ-941), a naturally occurring multifunctional antiangiogenic agent that is currently in Phase III clinical trials, on tissue and urokinase plasminogen activator activities. We found that in vitro, Neovastat at 100μg/ml markedly stimulates t-PA-mediated plasmin generation, while it slightly inhibits the generation of plasmin mediated by uPA. The stimulatory effect of Neovastat on t-PA activity was markedly increased by a heat treatment, resulting in a 15-fold increase in the rate of activation of plasminogen. Neovastat did not directly stimulate the activity of t-PA or plasmin towards exogenous substrates, suggesting that its effect requires the presence of plasminogen. Accordingly, kinetic analysis showed that Neovastat increases both the kcatof t-PA as well as its affinity for plasminogen by 10-fold. The stimulation of t-PA activity by Neovastat was also correlated with a direct interaction of Neovastat with plasminogen as monitored by the surface plasmon resonance technology. Overall, these results identify Neovastat as a potent stimulator of t-PA-dependent activation of plasminogen, further emphasizing its pleiotropic mechanism of action on several molecular events involved in angiogenesis.

Pyrethrins, the most economically important natural insecticide, comprise a group of six closely related monoterpene esters. The industrial production is based on their extraction from Chrysanthemum cinerariaefolium (Pyrethrum) capitula. The world production of natural pyrethrins still falls short of global market demand stimulating the research in in vitro production as an alternative to conventional cultivation methods. The different biotechnological alternatives such as callus cultures, shoot and root cultures, plant cell suspension cultures, and bioconversion of precursors by means of enzymatic synthesis or genetically engineered microorganisms, as well as the progress achieved in methods for the identification and quantitation of insecticidal compounds have been reviewed. Although technology for plant cell culture exists, industrial applications have, to date, been limited due to both the low economical viability and technological feasibility at large scale. Bioconversion of readily available precursors looks more attractive, but more research is needed before this technology is used for the industrial production of pyrethrins.

An efficient procedure using sucrose as cryoprotectant to confer freezing tolerance on high-pyrethrinproducing cell lines of Chrysanthemum cinerariaefoliumwas developed. The efficiency of sucrose depended on both its concentration in the pregrowth medium and on preculture duration. A low sucrose concentration (0.25 mol/L) induced insufficient dehydration of cells, and so failed to prevent damage during freezing and prevented regrowth. The high cell dehydration obtained by incubating cells for 10 days in a medium supplemented with 0.5 mol/L sucrose was essential for freezing tolerance, but was insufficient to induce cell cryoprotection on its own. At this concentration, the post-thaw viability race increased with culture duration. Twenty days of pretreatment afforded a 57% viability rate, which was correlated with a different partitioning of water into unfrozen and frozen fractions, without modifications in cell dehydration. To study the water sorption strengths in pretreated cells, sorption isotherms were constructed. The effects of sucrose concentration and preculture duration during pretreatments were investigated. Sorption isotherms were analysed according to the D'Arcy and Watt model. Sucrose acted by trapping intracellular water through modification of hydrogen and multimolecular linkages, and thus increased the unfrozen water fraction in cells.

A simple method for efficient cryopreservation of Chrysanthemum cinerariaefoliumshoot tips is described. It comprises the following steps: treatment of the explanted shoot tips with 0.55 mol/L sucrose plus 4 µmol/L ABA for 3 days, direct immersion in liquid nitrogen, rapid rewarming at 40 °C and recovery of the shoot tips on solid nutrient medium containing 11 µmol/L ANA and 4.5 µmol/L BAP About 75 % of the shoot tips withstood cryopreservation using this procedure. The morphogenic potential of frozen shoot tips, and total chlorophyll and pyrethrin contents of regenerated plants were preserved after the different treatments.

Callus from four clones of Chrysanthemum cinerariaefoliumhave been investigated for pyrethrins biosynthesis. Calli from disc-flowers, bud-flowers, stems and leaves of clone HY D were cultivated. Best growth was obtained by initiating the culture on Murashige and Skoog medium containing 3% sucrose, 1% agar, 4 ppm β-naphtoxy acetic acid (ANA) and 0.4 ppm 6- benzyl aminopurine (BAP) for 28 d, then transfering explants on $${\raise0.7ex\hbox{$1$} \!\mathord{\left/ {\vphantom {1 2}}\right.\kern-\nulldelimiterspace}\!\lower0.7ex\hbox{$2$}}$$ i.e.at the beginning of the stationnary phase. Disc-flowers calli from two high-producers clones (HY C and HY D) and two low-producers clones (SY A and SY B) were also cultivated. Pyrethrin amounts found in callus cells showed a direct relationship between parental clone synthesis ability and callus cell production. Selection of high-producers clones is thus essential for optimisation of pyrethrin synthesis.

A protease fromAspergillus niger LCF 8was purified in anion exchange mode using BioCAD Workstations and the process scaled up. The use of POROS 50 HQ media brings the benefits of Perfusion chromatography i.e. high resolution and high dynamic capacity with very short cycle times. Results obtained at pilot scale enable to purify at process scale 151g of protease/m3crude broth in 1h. The small column size needed (2.25 l media/m3crude broth) and the high processing speed of POROS*50 HQ compared with conventional media lead to greatly improved cost effectiveness and higher product recovery.

A high activity tannase (tannin acyl hydrolase EC 3.1.1.20) is synthetized in high yield by Aspergillus nigerLCF 8. At the production optimum, the tannase is strongly bound to the mycelium and detachment of the enzyme by classical physical and chemical means, largely failed. Enzymatic hydrolysis of cell walls using a chitinase from Streptomyces griseusfollowed by reverse micellar tannase extraction resulted in a recovery of 43% active enzyme, i.e. an improvement in yield of 2.5 from a previous process. Best conditions were enzymatic hydrolysis of mycelium with chitinase at pH 6.0 and 25°C for 2.5 h followed by tannase extraction at pH 7.5 with isooctane containing 80 mM cetyl trimethyl ammonium bromide and stripping at pH 4.0 in the presence of 0.35 M NaCl.

The production of high-content fructo-oligosaccharides from sucrose by a crude FTF from a new strain of Penicilliumisolated in our Laboratory was investigated. The optimum conditions for the production of the enzyme and for the enzymic reaction have been determined. It has been demonstrated that the crude enzyme acts as a mixed enzyme system of fructosyl transferase (FTF; Class 2 of Enzyme Nomenclature) and glycosidases (Class 3 of Enyme Nomenclature). Under optimum conditions: pH 5.5, temperature 55°C, sucrose concentration 750 g/l, enzyme concentration 5 FTF units/g sucrose, conversion yield up to 80% were obtained and high concentration of nystose (412 g/l) and fructofuranosyl-nystose (176 g/l) were accumulated.

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