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There is growing evidence that docetaxel, a microtubule-targeting agent like the other taxane paclitaxel, induces dual cytotoxicity mechanism according to dose level. Postgenomics screening technologies are now more and more applied to the elucidation of drug response mechanisms. Proton nuclear magnetic resonance spectroscopy-based pharmacometabolomics was here applied to get further insight into the response of human MCF7 breast carcinoma cells to docetaxel at high (clinical, 5 microM) and low (1 nM) doses. The global response to both doses was evaluated by nuclear morphology and DNA content, the latter as an index of cell proliferation and DNA ploidy. High dose provoked long-lasting cell cycle arrest in mitosis during the first 48 h of exposure to treatment and severe decrease in DNA content followed by significant amount of cell death. In contrast, at low dose, no long-lasting cell cycle arrest was observed on micrographies, and DNA content was decreased but less than at high dose (P < 0.05), without significant cell death. This response was compared to biochemical alteration assessed by pharmacometabolomics. Thirty metabolites were identified and quantified. Metabolite profiling at clinical dose revealed time-dependent disorders in derivatives of glycolysis, lipid metabolism and glutathione metabolism. Comparison between high and low doses was performed at 72 h and showed common traits including the accumulation of cytidinediphosphocholine (x 5.0 and x 6.9, respectively, P < 0.03), the decrease in phosphatidylcholine (x 0.3 and x 0.2, respectively, P < 0.03), and gluthathione (x 0.6 and x 0.6, respectively, P < 0.03). Despite that, significant dose-dependent differences were found in 12 of 30 measured metabolites. Among them, the most discriminant metabolites were polyunsaturated fatty acids (ratio of high-to-low dose of 14.8, P < 0.05), glutamate, myoinositol, and homocysteine (ratio < 0.4, P < 0.05). In addition, the mechanism for glutathione decrease was different. At high dose, it resulted from extensive consumption with precursor starvation (glutamate: -89%, P < 0.05) and increased glutathione S-transferase activity (x 5, P < 0.01), whereas at low dose, it resulted from glutathione biosynthesis blockade with homocysteine accumulation (+144%, P < 0.03) and decreased glutathione S-transferase activity (-70%, P < 0.01). Altogether, this pharmacometabolomics analysis provides further evidence of the varying cellular responses at high and low doses of docetaxel in MCF7 breast cancer cells.

The present study focuses on eudesmin (bicyclic lignan, 0.15 % of dry leaves) and diphyllin (arylnaphthalene lignan, 0.1 % of dry roots), both isolated from H. perforatum Kar. et Kir, a Rutaceae species endemic to Uzbekistan. We first compared their specificity for cancer cells with those of etoposide and podophyllotoxin by screening their cytotoxicity on 3 healthy cell-lines and 7 sensitive or resistant human solid cancer lines. We then tested their capacity to reverse P-glycoprotein-mediated multidrug resistance (MDR) by assaying dye and drug uptake in MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) and doxorubicine-resistant human breast carcinoma cells (MCF7/Dox). Eudesmin displays IC (50) values > 100 microM on all tested lines. Our data provide the first demonstration that this non-toxic lignan reverses Pgp-mediated drug efflux and supports the hypothesis that it may inhibit resistance mediated by MDR1 and MRP proteins. Even if its reversal activity is insufficient for clinical application, its capacity to accumulate [(3)H]-vinblastine in MDCK/MDR1 and MCF7/Dox cells suggests that eudesmin may positively affect the bioavailability and, thereby, the therapeutic potency of anticancer drugs in Pgp-overexpressing cells. Diphyllin exhibits IC (50) values ranging from 10 (- 6) to 10 (- 4) M. It is markedly less toxic than podophyllotoxin (IC (50) : 13 - 61 nM), but exhibits tumoricidal effects close to those of etoposide. Unfortunatly, it is 65-fold more toxic than etoposide on human primary fibroblasts. Consequently, it has no value as an anticancer drug. Its value as raw material for the hemisynthesis of anticancer drugs is discussed.

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 microM, 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.

Purpose: The quaternary benzophenanthridine alkaloid sanguinarine exhibits a broad range of activity, including cytotoxicity against various human tumour and normal cell lines. Here, we examined its potency as an anticancer drug., Methods: The differential cytotoxicity against cancer versus normal cells was assessed in vitro by two fluorimetric assays (RRT and Hoechst 33342 dye DNA assays, respectively) in a panel of human solid cancer cell lines and a human fibroblast primary culture. The ability to induce apoptosis was demonstrated in PC3 human prostatic adenocarcinoma cells by analysis of morphological changes, internucleosomal DNA fragmentation, cellular poly(ADP-ribose) polymerase cleavage and caspase 3/7 activation. Production of reactive oxygen species was evaluated by the 2',7'-dichlorofluorescin diacetate assay. Depletion of cellular glutathione content was assessed with the monochlorobimane assay., Results: Sanguinarine markedly inhibited the growth of all tested cells (IC(50) 0.9-3.3 microM) without differential cytotoxicity against normal versus cancer cells. In PC3 cells, continuous treatment with 5 microM sanguinarine induced an early (within 10 min) cellular reduced glutathione depletion insensitive to dithiothreitol or N-acetylcysteine treatment, followed by a caspase 3/7-dependent apoptotic response within 2 h. Complementary assays suggested that the glutathione depletion was initiated by direct reactivity of sanguinarine with reduced glutathione., Conclusions: Taken together, these results show that (1) sanguinarine exhibits no specificity for cancer cells, and (2) its strong cytotoxicity is probably due to a rapid apoptotic response induced by an early and severe glutathione-depleting effect. They also suggest that the clinical usefulness of this alkaloid as an anticancer drug is limited.