No abstract

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that can be activated by natural ligands such as 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ(2)) as well as synthetic drugs such as thiazolidinediones. The treatment of human breast cancer cell lines with PPARgamma agonists is known to have antiproliferative effects but the role of PPARgamma activation in the process remains unclear. In the present study, we investigated the effects of four PPARgamma agonists, Rosiglitazone (RGZ), Ciglitazone (CGZ), Troglitazone (TGZ) and the natural agonist 15d-PGJ(2), on estrogen receptor alpha (ERalpha) signalling pathway in two hormone-dependent breast cancer cell lines, MCF-7 and ZR-75-1. In both of them, TGZ, CGZ and 15d-PGJ(2) induced an inhibition of ERalpha signalling associated with the proteasomal degradation of ERalpha. ZR-75-1 cells were more sensitive than MCF-7 cells to these compounds. Treatments that induced ERalpha degradation inhibited cell proliferation after 24 h. In contrast, 24 h exposure to RGZ, the most potent activator of PPARgamma disrupted neither ERalpha signalling nor cell proliferation. 9-cis retinoic acid never potentiated the proteasomal degradation of ERalpha. PPARgamma antagonists (T0070907, BADGE and GW 9662) did not block the proteolysis of ERalpha in MCF-7 and ZR-75-1 cells treated with TGZ. ERalpha proteolysis still occurred in case of PPARgamma silencing as well as in case of treatment with the PPARgamma-inactive compound Delta2-TGZ, demonstrating a PPARgamma-independent mechanism. The use of thiazolidinedione derivatives able to trigger ERalpha degradation by a PPARgamma-independent pathway could be an interesting tool for breast cancer therapy.

Modified azasugar molecules have been synthesized and characterized as excellent pharmacological chaperone candidates to treat the neurodegenerative disorder Krabbe disease.

Numerous recent studies indicate that most anticancer effects of PPARγ agonists like thiazolidinediones are the result of PPARγ-independent pathways. These conclusions were obtained by several approaches including the use of thiazolidinedione derivatives like Δ2-Troglitazone (Δ2-TGZ) that does not activate PPARγ. Since biotinylation has been proposed as a mechanism able to increase the specificity of drug delivery to cancer cells which could express a high level of vitamin receptor, a biotinylated derivative of Δ2-TGZ (bΔ2-TGZ) has been synthetized. In the present article, we have studied the in vitro effects of this molecule on both hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cells. In both cell lines, bΔ2-TGZ was more efficient than Δ2-TGZ to decrease cell viability. bΔ2-TGZ was also more potent than Δ2-TGZ to induce the proteasomal degradation of cyclin D1 in both cell lines and those of ERα in MCF-7 cells. However, in competition experiments, the presence of free biotin in the culture medium did not decrease the antiproliferative action of bΔ2-TGZ. Besides, other compounds that had no biotin but that were substituted at the same position of the phenolic group of the chromane moiety of Δ2-TGZ decreased cell viability similarly to bΔ2-TGZ. Hence, we concluded that the increased antiproliferative action of bΔ2-TGZ was not due to biotin itself but to the functionalization of the terminal hydroxyl group. This should be taken into account for the design of new thiazolidinedione derivatives able to affect not only hormone-dependent but also hormone-independent breast cancer cells in a PPARγ-independent pathway.

The powerful chemo-enzymatic synthesis of the pentadecasaccharide hapten involved in the first synthetic carbohydrate-based vaccine candidate against endemic shigellosis is reported. The high yielding site-selective α-D-glucosylation of a lightly protected disaccharide by an engineered transglucosylase-sucrose system gave a trisaccharide, which was chemically elongated by an efficient [5+5] process.

Our aim was to get new information about the Peroxisome Proliferator Activated Receptor gamma (PPARγ)-independent pathway involved in the antiproliferative action of PPARγ ligands in breast cancer cells. We investigated the effects of Troglitazone (TGZ), Ciglitazone (CGZ), Rosiglitazone (RGZ) and, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ(2)) on the hormone-dependent breast cancer cell line MCF7. The early transcription factor EGR1 (Early Growth Response gene 1) mRNA and protein levels peaked after 3h of incubation with 25μM TGZ, CGZ or 15d-PGJ(2) and then gradually decreased. RGZ, the most potent activator of PPARγ, did not show this effect. The PPARγ antagonist GW 9662 did not block EGR1 mRNA induction which also still occurred in case of PPARγ silencing as well as in case of treatment with the PPARγ-inactive compound Δ2-TGZ. EGR1 mRNA induction required ERK1/2 phosphorylation which was not blocked by EGF Receptor (EGFR) inhibition. The ERK1/2 pathway was also involved in Δ2-TGZ-induced EGR1 mRNA expression in the hormone-independent breast cancer cell line MDA-MB-231. Using the fluorescent dye Fura2, we showed in MCF7 that TGZ or Δ2-TGZ induced an immediate increase in cytosolic calcium which was required for ERK1/2 phosphorylation and EGR1 mRNA induction as demonstrated by calcium chelation experiments. Furthermore, in MCF7 transfected with siRNA targeting EGR1, Δ2-TGZ inhibited less efficiently cell proliferation.

The exploration of chemo-enzymatic routes to complex carbohydrates has been hampered by the lack of appropriate enzymatic tools having the substrate specificity for new reactions. Here, we used a computer-aided design framework to guide the construction of a small, diversity-controlled library of amino acid sequences of an α-transglucosylase, the sugar binding subsites of which were re-engineered to enable the challenging 1,2-cis-glucosylation of a partially protected β-linked disaccharide allyl (2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-α-L-rhamnopyranoside, a potential intermediate in the synthesis of Shigella flexneri cell-surface oligosaccharides. The target disaccharide is not recognized by the parental wild-type enzyme and exhibits a molecular structure very distinct from that of the natural α-(1→4)-linked acceptor. A profound reshaping of the binding pocket had thus to be performed. Following the selection of 23 amino acid positions from the first shell, mutations were sampled using RosettaDesign leading to a subset of 1515 designed sequences, which were further analyzed by determining the amino acid variability among the designed sequences and their conservation in evolutionary-related enzymes. A combinatorial library of 2.7 × 10 4 variants was finally designed, constructed, and screened. One mutant showing the desired and totally new specificity was successfully identified from this first round of screening. Impressively, this mutant contained seven substitutions in the first shell of the active site leading to a drastic reshaping of the catalytic pocket without significantly perturbing the original specificity for sucrose donor substrate. This work illustrates how computer-aided approaches can undoubtedly offer novel opportunities to design tailored carbohydrate-active enzymes of interest for glycochemistry or synthetic glycobiology.

An epimer of the known glycosidase inhibitor noeurostegine, galacto-noeurostegine, was synthesised in 21 steps from levoglucosan and found to be a potent, competitive and highly selective galactosidase inhibitor of Aspergillus oryzae β-galactosidase. Galacto-noeurostegine was not found to be an inhibitor of green coffee bean α-galactosidase, yeast α-glucosidase and E. coli β-galactosidase, whereas potent but non-competitive inhibition against sweet almond β-glucosidase was established. The 2-deoxy-galacto-noeurostegine analogue was also prepared and found to be a less potent inhibitor of the same enzymes.