Crocin, a natural molecule with potentially beneficial effects against skin ageing.

OBJECTIVE: Oxidative stress and low-grade chronic inflammation stand out as key features of physiological skin ageing. The aim of this study was to examine in normal human epidermal keratinocytes (NHEK) and human dermal fibroblasts (HDF) grown in vitro, the antioxidant and anti-inflammatory properties of crocin, a carotenoid glycoside responsible for the colour of saffron. Moreover, considering the newly emerging field of skin glycobiology and the presence of two gentiobiosyl moieties in crocin, the effect of crocin on NHEK glycosylation pathways was for the first time investigated. METHODS: The anti-inflammatory and antioxidant activities of crocin were evaluated by in vitro assays of antioxidation activities, ELISA and microarray analysis. The effect of crocin on keratinocyte glycobiology was evaluated by proprietary GLYcoDiag lectin technologies and microarray analysis. RESULTS: Crocin is endowed with antioxidant potential against reactive oxygen species, protects squalene against UVA-induced peroxidation and prevents the release of inflammatory mediators. The expression of NF-kB-related genes and glycosylation-related genes is modulated in the presence of crocin. CONCLUSION: Results could designate this molecule as a promising skin ageing prevention cosmetic agent. Of note, some of these effects could be mediated by protein O-glycosylation and interaction of crocin with osidic receptors of keratinocytes.

Vichy Thermal Spring Water (VTSW), a cosmetic ingredient of potential interest in the frame of skin ageing exposome: an in vitro study.

OBJECTIVE: To study the effects of the very high minerality Vichy Thermal Spring Water (VTSW) on human keratinocytes grown in vitro. METHODS: The effect of VTSW was monitored by full genome transcriptomic technology and immunofluorescence microscopy. RESULTS: In the presence of 50% VTSW, the expression of a number of skin homoeostasis-related genes is increased, specifically with respect to dermal-epidermal junction, epidermal cohesion and communication, keratinocyte proliferation-differentiation balance, antioxidant mechanisms and DNA repair. CONCLUSION: This work suggests that VTSW could be considered as an ingredient of potential interest to address some of the deleterious effects of skin ageing exposome.

Effects of ketoconazole on the proliferation and cell cycle of human cancer cell lines.

The growth-inhibitory effects of ketoconazole, an antifungal agent which inhibits arachidonic acid lipoxygenases and cytochrome P-450 enzymes, were tested in human colon and breast cancer cell lines. In the serum independent HT29-S-B6 colon cell clone, ketoconazole reduced cell proliferation and [3H]thymidine incorporation in a dose-dependent fashion, with a 50% inhibitory concentration of approximately 2.5 microM. Flow cytometry showed an accumulation of cells in the G0-G1 phase of the cell cycle and a concomitant decrease of the percentage of cells in S phase. Ketoconazole also inhibited [3H]thymidine incorporation in the hormone-independent breast cancer cells MDA-MB-231 and Evsa-T, with respective 50% inhibitory concentration of approximately 13 and 2 microM. The mechanism of action of ketoconazole is unknown. However, another lipoxygenase inhibitor, BW755C, inhibited only weakly [3H]-thymidine incorporation and accumulated the cells in S and G2. Conversely, clotrimazole and SKF525A, inhibitors of cytochrome P-450 enzymes, had effects similar to those of ketoconazole on HT29-S-B6 cells whereas metronidazole and secnidazole, other azole derivatives which do not inhibit cytochrome P-450 enzymes, had no effect. The results suggest that cytochrome P-450 enzyme(s) activity(ies) could be implicated in the antiproliferative effects of ketoconazole.

Antimitogenic effects of dexamethasone in chemically transformed mouse fibroblasts.

Dexamethasone (a synthetic glucocorticoid) inhibited the entry into the S-phase of quiescent chemically transformed mouse fibroblasts (BP-A31) stimulated with 12-O-tetradecanoyl 13-acetate (TPA; a protein kinase-C activator) or with basic fibroblast growth factor. The basal rate of DNA synthesis was also strongly reduced by dexamethasone. In contrast, the mitogenic activity of insulin (acting via the insulin-like growth factor-I receptor) was little or not at all affected by dexamethasone. The antimitogenic activity of dexamethasone was enhanced when the steroid was included in the culture medium 24 h before the addition of mitogens. The effects of dexamethasone were glucocorticoid specific, partially reversed by the antiglucocorticoid RU 486, and prevented by cycloheximide (suggesting the involvement of glucocorticoid-induced protein synthesis in the antimitogenic activity of dexamethasone). Under the conditions of exponential growth in serum-free medium as well as in the presence of TPA, dexamethasone arrested the proliferation of sparsely seeded cells after a delay of 24-48 h. The BP-A31 cells are known to be constitutively competent and express at quiescence certain genes related to the G0/G1 transition in the original nontransformed A31 cell line. Of the transcripts corresponding to these genes, dexamethasone caused a rapid elimination of the JE mRNA, coding for a protein of the family of cytokines. The cell content of c-jun mRNA was also strongly reduced in the cells incubated at quiescence with dexamethasone (in the absence of mitogen). The presence of TPA along with dexamethasone prevented the elimination of c-jun, but not of JE mRNA. Short (30-min; together with the inducers) or long (24-h) treatment of the cells with dexamethasone did not prevent the induction of the c-fos gene expression by either TPA or basic fibroblast growth factor, indicating that dexamethasone does not interfere with mitogenic signal transduction. We conclude that in TPA-stimulated cells, the antiproliferative effect of dexamethasone is not due to interference with the expression of the c-jun gene, but may be related to the decreased level of the JE cytokine mRNA as well as to the synthesis of growth inhibitory protein(s).

Mitogenic signaling by prostaglandins in chemically transformed mouse fibroblasts: comparison with phorbol esters and insulin.

In the quiescent mouse BP-A31 fibroblasts, prostaglandin F2 alpha (PGF2 alpha) induces the expression of cell cycle-related genes c-fos, c-jun, and c-myc, and after a delay of approximately 12 h the entry into the phase of DNA replication. A weaker mitogenic effect was produced by certain other PGs (F1 alpha > D2), whereas the effects of PGs E and I were marginal or absent. The mitogenic effects of PGF2 alpha as well as of 12-O-tetradecanoyl phorbol 13-acetate (TPA; activator of protein kinase C) but not those of insulin (acting via the insulin-like growth factor 1 receptor) were abolished by a low concentration (7.5 nM) of staurosporine (inhibitor of protein kinase C). Moreover, long-time (24 h) preincubation with phorbol dibutyrate reduced the mitogenic effects of a subsequent exposure either TPA or PGF2 alpha. These observations favor the involvement of protein kinase C in the PGF2 alpha-dependent intracellular signal transduction. However, simultaneous stimulation of the quiescent cells with saturating concentrations of PGF2 alpha and TPA had a greater mitogenic effect than either drug alone, both in cells with and without down-regulation of protein kinase C, indicating that the protein kinase C-dependent signaling does not entirely account for the mitogenic activity of PGF2 alpha.

The arotinoid Ro 40-8757 has antiproliferative effects in drug-resistant human colon and breast cancer cell lines in vitro.

We have examined the antiproliferative effects of the arotinoid Ro 40-8757 in 3 drug-resistant human adenocarcinoma cell lines: the colonic cells HT29-5FU and CaCo2, and the mammary cells MCF-7mdr1. Whereas all-trans retinoic acid had no effect at the concentration of 10(-6) M, Ro 40-8757 was found to exert a high antiproliferative action with similar inhibitory potency (IC50) in drug-resistant and parental cell lines (range, 0.06 x 10(-6) to 0.57 x 10(-6) M). We conclude that: (1) thymidylate synthase is not involved in the mechanism of action of Ro 40-8757; (2) the mdr1 gene product does not recognize this retinoic derivative, and (3) Ro 40-8757, alone or in combinations with other cytotoxic drugs, can be very useful in patients with progressive disease after conventional chemotherapy.

The effect of age and individual differences in attentional control: a sample case using the Hayling test.

Individual differences in working memory (WM) have been shown to reflect the ability to control attention in order to prevent interference. This study examines the role of WM capacity in resisting interference in the Hayling task, in samples of younger and older adults. In each age group, high and low WM span individuals had to complete high-cloze sentences with either expected words (initiation) or words providing no meaning to the sentences (interference). Results showed increased response times and decreased correct responses in interference, as compared to initiation. As interference increased, older adults demonstrated lower accuracy than younger ones. Further, low spans demonstrated higher interference costs than high spans on accuracy, while the reverse pattern was found for response times. Our findings suggest that both age and individual differences in WM capacity need to be considered to account for differences in the ability to resist to interference.

Pharmacological control of the human gastric histamine H2 receptor by famotidine: comparison with H1, H2 and H3 receptor agonists and antagonists.

Histamine 0.1 microM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 microM. The histamine dose-response curve was mimicked by the H3 receptor agonist (R) alpha-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H2, H1 and H3 receptor antagonists, according to the following order of potency IC50: famotidine (0.3 microM) greater than triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (beta 2-type), PGE2 and VIP. The Schild plot was linear for famotidine (P less than 0.01) with a regression coefficient r = 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H2 receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H2 receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) alpha-MeHA and thioperamide are two selective ligands at histamine H3 receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H3 receptors in the regulation of gastric secretion is proposed.

Mitogenic activity of phorbol esters and insulin-like growth factor 1 in chemically transformed mouse fibroblasts BP-A31: independent effects and differential sensitivity to inhibition by 3-isobutyl-1-methyl xanthine.

Mitogenic effects of agents activating either the protein kinase C (PDGF; phorbol esters) or the insulin-like growth factor 1 (IGF1)-receptor pathway were studied in quiescent chemically transformed mouse fibroblasts (BP-A31), by evaluating the rate of [3H]thymidine incorporation. Each of these pathways alone was found to be sufficient to sustain progression through the entire cell division cycle. The mitogenic activity of phorbol 12-myristate 13-acetate (PMA) but not that of insulin was blocked by staurosporine (an inhibitor of protein kinase C), in support of the notion that protein kinase C activation was required for the PMA-induced cell cycle progression. The mitogenic effects of PMA were potentiated by cycloheximide pretreatment, and they were abolished by 3-isobutyl-1-methyl xanthine (IBMX; a cyclic nucleotide phosphodiesterase inhibitor). PDGF (known to activate the phospholipase C-protein kinase C pathway) also displayed mitogenic activity in the cycloheximide-pretreated BP-A31 cells, and its effects were prevented by IBMX. In contrast, the mitogenic effects of insulin (at concentrations where it activates the IGF1 receptor) or of IGF1 neither were notably influenced by cycloheximide pretreatment nor were inhibited by IBMX (in the presence of IBMX, the onset of S-phase was delayed by several hours). The expression of the c-fos gene was absent at quiescence; its induction by growth factors was not proportional to their mitogenic potency. Thus, c-fos expression was strongly induced by PMA but only weakly by insulin. IBMX was a powerful inducer of c-fos gene expression but caused a decrease in the level of c-myc mRNA.

Antiproliferative effects of sodium butyrate in adriamycin-sensitive and -resistant human cancer cell lines.

The proliferation of the MCF7 and MCF7-A (adriamycin-resistant) and KB-3-1 and KB-A (adriamycin-resistant) cell lines was arrested by sodium butyrate (NaBut) at 1 mM or higher concentrations. In the MCF7 and MCF7-A cell lines, an accumulation in the G1 phase was observed, whereas the KB-3-1 and KB-A cell lines accumulated in both G1 and G2/M phases. The level of the mRNA coded by the 'early G1' gene c-myc was high in all these cell lines, and was only transiently decreased by NaBut treatment. The 'late' mRNA coding for the proliferating cell nuclear antigen (PCNA) was also strongly expressed in all the cell lines studied; incubation with NaBut caused a decrease of the PCNA mRNA in the MCF7 and MCF7-A cells but not in the KB-3-1 and KB-A cells. The anti-oncoprotein p105RB was undetectable in the MCF7 and MCF7-A cells, while the KB-3-1 as well as KB-A cells contained a high level of this protein. Neither the content nor the apparent state of phosphorylation of the RB protein were affected by incubation (up to 48 h) with NaBut.

Cell cycle dependent regulation of cdc2 mRNA in mouse fibroblasts: requirement of protein synthesis and of continued mitogenic stimulation.

In the chemically transformed mouse fibroblasts (BP-A31) placed in a serum-free medium, the cdc2 mRNA content decreases in parallel with the cessation of [3H]thymidine incorporation. Extinction of the cdc2 gene expression is also observed in BP-A31 cells overexpressing the human c-myc oncogene. At quiescence, the cdc2 gene expression can be reinduced with serum or with other mitogens such as insulin or 12-O-tetradecanoyl phorbol 13-acetate (TPA). The kinetics of induction is characterized by a lag period which differs according to the mitogen used and reflects the length of the G1 phase (4-6 h with insulin or serum, 9-12 h with TPA). The cdc2 mRNA accumulation is prevented when protein synthesis is blocked with cycloheximide, also if the drug is added at a time when the synthesis of cdc2 mRNA is already under way. Similarly, removal of the mitogen leads to a cessation of the cdc2 mRNA accumulation. These results suggest that the increased expression of the cdc2 gene is mediated by (a) short-lived, growth factor-regulated protein(s).

Pharmacological control of the histamine H2 receptor-adenylate cyclase system by famotidine and ranitidine in normal and cancerous human gastric epithelia.

In human fundic glands, famotidine was about 17 times more potent than ranitidine as an inhibitor of histamine - stimulated cAMP generation. This H2-receptor antagonist had no effect on the receptor-adenylate cyclase systems sensitive to PGE2, isoproterenol (beta 2-receptor), VIP and on forskolin-induced activation of the Gs/catalytic units of the membrane-bound enzyme prepared from human fundic glands. In the HGT-1 human gastric cancer cell line, famotidine and ranitidine showed long lasting, irreversible actions probably related to a slow rate of dissociation from the histamine H2-receptor.

Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases.

Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.