Galectin-1 sensitizes carcinoma cells to anoikis via the fibronectin receptor α5ß1-integrin.

Anoikis resistance is a hallmark of transformed epithelial cells. Here, we show that treatment of anoikis-resistant carcinoma cell lines with the endogenous lectin galectin-1 (Gal-1) promoted apoptosis via interaction with the unligated fibronectin receptor α(5)ß(1)-integrin. Gal-1 efficiency correlated with expression of α(5)ß(1)-integrin, and transfection of the α(5)-subunit into deficient cell lines conferred Gal-1 binding and anoikis stimulation. Furthermore, Gal-1 and the α(5)- and ß(1)-integrin subunits co-precipitated in Gal-1-stimulated cells undergoing anoikis. Other members of the galectin family failed to be active. The functional interaction between Gal-1 and α(5)ß(1)-integrin was glycan dependent with α2,6-sialylation representing a switch-off signal. Desialylation of cell surface glycans resulted in increased electrophoretic mobility of α(5)ß(1)-integrin and facilitated Gal-1 binding and anoikis stimulation. On the level of signaling, Gal-1-stimulated anoikis was prevented by filipin, which impaired the internalization of α(5)ß(1)-integrin via cholesterol-enriched microdomains, and by pretreatment with a caspase-8 inhibitor. We propose that Gal-1/α(5)ß(1)-integrin interaction participates in the control of epithelial integrity and integrin sialylation may enable carcinoma cells to evade this Gal-1-dependent control mechanism.

Shower PUVA: a novel variant of photochemotherapy. Distribution of photosensitivity and accumulation of trioxsalen in the skin.

Shower PUVA is a new variant of photochemotherapy suitable for therapy of various skin disorders. Psoralen, e.g. trioxsalen-containing water recirculates in a closed shower system and wets the skin continuously. After showering, whole-body UVA irradiation (320-400 nm) is performed. In order to prove the equal distribution of photosensitivity in vivo minimal phototoxic dose (MPD) was determined in different skin areas of healthy individuals. Additionally, we investigated the accumulation of trioxsalen in psoriasis lesions under the conditions described by quantifying psoralen in scales collected after showering. In a randomized study 20 healthy volunteers (skin type I-III) took showers for 5 and 10 min in trioxsalen (0.27 mg/l)-containing water at 37 degrees C. Immediately afterwards, MPD was tested on the inside of the upper arms and on the buttocks by using a polychromator light source (315-400 nm). The applied UVA doses were 0.06-0.75 J/cm(2) with steps of 0.125 J/cm(2). MPD was evaluated after 72 h. Equal distribution of photosensitivity was defined as equal MPD on the insides of the upper arm and the buttocks (+/-0.125 J/cm(2)). Skin scales of 21 patients with psoriasis were collected by scratching after showering with trioxsalen-containing water (0.27 mg/l) for 5 min. For quantification of trioxsalen in the scales HPLC was performed. An equal distribution of photosensitivity was achieved in 70% (14/20) cases after 10-min showering in trioxsalen-containing water. Showering for 5 min only revealed a 30% (6/20) rate of equal distributed photosensitivity. After 10-min shower time MPD was 0.325 J/cm(2) (median; range: 0.06-0.625 J/cm(2)). The average amount of trioxsalen found in the scales was 2.03 ng/mg scales (range: 0.38-7.2 ng/mg). For shower PUVA using trioxsalen, 10 min shower time is recommended to achieve sufficient distribution of photosensitivity on the skin. Clinical efficacy of shower PUVA can be explained by skin accumulation of trioxsalen which enters from the aqueous phase into the upper skin layers in detectable amounts. This is the first report demonstrating the efficacy of shower PUVA which in short shower time allows an uptake of psoralen by the skin.

Trioxsalen in the presence of UVA is able to induce nuclear factor kappa B binding activity in HaCaT keratinocytes.

It has been described that treatment of cells with high dose psoralen and UVA induce the production of reactive oxygen species (ROS) leading to DNA damage. Transcription factor nuclear factor kappa B (NFkappaB) plays a crucial role in regulating not only cell growth but also cell differentiation, and ROS seem to be partly involved in these mechanisms. The aim of this research was to find out the effect of a combined treatment with trioxsalen (TMP)/UVA on NFkappaB binding activity in HaCaT keratinocytes. HaCaT keratinocytes were treated with 27 microg/l TMP. This concentration did not affect the proliferation rates, nor was it toxic, as shown by cytotoxicity assays. After treatment with TMP with or without UVA (1 J/cm(2)), NFkappaB binding activity in nuclear protein extracts was measured by electrophoretic mobility shift assays. The effect on cytokines and cytokine receptor genes was investigated using cDNA expression arrays. An inhibitory effect on NFkappaB binding activity was found between 30 and 60 min after TMP supplementation of the culture media. UVA irradiation induced a 2-fold increase in NFkappaB binding activity in TMP supplemented HaCaT keratinocytes compared with the non-irradiated control. In addition, NFkappaB binding activity was higher after UVA irradiation with TMP than in UVA irradiated cells in the absence of TMP. TGF-alpha, IL-1R, IL-2Ralpha, IL-12beta and PDGF expression was induced by UVA. However, all of them except PDGF were inhibited by combined TMP/UVA treatment. Using an inhibitor of NFkappaB activation, we found out that under these conditions, these cytokines or cytokine receptor genes are apparently not regulated by NFkappaB. Our results indicate that a combined TMP/UVA treatment of HaCaT keratinocytes induces NFkappaB binding activity, and that this is a synergistic effect. The investigated cytokines, and cytokine receptor genes do not seem to be NFkappaB regulated; however, TMP shows anti-inflammatory capacities in vitro.

Effects of UVA and L-ascorbic acid on nuclear factor-kappa B in melanocytes and in HaCaT keratinocytes.

Nuclear factor-kappaB (NFkappaB) is a pleiotropic transcriptional activator, which is a sensitive transcriptional factor for free radicals and activates multiple target genes. UVA is very efficient in inducing free radicals in human skin cells. L-ascorbic acid is regarded as a scavenger of UVA-induced free radicals in human keratinocytes. In epidermis, melanocytes and keratinocytes play an important protective role against skin photodamage. In the present study, we aimed to investigate the role of NFkappaB on photodamage in melanocytes and keratinocytes. Normal human melanocytes (NHM) and HaCaT keratinocytes were treated with UVA (500 mJ/cm(2), 1,000 mJ/cm(2)) and/or L-ascorbic acid (100 microM, 250 microM). NFkappaB binding activity was analysed by electrophoretic mobility shift assay. NFkappaB binding activity was increased by UVA irradiation in HaCaT keratinocytes, but it was not affected in NHM. On the other hand, L-ascorbic acid decreased NFkappaB binding activity both in UVA-irradiated and in non-irradiated NHM. In contrast, NFkappaB binding activity in HaCaT keratinocytes was increased after treatment with L-ascorbic acid. In addition, L-ascorbic acid synergistically induced NFkappaB binding activity with UVA irradiation. The contrary response on NFkappaB binding activity in NHM and HaCaT keratinocytes indicated that the redox regulation might be different on photoprotective action in melanocytes and keratinocytes.