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G Ghanem

Publications and source records attributed to G Ghanem.

At least 19 recordsLinked to original sources
PubMed2006

High serum galectin-3 in advanced melanoma: preliminary results.

BACKGROUND: Galectin-3 (Gal-3) is a member of the family of beta-galactoside-binding mammalian lectins, and has been implicated in tumour invasion and metastatic process in vitro and in vivo. AIM: To determine whether an increase in serum Gal-3 production could be found in patients with advanced metastatic melanoma. METHODS: We collected 18 sera from patients with AJCC stage IV metastatic melanomas and 20 sera from healthy volunteers. Determination of Gal-3 was performed by ELISA, and in the group of patients with melanoma, these results were compared with the serum lactate dehydrogenase (LDH) and the C-reactive protein (CRP) concentrations. RESULTS: Gal-3 concentration was shown to be significantly higher in the group of patients with melanoma compared with healthy volunteers, and Gal-3 concentration was significantly correlated with both LDH and CRP in the melanoma group. We also selected four patients in the melanoma group for Gal-3 retrospective immunostaining analysis on cutaneous metastases. Two of these patients, who had a higher Gal-3 serum level, showed more intense staining and the other two patients, with a lower serum level of Gal-3, had moderate immunostaining, suggesting that at least part of serum Gal-3 might be produced by metastatic melanoma tissue. CONCLUSIONS: Gal-3 might play a role in melanoma progression and/or inflammation, and warrants further study.

Adult↗
PubMed2004

Cysteine but not glutathione modulates the radiosensitivity of human melanoma cells by affecting both survival and DNA damage.

Glutathione (GSH) and its precursor cysteine (Cys) are both known to react within any cells with oxidative species and thus play an important role in cellular defense mechanisms against oxidative stress. In melanocytes, these are also important precursors of melanogenesis by reacting non-enzymatically with l-dopaquinone to form the sulfur-containing pheomelanin. Our aim was to assess pigment role in the cellular radioprotection mechanism using a human melanoma cell model of mixed-type melanin under GSH depletion to obtain a radiosensitizing effect. The latter has been achieved either by Cys deprivation or GSH specific depletion. We first compared cell survival of Cys-deprived and GSH-depleted cells vs. control cells. Cys deprivation was achieved by decreasing Cys concentration in the culture medium for 24 h. In this condition, no toxicity was observed, Cys and GSH levels decreased, melanogenesis switched to a higher eumelanin synthesis and cells were significantly more resistant to 10-Gy dose of ionizing radiations than untreated cells. Glutathione depletion was achieved with the gamma-glutamylcysteine synthetase inhibitor buthionine-S-sulfoximine (BSO) for 24 h at 50 microM, a concentration yielding no toxicity. In this condition, intracellular GSH level decreased but no change in pigmentation was observed and cells were slightly but significantly more sensitive to radiation than the control. We then compared DNA radio-induced damages by Comet assay in control cells, cells treated as above and cells with stimulated pigmentation by increasing Tyr concentration in the medium. Our results showed that, when intracellular eumelanin content increased, DNA damage decreased. By contrast, DNA damage increased in cells treated with BSO alone. It is concluded that increasing the intracellular eumelanin content by the melanin precursor Tyr or by favoring the Pheo- to Eumelanin switch, compensates for the loss of the two intracellular radioprotectors that are GSH and Cys.

Buthionine Sulfoximine↗
PubMed2003

Anti-inflammatory and anti-invasive effects of alpha-melanocyte-stimulating hormone in human melanoma cells.

Alpha-melanocyte stimulating hormone (alpha-MSH) is known to have pleiotrophic functions including pigmentary, anti-inflammatory, antipyretic and immunoregulatory roles in the mammalian body. It is also reported to influence melanoma invasion with levels of alpha-, beta- and gamma-MSH correlated clinically with malignant melanoma development, but other studies suggest alpha-MSH acts to retard invasion. In the present study, we investigated the action of alpha-MSH on three human melanoma cell lines (HBL, A375-SM and C8161) differing in metastatic potential. alpha-melanocyte-simulating hormone reduced invasion through fibronectin and also through a human reconstructed skin composite model for the HBL line, and inhibited proinflammatory cytokine-stimulated activation of the NF-kappaB transcription factor. However, A375-SM and C8161 cells did not respond to alpha-MSH. Immunofluorescent microscopy and Western blotting identified melanocortin-1 receptor (MC-1R) expression for all three lines and MC-2R on HBL and A375-SM lines. Receptor binding identified a similar affinity for alpha-MSH for all three lines with the highest number of binding sites on HBL cells. Only the HBL melanoma line demonstrated a detectable cyclic adenosine monophosphate (cAMP) response to alpha-MSH, although all three lines responded to acute alpha-MSH addition (+(-)-N(6)-(2-phenylisopropyl)-adenosine (PIA)) with an elevation in intracellular calcium. The nonresponsive lines displayed MC-1R polymorphisms (C8161, Arg (wt) 151/Cys 151; A375-SM, homozygous Cys 151), whereas the HBL line was wild type. Stable transfection of the C8161 line with wild-type MC-1R produced cells whose invasion was significantly inhibited by alpha-MSH. From this data, we conclude that alpha-MSH can reduce melanoma cell invasion and protect cells against proinflammatory cytokine attack in cells with the wild-type receptor (HBL).

Cytokines↗
PubMed2002

Melanoma progression and serum L-dopa/L-tyrosine ratio: a comparison with S100B.

The challenge to find a reliable tumour marker for the management of melanoma patients still remains. In this study, the serum L-dopa/L-tyrosine ratio was compared with serum S100B as a reference marker. A total of 89 melanoma patients were sampled and staged according to the American Joint Committee on Cancer (AJCC) classification. Of these, 19 stage III and 28 stage IV patients were evaluated for disease progression at 1.5 years and 6 months post-sampling, respectively. Serum L-dopa and L-tyrosine were measured by high performance liquid chromatography (HPLC) (normal value for ratio < 16 x 10(-5)) and S100B using the LIA-mat Sangtec 100 assay (normal value < 0.10 microg/l). Non-parametric tests (Kruskal-Wallis analysis of variance, Dunn's and Spearman) were used for the statistical analysis. The median serum L-dopa/L-tyrosine ratio was 16.0 x 10(-5) (range 2.7-545.1 x 10-5 and the median S100B level was 0.15 microg/l (range < 0.10-13.8 microg/l), with a sensitivity of 51% for the ratio and 66% for S100B. There was a 47% discordance and no correlation between the two markers (r = 0.149). The ratio was higher in stage IV than in other stages (P < 0.05), as was the S100B level (P < 0.0001). Both markers were higher in patients with evolutive disease (n = 23) than in stable patients (n = 24), with values of 20.8 x 10(-5) versus 13.1 x 10(-5) for the ratio (P < 0.05) and 0.89 microg/l versus 0.16 microg/l for S100B (P < 0.001); for the ratio, this difference was more pronounced in stage III than in stage IV patients. The overall sensitivity and specificity of the markers to predict disease progression were 78% and 67%, respectively, for the ratio, and 74% and 83%, respectively, for S100B (using an ROC cut-off of 0.38 microg/l). In conclusion, the serum L-dopa/L-tyrosine ratio correlates with melanoma progression and has predictive value, especially in stage III patients. This tumour marker, like S100B, could serve as an additional tool in the management of melanoma.

Adult↗
PubMed2002

Urinary excretion of epidermal-type fatty acid-binding protein and S100A7 protein in patients with cutaneous melanoma.

Epidermal-type fatty acid-binding protein (E-FABP), a protein related to the intracellular trafficking of fatty acids, is expressed in melanocytic tumours but not in normal human melanocytes. E-FABP interacts with S100A7. The presence of these two proteins was investigated in the urine of patients with cutaneous melanoma or other types of cancer, and healthy controls. The first voided morning urine samples of 31 patients with melanoma, 73 patients with other types of cancer and 17 healthy controls were concentrated and submitted to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting for protein detection. In the healthy controls, the incidences of urinary detection of these proteins were higher in females than in males, being 50% (five out of 10) versus 0% (zero out of seven) for E-FABP ( < 0.05), and 70% (seven out of 10) versus 0% (zero out of seven) for S100A7 ( < 0.05). Both proteins were detected in the urine of patients with melanoma. The incidence of S100A7 was higher in the urine of patients with melanoma (77%, 24 out of 31) compared with healthy controls (41%, seven out of 17) and patients with other types of cancer (53%, 39 out of 73) ( < 0.03). In contrast, the incidence of E-FABP was the same among the melanoma group (39%, 12 out of 31), healthy controls (29%, five out of 17) and patients with other types of cancer (23%, 17 out of 73). Surprisingly, E-FABP was always detected in the urine of females with stage I/II or III melanoma, but was no longer detectable in the urine of patients with stage IV melanoma. Urinary S100A7 may have some specificity to the host response to melanoma since its incidence was not increased in other cancers. The lack of E-FABP detection in the urine of patients with distant metastases suggests an inverse relationship between E-FABP release and the spread of melanoma.

Adult↗
PubMed2001

On the release and half-life of S100B protein in the peripheral blood of melanoma patients.

The aim of the present work was to investigate the origin and half-life of endogenous S100B protein reported by many investigators as a useful melanoma serum marker. Within cells, S100B protein exists in homo- or heterodimer form containing mainly Ca(++), having a substantial fraction bound to membranes. As such, S100B is believed to be involved in the regulation of cytoskeleton. Also, a role in the cell cycle progression has been suggested. Although S100B appears having important intracellular functions, proofs of its secretion, at least at concentrations such as the ones measured in melanoma patients, are still lacking. Consistent with this view is the fact that immunohistology for S100 protein reported by numerous authors clearly indicate an exclusive intracellular staining. For these reasons, it was of a major interest to investigate how and when S100B is shed to the blood. Knowing that significant S100B levels are seen only in stage IV patients, we hypothesized that cell death may be the major source of circulating S100B protein in these patients. This hypothesis was studied in an in vitro model simulating cell death and in vivo in melanoma and other cancer patients undergoing highly cytotoxic regional immunochemotherapy using isolated limb perfusion with tumor necrosis factor and melphalan, as well as in tumor exudates and pleural fluids. Our results strongly suggest melanoma and endothelial cell death and subsequent continuous drainage to the blood as the major mechanism behind S100B release to the blood circulation. We estimated the endogenous S100B protein half-life to be about 30 min.

Biomarkers, Tumor↗
PubMed2001

Transcriptional repression of the microphthalmia gene in melanoma cells correlates with the unresponsiveness of target genes to ectopic microphthalmia-associated transcription factor.

In the melanocyte, expression of genes required for pigment formation is mediated by the microphthalmia transcription factor, which is also critical for the development and survival of normal melanocytes during embryogenesis. Here we show that the expression of the melanocyte-specific isoform of microphthalmia transcription factor is lost in a subset of human melanoma cell lines, accompanied by the repression of tyrosinase and tyrosinase-related proteins 1 and 2, the three transcriptional target genes for microphthalmia. After the forced expression of microphthalmia transcription factor in melanoma cells where the expression of endogenous microphthalmia gene was found to be extinguished, no restoration of the melanogenic phenotype occurred and the transcription of the three microphthalmia transcription factor target genes remained silent. The transcription activation domain of microphthalmia transcription factor, tested as a GAL-MITF fusion protein, remained fully functional in these cells, however, and ectopic microphthalmia transcription factor localized normally to the nucleus and bound to the tyrosinase initiator E-box in gel retardation assays. Thus, the block of differentiation in microphthalmia-transcription-factor-negative melanomas extended the transcriptional repression of the microphthalmia transcription factor gene alone, and endogenous promoters in these melanoma cells became no longer responsive to microphthalmia transcription factor when this was substituted exogenously. The data presented suggest that a specific nuclear context is required for the transcriptional activation of the melanocyte markers by the microphthalmia transcription factor in malignant melanocytes and this specificity is lost concomitantly with the transcriptional repression of microphthalmia transcription factor.

Cell Division↗
PubMed2001

Tips and step-by-step protocol for the optimization of important factors affecting cellular enzyme-linked immunosorbent assay (CELISA).

CELISA, or cellular enzyme-linked immunosorbent assay, is a powerful and easy to use technique to study cell surface antigens under different stimulations. Nevertheless, some factors must be discussed and optimized prior to reaching a reproducible CELISA. These include the choice of cell density, fixative agent, blocking agent, culture medium, optimal antibody dilutions, and incubation time. In this paper, we first present a short review of some references devoted to CELISA by means of a comparison of these parameters, followed by their description. Then, we describe and study these different parameters using practical examples comparing TNF-induced ICAM-1 expression as an end point, on HBL melanoma and HUVEC. These cell lines were also chosen because they differ in their ability to grow as discontinuous and continuous layers, respectively. Furthermore, we designed a comprehensive flow chart, as well as a complete step-by-step protocol for CELISA optimization.

Antigens, Surface↗
PubMed2000

Alpha-melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma cell lines.

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Oestrogenic steroids and melanoma cell interaction with adjacent skin cells influence invasion of melanoma cells in vitro.

The invasion of melanoma is complex and multi-staged and involves changes in both cell/extracellular matrix (ECM) and cell/cell interactions. Female steroids and alpha-MSH have also been reported to influence metastatic melanoma progression, but their mechanisms of action are unknown. Accordingly, our aim was to establish in vitro models to examine (a) the influence of sex steroids and alpha-melanocyte-stimulating hormone (alpha-MSH) on tumour invasion and the influence of (b) ECM proteins and (c) adjacent cells on melanoma invasion. In the first model, melanoma cell invasion through fibronectin over 20 hr under serum-free conditions was used to investigate the effects of 17beta-oestradiol and oestrone on the invasion of human melanoma cell lines, A375-SM and HBL. A375-SM, but not HBL cells, proved very susceptible to inhibition by female steroids. However, invasion of the HBL line was inhibited by alpha-MSH. Using the second model of reconstructed human skin based on de-epidermised acellular dermis, we found that the HBL cells on their own failed to invade into the dermis (irrespective of the presence or absence of the basement membrane). However, there was a significant synergistic interaction between keratinocytes, fibroblasts and HBL cells, such that a modest invasion of HBLs into the dermis was seen within 2 weeks when other skin cells were present. In contrast, A375-SM cells showed a significant ability to invade the dermis in the absence of other cells, with less invasion when other skin cells were present. In summary, these models have provided new information on the extent to which melanoma cell invasion is sensitive to oestrogenic steroids and to alpha-MSH and to interaction, not only with adjacent skin cells but also to the presence of basement membrane antigens.

Estradiol↗
PubMed2000

Characterization of an in vitro model of human melanoma invasion based on reconstructed human skin.

The purpose of this study was to compare the invasive properties of normal human cutaneous melanocytes and of a cutaneous melanoma cell line (HBL) in a three-dimensional model of reconstructed human skin. Specifically, we asked to what extent the pigmentary and invasive behaviour of both cells is influenced by their interaction with adjacent skin cells (keratinocytes and fibroblasts) and the basement membrane (BM). In the presence of a BM, normal human melanocytes within this model remained within the basal layer of keratinocytes and did not pigment spontaneously. When the BM was removed, melanocytes were found suprabasally and pigmented extensively. No significant invasion of melanocytes into the dermis was detected in the presence or absence of the BM. HBL melanoma cells showed no significant ability to invade into the dermis in the absence of other cells, irrespective of the presence or absence of the BM. However, when added to keratinocytes and fibroblasts, HBL cells showed a capacity to invade into the dermis, both in the presence and absence of the BM. Associated with HBL invasion into the dermis, we noted significant keratinocyte entry into the dermis. On their own, keratinocytes entered the dermis in the absence of the BM but showed no significant penetration into the dermis when the BM was present. In summary, this model demonstrates clear differences between melanocytes and a melanoma cell line with respect to their invasive properties. It also allows demonstration of interactions between cells, and between cells and the BM. The study also provides evidence for a synergistic interaction between this melanoma cell line and keratinocytes in penetrating the BM.

Basement Membrane↗
PubMed2000

Alpha-melanocyte stimulating hormone can reduce T-cell interaction with melanoma cells in vitro.

This study was undertaken to investigate whether alpha-melanocyte stimulating hormone (alphaMSH) influences the interaction of melanoma cells with T-lymphocytes in the light of previous work from our laboratories showing that alphaMSH can reduce tumour necrosis factor-alpha (TNFalpha) stimulated ICAM-1 upregulation in both normal and transformed melanocytes. Two cutaneous melanoma cell lines--A375-SM and HBL--were examined initially. A375-SM cells gave only a two-fold increase in T-cell proliferation, which was not much improved by the pretreatment of the melanoma cells with cytokines. HBL cells induced a three-fold increase in T-cell proliferation, which was slightly enhanced by the addition of cytokines. Neither cell line expressed B7(1), HBL cells expressed a low level of B7(2), whereas A375-SM cells had little, if any, B7(2) expression. Addition of alphaMSH reduced the interaction between these cutaneous melanoma cells and T-lymphocytes in some, but not all, conditions. An ocular melanoma cell line transfected with B7 showed a modest interaction with T-cells (in two out of three donors) and this response was reduced by the addition of alphaMSH. Pretreatment of the transfected line with cytokines markedly enhanced stimulation of T-cell proliferation by these tumour cells, and alphaMSH reduced the interaction between melanoma cells and T-cells for two out of three donors. In summary, under experimental conditions where melanoma cell stimulation of T-cells occurred (generally pretreatment of the cells with interferon-gamma gave the most convincing response), alphaMSH reduced this response in the majority of experiments, providing preliminary evidence to confirm the hypothesis that MSH may assist melanoma cells to evade interaction with immune cells.

Antibodies, Monoclonal↗
PubMed2000

The degree of pigmentation modulates the radiosensitivity of human melanoma cells.

The relationship between cell pigmentation and radiosensitivity was investigated in two selected human melanoma cell lines with different melanin content (mixed type: eumelanin and pheomelanin, and pheomelanotic phenotypes). The same study was also done after stimulation of melanogenesis (1) by addition of the melanin precursor l-tyrosine to each of the cell lines separately and (2) by irradiation alone with doses ranging from 0 to 10 Gy. We found that a decrease in cell radiosensitivity was correlated with the type of melanin, with a clear involvement of eumelanin rather than pheomelanin. Increasing the intracellular content of both melanins promoted the growth of irradiated cells. Moreover, at a dose of 10 Gy, both tyrosinase activity and melanin cell content were significantly increased in the absence of any other melanogenesis promoter. Our data suggest that the amount of intracellular melanin is inversely related to the radiosensitivity of melanoma cells and may explain at least in part the controversial responses to ionizing radiations reported for melanoma.

Cell Survival↗
PubMed1999

Alpha-melanocyte-stimulating hormone inhibits NF-kappaB activation in human melanocytes and melanoma cells.

Alpha-melanocyte-stimulating hormone is produced by several different cell types including neural cells, endothelial cells, monocytes, and keratinocytes. A biologic role in melanocyte pigmentation is widely recognized, but more recent studies describe a part in modulating inflammatory and immune responses. The aim of the this study was to investigate the mechanism by which alpha-melanocyte-stimulating hormone antagonizes proinflammatory cytokine action. We report that alpha-melanocyte-stimulating hormone (10-9 M) was effective in opposing a tumor necrosis factor-alpha stimulated increase in NF-kappaB DNA binding activity in: (i) normal ocular melanocytes; (ii) cells cultured from ocular melanoma tumors; and (iii) two cutaneous melanoma cell lines. NF-kappaB is activated by many inflammatory mediators and controls transcription of genes required for immune and inflammatory responses. The transcription factor complex was positively identified as the p50/p65 heterodimer, recognized to have transcriptional activating potential. Maximum reduction of NF-kappaB DNA binding activity with alpha-melanocyte-stimulating hormone was detected 2 h after cellular stimulation and varied from between 53% and 18% depending on cell type. Whereas the acute inhibitory effects could be mimicked by elevating cyclic adenosine monophosphate, alpha-melanocyte-stimulating hormone was not found to have any effect on the relative level of IkappaBalpha protein expression over 24 h. These data show that alpha-melanocyte-stimulating hormone has a pronounced effect on NF-kappaB activity in melanocytes and melanoma cells, identifying a specific dimeric complex, and suggest this to be a key pathway by which immunomodulation/anti-inflammation may operate. The results may also be considered in the broader context of general inflammatory pathologies concerning cells which express alpha-melanocyte-stimulating hormone receptors and utilize the NF-kappaB signaling pathway.

Cells, Cultured↗
PubMed1999

SODs are involved in the regulation of ICAM-1 expression in human melanoma and endothelial cells.

It is well known that ICAM-1 expression can be stimulated by TNF and by oxidative stress, via the activation of specific transcription factors. Two of these--NFkappaB and AP-1--can also be activated by reactive oxygen species, including the superoxide anion (also produced under TNF challenge). The latter is inactivated by superoxide dismutase of which two forms exist: Cu/Zn-SOD (cytoplasmic) and Mn-SOD (mitochondrial). We investigated whether superoxide anion direct generation or accumulation through specific SOD inhibition, may affect ICAM-1 expression in human melanoma and endothelial cells. Our results show a 20-50% increase in both SOD activities when cells were exposed to TNF or to an oxidative stress produced by Paraquat (a generator of superoxide anion radicals), both in terms of enzymes activity (zymogram) and protein levels (Western blotting and ELISA). Either with TNF or Paraquat, we could measure a significant increase of ICAM-1 expression with maxima ranging from 140 to 200%, depending on the cell line. Specific inhibition of Cu/Zn-SOD activity by DTIC (diethyldithiocarbamic acid), in presence of Paraquat or TNF, was followed by an upregulation of ICAM-1 expression (60 and 20%, respectively). In contrast, the addition of a SOD mimetic (MnTMPyP) completely inhibited Paraquat-stimulated ICAM-1 expression in melanoma cells and significantly decreased it in HUVEC (50%). In presence of TNF however, the same SOD mimetic inhibited TNF-stimulated ICAM-1 expression by 25% in melanoma and 17% in endothelial cells. In conclusion, these data provide evidence that melanoma and endothelial cells exposure to TNF or oxidative stress results in a significant increase of both Mn- and Cu/Zn-SOD activities. This increase seems to be associated with a reduction in the stimulation of ICAM-1 expression by TNF or oxidative stress.

Cells, Cultured↗