Renato Morandini

TRP-1 expression correlates with eumelanogenesis in human pigment cells in culture

We have investigated the relationship in human cultured normal and malignant melanocytes between the accumulation of mRNAs encoding tyrosinase and tyrosinase‐related protein‐1 (TRP‐1), the activity of tyrosinase and the presence of melanin. Tyrosinase mRNA correlates with tyrosinase activity and with the presence of pheomelanin, eumelanin or both melanin types. In contrast TRP‐1 mRNA is only detectable in cells containing eumelanin, which suggests a role for TRP‐1 in the eumelanin synthesis pathway.

Genotypic and Gene Expression Studies in Congenital Melanocytic Nevi: Insight into Initial Steps of Melanotumorigenesis

Large congenital melanocytic nevi (CMNs) are said to have a higher propensity to malignant transformation compared with acquired nevi. Thus, they represent a good model for studying initial steps of melanotumorigenesis. We have performed genotypic (karyotype, fluorescence in situ hybridization, and mutational analyses) and differential expression studies on a large cohort of medium (n=3) and large (n=24) CMN. Chromosomal abnormalities were rare and single, a feature probably reflecting the benignity of these lesions. Mutational screening showed a high frequency of NRAS mutations in our series (19/27 cases, 70%), whereas BRAF mutations were less common (4/27 cases, 15%). Differential did not show significant alterations of cellular processes such as cell proliferation, cell migration/invasion, angiogenesis, apoptosis, and immune/inflammatory responses. However, significant downregulation of genes involved in pigmentation and upregulation of genes playing a role in DNA protection were observed. Lastly, our microarrays displayed upregulation of genes mediating chemoresistance in cancer. As alteration of pigmentation mechanisms can trigger oxidative damage, increased expression of genes involved in maintenance of DNA integrity might reflect the ability of nevocytic cells to self-protect against cellular stress. Furthermore, the observed alterations linked to chemoresistance might partially account for the well-known inefficacy of chemotherapy in malignant melanoma.

Evaluation of the prognostic significance of serum galectin-3 in American Joint Committee on Cancer stage Iii and stage Iv melanoma patients

Galectin-3 (Gal-3) is a member of the &bgr;-galactoside-binding lectins family and has been implicated in angiogenesis, tumor invasion, and metastatic process in vitro and in vivo. As we showed recently that advanced melanoma patients presented high serum level of Gal-3, we investigated the association of this protein with the outcome of melanoma patients. Whether this protein could be a biomarker has not been assessed, and we compared the prognostic value of serum Gal-3 in multivariate analysis with lactate dehydrogenase, C-reactive protein and S100B. We conclude that Gal-3 could be of prognostic value in melanoma patients; more precisely, this protein has a strong independent prognostic signification with a cut-off value of 10 ng/ml. After these data, we believe that serum Gal-3 measurement can have an important role in the follow-up and management of advanced American Joint Commission on Cancer stage III and stage IV melanoma patients. Further studies will uncover whether Gal-3 will be able to open new therapeutic perspectives.

p53 Reactivation by PRIMA-1(Met) (APR-246) sensitises (V600E/K)BRAF melanoma to vemurafenib.

Intrinsic and acquired resistance of metastatic melanoma to (V600E/K)BRAF and/or MEK inhibitors, which is often caused by activation of the PI3K/AKT survival pathway, represents a major clinical challenge. Given that p53 is capable of antagonising PI3K/AKT activation we hypothesised that pharmacological restoration of p53 activity may increase the sensitivity of BRAF-mutant melanoma to MAPK-targeted therapy and eventually delay and/or prevent acquisition of drug resistance. To test this possibility we exposed a panel of vemurafenib-sensitive and resistant (innate and acquired) (V600E/K)BRAF melanomas to a (V600E/K)BRAF inhibitor (vemurafenib) alone or in combination with a direct p53 activator (PRIMA-1(Met)/APR-246). Strikingly, PRIMA-1(Met) synergised with vemurafenib to induce apoptosis and suppress proliferation of (V600E/K)BRAF melanoma cells in vitro and to inhibit tumour growth in vivo. Importantly, this drug combination decreased the viability of both vemurafenib-sensitive and resistant melanoma cells irrespectively of the TP53 status. Notably, p53 reactivation was invariably accompanied by PI3K/AKT pathway inhibition, the activity of which was found as a dominant resistance mechanism to BRAF inhibition in our lines. From all various combinatorial modalities tested, targeting the MAPK and PI3K signalling pathways through p53 reactivation or not, the PRIMA-1(Met)/vemurafenib combination was the most cytotoxic. We conclude that PRIMA-1(Met) through its ability to directly reactivate p53 regardless of the mechanism causing its deactivation, and thereby dampen PI3K signalling, sensitises (V600E/K)BRAF-positive melanoma to BRAF inhibitors.

Tyrosinase modulation by five Rwandese herbal medicines traditionally used for skin treatment

ETHNOPHARMACOLOGICAL RELEVANCE Traditional herbal medicines provide an interesting, largely unexplored source for the development of potential new drugs and skin-care cosmetics. Some herbal extracts are known to be inhibitors of melanin formation, sometimes more potent than the classical inhibitors, hydroquinone/arbutin or kojic acid, and are not associated with melanocytes cytotoxicity or mutagenicity. Such plants are used in traditional medicine in many countries, particularly in Africa, for skin lightening. AIM OF THE STUDY To evaluate in vitro the ability of Rwandese medicinal plants, traditionally used for the treatment of skin (discoloration and attenuation of discolored spots), to modulate pigmentation and tyrosinase activity. MATERIALS AND METHODS Based on an ethnopharmacological survey, five herbs [Brillantaisia cicatricosa Lindau (Acanthaceae), Chenopodium ugandae (Aellen) Aellen (Chenopodiaceae), Dolichopentas longiflora Oliv. (Rubiaceae), Protea madiensis Oliv. (Proteaceae) and Sesamum angolense Welw. (Pedaliaceae)] were selected. Twenty-seven extracts, obtained by treating the herbs with increasing polarities solvents, were investigated for their effects on cell viability (MTT test) and on pigmentation: inhibition of the enzyme tyrosinase (colorimetry of reaction products, measurement of enzyme activity, TLC-autography; studies on crude cellular extracts obtained from normal melanocytes and on a mushroom tyrosinase) and measurement of melanogenesis by human melanoma cells. RESULTS None of the tested plant extracts were cytotoxic on tested human melanoma cell lines, except for Dolichopentas longiflora (IC50 of leaves n-hexane extract, 4μg/ml for MM028 and 4.5μg/ml for MM001; IC50 of roots ethyl acetate extract, 0.8μg/ml for MM028 and 3.9μg/ml for MM001). Almost all extracts inhibited melanogenesis in a melanoma whole cells overall pigmentation assay, a model reflecting the entire cycle of melanogenesis. All the Protea madiensis extracts quite strongly inhibited melanogenesis and, surprisingly, one of the Dolichopentas longiflora leaves extracts was found to increase melanogenesis. These results were confirmed by the modulation of pigmentation reactions by crude cellular extracts obtained from normal melanocytes; interestingly, one of the extracts (Dolichopentas longiflora ethyl acetate extract) is even more active (61% at 500μg/ml) than kojic acid (<3% at 142μg/ml and 68% at 1421μg/ml). In a mushroom tyrosinase inhibition assay, data obtained on some extracts fairly agree with pigmentation inhibition measured on melanocytes proteins as, for example, the methanol extract of Protea madiensis. While a few others extract display discording data, this probably reflects either differences between human and mushroom tyrosinase, interference with melanocytes enzymes at later steps than tyrosinase or the simultaneous presence of compounds with conflicting activities in a given extract. CONCLUSIONS Ethnopharmacological data represent an efficient approach to discover active herbs. Some of the selected medicinal plants clearly show potent tyrosinase inhibitions while one extract significantly increases cell pigmentation; one extract contains potent growth melanocytes inhibitors.

Tumor type and vascularity: Important variables in infusional brachytherapy with colloidal 32P

PURPOSE This study investigated the role of histologic tumor characteristics, in comparison with a normal tissue, and of tumor vascularization on the uptake and retention of colloidal 32P used in infusional brachytherapy of solid cancers. The cytotoxicity of colloidal 32P was also evaluated for two tumors of different radiosensitivity, a melanoma, and a squamous cell carcinoma. METHODS AND MATERIALS An in vitro analysis of colloidal 32P uptake was carried out on a human melanoma cell line, HBL, a human squamous cell carcinoma, SCC1, and normal fibroblasts, F-NBB. Tumor retention of colloidal 32P was studied in vivo for the HBL and the SCC1 tumors implanted subcutaneously in nude mice. Tumor vascular density was determined by microscopic study of Massons trichrome slides of HBL and SCC1 tumors of about 1 cm diameter. RESULTS In vitro studies showed that the time required for maximal cell uptake of colloidal 32P was only 10-20 min for the SCC1 and HBL tumors, while it took at least 60 min for the fibroblasts. After intratumoral injection of macroaggregated albumin (MAA), followed by 50 microCi of colloidal 32P, Bremsstrahlung imaging performed at 6 and 24 h showed that the activity remained in the HBL tumor while some of the radiocolloids leaked from the SCC1 tumor and was trapped in the reticuloendothelial system of the liver. Organ activity counting confirmed this finding: 32P activity was three to four times higher in the HBL than in the SCC1 tumor, whereas the activity in the liver, insignificant in the HBL mice (less than 0.1 microCi/g), was as high as 24 microCi/g in the SCC1 mice. This phenomenon may be explained by the difference in tumor vascular density, estimated for the HBL to be about four times less than that of the SCC1 tumor (5.7 vs. 21.4 blood vessels per mm2 for the HBL and the SCC1 tumors, respectively). CONCLUSION Intratumoral infusion of colloidal 32P may be a useful complement of radiation therapy in the treatment of nonresectable but accessible solid tumors. Tumor vascularization must be taken into account for a successful vascular blockade by MAA prior to the infusion of colloidal 32P.

Prominent role of cyclic adenosine monophosphate signalling pathway in the sensitivity of WTBRAF/WTNRAS melanoma cells to vemurafenib

Vemurafenib improves survival in patients with melanoma bearing the (V600E)BRAF mutation, but it did not show any benefit in clinical trials focusing on wild type tumours while it may well inhibit (WT)BRAF considering the dosage used and the bioavailability of the drug. As tumours may contain a mixture of mutant and wild type BRAF cells and this has been also put forward as a resistance mechanism, we aimed to evaluate the sensitivity/resistance of six, randomly selected, (WT)BRAF/(WT)NRAS lines to vemurafenib and found four sensitive. The sensitivity to the drug was accompanied by a potent inhibition of both phospho-ERK and phospho-AKT, and a significant induction of apoptosis while absent in lines with intrinsic or acquired resistance. Phospho-CRAF expression was low in all sensitive lines and high in resistant ones, and MEK inhibition can effectively potentiate the drug effect. A possible explanation for CRAF modulation is cyclic adenosine monophosphate (cAMP), a mediator of melanocortin receptor 1 (MC1R) signalling, since it can actually inhibit CRAF. Indeed, we measured cAMP and found that all four sensitive lines contained significantly higher constitutive cAMP levels than the resistant ones. Consequently, vemurafenib and cAMP stimulator combination resulted in a substantial synergistic effect in lines with both intrinsic and acquired resistance but only restricted to those where cAMP was effectively increased. The use of a cAMP agonist overcame such restriction. In conclusion, we report that (WT)BRAF/(WT)NRAS melanoma lines with low phospho-CRAF and high cAMP levels may be sensitive to vemurafenib and that CRAF inhibition through cAMP stimulation may overcome the resistance to the drug.

Improving the spectrophotometric determination of the alkylating activity of anticancer agents: a new insight into the mechanism of the NBP method.

In this paper, the mechanism of the nitrobenzylpyridine (NBP) method to measure the alkylating activity of drugs originally described by Epstein et al. [J. Epstein, R.W. Rosenthal, R.J. Ess, Anal. Chem. 27 (1955) 1435-1439] and modified later by others was revisited using melphalan, m-sarcolysin, chlorambucil, cyclophosphamide and ifosfamide. Its direct application to determine the activity of these drugs in human serum and aqueous media is described and discussed. This method, based on the formation of a chromophore due to the reaction between the alkylating agent and NBP, was significantly improved by extracting as quickly as possible the reaction product(s) into chloroform before adding alkali to develop the color. This significantly limited the degradation by hydrolysis of the products and enhanced the yield of the end chromophore in the organic phase. The reaction time was optimized by monitoring each compound color development. The best reaction time for each compound was selected and a higher stability of the extracted color over at least 1h was obtained (compared to a couple of minutes in previous studies). Most interestingly, water evaporation due to heating had little or no effect on the linearity of standard curves evaluated in the micromolar concentration range. Both the sensitivity and reproducibility of the method were therefore significantly improved. There appears to be a direct correlation between compound hydrolysis and alkylation activity; the relative reactivity is different among the compounds owing to the rate of (i) production, (ii) the relative proportions and (iii) the hydrolysis of the intermediates. A general mechanism for the nucleophilic competitive substitution is proposed.

Survey on skin-lightening practices and cosmetics in Kigali, Rwanda.

The use of skin‐lightening (SL) cosmetics appears to be common throughout the world, especially among dark‐skinned women from sub‐Saharan Africa.

Influence of paramagnetic melanin on the MRI contrast in melanoma: a combined high-field (11.7 T) MRI and EPR study.

Melanoma is the most dangerous form of skin cancer and its incidence is rising each year. Because the current methods of diagnosis based on the visual aspect of the tumor show limitations, several new techniques are emerging to help in this diagnosis, amongst which are magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR). The origin of the typical contrast pattern observable in melanoma in T1 - and T2 -weighted images remains to be elucidated and is a source of controversy. In addition, melanin could create sufficient magnetic inhomogeneities to allow its visualization on T2 *-weighted images using high-field MRI. In order to elucidate the possible role of melanin in the MRI contrast of melanoma, the present study was designed to correlate the paramagnetic content in melanin pigment to the contrast on T1 -, T2 - and T2 *-weighted images. MR images were obtained in vivo at 11.7 T using four types of experimental tumors with different pigmentations (B16, HBL, LND1 melanomas and KHT sarcomas). The paramagnetic content in melanin pigment was measured by EPR. No significant correlation was observed between the content in melanin and the relaxation times T1 , T2 and T2 *, emphasizing that the presence of pigment alone has negligible effect on the MRI contrast.