Iodinated 4,4'-Bipyridines with Antiproliferative Activity Against Melanoma Cell Lines.

In the last decade, biological processes involving halogen bond (HaB) as a leading interaction attracted great interest. However, although bound iodine atoms are considered powerful HaB donors, few iodinated new drugs were reported so far. Recently, iodinated 4,4'-bipyridines showed interesting properties as HaB donors in solution and in the solid state. In this paper, a study on the inhibition activity of seven halogenated 4,4'-bipyridines against malignant melanoma (MM) cell proliferation is described. Explorative dose/response proliferation assays were first performed with three 4,4'-bipyridines by using four MM cell lines and the normal BJ fibroblast cell line as control. Among them, the A375 MM cell line was the most sensitive, as determined by MTT assays, which was selected to evaluate the antiproliferative activity of all 4,4'-bipyridines. Significantly, the presence of an electrophilic iodine impacted the biological activity of the corresponding compounds. The 3,3',5,5'-tetrachloro-2-iodo-4,4'-bipyridine showed significant antiproliferation activity against the A375 cell line, and lower toxicity on BJ fibroblasts. Through in silico studies, the stereoelectronic features of possible sites determining the bioactivity were explored. These results pave the way for the utilization of iodinated 4,4'-bipyridines as templates to design new promising HaB-enabled inhibitors of MM cell proliferation.

Inhibitory Effect of Curcumin-Inspired Derivatives on Tyrosinase Activity and Melanogenesis.

Tyrosinase is a well-known copper-containing metalloenzyme typically involved in the synthesis of melanin. Recently, curcumin and several synthetic derivatives have been recognized as tyrosinase inhibitors with interesting anti-melanogenic therapeutic activity. In this study, three curcumin-inspired compounds 1, 6 and 7 were prepared in yields ranging from 60 to 88 % and spectrophotometric, electrochemical, in vitro and in silico analyses were carried out. The viability of PC12 cells, a rat pheochromocytoma derived-cell line, with compounds 1, 6 and 7, showed values around 80% at 5 µM concentration. In cell proliferation assays, compounds 1, 6 and 7 did not show significant toxicity on fibroblasts nor melanoma cells up to 10 µM with viability values over 90%. The inhibition of tyrosinase activity was evaluated both by a UV-Vis spectroscopic method at two different concentrations, 0.2 and 2.0 µM, and by amperometric assay with IC50 for compounds 1, 6 and 7 ranging from 11 to 24 nM. Melanin content assays on human melanoma cells were performed to test the capability of compounds to inhibit melanin biosynthesis. All compounds exerted a decrease in melanin content, with compound 7 being the most effective by showing a melanogenesis inhibition up to four times greater than arbutin at 100 µM. Moreover, the antioxidant activity of the selected inhibitors was evaluated against H2O2 in amperometric experiments, whereby compound 7 was about three times more effective compared to compounds 1 and 6. The tyrosinase X-ray structure of Bacterium megaterium crystal was used to carry out molecular docking studies in the presence of compounds 1, 6 and 7 in comparison with that of kojic acid and arbutin, two conventional tyrosinase inhibitors. Molecular docking of compounds 6 and 7 confirmed the high affinity of these compounds to tyrosinase protein.

Anticancer Activity of Two Novel Hydroxylated Biphenyl Compounds toward Malignant Melanoma Cells.

Melanoma, the deadliest form of skin cancer, is still one of the most difficult cancers to treat despite recent advances in targeted and immune therapies. About 50% of advanced melanoma do not benefit of such therapies, and novel treatments are requested. Curcumin and its analogs have shown good anticancer properties and are being considered for use in combination with or sequence to recent therapies to improve patient outcomes. Our group previously published the synthesis and anticancer activity characterization of a novel curcumin-related compound against melanoma and neuroblastoma cells (D6). Here, two hydroxylated biphenyl compounds-namely, compounds 11 and 12-were selected among a small collection of previously screened C2-symmetric hydroxylated biphenyls structurally related to D6 and curcumin, showing the best antitumor potentiality against melanoma cells (IC50 values of 1.7 ± 0.5 µM for 11 and 2.0 ± 0.7 µM for 12) and no toxicity of normal fibroblasts up to 32 µM. Their antiproliferative activity was deeply characterized on five melanoma cell lines by performing dose-response and clonal growth inhibition assays, which revealed long-lasting and irreversible effects for both compounds. Apoptosis induction was ascertained by the annexin V and TUNEL assays, whereas Western blotting showed caspase activation and PARP cleavage. A cell cycle analysis, following cell treatments with either compound 11 or 12, highlighted an arrest in the G2/M transition. Taking all this evidence together, 11 and 12 were shown to be good candidates as lead compounds to develop new anticancer drugs against malignant melanoma.

Are Molecular Alterations Linked to Genetic Instability Worth to Be Included as Biomarkers for Directing or Excluding Melanoma Patients to Immunotherapy?

The improvement of the immunotherapeutic potential in most human cancers, including melanoma, requires the identification of increasingly detailed molecular features underlying the tumor immune responsiveness and acting as disease-associated biomarkers. In recent past years, the complexity of the immune landscape in cancer tissues is being steadily unveiled with a progressive better understanding of the plethora of actors playing in such a scenario, resulting in histopathology diversification, distinct molecular subtypes, and biological heterogeneity. Actually, it is widely recognized that the intracellular patterns of alterations in driver genes and loci may also concur to interfere with the homeostasis of the tumor microenvironment components, deeply affecting the immune response against the tumor. Among others, the different events linked to genetic instability-aneuploidy/somatic copy number alteration (SCNA) or microsatellite instability (MSI)-may exhibit opposite behaviors in terms of immune exclusion or responsiveness. In this review, we focused on both prevalence and impact of such different types of genetic instability in melanoma in order to evaluate whether their use as biomarkers in an integrated analysis of the molecular profile of such a malignancy may allow defining any potential predictive value for response/resistance to immunotherapy.

Synthesis of Hydroxylated Biphenyl Derivatives Bearing an α,ß-Unsaturated Ketone as a Lead Structure for the Development of Drug Candidates against Malignant Melanoma.

A small collection of C2 -symmetric hydroxylated biphenyl derivatives featuring an α,ß-unsaturated ketone as a lead structure was prepared, and the capacity of these compounds to act as antiproliferative agents against four human malignant melanoma cell lines was assayed. The prodrug approach was applied in order to improve the delivery of compounds into the cell by modulation of the phenolic hydroxy protecting group. The hydroxylated biphenyl structure bearing an α,ß-unsaturated ketone and a phenolic-O-prenylated chain was found to facilitate the delivery of the molecule and interactions with biological targets. Four compounds showed antiproliferative activity resulting in IC50 values in the range of 1.2 to 2.8 µM.

Quality assessment of a clinical next-generation sequencing melanoma panel within the Italian Melanoma Intergroup (IMI).

BACKGROUND: Identification of somatic mutations in key oncogenes in melanoma is important to lead the effective and efficient use of personalized anticancer treatment. Conventional methods focus on few genes per run and, therefore, are unable to screen for multiple genes simultaneously. The use of Next-Generation Sequencing (NGS) technologies enables sequencing of multiple cancer-driving genes in a single assay, with reduced costs and DNA quantity needed and increased mutation detection sensitivity. METHODS: We designed a customized IMI somatic gene panel for targeted sequencing of actionable melanoma mutations; this panel was tested on three different NGS platforms using 11 metastatic melanoma tissue samples in blinded manner between two EMQN quality certificated laboratory. RESULTS: The detection limit of our assay was set-up to a Variant Allele Frequency (VAF) of 10% with a coverage of at least 200x. All somatic variants detected by all NGS platforms with a VAF ≥ 10%, were also validated by an independent method. The IMI panel achieved a very good concordance among the three NGS platforms. CONCLUSION: This study demonstrated that, using the main sequencing platforms currently available in the diagnostic setting, the IMI panel can be adopted among different centers providing comparable results.

Comparison of BRAF Mutation Screening Strategies in a Large Real-Life Series of Advanced Melanoma Patients.

Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation status plays a predominant role in the management of MM patients. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of the BRAF and NRAS genes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused on BRAF mutation detecting approaches only. BRAF wild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla™) and NGS assays. Globally, 319 patients were included in the study; pathogenic BRAF mutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additional BRAF mutations after SS and pyrosequencing, respectively. NGS detected one additional BRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods; therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection.

Antiproliferative activity of vanadium compounds: effects on the major malignant melanoma molecular pathways.

Malignant melanoma (MM) is the most fatal skin cancer, whose incidence has critically increased in the last decades. Recent molecular therapies are giving excellent results in the remission of melanoma but often they induce drug resistance in patients limiting their therapeutic efficacy. The search for new compounds able to overcome drug resistance is therefore essential. Vanadium has recently been cited for its anticancer properties against several tumors, but only a few data regard its effect against MM. In a previous work we demonstrated the anticancer activity of four different vanadium species towards MM cell lines. The inorganic anion vanadate(v) (VN) and the oxidovanadium(iv) complex [VO(dhp)2] (VS2), where dhp is 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate, showed IC50 values of 4.7 and 2.6 µM, respectively, against the A375 MM cell line, causing apoptosis and cell cycle arrest. Here we demonstrate the involvement of Reactive Oxygen Species (ROS) production in the pro-apoptotic effect of these two V species and evaluate the activation of different cell cycle regulators, to investigate the molecular mechanisms involved in their antitumor activity. We establish that VN and VS2 treatments reduce the phosphorylation of extracellular-signal regulated kinase (ERK) by about 80%, causing the deactivation of the mitogen activated protein kinase (MAPK) pathway in A375 cells. VN and VS2 also induce dephosphorylation of the retinoblastoma protein (Rb) (VN 100% and VS2 90%), together with a pronounced increase of cyclin-dependent kinase inhibitor 1 p21 (p21Cip1) protein expression up to 1800%. Taken together, our results confirm the antitumor properties of vanadium against melanoma cells, highlighting its ability to induce apoptosis through generation of ROS and cell cycle arrest by counteracting MAPK pathway activation and strongly inducing p21Cip1 expression and Rb hypo-phosphorylation.

Antitumoral effect of vanadium compounds in malignant melanoma cell lines.

In this study we evaluated the anticancer activity against malignant melanoma (MM) of four different vanadium species: the inorganic anion vanadate(V) (indicated with VN), and three oxidovanadium(IV) complexes, [VIVO(dhp)2] where dhp- is the anion 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS2), [VIVO(mpp)2] where mpp- is 1-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS3), and [VIVO(ppp)2] where ppp- is 1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS4). The antitumor effects of these compounds were studied against two different MM cell lines (A375 and CN-mel) and a fibroblast cell line (BJ) as normal control. All tested V compounds exert antiproliferative activity on MM cells in a dose dependent manner (IC50 ranges from 2.4µM up to 14µM) being A375 the most sensitive cell line. VN and VS2 were the two most active compounds against A375 (IC50 of 4.7 and 2.6µM, respectively), causing apoptosis and cell cycle block. The experimental data indicate that the cell cycle arrest occurs at different phases for the two V species analyzed (G2 checkpoint for VN and G0/G1 for VS2), showing the importance of the chemical form in determining their mechanism of action. These results add more insights into the landscape of vanadium versatility in biological systems and into its role as a potential cancer therapeutic agent.

Vitamin D status and risk for malignant cutaneous melanoma: recent advances.

Cutaneous malignant melanoma, whose incidence is increasing steadily worldwide, is the result of complex interactions between individual genetic factors and environmental risk factors. Ultraviolet radiation represents the most important environmental risk factor for the development of skin cancers, including melanoma. Sun exposure and early sunburn during childhood are the principal causes of cutaneous melanoma insurgence in adults, with double the risk relative to a nonexposed population. Consequently, ultraviolet protection has long been recognized as an important measure to prevent such a malignancy. Biological and epidemiological data suggest that vitamin D status could affect the risk of cancer and play a role in cancer prevention by exerting antiproliferative effects. Solar radiations are critical for vitamin D synthesis in humans; however, uncontrolled and intensive sun exposure is dangerous to skin health and may contribute toward the development of cutaneous malignant melanoma. An optimum balance between sun protection and exposure is thus advocated. Additional research is required to confirm the preventive role of vitamin D in melanoma incidence or a positive influence on patient outcome.

Protein expression changes induced in a malignant melanoma cell line by the curcumin analogue compound D6.

BACKGROUND: We have previously demonstrated that the hydroxylated biphenyl compound D6 (3E,3'E)-4,4'-(5,5',6,6'-tetramethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one), a structural analogue of curcumin, exerts a strong antitumor activity on melanoma cells both in vitro and in vivo. Although the mechanism of action of D6 is yet to be clarified, this compound is thought to inhibit cancer cell growth by arresting the cell cycle in G2/M phase, and to induce apoptosis through the mitochondrial intrinsic pathway. To investigate the changes in protein expression induced by exposure of melanoma cells to D6, a differential proteomic study was carried out on D6-treated and untreated primary melanoma LB24Dagi cells. METHODS: Proteins were fractionated by SDS-PAGE and subjected to in gel digestion. The peptide mixtures were analyzed by liquid chromatography coupled with tandem mass spectrometry. Proteins were identified and quantified using database search and spectral counting. Proteomic data were finally uploaded into the Ingenuity Pathway Analysis software to find significantly modulated networks and pathways. RESULTS: Analysis of the differentially expressed protein profiles revealed the activation of a strong cellular stress response, with overexpression of several HSPs and stimulation of ubiquitin-proteasome pathways. These were accompanied by a decrease of protein synthesis, evidenced by downregulation of proteins involved in mRNA processing and translation. These findings are consistent with our previous results on gene expression profiling in melanoma cells treated with D6. CONCLUSIONS: Our findings confirm that the curcumin analogue D6 triggers a strong stress response in melanoma cells, turning down majority of cell functions and finally driving cells to apoptosis.

4-Substituted-2-Methoxyphenol: Suitable Building Block to Prepare New Bioactive Natural-like Hydroxylated Biphenyls.

A small collection of eugenol- and curcumin-analog hydroxylated biphenyls was prepared by straightforward methods starting from natural 4-substituted-2-methoxyphenols and their antitumoral activity was evaluated in vitro. Two curcumin-biphenyl derivatives showed interesting growth inhibitory activities on different malignant melanoma cell lines with IC50 ranging from 13 to 1 µM. Preliminary molecular modeling studies were carried out to evaluate conformations and dihedral angles suitable for antiproliferative activity in hydroxylated biphenyls bearing a side aliphatic chain.

Molecular changes induced by the curcumin analogue D6 in human melanoma cells.

BACKGROUND: In a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours. In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells. RESULTS: To shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition. A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound. The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms. In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways. Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis. CONCLUSIONS: Our findings contribute to the understanding of the mechanisms of action of D6, through a comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells.

In vitro activity of the αvß3 integrin antagonist RGDechi-hCit on malignant melanoma cells.

BACKGROUND: In malignant melanoma (MM), overexpression of αvß3 integrin is linked to a more metastatic phenotype. Development of anti-αvß3 agents able to counteract melanoma progression would be helpful for disease treatment. A new selective ligand of αvß3, RGDechi-hCit, has anti-angiogenic properties against endothelial cells in animal angiogenesis models. The aim of this study was to evaluate the in vitro effects of the RGDechi-hCit peptide on MM cell lines. MATERIALS AND METHODS: Cytofluorimetric analysis characterized the cell surface expression of αvß3 integrin on seven MM cell lines: A375, WM266-4, SK-Mel-28, Sbcl2, LB24Dagi, PR-Mel and PNP-Mel. Cell proliferation, adhesion, and migration assays were carried out using the αvß3-antagonist RGDechi-hCit. RESULTS: Proliferation was not significantly inhibited by RGDechi-hCit, although striking morphological changes were detected in MM cell lines highly expressing αvß3. Conversely, assays on fibronectin-coated plates showed a significant RGDechi-hCit dose-dependent inhibitory effect on both adhesion and migration. CONCLUSION: The data demonstrate anti-adhesion and anti-migration, but not antiproliferative, activities of RGDechi-hCit against MM cells.

Proteomic profiling of human melanoma metastatic cell line secretomes.

During the last few years, the incidence and mortality of human melanoma have rapidly increased. Metastatic spread of malignant melanoma is often associated with cancer progression with poor prognosis and survival. These processes are controlled by dynamic interactions between tumor melanocytes and neighboring stromal cells, whose deregulation leads to the acquisition of cell proliferation capabilities and invasiveness. It is increasingly clear that a key role in carcinogenesis is played by secreted molecules either by tumor and surrounding stromal cells. To address the issue of the proteins secreted during cancer progression, the proteomic profiling of secretomes of cancer cell lines from different melanoma metastases of the same patient (PE-MEL-41, PE-MEL-47, and PE-MEL-43) was performed by applying a shotgun LC-MS/MS-based approach. The results provide a list of candidate proteins associated with the metastatic potential of PE-MEL melanoma cell lines. Among them, several matricellular proteins previously reported as involved in melanoma aggressiveness were identified (i.e., SPARC, osteopontin). In addition, the extracellular matrix protein 1 that stimulates proliferation and angiogenesis of endothelial cells as well as the fibronectin, involved in cell adhesion and motility, were identified. The present work provides the basis to clarify the complex extracellular protein networks implicated in human melanoma cell invasion, migration, and motility.

Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells.

Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells.

BACKGROUND: Sharing the common neuroectodermal origin, melanoma and neuroblastoma are tumors widely diffused among adult and children, respectively. Clinical prognosis of aggressive neuroectodermal cancers remains dismal, therefore the search for novel therapies against such tumors is warranted. Curcumin is a phytochemical compound widely studied for its antioxidant, anti-inflammatory and anti-cancer properties. Recently, we have synthesized and tested in vitro various curcumin-related compounds in order to select new anti-tumor agents displaying stronger and selective growth inhibition activity on neuroectodermal tumors. RESULTS: In this work, we have demonstrated that the new alpha,beta-unsaturated ketone D6 was more effective in inhibiting tumor cells growth when compared to curcumin. Normal fibroblasts proliferation was not affected by this treatment. Clonogenic assay showed a significant dose-dependent reduction in both melanoma and neuroblastoma colony formation only after D6 treatment. TUNEL assay, Annexin-V staining, caspases activation and PARP cleavage unveiled the ability of D6 to cause tumor cell death by triggering apoptosis, similarly to curcumin, but with a stronger and quicker extent. These apoptotic features appear to be associated with loss of mitochondrial membrane potential and cytochrome c release. In vivo anti-tumor activity of curcumin and D6 was surveyed using sub-cutaneous melanoma and orthotopic neuroblastoma xenograft models. D6 treated mice exhibited significantly reduced tumor growth compared to both control and curcumin treated ones (Melanoma: D6 vs control: P < 0.001 and D6 vs curcumin P < 0.01; Neuroblastoma: D6 vs both control and curcumin: P < 0.001). CONCLUSIONS: Our data indicate D6 as a good candidate to develop new therapies against neural crest-derived tumors.

A point mutation (G574A) in the chemokine receptor CXCR4 detected in human cancer cells enhances migration.

The chemokine receptor CXCR4 is widely expressed in human cancers and regulates cell invasion, proliferation and survival. Because mutations in the CXCR4 gene could regulate its function we sequenced the coding region of the CXCR4 gene in 18 human melanoma and 3 human colon carcinoma cell lines. The same somatic point mutation (G574A; V160I) in the fourth transmembrane region of CXCR4 was detected in one colon cancer cell line (PD) and one melanoma cell line (LB). CXCR4 was expressed and functional in both PD and LB cells, PD and LB cells migrated specifically toward the receptor ligand, CXCL12 and P-Erk was specifically induced by CXCL12. To give insight into the function of the mutant CXCR4 receptor, human A431, epidermoid carcinoma cells, were stably transfected with both mutant and wild type CXCR4. In vitro, A431 cells harboring CXCR4(G574A) migrated specifically toward CXCL12 and CXCL12 induced ERK phosphorylation. Interestingly, in vivo studies showed that the growth of A431 tumors harboring CXCR4(G574A) was delayed compared to those harboring WT CXCR4. As expected, treatment with AMD3100, a specific CXCR4 inhibitor, reduced the in vivo growth of CXCR4(G574A) tumor b(G574A) but surprisingly, increased the growth of CXCR4(G574A) A431 cells. This is the first report of a spontaneously occurring, functionally active CXCR4 mutation in human cancer cells. While the mutation impairs cell growth in vivo, the CXCR4 inhibitor, AMD3100, stimulated the growth of cells harboring CXCR4(G574A).

Antiproliferative and pro-apoptotic activity of eugenol-related biphenyls on malignant melanoma cells.

BACKGROUND: Malignant melanoma is one of the most aggressive skin cancer and chemotherapeutic agents currently in use are still unsatisfactory. Prevention and early diagnosis are the only effective tools against this tumour whose incidence and mortality rates are highly increased during the last decades in fair skin populations. Therefore the search for novel therapeutic approaches is warranted. Aim of this work was to identify and test new compounds with antiproliferative and cytotoxic activity on melanoma cells. We tested eugenol together with six natural and synthetic eugenol-related compounds for their capability to inhibit cell growth on primary melanoma cell lines established from patients' tissue samples. RESULTS: Eugenol and isoeugenol monomers and their respective O-methylated forms did not show to inhibit melanoma cells proliferation. Conversely, the dimeric forms (biphenyls) showed some antiproliferative activity which was mild for dehydrodieugenol, higher for its O,O'-methylated form (O,O'-dimethyl-dehydrodieugenol), and markedly pronounced for the racemic mixture of the brominated biphenyl (6,6'-dibromo-dehydrodieugenol) (S7), being its enantiomeric form (S) the most effective compared to the other compounds. Such activity resulted to be selective against tumour cells, without affecting cultured normal human skin fibroblasts. Dose and time dependence curves have been obtained for the enantiomeric form S7-(S). Then IC50 and minimal effective doses and times have been established for the melanoma cell lines tested. TUNEL and phosphatidylserine exposure assays demonstrated the occurrence of apoptotic events associated with the antiproliferative activity of S7-(S). Cytotoxic activity and apoptosis induced by treating melanoma cells with eugenol-related biphenyls was partially dependent by caspase activation. CONCLUSION: Our findings demonstrate that the eugenol related biphenyl (S)-6,6'-dibromo-dehydrodieugenol elicits specific antiproliferative activity on neuroectodermal tumour cells partially triggering apoptosis and its activity should be further investigated on in vivo melanoma models in order to evaluate the real anticancer effectiveness on such tumour.

Identification of a novel candidate gene, CASC2, in a region of common allelic loss at chromosome 10q26 in human endometrial cancer.

Allelic deletions, which are suggestive for the presence of tumor suppressor genes, represent a common event in endometrial cancer (EC). Previous loss-of-heterozygosity studies for human chromosome 10q identified a candidate deletion interval at 10q25-q26, which we further narrowed to a 160-kb region at 10q26, bounded by markers D10S1236 and WIAF3299. Using a positional candidate approach, we identified three alternative transcripts of a novel human gene, CASC2 (cancer susceptibility candidate 2; formely C10orf5). One of such transcripts, CASC2a, encodes a short protein of 102 amino acids with no similarity to any other known gene product. Three (7%) CASC2a mutations were identified in tumor DNA from 44 EC patients. While c.-156G>T and c.22C>T (p.Pro8Ser) are sequence variants with unknown functional significance, c.84delA is a mutation with a truncation effect on the predicted protein (p. Asn28fsX50). Expression studies by real-time RT-PCR on several normal and tumor cells revealed that CASC2a mRNA is downregulated in cancer, suggesting that it may act as a potential tumor suppressor gene. The very low mutation rate seems to also indicate that inactivation of CASC2a might probably be due to mechanisms different from genetic alterations.