Catecholamine effects on human melanoma cells evoked by α1-adrenoceptors

The biological effects of catecholamines in mammalian pigment cells are poorly understood. Our previous results showed the presence of α1-adrenoceptors in SK-Mel 23 human melanoma cells. The aims of this work were to (1) characterize catecholamine effects on proliferation, tyrosinase activity and expression, (2) identify the α1- adrenoceptor subtypes, and (3) verify whether chronic norepinephrine (NE) treatment modified the types and/or pharmacological characteristics of adrenoceptors present in SK-Mel 23 human melanoma cells. Cells treated with the aradrenergic agonist, phenylephrine (PHE, 10-5 or 10-4 M), for 24-72 h, exhibited decreased cell proliferation and enhanced tyrosinase activity, but unaltered tyrosinase expression as compared with the control. The proliferation and tyrosinase activity responses were inhibited by the α1-adrenergic antagonist prazosin, suggesting they were evoked by α1-adrenoceptors. The presence of actinomycin D, a transcription inhibitor, did not diminish PHE-induced effects. RT-PCR assays, followed by cloning and sequencing, demonstrated the presence of α1A- and α1B-adrenoceptor subtypes. NE-treated cells (24 or 72 h) were used in competition assays, and showed no significant change in the competition curves of α1-adrenoceptors as compared with control curves. Other adrenoceptor subtypes were not identified in these cells, and NE pretreatment did not induce their expression. In conclusion, the activation of SK-Mel 23 human melanoma α1- radrenoceptors elicit biological effects, such as proliferation decrease and tyrosinase activity increase. Desensitization or expression of other adrenoceptor subtypes after chronic NE treatment were not observed.

Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

Role of versican V0/V1 and CD44 in the regulation of human melanoma cell behaviour

Versican is a hyaluronan-binding, large extracellular matrix chondroitin sulfate proteoglycan whose expression is increased in malignant melanoma. Binding to hyaluronan allows versican to indirectly interact with the hyaluronan cell surface receptor CD44. The aim of this work was to study the effect of silencing the large versican isoforms (V0 and V1) and CD44 in the SK-mel-131 human melanoma cell line. Versican V0/V1 or CD44 silencing caused a decrease in cell proliferation and migration, both in wound healing assays and in Transwell chambers. Versican V0/V1 silencing also caused an increased adhesion to type I collagen, laminin and fibronectin. These results support the proposed role of versican as a proliferative, anti-adhesive and pro-migratory molecule. On the other hand, CD44 silencing caused a decrease in cell adhesion to vitronectin, fibronectin and hyaluronan. CD44 silencing inhibited the binding of a FITC-hyaluronan complex to the cell surface and its internalization into the cytoplasm. Our results indicate that both versican and CD44 play an important role regulating the behavior of malignant melanoma cells.

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^

Comparison of radiobiological effects induced by ultra-high and standard dose rate of x-rays on a radio-resistant cell line

Radiotherapy (RT) has recently evolved with the emergence of heavy ion radiations or new fractionation schemes of photon therapy, which modify the dose rate of treatment delivery. The aim of the present study was then to evaluate the in vitro influence of a ultra-high dose rate comparing them with standard dose rate. In this regard, a radioresistant SK-MEL-28 cell line were irradiated with x-ray in order to have a total dose of 2 and 4 Gy, at two different dose rate. The ultra-high dose rate is a specific property of the dense plasma focus (DPF) device, which has pulsed operation and thus gives short and highly energetic pulses of multiple types of rays and particles, in this case, we focused our study on the influence of X-rays. While a low dose rate is obtained with conventional X-ray tube. In this study it results that a ultra-high dose rate enhances radiosensitivity of melanoma cells while reducing the adhesion, proliferation and migration ability of cells.

p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the 'high-p53'Mdm4+/- mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/- background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/- mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and β-catenin, localized increased levels of vimentin and redistribution of α6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

Micromolar concentration of pentoxifylline improves development in vitro of hamster 8-cell embryos: conrmation of biological viability by embryo transfer

The inßuence of the sperm motility stimulant pentoxifylline (PF) on preimplantation embryo development in hamsters was evaluated. Eight-cell embryos were cultured in hamster embryo culture medium (HECM)-2, with or without PF (0· 0233·6 mM). There was 90%, 37% and 29% inhibition of blastocyst development by 3·6 (used for human sperm), 0·9 and 0 ·45 mM PF, respectively. However, 23 µM PF (exposed to hamster oocytes during IVF) signicantly (P < 0·05) improved blastocyst development (63· 6% v. 51· 8%); morulae development was, however, not curtailed by 0·45 mM or 0·9 mM PF (51·8%±6·0 or 50·5%±11·3, respectively). Post-implantation viability of PF-treated embryos was assessed by embryo transfer; 43% of 80 PF-treated embryos implanted compared with 40% of 79 control embryos. Of the 9 recipients, 6 females delivered pups (19, i.e. 16% of transferred embryos or 53% of implanted embryos). These data show that in hamsters, continuous presence of PF at 0·45-3·6 mM is detrimental to 8-cell embryo development whereas 23 µM PF improves the development of embryos to viable blastocysts which produce live offspring.

A new heteroleptic ruthenium sensitizer enhances the absorptivity of mesoporous titania film for a high efficiency dye-sensitized solar cell

A heteroleptic polypyridyl ruthenium complex, cis-Ru(4,4'-bis(5-octylthieno[3,2-b]thiophen-2-yl)-2,2'-bipyridine)(4,4'-dicarboxyl-2,2'-bipyridine)(NCS) 2, with a high molar extinction coefficient of 20.5 x 10(3) M-1 cm(-1) at 553 nm has been synthesized and demonstrated as a highly efficient sensitizer for a dye-sensitized solar cell, giving a power conversion efficiency of 10.53% measured under an irradiation of air mass 1.5 global ( AM 1.5G) full sunlight.

Preparation of rare earth ion induced conformation-specific anti-calmodulin monoclonal antibody

Monoclonal antibody technique was employed to detect the conformational change of calmodulin induced by metal ions. Bovine calmodulin was firstly modified by 2,4-dinitrofluorobenzene to improve its immunogenicity, then, the derived protein was saturated with trivalent europium ions and injected to Balb/c mice as antigen. After four times of immunization, a corresponding antibody was detected and its titer in serum was determined as 1 : 12 000. By fusing of the spleen cells with hybridoma cells, a europium induced conformation-specific anti-calmodulin monoclonal antibody cell strain named as 2C3 was produced successfully. The molecular recognition ability of antibody to apocalmodulin and holocalmodulin showed a significant difference, indicating that this antibody could be applied to the studies of different effects of metal ions on the conformational change of calmodulin and its interaction with target molecules.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Oxidation and glycoxidation of phosphatidylserine and their effect in immune response : a lipidomic approach

Phosphatidylserine (PS) is a member of the class of phospholipids, and is distributed among all cells of mammalians, playing important roles in diverse biological processes, including blood clotting and apoptosis. When externalized, PS is a ligand that is recognized on apoptotic cells. It has been considered that before externalization PS is oxidized and oxPS enhance the recognition by macrophages receptors, however the knowledge about oxidation of PS is still limited. PS, like others phospholipids, has two fatty acyl chains and one polar head group, in this case is the amino acid serine. The modifications in PS structure can occur by oxidation of the unsaturated fatty acyl chains and by glycation of the polar head group, due to free amine group, thus increasing the susceptibility to oxidative events. The main goal of this work was to characterize and identify oxidized and glycoxidized PS, contributing to the knowledge of the biological role of oxidation products of PS, as well as of glycated PS, in immune and inflammatory processes. To achieve this goal, PS standards (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (POPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS) and 1-palmitoyl-2- arachidonoyl-sn-glycero-3-phospho-L-serine (PAPS)) and glycated PS (PAPS and POPS) were induced to oxidize in model systems, using different oxidant reagents: HO• and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) . The detailed structural characterization of the oxidative products was performed by ESI-MS and MS/MS coupled to separation techniques such as off line TLC-MS and on line LC-MS, in order to obtained better characterization of the larger number of PS and glycated PS oxidation products. The results obtained in this work allowed to identify several oxidation products of PS and glycated PS with modifications in unsaturated fatty acyl chain. Also, oxidation products formed due to structural changes in the serine polar head with formation of terminal acetamide, terminal hydroperoxyacetaldehyde.and terminal acetic acid (glycerophosphacetic acid, GPAA) were identified. The mass spectrometric specific fragmentation pathway of each type of oxidation product was determined using different mass spectrometry approaches. Based on the identified fragmentation pathways, targeted lipidomic analysis was performed to detect oxidation products modified in serine polar head in HaCaT cell line treated with AAPH. The GPAA was detected in HaCaT cells treated with AAPH to induce oxidative stress, thus confirming that modifications in PS polar head is possible to occur in biological systems. Furthermore, it was found that glycated PS species are more prone to oxidative modifications when compared with non glycated PS. During oxidation of glycated PS, besides the oxidation in acyl chains, new oxidation products due to oxidation of the glucose moiety were identified, including PS advanced glycation end products (PSAGES). To investigate if UVA oxidative stress exerted changes in the lipidome of melanoma cell lines, particularly in PS profile, a lipidomic analysis was performed. The lipid profile was obtained using HILIC-LC-MS and GC-MS analysis of the total lipid extracts obtained from human melanoma cell line (SKMEL- 28) after UVA irradiation at 0, 2 and 24 hours. The results did not showed significant differences in PS content. At molecular level, only PS (18:0:18:1) decreased at the moment of irradiation. The most significant changes in phospholipids content occurred in phosphatidylcholines (PC) and phosphatidylinositol (PI) classes, with an increase of mono-unsaturated fatty acid (MUFA), similarly as observed for the fatty acid analysis. Overall, these data indicate that the observed membrane lipid changes associated with lipogenesis after UVA exposure may be correlated with malignant transformations associated with cancer development and progression. Despite of UVA radiation is associated with oxidative damage, in this work was not possible observe oxidation phospholipids. The anti/pro-inflammatory properties of the oxidized PLPS (oxPLPS) versus non-oxidized PLPS were tested on LPS stimulated RAW 264.7 macrophages. The modulation of intracellular signaling pathways such as NF-kB and MAPK cascades by oxPLPS and PS was also examined in this study. The results obtained from evaluation of anti/pro-inflammatory properties showed that neither PLPS or oxPLPS species activated the macrophages. Moreover only oxidized PLS were found to significantly inhibit NO production and iNOS and il1β gene transcription induced by LPS. The analysis at molecular level showed that this was the result of the attenuation of LPS-induced c-Jun-N-terminal kinase (JNK) and p65 NF-kB nuclear translocation. Overall these data suggest that oxPLPS, but not native PLPS, mitigates pro-inflammatory signaling in macrophages, contributing to containment of inflammation during apoptotic cell engulfment. The results obtained in this work provides new information on the modifications of PS, facilitating the identification of oxidized species in complex samples, namely under physiopathologic conditions and also contributes to a better understanding of the role of oxPS and PS in the inflammatory response, in the apoptotic process and other biological functions.