Amelanotic Melanoma Cell Line Research Articles

Abstract 2783: The exosomes from cord blood stem cells, containing novel miRNAs, induce apoptosis in melanoma cells and enhance T cell anticancer function

Abstract Regulatory T (Treg) cells expressing FOXP3 play a critical role in suppressing immune responses against anti-tumor immune responses. However, their presence in tumor tissues is often associated with poor prognosis. Cord blood stem cell-derived exosome vesicles (CBSC-EV) constitute a valuable source of lipids, microRNAs, and proteins, that are essential for cell-to-cell communication and provide therapeutic benefits. This study aimed to investigate the contents of CBSC-EV, identifying several known miRNAs (from Let7 and Let5 families) and two unknown miRNAs. Healthy lymphocytes, fibroblasts, and CHL1 amelanotic melanoma cell lines were transfected with these two novel miRNA inhibitors, and the changes before and after treatment compared to CBSC-EV and H2O2 were examined. Following treatment, the cells underwent a 24-hour incubation period, during which the level of cytotoxicity was assessed using CCK8, and the degree of DNA damage incurred was quantified with the Fast Microplate DNA damage assay. To further investigate the effects of treatment, RNA sequencing was conducted on both healthy and cancerous cells. The results demonstrated that CBSC-EV and novel miRNAs induced cytotoxicity in cancer cells, while in healthy cells, the DNA damage was repaired and cell viability was significantly increased (p<0.001). RNA sequencing revealed that following treatment with CBSC-EV and both CBSC-EV+N1 and N2 inhibitors in malignant melanoma cells, the expression of genes involved in the cytokine-mediated signaling pathway, cytokine-cytokine interaction, and cytokine activity were enhanced. In healthy cells treated with CBSC-EV and novel miRNA inhibitor transfection, the MT-CO1 gene was upregulated, contributing to cytochrome-c oxidase activity. This study presents evidence of the potential use of novel miRNAs as genetic material for cancer therapeutics in the future. Additionally, the findings suggest that these miRNAs could be used as an immune system modulator against melanoma. These findings demonstrate that miRNAs can effectively suppress resistance to anticancer cytotoxic therapy, which is a common feature of cancer cells. This suggests that miRNAs can potentially enhance current cancer therapies. Citation Format: Mojgan Mojgan Najafzadeh, Adi Baumgartner, Shohreh Jafarinejad, Zahra Karimi, Mohammad Isreb, Pouria Akhbari, Nader Ghaderi, Farshid Sefat, Saeed Heidari Keshel, Parisa Naeem, Rojan Ghaderi, Jacobo Elis Gomez, Diana Anderson, Andrew Wright. The exosomes from cord blood stem cells, containing novel miRNAs, induce apoptosis in melanoma cells and enhance T cell anticancer function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2783.

The combination of pro-oxidative acting vanicosides and GLUT1 inhibitor (WZB117) exerts a synergistic cytotoxic effect against melanoma cells

Vanicosides A and B isolated from Reynoutria sachalinensis rhizomes are disaccharide phenylpropanoid esters with proven antioxidant activity. Our earlier study showed the cytotoxic activity of vanicosides against melanoma cells, but the mechanism of cell death has not been elucidated.Based on the chemical structure of vanicosides, we proposed that they may induce cell death by generating reactive oxygen species (ROS) into melanoma cells. Moreover, the glucose molecule in their structure can affect the glucose transporters (GLUTs), upregulated in cancer cells.The A375 (melanotic) and C32 (amelanotic) melanoma cell lines were applied. Cell viability assay and ROS-Glo™ assay were performed before and after blocking of Glucose Transporter Type 1 (GLUT1) by WZB117. Fibroblasts and the SKOV-3 line were included in the study to test selectivity in the action of vanicosides and help to elucidate the mechanism of action.Upon incubation with vanicosides, high production of ROS occured, especially inside C32 cells, which was significantly reduced after GLUT-1 blocking. The A375 cells produced less ROS. Melanoma cells were simillary sensitive to the cytotoxic effects of vanicosides, which was clearly enhanced when vanicosides were used together with the WZB117 (GLUT1 inhibitor). The SKOV-3 line and the fibroblasts showed much less sensitivity to the cytotoxicity of vanicosides, also used together with WZB117. Moreover, no significant ROS formation was observed in these lines.The study proved that vanicosides generate ROS inside melanoma cells. These findings suggest that the combination of pro-oxidative acting vanicosides and GLUT1 inhibitors exerts a synergistic cytotoxic effect on melanoma cells.

The Assessment of Anti-Melanoma Potential of Tigecycline-Cellular and Molecular Studies of Cell Proliferation, Apoptosis and Autophagy on Amelanotic and Melanotic Melanoma Cells.

High mortality, aggressiveness, and the relatively low effectiveness of therapy make melanoma the most dangerous of skin cancers. Previously published studies presented the promising therapeutic potential of minocycline, doxycycline, and chlortetracycline on melanoma cells. This study aimed to assess the cytotoxicity of tigecycline, a third-generation tetracycline, on melanotic (COLO 829) and amelanotic (A375) melanoma cell lines. The obtained results showed that tigecycline, proportionally to the concentration and incubation time, efficiently inhibited proliferation of both types of melanoma cells. The effect was accompanied by the dysregulation of the cell cycle, the depolarization of the mitochondrial membrane, and a decrease in the reduced thiols and the levels of MITF and p44/42 MAPK. However, the ability to induce apoptosis was only found in COLO 829 melanoma cells. A375 cells appeared to be more resistant to the treatment with tigecycline. The drug did not induce apoptosis but caused an increase in LC3A/B protein levels-an autophagy marker. The observed differences in drug action on the tested cell lines also involved an increase in p21 and p16 protein levels in melanotic melanoma, which was related to cell cycle arrest in the G1/G0 phase. The greater sensitivity of melanotic melanoma cells to the action of tigecycline suggests the possibility of considering the use of the drug in targeted therapy.

The Anticancer Potential of Doxycycline and Minocycline—A Comparative Study on Amelanotic Melanoma Cell Lines

Malignant melanoma is still a serious medical problem. Relatively high mortality, a still-growing number of newly diagnosed cases, and insufficiently effective methods of therapy necessitate melanoma research. Tetracyclines are compounds with pleiotropic pharmacological properties. Previously published studies on melanotic melanoma cells ascertained that minocycline and doxycycline exerted an anti-melanoma effect. The purpose of the study was to assess the anti-melanoma potential and mechanisms of action of minocycline and doxycycline using A375 and C32 human amelanotic melanoma cell lines. The obtained results indicate that the tested drugs inhibited proliferation, decreased cell viability, and induced apoptosis in amelanotic melanoma cells. The treatment caused changes in the cell cycle profile and decreased the intracellular level of reduced thiols and mitochondrial membrane potential. The exposure of A375 and C32 cells to minocycline and doxycycline triggered the release of cytochrome c and activated initiator and effector caspases. The anti-melanoma effect of analyzed drugs appeared to be related to the up-regulation of ERK1/2 and MITF. Moreover, it was noticed that minocycline and doxycycline increased the level of LC3A/B, an autophagy marker, in A375 cells. In summary, the study showed the pleiotropic anti-cancer action of minocycline and doxycycline against amelanotic melanoma cells. Considering all results, it could be concluded that doxycycline was a more potent drug than minocycline.

Mechanisms of curcumin-based photodynamic therapy and its effects in combination with electroporation: An in vitro and molecular dynamics study

Photodynamic therapy (PDT) and electrochemotherapy (ECT) are two methods designed to enhance the anticancer potential of various drugs. Various clinical trials proved the efficacy of both ECT and PDT in melanoma treatment. Curcumin is a natural polyphenolic compound with high anticancer potential against melanoma due to its light absorption properties and toxicity towards cancer cells; however, high reactivity and amphipathic structure of curcumin are limiting its utility. This study aimed to propose the most effective protocol for antimelanoma combination of both therapies (PDT and ECT) in the context of curcumin. The in vitro studies were carried on melanotic melanoma (A375), amelanotic melanoma (C32) and fibroblast (HGF) cell lines. In molecular dynamics studies curcumin presented the single-layer localization in the water-membrane interphase. Further, the mass spectrometry studies exposed that during the PDT treatment curcumin is degraded to vanillin, feruloylmethane, and ferulic acid. Instant ECT with curcumin followed by PDT is the most efficient approach due to its selective genotoxicity towards malignant cells. The metabolic activity of fibroblasts decreased, however, at the same time the fragmentation of DNA did not occur. Additionally, instant PDT with curcumin followed by ECT after 3 h of incubation was a therapy selective towards melanotic melanoma.

In Vitro Anticancer Potential of Jasione montana and Its Main Components against Human Amelanotic Melanoma Cells.

Jasione montana L. (Campanulaceae) is used in traditional Belarusian herbal medicine for sleep disorders in children, but the chemical composition and biological activity have not been investigated. In this study, the activities of J. montana extracts, their fractions and main compounds were evaluated in amelanotic melanoma C32 (CRL-1585) cells and normal fibroblasts (PCS-201-012). The extracts and fractions were analyzed using liquid chromatography–photodiode array detection–electrospray ionization–mass spectrometry (LC–PDA–ESI–MS/TOF) to characterize 25 compounds. Further, three major and known constituents, luteolin (22) and its derivatives such as 7-O-glucoside (12) and 7-O-sambubioside (9) were isolated and identified. The cytotoxic activities against fibroblasts and the amelanotic melanoma cell line were determined using the fixable viability stain (FVS) assay. The influence of diethyl ether (Et2O) fraction (JM4) and 22 on apoptosis induction was investigated using an annexin V binding assay. The obtained results showed significant cytotoxicity of JM4 and 22 with IC50 values of 119.7 ± 3.2 and 95.1 ± 7.2 μg/mL, respectively. The proapoptotic potential after 22 treatment in the C32 human amelanotic melanoma cell line was comparable to that of vinblastine sulfate (VLB), detecting 29.2 ± 3.0% apoptotic cells. Moreover, 22 displayed less necrotic potential against melanoma cells than VLB. In addition, the influences of JM4 and 22 on the dysfunction of the mitochondrial membrane potential (MMP), cell cycle and activity of caspases 3, 8, 9, and 10 were established. The effects of JM4 on MMP change (74.5 ± 3.0% of the cells showed a reduced MMP) corresponded to the results obtained from the annexin V binding assay and activation of caspase-9. JM4 and 22 displayed a significant impact on caspase-9 (40.9 ± 2.4% of the cells contained active caspase-9 after JM4 treatment and 16.6 ± 0.8% after incubation with 22) and the intrinsic (mitochondrial) apoptotic pathway. Moreover, studies have shown that JM4 and 22 affect the activation of external apoptosis pathways by inducing the caspase-8 and caspase-10 cascades. Thus, activation of caspase-3 and DNA damage via external and internal apoptotic pathways were observed after treatment with JM4 and 22. The obtained results suggest that J. montana extracts could be developed as new topical preparations with potential anticancer properties due to their promising cytotoxic and proapoptotic potential.

Mitochondrial function is controlled by melatonin and its metabolites in vitro in human melanoma cells

Melanoma is a leading cause of cancer deaths worldwide. Although immunotherapy has revolutionized the treatment for some patients, resistance towards therapy and unwanted side effects remain a problem for numerous individuals. Broad anti-cancer activities of melatonin are recognized; however, additional investigations still need to be elucidated. Herein, using various human melanoma cell models, we explore in vitro the new insights into the regulation of melanoma by melatonin and its metabolites which possess, on the other side, high safety profiles and biological meaningful. In this study, using melanotic (MNT-1) and amelanotic (A375, G361, Sk-Mel-28) melanoma cell lines, the comparative oncostatic responses, the impact on melanin content (for melanotic MNT-1 melanoma cells) as well as the mitochondrial function controlled by melatonin, its precursor (serotonin), a kynuric (N1 -acetyl-N2 -formyl-5-methoxykynuramine, AFMK) and indolic pathway (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) metabolites were assessed. Namely, significant disturbances were observed in bioenergetics as follows: (i) uncoupling of oxidative phosphorylation (OXPHOS), (ii) attenuation of glycolysis, (iii) dissipation of mitochondrial transmembrane potential (mtΔΨ) accompanied by (iv) massive generation of reactive oxygen species (ROS), and (v) decrease of glucose uptake. Collectively, these results together with previously published reports provide a new biological potential and make an imperative to consider using melatonin or its metabolites for complementary future treatments of melanoma-affected patients; however, these associations should be additionally investigated in clinical setting.

Melanization as unfavorable factor in amelanotic melanoma cell biology

The biology of three amelanotic melanoma cell lines (Ab, B16F10, and A375) of different species origin was analyzed during in vitro induced melanization in these cells. Melanin production was induced by DMEM medium characterized by a high level of L-tyrosine (a basic amino acid for melanogenesis). The biodiversity of amelanotic melanoma cells was confirmed by their different responses to melanogenesis induction; Ab hamster melanomas underwent intensive melanization, mouse B16F10 darkened slightly, while human A375 cells did not show any change in melanin content. Highly melanized Ab cells entered a cell death pathway, while slight melanization did not influence cell biology in a significant way. The rapid and high melanization of Ab cells induced apoptosis documented by phosphatidylserine externalization, caspase activation, and mitochondrial energetic state decrease. Melanoma cell type, culture medium, and time of incubation should be taken into consideration during amelanotic melanoma cell culture in vitro. L-tyrosine, as a concentration-dependent factor presented in the culture media, could stimulate some amelanotic melanoma cell lines (Ab, B16F10) to melanin production. The presence of melanin should be considered in the examination of antimelanoma compounds in vitro, because induction of melanin may interfere or be helpful in the treatment of amelanotic melanoma.

UVA Radiation Enhances Lomefloxacin-Mediated Cytotoxic, Growth-Inhibitory and Pro-Apoptotic Effect in Human Melanoma Cells through Excessive Reactive Oxygen Species Generation.

Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths. Thus, searching for new melanoma treatment options is an important field of study. The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation. C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system. The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells. The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells’ viability in a concentration-dependent manner. This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug. For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells’ viability by 50% (EC50) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively. Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells’ cell cycle distribution (including G2/M, S as well as Sub-G1-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation. This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy. The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.

Effects of curcumin based PDT on the viability and the organization of actin in melanotic (A375) and amelanotic melanoma (C32) – in vitro studies

Curcumin is a turmeric, antioxidative compound, well-known of its anti-cancer properties. Nowadays more and more effort is made in the field of enhancing the efficiency of the anticancer therapies. Combining the photoactive properties of curcumin with the superficial localization of melanoma and photodynamic therapy (PDT) seems to be a promising treatment method.The research focused on the evaluation of the curcumin effectiveness as an anticancer therapeutic agent in the in vitro treatment of melanotic (A375) and amelanotic (C32) melanoma cell lines. Keratinocytes (HaCat) and fibroblasts (HGF) were used to assess the impact of the therapy on the skin tissue. The aim of the study was to investigate the cell death after exposure to light irradiation after preincubation with curcumin. Additionaly the authors analized the interactions between curcumin and the actin cytoskeleton.The cytotoxic effect initiated by curcumin and increased by irradiation confirm the usefulness of the flavonoid in the PDT approach. Depending on curcumin concentration and incubation time, melanoma cells survival rate ranged from: 93.68 % (C32 cell line, 10 μM, 24 h) and 83.47 % (A375 cell line, 10 μM, 24 h) to 8.98 % (C32 cell line, 50 μM, 48 h) and 12.42 % (A375 cell line, 50 μM, 48 h). Moreover, photodynamic therapy with curcumin increased the number of apoptotic and necrotic cells in comparison to incubation with curcumin without irradiation. The study demonstrated that PDT induced caspase-3 overexpression and DNA cleavage in the studied cell lines. The cells revealed decreased proliferation after the therapy due to the actin cytoskeleton rearrangement. Although effective, the therapy remains not selective towards melanoma cells.

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis Against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1.

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family. So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma. In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines—keratinocytes (HaCaT) and the primary fibroblast line. Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed. Cell viability was studied using an MTT assay. A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD). Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B. This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A. No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence. Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future. Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.

Synthesis and Biological Evaluation of Acridine/Acridone Analogs as Potential Anticancer Agents.

The lack of efficacious therapy for advanced melanoma and neuroblastoma makes new approaches necessary. Therefore, many scientists seek new, more effective, more selective and less toxic anticancer drugs. We propose the synthesis of the new functionalized analogs of 1-nitroacridine/4- nitroacridone connected to tuftsin/retro-tuftsin derivatives as potential anticancer agents. Acridine and acridone analogues were prepared by Ullmann condensation and then cyclization reaction. As a result of nucleophilic substitution reaction 1-nitro-9-phenoxyacridine or 1- chloro-4-nitro-9(10H)-acridone with the corresponding peptides, the planned acridine derivatives (10a-c, 12, 17-a-d, 19) have been obtained. The cytotoxic activity of the newly obtained analogs were evaluated against melanotic (Ma) and amelanotic (Ab) melanoma cell lines and neuroblastoma SH-SY5Y by using the XTT method. Apoptosis and cell cycle were analyzed by flow cytometry. Among the investigated analogs compound 12 exhibited the highest potency comparable to dacarbazine action for amelanotic Ab melanoma cells. FLICA test (flurochrome-labeled inhibitors of caspases) showed that this analog significantly increased the content of cells with activated caspases (C+) among both neuroblastoma lines and only Ab melanoma line. Using phosphatidylserine (PS) externalization assay, 12 induced changes in the Ab melanoma plasma membrane structure as the externalization of phosphatidylserine (An+ cells). These changes in neuroblastoma cells were less pronounced. Analog 12 could be proposed as the new potential chemotherapeutic against amelanotic melanoma form especially.

Abstract 881: A role for microsomal glutathione transferase 1 in melanin biosynthesis in melanoma

Abstract Higher organisms have multiple defenses against toxic chemical insult, ultraviolet light (UVR) and oxidative stress. These include thioredoxin and glutathione dependent enzymes, some of which are selenium (Se) dependent, including thioredoxin reductases, Se-dependent glutathione peroxidases, Se-independent glutathione transferases and peroxiredoxins. Microsomal glutathione transferase 1 (MGST1) shares an important and unique characteristic with Se-dependent glutathione peroxidase GPX4, namely the ability to reduce lipid hydroperoxides directly in membranes. In contrast to most other glutathione transferases and peroxidases, we previously found that deletion of MGST1 in mice was embryonic lethal, signifying some essential functional importance. We found that MGST1 was crucial for embryonic development through regulating early hematopoiesis in both zebrafish and mouse derived hematopoietic stem cells. Interestingly, after partial knock-down MGST1, the number of melanocytes located at the midline of zebrafish embryos was significantly reduced. Melanocytes are specialized cells that produce melanin, contributing to skin color, but also providing an antioxidant defense system that includes UVR - causative in the initiation of melanoma. Both mRNA and protein levels of MGST1 were markedly decreased in several amelanotic melanoma cell lines and melanogenesis was significantly decreased in B16 melanoma cells when MGST1 expression was knocked down by 60%. This was accompanied by increased UVB-induced cell death in these B16 cells. In contrast, in normal human melanocytes, recombinant adenovirus induced over-expression of MGST1 induced melanogenesis, concomitant with an increase of tyrosinase protein levels. UVB light was used to show that stimulus of melanin biosynthesis occurred in different melanoma cells in quantitative agreement with their levels of MGST1. We conclude that within the melanin synthetic pathway, MGST1 is critical in regulating conversion of dopaquinone to glutathionyl-DOPA, a precursor of pheomelanin. In the absence of MGST1, dopaquinone can cause toxicities and result in reduced or absent levels of melanin, leading to a deficiency in protection against UVR-induced DNA damage. Thus, absence of MGST1 produces embryonic lethality and influences the susceptibility of skin melanocytes to melanoma. Citation Format: Jie Zhang, Zhi-wei Ye, Danyelle M. Townsend, Ralf Morgenstern, Kenneth D. Tew. A role for microsomal glutathione transferase 1 in melanin biosynthesis in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 881.

Caffeine modulates growth and vitality of human melanotic COLO829 and amelanotic C32 melanoma cells: Preliminary findings

Coffee is one of the most popular beverages consumed worldwide and therefore even small effects of coffee bioactive compounds on cellular homeostasis could have a large impact on public health. Recent cohort health studies have shown an impressive inverse correlation between caffeinated coffee consumption and malignant melanoma risk. The aim of presented study was to examine the effect of caffeine on cell viability and glutathione status of melanotic (COLO829) and amelanotic (C32) melanoma cell lines as well as normal human melanocytes. Cell viability was determined by WST-1 colorimetric assay and then confirmed by the cytometry analysis with NucleoCounter® NC-3000™. The intracellular level of reduced thiols was determined by the use of the fluorescence-based image cytometer. The obtained results indicate the ability of caffeine to induce reduced thiols depletion and pro-apoptotic effect selectively in melanoma cells. The COLO829 cell line was shown to be more susceptible to studied methylxanthine in comparison to C32 cells. These data may support, for the first time, the recent epidemiological studies and provide the basis for further research into the mechanism of chemopreventive properties of caffeine.

UVA radiation augments cytotoxic activity of psoralens in melanoma cells

Purpose: Melanoma is an aggressive form of skin cancer. The aim of the study was to evaluate the influence of UVA radiation and psoralens: 5-methoxypsoralen (5-MOP) or 8-methoxypsoralen (8-MOP) on melanoma cells viability.Materials and methods: The amelanotic C32 and melanotic COLO829 human melanoma cell lines were exposed to increasing concentrations of psoralens (0.1–100 μM) in the presence or absence of UVA radiation. Cell viability was evaluated by the WST-1 assay.Results: We demonstrated that 8-MOP, in contrast to 5-MOP, has no cytotoxic effect on both melanoma cell lines. Simultaneous exposure of cells to 8-MOP and UVA radiation caused significant cytotoxic response in C32 cells where the EC50 value was estimated to be 131.0 μM (UVA dose: 1.3 J/cm2) and 105.3 μM (UVA dose: 2.6 J/cm2). The cytotoxicity of 5-MOP on both C32 and COLO829 cells was significantly augmented by UVA radiation – the EC50 was estimated to be 22.7 or 7.9 μM (UVA dose: 1.3 J/cm2) and 24.2 or 7.0 μM (UVA dose: 2.6 J/cm2), respectively.Conclusions: The demonstrated high cytotoxic response after simultaneous exposure of melanoma cells to psoralens and UVA radiation in vitro suggests the usefulness of PUVA therapy to treat melanoma in vivo.

In Vivo Imaging of Experimental Melanoma Tumors using the Novel Radiotracer 68Ga-NODAGA-Procainamide (PCA).

Purpose: The most aggressive form of skin cancer is the malignant melanoma. Because of its high metastatic potential the early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of the disease. Previous studies have already shown that benzamide derivatives, such as procainamide (PCA) specifically bind to melanin pigment. The aim of this study was to synthesize and investigate the melanin specificity of the novel 68Ga-labeled NODAGA-PCA molecule in vitro and in vivo using PET techniques.Methods: Procainamide (PCA) was conjugated with NODAGA chelator and was labeled with Ga-68 (68Ga-NODAGA-PCA). The melanin specificity of 68Ga-NODAGA-PCA was tested in vitro, ex vivo and in vivo using melanotic B16-F10 and amelanotic Melur melanoma cell lines. By subcutaneous and intravenous injection of melanoma cells tumor-bearing mice were prepared, on which biodistribution studies and small animal PET/CT scans were performed for 68Ga-NODAGA-PCA and 18FDG tracers.Results: 68Ga-NODAGA-PCA was produced with high specific activity (14.9±3.9 GBq/µmol) and with excellent radiochemical purity (98%<), at all cases. In vitro experiments showed that 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than Melur cells. Ex vivo biodistribution and in vivo PET/CT studies using subcutaneous and metastatic tumor models showed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake in B16-F10 primary tumors and lung metastases in comparison with amelanotic Melur tumors. In experiments where 18FDG and 68Ga-NODAGA-PCA uptake of B16-F10 tumors was compared, we found that the tumor-to-muscle (T/M) and tumor-to-lung (T/L) ratios were significantly (p≤0.05 and p≤0.01) higher using 68Ga-NODAGA-PCA than the 18FDG accumulation.Conclusion: Our novel radiotracer 68Ga-NODAGA-PCA showed specific binding to the melanin producing experimental melanoma tumors. Therefore, 68Ga-NODAGA-PCA is a suitable diagnostic radiotracer for the detection of melanoma tumors and metastases in vivo.

Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression

l-Dopachrome tautomerase (l-DCT), also called tyrosinase-related protein-2 (TRP-2), is a melanoma antigen overexpressed in most chemo-/radiotherapeutic stress-resistant tumor clones, and caveolin-1 (CAV1) is a main regulator of numerous signaling processes. A structural and functional relationship between DCT and CAV1 is first presented here in two human amelanotic melanoma cell lines, derived from vertical growth phase (MelJuSo) and metastatic (SKMel28) melanomas. DCT co-localizes at the plasma membrane with CAV1 and Cavin-1, another molecular marker for caveolae in both cell phenotypes. Our novel structural model proposed for the DCT-CAV1 complex, in addition to co-immunoprecipitation and mass spectrometry data, indicates a possible direct interaction between DCT and CAV1. The CAV1 control on DCT gene expression, DCT post-translational processing, and subcellular distribution is cell phenotype-dependent. DCT is a modulator of CAV1 stability and supramolecular assembly in both cell phenotypes. During autocrine stimulation, the expressions of DCT and CAV1 are oppositely regulated; DCT increases while CAV1 decreases. Sub-confluent MelJuSo clones DCT(high)/CAV1(low) are proliferating and acquire fibroblast-like morphology, forming massive, confluent clusters as demonstrated by immunofluorescent staining and TissueFAXS quantitative image cytometry analysis. CAV1 down-regulation directly contributes to the expansion of MelJuSo DCT(high) subtype. CAV1 involved in the perpetuation of cell phenotype-overexpressing anti-stress DCT molecule supports the concept that CAV1 functions as a tumor suppressor in early stages of melanoma. DCT is a regulator of the CAV1-associated structures and is possibly a new molecular player in CAV1-mediated processes in melanoma.

Anthemis wiedemanniana essential oil prevents LPS-induced production of NO in RAW 264.7 macrophages and exerts antiproliferative and antibacterial activities in vitro

Anthemis wiedemanniana is known in folk medicine for the treatment of microbial infections, cancer and also urinary and pulmonary problems. In this study, the chemical composition of the essential oil from A. wiedemanniana was evaluated and its antibacterial activity was tested against 10 bacterial strains. The oil was also tested for its potentiality to inhibit nitric oxide production in RAW 264.7 macrophages and for its cytotoxicity against four human cancer cell lines. A. wiedemanniana oil, rich of oxygenated monoterpenes (25.4%), showed a good antibacterial activity against Gram-positive bacteria and a good activity against the two Gram-negative bacteria, Escherichia coli and Proteus vulgaris. Besides that, it exhibited a high inhibitory effect on the LPS-induced nitrite production and a strong cytotoxic activity, especially against amelanotic melanoma (C32) and large lung cell carcinoma (COR-L23) cell lines.

Impact of NAD(P)H:Quinone Oxidoreductase-1 on Pigmentation

We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription-PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process.

Antiproliferative Activity on Human Cancer Cell Lines after Treatment with Polyphenolic Compounds Isolated from Iris pseudopumila Flowers and Rhizomes

The present study describes the antiproliferative properties of Iris pseudopumila flowers and rhizomes extracts and fourteen constituents isolated from them. The in vitro cytotoxic activity assay against two human cancer cell lines, large lung carcinoma (CORL-23) and amelanotic melanoma (C32), showed that the most antiproliferative extract was the MeOH extract from flowers with a percentage of inhibition of 50.9 at 100 microg/ml against amelanotic melanoma cells. The most antiproliferative compounds against amelanotic melanoma cells were kaempferol-3-O-beta-D-glucopyranoside and irisolidone with a percentage of inhibition of 100 and 96.6, respectively, and against large lung carcinoma cells with a percentage of inhibition of 82.1 and 84.6, respectively. Significant activity on the amelanotic melanoma cell line was also showed by irigenin-7-O-beta-D-glucopyranoside, with a percentage of inhibition of 89.3. The compounds isovitexin and isoorientin-6-O''-beta-D-glucopyranoside showed a selective activity against amelanotic melanoma cells with a percentage of inhibition of 83.2 and 79.8, respectively.