Melanoma Cell Death Research Articles

Enhanced anti-melanoma efficacy of interferon α-2b via overexpression of ING4 by enhanced Fas/FasL-mediated apoptosis.

Melanoma, is a highly aggressive and the most lethal form of skin cancer, and is known to be resistant to current therapeutic modalities. Interferon (IFN)-α2b is an immunostimulatory cytokine and is used to treat melanoma by inhibiting proliferation and promoting apoptosis of cells. However, there is a need to improve the efficacy of IFN-α2b. Inhibitor of growth family member 4 (ING4) has been reported to function as a tumor suppressor and is involved in regulating cell cycle progression, apoptosis, cell migration and invasion. Previously studies have also reported that caspase-3, caspase-8, poly (ADP-ribose) polymerase (PARP) and Fas/Fas ligand (FasL) pathways are involved in the process of apoptosis. In the present study, it was investigated whether overexpression of ING4 is able to enhance IFN-α2b response in human melanoma cells. It was determined that the overexpression of ING4 was able to increase the effects of IFN-α2b, and induce cell death and apoptosis in melanoma cells. Furthermore, the overexpression of ING4 resulted in decreased expression of PARP, caspase-3 and -8. The expression of cleaved PARP, cleaved caspase-3, cleaved caspase-8, Fas and FasL was increased in the A375 melanoma cell line. These results demonstrate that the overexpression of ING4 is able to enhance the anti-melanoma activity of IFN-α2b. These findings provide a potential therapeutic strategy where a combination of ING4 overexpression and IFN-α2b treatment may lead to higher levels of apoptosis in melanoma cells.

Extracellular acidification by lactic acid suppresses glucose deprivation-induced cell death and autophagy in B16 melanoma cells

In solid tumors, cancer cells survive and proliferate under conditions of microenvironment stress such as poor nutrients and hypoxia due to inadequate vascularization. These stress conditions in turn activate autophagy, which is important for cancer cell survival. However, autophagy has a contrary effect of inducing cell death in cancer cells cultured in vitro under conditions of glucose deprivation. In this study, we hypothesized that supplementation of lactic acid serves as a means of cell survival under glucose-deprived conditions. At neutral pH, cell death of B16 murine melanoma cells by autophagy under glucose-deprived conditions was observed. However, supplementation of lactic acid suppressed cell death and autophagy in B16 melanoma cells when cultured in glucose-deprived conditions. Sodium lactate, which does not change extracellular pH, did not inhibit cell death, while HCl-adjusted acidic pH suppressed cell death under glucose-deprived conditions. These results suggested that an acidic pH is crucial for cell survival under glucose-deprived conditions.

Mitochondrial Complex IV Inhibition and Exogenous Oxidants Potently Synergize in Melanoma Cell Death Induction

Due to its functional duality in toxicity as well as signaling, research on therapeutic manipulation of reactive oxygen species (ROS) in cancer has been futile. Targeting melanoma cell death with excess amounts ROS, however, is difficult as these cells are highly resistant towards oxidative stress, creating a need for alternative and combinational approaches. Tumor metabolism mainly relies on aerobic glycolysis. We investigated a potential synergism of mitochondrial complex IV inhibition and exogenous-derived oxidants with both single treatment modalities being essentially non-toxic in melanoma cells. In this study, we investigated murine (metastatic B16F10 and non-metastatic B16F0) as well as human (SK-Mel 28) melanoma cells, and compared them with murine (primary fibroblasts) and human (HaCaT keratinocytes) non-cancer cells. Mitochondrial complex IV inhibition was realized using sodium azide (NaN3) and potassium cyanide (KCN) and pharmacological inhibitor ADDA5. Exogenous oxidants were generated using cell culture medium that has been treated with the cold physical plasma effluent of an atmospheric pressure argon plasma jet (kINPen). This plasma-treated medium was subsequently applied to different cells. Metabolic activity was assessed by fluorescence quantification of resazurin-to-resorufin transformation. ATP levels were monitored by a luminescence assay. Total cell counts, intracellular ROS levels (DCF), mitochondria membrane potential (TMRE), cell membrane integrity (sytox green) and cellular morphology were quantitatively assessed using live cell and high throughput confocal imaging (Operatta; Perkin Elmer). For tumor cells, also 3D spheroid models were investigated for viability by sytox green staining. ADDA5, NaN3 and KCN treatment alone failed to induce a significant alteration in metabolic activity in cancer cells. We then exposed the cells pretreated with NaN3 (100-500 µM) or KCN (250-500 µM) to plasma-treated medium. There was a significant inhibition of metabolic activity and rapid induction of cell death 6 h following treatment in melanoma cells compared to cell receiving plasma-treated medium alone. Increase in cellular superoxide levels and decrease in mitochondrial membrane potential and ATP levels was observed preceding cell death. However, no caspase 3 cleavage was observed upon immunoblotting. siRNA mediated knockdown of COX4a led to increased sensitivity of tumor cells towards plasma-treated medium. Furthermore, we validated our findings using a pharmacological inhibitor of complex IV ADDA5 in a tumor spheroid model and demonstrated that complex IV inhibition supplemented with plasma-treated medium, triggered significant cell death via caspase independent mechanism in melanoma cells.

Role of ubenimex as an anticancer drug and its synergistic effect with Akt inhibitor in human A375 and A2058 cells.

BackgroundMalignant melanoma (MM) is a malignant tumor produced by changes in melanocytes in the skin or other organs. In the classification of skin tumor mortality, skin melanoma ranks the highest. Ubenimex, an Aminopeptidase N (APN) inhibitor, is now widely used for cancer as an adjunct therapy, conferring antitumor effects. Apoptosis and the induction of autophagy have both been found to be closely associated with tumor cell death.MethodsIn this study, the A375 and A2058 cell lines were treated with ubenimex. Cell viability was measured using the Cell Counting Kit 8 assay. Apoptosis and autophagic cell death were assessed using flow cytometry and acridine orange/ethidium bromide staining. Protein expression was assessed by Western blot analyses and immunofluorescence. Matrigel invasion and migration assays were used to examine the metastatic ability of melanoma cells.ResultsThe results revealed that ubenimex inhibited the expression of APN in melanoma cells, which may be connected with the inhibition of metastasis. In addition, it increased melanoma cell death by inducing apoptosis and autophagic cell death. This effect was accompanied by increased levels of p-JNK. Moreover, treatment with ubenimex induced protective Akt activation, and combined use of an Akt inhibitor with ubenimex provided a better effect for inducing tumor cell death.ConclusionAs an effective anti-tumor drug in vitro, ubenimex might be an excellent adjunctive therapy for the treatment of melanoma, with greater effects when combined with the use of an Akt inhibitor.

Abstract A50: 7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma

Abstract Background: The exposure of phosphatidylserine (PS) is one of the first steps of programmed cell death. Phagocytosis on cancer microenvironment is well described in tumors and is associated with malignancy and poor prognosis. Tumor associated macrophages (TAMs) act suppressing the anticancer immune response. The tumor parenchymal cells are also capable of phagocytosis cells in apoptosis. In a previous study we observed that 7-ketocholesterol is capable of inducing autophagy on melanoma cell. Aims: Evaluate the activities of a 7-ketocholesterol loaded-phosphatidylserine liposome on autophagy and phagocytosis of tumor microenvironment. Methods: Liposomes were constituted by 20 mg commercial Phosphatidylserine (PS) and PS associated with 5 mg of 7-ketocholesterol extracted with chloroform/methanol (10: 1), dried, resuspended in 10 mL phosphate buffer, homogenized and sonicated for 6 minutes. The size and Zeta Pontencial (ZP) of liposomes were evaluated. Antiinflammatory activity of liposomes was evaluated by paw edema induced by carrageenan. A dependent-dose effect of liposomes on J774 macrophages, B16F10 melanoma cells, and 4T1 breast cancer cells was assessed by MTT. Cell death evaluations, for the same cells, were performed by flow cytometry with propidium iodide (PI) staining. The presence of acid vacuoles related to autophagy was evaluated by flow cytomery by acridine orange staining. The effects of the liposomes in vivo were evaluated by B16F10 melanoma-bearing C57/bl6 mice and 4T1 breast cancer-bearing Balb c mice. Endocytosis efficiency of the liposomes was observed by labeling it with PKH26 fluorescent staining and evaluated in 4T1 cells after 12 h. Liposomes were radiolabeled by adding 1 to 30 mCi of 99mTc radiopharmaceuticals (99mTcO4-, 99mTc-dextran-70, 99mTc-MIBI, 99mTc-DISIDA) and 18FDG; the solution was homogenized and sonicated for 6 minutes. The samples were centrifuged and part of the supernatant was added to an Amicon® filter (10kD) and concentrated, the concentrated was diluted with 400 uL of PBS and concentrated again. Liposome incorporation was determined by quotient of the radioactivity in the Amicon® by sum of Amicon® and filtrated solutions. Furthermore, lipophilicity (L), hydrophilicity (H), and charge (-/0/+) of the radioactive material were considered in the final analysis. Results: PS liposomes presented 141,9nm + 9,101 size with a -25,2 ZP; PS-7-ketocholesterol (PS/7KC) liposomes presented 153,9 nm + 10,35 size with a -29,1 ZP. The paw edema was inhibited by both liposomes after 240 min of the carrageenan induction. The concentration of 26 uM/mL of PS and PS/7KC liposomes stimulated cell proliferation. PS/7KC at the concentrations above 84 uM/mL inhibited the cell proliferation. PS/7KC showed intense antiproliferative activity in melanoma cells and breast adenocarcinoma cells, assessed by the MTT method and by flow cytometry with PI. It was observed 10% more autophagic vacuoles on melanoma cells treated with PS/7KC than the control groups. Both in vivo tumor models had the same antiproliferative effect of the PS/7KC liposomes with daily doses. Daily doses of PS liposomes induced a high size of tumors. 99mTc-MIBI was efficiently and strongly incorporated to liposomes than the other proposed formulations. Conclusion: PS liposomes have effects in vivo and in vitro and must be related to phagocyte and autophagy activities. PS/7KC impairs J774 macrophage, B16F10 melanoma, and 4T1 breast adenocarcinoma cell growth. PS/7KC induces the presence of acid vacuoles corresponding to autophagy. The liposomes had a high endocytosis evaluated by PKH 26 labelled particles. PS keeps the tumor proliferation and PS/7KC inhibits tumor growth after ten days of daily doses. Supported by CNPq and Fundação Araucária. Citation Format: Giovani Marino Favero, Tharcisio Citrangulo Tortelli, Jr., Daniel Fernandes, Ana Paula Prestes, Louise N.B. Kmetiuk, Andreia Hanada Otake, Luciana N.S. Andrade, Daniele de Paula Faria, Camila de Godoi Carneiro, Alexandre Teles Garcez, Fabio L.N. Marques, Roger Chammas. 7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A50.

Oncolytic Properties of a Mumps Virus Vaccine Strain in Human Melanoma Cell Lines

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.

Synthesis and characterization of copper nanoparticles stabilized with Quisqualis indica extract: Evaluation of its cytotoxicity and apoptosis in B16F10 melanoma cells

Green synthesis of metallic nanoparticles is a cost-effective environment-friendly technique and Quisqualis indica has ethnomedicinal values. With this background in this study, the floral extract of Q. indica was used to fabricate copper nanoparticles (QCuNPs) from copper acetate. Biophysical analysis revealed the formation of spherical, monodisperse, crystalline QCuNPs. Significant cytotoxic potentials of the nanoformulation were determined by MTT and lactate dehydrogenase (LDH) assay on B16F10 melanoma cells. Estimation of GSH and ROS demonstrated that QCuNPs induced melanoma cell death by induction of oxidative stress. Gene transcript analysis showed up-regulation of caspase-dependent as well as caspase-independent (AIF) apoptotic genes in treated cells. Comparative proteomics study mostly showed the abundance of apoptotic and cell cycle arrest proteins in treated samples. The in vivo therapeutic efficacy was studied in mice bearing B16F10 melanoma tumor where a significant decrease in tumor growth was observed in nanoparticles treated animal model. In conclusion, QCuNPs caused cytotoxicity and apoptosis in melanoma cells and its mechanism was established from gene expression and proteomic studies. QCuNPs exhibited potential suppression of B16F10 melanoma cell proliferation and substantial inhibition of tumor growth in animals. As per our information, this is the first study exploring the potential of Q. indica for the formulation of eco-friendly copper nanoparticle which will have great future application in the medicinal field.

Dendrogenin A drives LXR to trigger lethal autophagy in cancers

Dendrogenin A (DDA) is a newly discovered cholesterol metabolite with tumor suppressor properties. Here, we explored its efficacy and mechanism of cell death in melanoma and acute myeloid leukemia (AML). We found that DDA induced lethal autophagy in vitro and in vivo, including primary AML patient samples, independently of melanoma Braf status or AML molecular and cytogenetic classifications. DDA is a partial agonist on liver-X-receptor (LXR) increasing Nur77, Nor1, and LC3 expression leading to autolysosome formation. Moreover, DDA inhibited the cholesterol biosynthesizing enzyme 3β-hydroxysterol-Δ8,7-isomerase (D8D7I) leading to sterol accumulation and cooperating in autophagy induction. This mechanism of death was not observed with other LXR ligands or D8D7I inhibitors establishing DDA selectivity. The potent anti-tumor activity of DDA, its original mechanism of action and its low toxicity support its clinical evaluation. More generally, this study reveals that DDA can direct control a nuclear receptor to trigger lethal autophagy in cancers.

The natural product mensacarcin induces mitochondrial toxicity and apoptosis in melanoma cells

Mensacarcin is a highly oxygenated polyketide that was first isolated from soil-dwelling Streptomyces bacteria. It exhibits potent cytostatic properties (mean of 50% growth inhibition = 0.2 μm) in almost all cell lines of the National Cancer Institute (NCI)-60 cell line screen and relatively selective cytotoxicity against melanoma cells. Moreover, its low COMPARE correlations with known standard antitumor agents indicate a unique mechanism of action. Effective therapies for managing melanoma are limited, so we sought to investigate mensacarcin's unique cytostatic and cytotoxic effects and its mode of action. By assessing morphological and biochemical features, we demonstrated that mensacarcin activates caspase-3/7-dependent apoptotic pathways and induces cell death in melanoma cells. Upon mensacarcin exposure, SK-Mel-28 and SK-Mel-5 melanoma cells, which have the BRAFV600E mutation associated with drug resistance, showed characteristic chromatin condensation as well as distinct poly(ADP-ribose)polymerase-1 cleavage. Flow cytometry identified a large population of apoptotic melanoma cells, and single-cell electrophoresis indicated that mensacarcin causes genetic instability, a hallmark of early apoptosis. To visualize mensacarcin's subcellular localization, we synthesized a fluorescent mensacarcin probe that retained activity. The natural product probe was localized to mitochondria within 20 min of treatment. Live-cell bioenergetic flux analysis confirmed that mensacarcin disturbs energy production and mitochondrial function rapidly. The subcellular localization of the fluorescently labeled mensacarcin together with its unusual metabolic effects in melanoma cells provide evidence that mensacarcin targets mitochondria. Mensacarcin's unique mode of action suggests that it may be a useful probe for examining energy metabolism, particularly in BRAF-mutant melanoma, and represent a promising lead for the development of new anticancer drugs.

Dihydroxyacetone induces G2/M arrest and apoptotic cell death in A375P melanoma cells.

The active ingredient in sunless tanning products (STPs) is a simple sugar, dihydroxyacetone (DHA). Several studies have demonstrated that DHA is absorbed within the viable layers of skin and not fully contained within the stratum corneum. Additionally, spray tanning and other aerosolized application methods have increased the risk of internal exposure through mucous membranes and inhalation. Beyond its presence in STPs, DHA also occurs as an endogenous by-product of fructose metabolism, and an excess of DHA in cells can induce advanced glycation end (AGE) products and oxidative stress. Therefore, exogenous and endogenous exposures to DHA may be harmful to cells, and it has already been demonstrated that exogenous exposure to DHA is cytotoxic in immortalized keratinocytes. Still, little is known about the exogenous DHA exposure effects on other skin components. In this study, we explore the effects of exogenous DHA exposure in a human melanoma cell line, A375P. Melanoma cells were sensitive to DHA and displayed a transient burst of reactive oxygen species within an hour of exposure. Cell cycle arrest at G2/M was observed within 24 h of exposure, and apoptosis, monitored by the cleavage of PARP-1 and Caspase-3, was detected within 72 h of exposure to DHA. Together, these demonstrate that exogenous exposure to DHA has cytotoxic effects in our selected cell model and indicates the need to further investigate the exogenous exposure effects of DHA in other relevant exposure models.

Cytotoxic effect of the serotonergic drug 1-(1-Naphthyl)piperazine against melanoma cells

1-(1-Naphthyl)piperazine (1-NPZ) is a serotonergic derivative of quipazine acting both as antagonist and agonist of different serotonin receptors, with promising results for the management of skin cancer. In this work, we studied the effect of 1-NPZ on human MNT-1 melanoma cells by evaluating its effects on cell viability, ability to form colonies, cell cycle dynamics, reactive oxygen species (ROS) production and apoptosis. Treatment of MNT-1 cells with 1-NPZ for 24h decreased cell viability and induced apoptosis in a dose-dependent manner. Activity against melanoma was confirmed with a different melanoma cell line, SK-MEL-28. Simultaneously, 1-NPZ affected cell cycle progression by mediating a S-phase delay. Higher levels of ROS were also detected in MNT-1 cells after treatment with 1-NPZ. Furthermore, 1-NPZ significantly increased the expression of cyclooxygenase-2 in MNT-1 cells. These findings suggest that 1-NPZ pretreatment is able to induce oxidative stress, and consequently apoptotic cell death in melanoma cells. In conclusion, this study demonstrates the cytotoxic and genotoxic potential of 1-NPZ against melanoma cells.

Examining the In Vitro Efficacy of the IAP Antagonist Birinapant as a Single Agent or in Combination With Dacarbazine to Induce Melanoma Cell Death

Antagonists of inhibitors of apoptosis proteins (IAPs), alone or in combination with genotoxic therapeutics, have been shown to efficiently induce cell death in various solid tumors. The IAP antagonist birinapant is currently being tested in phase II clinical trials. We herein aimed to investigate the antitumor efficacy of dacarbazine in vitro, both as a single agent and in combination with birinapant, in melanoma cell lines. Covering clinically relevant drug concentration ranges, we conducted a total of 5,400 measurements in a panel of 12 human melanoma cell lines representing different stages of disease progression. Surprisingly, functionally relevant synergies or response potentiation in combination treatments was not observed, and only one cell line modestly responded to birinapant single treatment (approximately 16% cell death). Although we did not study the underlying resistance mechanisms or more complex in vivo scenarios in which dacarbazine/birinapant response synergies may still possibly manifest, our findings are nevertheless noteworthy because IAP antagonists were demonstrated to strongly enhance responses to DNA-damaging agents in cell lines of other cancer types under comparable experimental conditions in vitro.

108 - Cytochrome C oxidase Inhibition and Exogenous Oxidants Synergize in Melanoma Cell Death Induction

Despite striking advances with new therapies in the treatment of metastasized melanoma, the disease is often still fatal. To further increase treatment efficacy increasing attention is therefore paid towards combinational regimens with additive effects. To this end, oxidants like hydrogen peroxide endogenously produced in mitochondria were shown to be an interesting target in pre-clinical and clinical studies. We here examined cytotoxic synergism of mitochondrial cytochrome c oxidase (CcO) inhibition in conjunction with addition of exogenous oxidants in 2D and 3D melanoma cell culture models. Murine (B16) and human SK-MEL-28 melanoma cells exposed to low dose CcO inhibitors (potassium cyanide or sodium azide) or exogenous oxidants alone showed no decrease in cell viability. However, we identified a potent cytotoxic synergism upon CcO inhibition and exogenous addition of oxidants that led to rapid onset of caspase-independent melanoma cell death. Cell death was mediated by mitochondrial dysfunction induced by superoxide elevation and ATP depletion. This observation was further validated by siRNA-mediated knockdown of COX4I1 in SK-MEL-28 cells that increased cytotoxicity in the presence of exogenous oxidants. The same effects were obtained with ADDA 5, a recently identified specific inhibitor of CcO activity showing low toxicity in vivo. Human HaCaT keratinocytes were not affected by this combinational treatment, suggesting selective effects on melanoma cells. Hence, targeting mitochondrial CcO activity in conjunction with exogenous pro oxidant therapies may constitute a new and effective melanoma treatment modality.

Structure–Activity Relationships within a Series of σ1 and σ2 Receptor Ligands: Identification of a σ2 Receptor Agonist (BS148) with Selective Toxicity against Metastatic Melanoma

A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1 R; three compounds were shown to be σ1 R agonists, while another proved to be the only σ1 R antagonist. Only one of the σ1 R agonists (BS148) also exhibited σ2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis.

Etlingera elatior Extract promotes cell death in B16 melanoma cells via down-regulation of ERK and Akt signaling pathways

BackgroundTorch ginger (Etlingera elatior, EE) is a ginger plant that found in Southeast Asia. Previous study showed its flowers and leaves composed of several flavonoids with anti-cancer activity. This study aims to investigate the mechanism of EE extract on cell death induction in melanoma cells.MethodsTo carry out this study, the cytotoxic effect of EE extract was performed using MTT assay. Nuclear morphological change and loss of mitochondrial membrane potential were observed using Hoechst 33,342 and JC-1 staining. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Caspase activity was detected by caspase activity kits. The expression of Bcl-2 family proteins, ERK and Akt signaling pathways were examined by Western blot analysis.ResultsThe treatment of EE extract resulted in a dose- and time-dependent reduction in cell viability in B16 cells. It also induced nuclear condensation, phosphatidylserine exposure, and loss of mitochondrial membrane potential, which are markers of apoptosis. Furthermore, the expression of Bim was increased instead of Bax and Bcl-2. The results also showed caspase-independent activity and the down-regulation of ERK and Akt signaling pathway.ConclusionThe results suggest that EE extract induced caspase-independent cell death via down-regulation of ERK and Akt pathways in B16 cells. This may be beneficial as a chemopreventive or chemotherapeutic agent in melanoma treatment.

Recent Advances in Hydrogel-Based Drug Delivery for Melanoma Cancer Therapy: A Mini Review.

The purpose of this study is to describe some of the latest advances in using hydrogels for cancer melanoma therapy. Hydrogel formulations of polymeric material from natural or synthetic sources combined with therapeutic agents have gained great attention in the recent years for treating various maladies. These formulations can be categorized according to the strategies that induce cancer cell death in melanoma. First of all, we should note that these formulations can only play a supporting role that releases bioactive agents against cancer cells rather than the main role. This strategy involves delivering the drug via transdermal pathways, resulting in the death of cancerous cells. Another strategy utilizes magnetic gel composites to combat melanoma via hyperthermia therapy. This review discusses both transdermal and hyperthermia therapies and the recent advances that have occurred in the field.

Inactivation/deficiency of DHODH induces cell cycle arrest and programed cell death in melanoma.

Malignant melanoma (MM) is one of the most malignant tumors and has a very poor prognosis. However, there are no effective drugs to treat this disease. As a kind of iron flavin dependent enzyme, dihydroorotate dehydrogenase (DHODH, EC 1.3.3.1) is the fourth and a key enzyme in the de novo biosynthesis of pyrimidines. Herein, we found that DHODH inactivation/deficiency inhibited melanoma cell proliferation, induced cell cycle arrest at S phase and lead to autophagy in human melanoma cells. Meanwhile, leflunomide treatment induced cell apoptosis and deficiency of DHODH sensitized cells to drug-induced apoptosis in BCL-2 deficient melanoma cells, while not in BCL-2 abundant melanoma cells. Then we found that BCL-2 could rescue apoptosis induced by DHODH inactivation/deficiency. Moreover, BCL-2 also showed to promote cell cycle arrest and to inhibit autophagy induced by leflunomide. To explore the mechanisms underlying autophagy induced by DHODH inhibition, we found that AMPK-Ulk1 axis was activated in this process. Besides, JNK was phosphorylated and activated to phosphorylate BCL-2, which abrogated the interaction between BCL-2 and Beclin1 and then abolished autophagy. Our findings provided evidences for the potential of DHODH used as a drug target for melanoma treatment.

Abstract 3683: Melphalan stimulates dendritic cell and CD8+ T cell expansion by inducing immunogenic cell death in melanoma cells

Abstract Background: Regional hyperthermic perfusion with the alkylating agent melphalan is a treatment option for patients with metastatic melanoma confined to the limbs or the liver. Following a single perfusion, tumors often decrease gradually in size during several months, suggesting an immune-mediated mechanism of action, in addition to the direct cytotoxic effects of melphalan. This study was designed to characterize the immunogenic effects of melphalan. Materials and methods: We have established an in vitro model of regional hyperthermic perfusion where human melanoma cell lines are exposed to melphalan at 40oC for 1 h, thus mimicking the currently employed clinical protocol. The melphalan-exposed melanoma cells were analyzed for markers of immunogenic cell death and were co-cultured with peripheral blood mononuclear cells (PBMCs) in the presence or absence of IL-2. The number and activation status of various immune populations were analyzed by flow cytometry. Results: Melphalan exposure triggered the expression of several immune-related markers on melanoma cells, including calreticulin, MHC class I, Hsp70 and PD-L1. Melphalan-treated, but not untreated melanoma cells, triggered an increase in dendritic cell (DC) numbers along with a dramatic expansion of CD8+ T cells in co-cultured PBMCs. The expanded CD8+ T cells showed an activated phenotype with the majority of cells belonging to the effector memory subtype. Conclusions: Melanoma cells exposed to melphalan undergo immunogenic cell death and trigger DC expansion with subsequent expansion and activation of CD8+ T cells. We propose that these events may contribute to the anti-tumor efficacy of regional hyperthermic perfusion with melphalan in metastatic melanoma. Citation Format: Junko Johansson, Roberta Kiffin, Per Lindnér, Peter Naredi, Roger Olofsson Bagge, Anna Martner. Melphalan stimulates dendritic cell and CD8+ T cell expansion by inducing immunogenic cell death in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3683. doi:10.1158/1538-7445.AM2017-3683

Different Concentrations of Titanium Dioxide Nanoparticles Induce Autophagy Followed by Growth Inhibition or Cell Death in A375 Melanoma Cells

: Dysregulation of autophagy is currently known as a contributing factor to tumorigenesis. Accordingly, exploring novel compounds with autophagy - modulating properties is a priority in cancer research. While a number of synthetic compounds have been developed, autophagy - modulating Nanoparticles (NPs), such as titanium dioxide (TiO2), have been recently considered. This research investigated the effects of a well - dispersed form of TiO2 NPs on autophagic vesicle formation in human melanoma A375 cells. The cells were treated with different concentrations of TiO2 NPs (1 - 100 µg/mL) for 24 hours, then growth, cell death, and autophagy were, respectively, evaluated by trypan blue exclusion test, MTT assay, and acridine orange staining. The results showed that well - dispersed TiO2 NPs induced growth inhibition in a dose dependent manner, while cell death was only observed at high concentrations (100 µg/mL). In addition, a significant increase in Acidic Vesicular Organelles (AVO) formation was detected at all concentrations of NPs after 24 hours, suggesting that TiO2 NPs may induce both cytostatic and cytotoxic autophagic responses, depending on the concentration.

Treatment with diphenyl–pyrazole compound anle138b/c reveals that α-synuclein protects melanoma cells from autophagic cell death

Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson's disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson's disease-related proteins-α-synuclein, LRRK2, and Parkin-α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell's survival.