Melanoma Cell Death Research Articles

A Sulfur Containing Melanogenesis Substrate, N-Pr-4-S-CAP as a Potential Source for Selective Chemoimmunotherapy of Malignant Melanoma

N-propionyl-4-S-cysteaminylphenol (N-Pr-4-S-CAP) is a substrate for tyrosinase, which is a melanin biosynthesis enzyme and has been shown to be selectively incorporated into melanoma cells. It was found to cause selective cytotoxicity against melanocytes and melanoma cells after selective incorporation, resulting in the induction of anti-melanoma immunity. However, the underlying mechanisms for the induction of anti-melanoma immunity remain unclear. This study aimed to elucidate the cellular mechanism for the induction of anti-melanoma immunity and clarify whether N-Pr-4-S-CAP administration could be a new immunotherapeutic approach against melanoma, including local recurrence and distant metastasis. A T cell depletion assay was used for the identification of the effector cells responsible for N-Pr-4-S-CAP-mediated anti-melanoma immunity. A cross-presentation assay was carried out by using N-Pr-4-S-CAP-treated B16-OVA melanoma-loaded bone marrow-derived dendritic cells (BMDCs) and OVA-specific T cells. Administration of N-Pr-4-S-CAP induced CD8+ T cell-dependent anti-melanoma immunity and inhibited the growth of challenged B16F1 melanoma cells, indicating that the administration of N-Pr-4-S-CAP can be a prophylactic therapy against recurrence and metastasis of melanoma. Moreover, intratumoral injection of N-Pr-4-S-CAP in combination with BMDCs augmented the tumor growth inhibition when compared with administration of N-Pr-4-S-CAP alone. BMDCs cross-presented a melanoma-specific antigen to CD8+ T cells through N-Pr-4-S-CAP-mediated melanoma cell death. Combination therapy using N-Pr-4-S-CAP and BMDCs elicited a superior anti-melanoma effect. These results suggest that the administration of N-Pr-4-S-CAP could be a new strategy for the prevention of local recurrence and distant metastasis of melanoma.

Acquired non-thermal plasma resistance mediates a shift towards aerobic glycolysis and ferroptotic cell death in melanoma.

To gain insights into the underlying mechanisms of NTP therapy sensitivity and resistance, using the first-ever NTP-resistant cell line derived from sensitive melanoma cells (A375). Melanoma cells were exposed to NTP and re-cultured for 12 consecutive weeks before evaluation against the parental control cells. Whole transcriptome sequencing analysis was performed to identify differentially expressed genes and enriched molecular pathways. Glucose uptake, extracellular lactate, media acidification, and mitochondrial respiration was analyzed to determine metabolic changes. Cell death inhibitors were used to assess the NTP-induced cell death mechanisms, and apoptosis and ferroptosis was further validated via Annexin V, Caspase 3/7, and lipid peroxidation analysis. Cells continuously exposed to NTP became 10 times more resistant to NTP compared to the parental cell line of the same passage, based on their half-maximal inhibitory concentration (IC50). Sequencing and metabolic analysis indicated that NTP-resistant cells had a preference towards aerobic glycolysis, while cell death analysis revealed that NTP-resistant cells exhibited less apoptosis but were more vulnerable to lipid peroxidation and ferroptosis. A preference towards aerobic glycolysis and ferroptotic cell death are key physiological changes in NTP-resistance cells, which opens new avenues for further, in-depth research into other cancer types.

Redox phospholipidomics discovers pro-ferroptotic death signals in A375 melanoma cells in vitro and in vivo

Growing cancer cells effectively evade most programs of regulated cell death, particularly apoptosis. This necessitates a search for alternative therapeutic modalities to cause cancer cell's demise, among them – ferroptosis. One of the obstacles to using pro-ferroptotic agents to treat cancer is the lack of adequate biomarkers of ferroptosis. Ferroptosis is accompanied by peroxidation of polyunsaturated species of phosphatidylethanolamine (PE) to hydroperoxy- (-OOH) derivatives, which act as death signals. We demonstrate that RSL3-induced death of A375 melanoma cells in vitro was fully preventable by ferrostatin-1, suggesting their high susceptibility to ferroptosis. Treatment of A375 cells with RSL3 caused a significant accumulation of PE-(18:0/20:4-OOH) and PE-(18:0/22:4-OOH), the biomarkers of ferroptosis, as well as oxidatively truncated products - PE-(18:0/hydroxy-8-oxo-oct-6-enoic acid (HOOA) and PC-(18:0/HOOA). A significant suppressive effect of RSL3 on melanoma growth was observed in vivo (utilizing a xenograft model of inoculation of GFP-labeled A375 cells into immune-deficient athymic nude mice). Redox phospholipidomics revealed elevated levels of 18:0/20:4-OOH in RSL3-treated group vs controls. In addition, PE-(18:0/20:4-OOH) species were identified as major contributors to the separation of control and RSL3-treated groups, with the highest variable importance in projection predictive score. Pearson correlation analysis revealed an association between tumor weight and contents of PE-(18:0/20:4-OOH) (r = −0.505), PE-18:0/HOOA (r = −0.547) and PE 16:0-HOOA (r = −0.503). Thus, LC-MS/MS based redox lipidomics is a sensitive and precise approach for the detection and characterization of phospholipid biomarkers of ferroptosis induced in cancer cells by radio- and chemotherapy.

Comparison of direct and indirect low‐temperature plasma triggering immunogenic cell death in B16F10 melanoma

AbstractThe potential antitumor application of low‐temperature plasma (LTP) in immunotherapy has attracted wide attention from researchers. The induction of immunogenic cell death (ICD) is one of the possible mechanisms underlying this event. In this work, the immunogenicity of B16F10 melanoma cells treated with direct and indirect LTP protocols was investigated. The results revealed the concentrations of H2O2, NO, and NO2− increased as LTP treatment time was longer, while the cell viability decreased. By comparison, the direct treatment induced a stronger expression of ICD markers. In vivo, the LTP‐treated B16F10 reduced tumor burden in C57BL/6 mice. This study demonstrated both direct and indirect LTP treatments triggered immunogenic cell death and were expected to provide an effective immunotherapy strategy.

RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma.

Radiotherapy induces DNA damage, resulting in cell-cycle arrest and activation of cell-intrinsic death pathways. However, the radioresistance of some tumour entities such as malignant melanoma limits its clinical application. The innate immune sensing receptor retinoic acid-inducible gene I (RIG-I) is ubiquitously expressed and upon activation triggers an immunogenic form of cell death in a variety of tumour cell types including melanoma. To date, the potential of RIG-I ligands to overcome radioresistance of tumour cells has not been investigated. Here, we demonstrate that RIG-I activation enhanced the extent and immunogenicity of irradiation-induced tumour cell death in human and murine melanoma cells in vitro and improved survival in the murine B16 melanoma model invivo. Transcriptome analysis pointed to a central role for p53, which was confirmed using p53-/- B16 cells. In vivo, the additional effect of RIG-I in combination with irradiation on tumour growth was absent in mice carrying p53-/- B16 tumours, while the antitumoural response to RIG-I stimulation alone was maintained. Our results identify p53 as a pivotal checkpoint that is triggered by RIG-I resulting in enhanced irradiation-induced tumour cell death. Thus, the combined administration of RIG-I ligands and radiotherapy is a promising approach to treating radioresistant tumours with a functional p53 pathway, such as melanoma.

An endoplasmic reticulum-targeting iridium(iii) complex induces immunogenic cell death in melanoma cells and enhances anti-PD-1 immunotherapy by remodeling tumor microenvironment

IrC induces immunogenic cell death in melanoma cells and enhances anti-PD-1 immunotherapy by remodeling the tumor microenvironment in mice.

Cytotoxic Effects of Ethanolic Extract of Polypodium Vulgare on Human Malignant Melanoma Cell Line.

Melanoma is a malignant tumor that originates from the skin's melanocytes and has the highest death rate from skin cancer. Developing more efficacious anticancer medications with fewer adverse effects is the key to effective cancer management. Natural products are considered relevant and cost-effective sources of treatment. The plant (Polypodium vulgare) is a small and evergreen fern. One of the most important chemical compounds in the extract of this herb is flavonoids, which are thought to have beneficial effects in the treatment of melanoma through antioxidant properties. Due to the limitations of current cancer management and cytotoxic drugs available in the country, the need to study drugs of natural origin has become more prominent. In this regard, the present study aims to investigate the cytotoxic effects of the ethanolic extract of Polypodium vulgare on A375 melanoma cells. Polypodium vulgare was extracted in 80% ethanol by the maceration. Then, its effects on the cell death of the melanoma cell line A375 compared to the AGO-1522 cell line as control were measured using the MTT-assay technique. The amount of cellular lipid peroxidation was estimated by TBARS assay. The amount of cellular ROS was calculated by fluorescent reagent 2,7-dichlorofluorescein diacetate. Cytochrome c concentration was measured by a cytochrome c immunoassay kit. In this experiment, the anticancer effects of Polypodium vulgare ethanolic extract on human melanoma cell lines were investigated for the first time. Herb extract with a concentration of 0.123 mg/ml significantly increased the death of A375 melanoma cells (p < 0.001), lipid peroxidation (p < 0.01), and reactive oxygen species (ROS) (p < 0.01) and cytochrome c concentration (p < 0.001). Meanwhile, the same amount was ineffective and safe on AGO-1522 normal fibroblast cells. A 0.123 mg/ml concentration of Polypodium vulgare increases apoptosis in melanoma cells. Meanwhile, the same amount was safe on healthy cells. So, it could be considered an effective treatment without side effects in human melanoma.

A mitochondria-localized iridium(iii) photosensitizer for two-photon photodynamic immunotherapy against melanoma.

Conventional photodynamic therapy mainly causes a therapeutic effect on the primary tumor through the localized generation of reactive oxygen species, while metastatic tumors remain poorly affected. Complementary immunotherapy is effective in eliminating small, non-localized tumors distributed across multiple organs. Here, we report the Ir(iii) complex Ir-pbt-Bpa as a highly potent immunogenic cell death inducing photosensitizer for two-photon photodynamic immunotherapy against melanoma. Ir-pbt-Bpa can produce singlet oxygen and superoxide anion radicals upon light irradiation, causing cell death by a combination of ferroptosis and immunogenic cell death. In a mouse model with two physically separated melanoma tumors, although only one of the primary tumors was irradiated, a strong tumor reduction of both tumors was observed. Upon irradiation, Ir-pbt-Bpa not only induced the immune response of CD8+ T cells and the depletion of regulatory T cells, but also caused an increase in the number of the effector memory T cells to achieve long-term anti-tumor immunity.

Antiproliferative and Proapoptotic Effects of Erucin, a Diet-Derived H2S Donor, on Human Melanoma Cells.

Melanoma is the most dangerous form of skin cancer and is characterized by chemotherapy resistance and recurrence despite the new promising therapeutic approaches. In the last years, erucin (ERU), the major isothiocyanate present in Eruca sativa, commonly known as rocket salads, has demonstrated great efficacy as an anticancer agent in different in vitro and in vivo models. More recently, the chemopreventive effects of ERU have been associated with its property of being a H2S donor in human pancreatic adenocarcinoma. Here, we investigated the effects of ERU in modulating proliferation and inducing human melanoma cell death by using multiple in vitro approaches. ERU significantly reduced the proliferation of different human melanoma cell lines. A flow cytometry analysis with annexin V/PI demonstrated that ERU was able to induce apoptosis and cell cycle arrest in A375 melanoma cells. The proapoptotic effect of ERU was associated with the modulation of the epithelial-to-mesenchymal transition (EMT)-related cadherins and transcription factors. Moreover, ERU thwarted the migration, invasiveness and clonogenic abilities of A375 melanoma cells. These effects were associated with melanogenesis impairment and mitochondrial fitness modulation. Therefore, we demonstrated that ERU plays an important role in inhibiting the progression of melanoma and could represent a novel add-on therapy for the treatment of human melanoma.

Methods for Investigating Transient Receptor Potential Melastatin-2 (TRPM2): A Cation Channel Activated by ADP-Ribose and Involved in Cell Death.

Transient receptor potential melastatin-2 (TRPM2) is an emerging chemotherapeutic target due to its involvement in poly(ADP-ribose) metabolism and the ability to induce anticancer effects after antagonism of its functions. Normally functioning as a nonspecific cation channel that is activated by free ADP-ribose, TRPM2 is involved with many cellular processes, including the induction of cell death after oxidative stress. What is becoming clear is that antagonism of TRPM2 selectively induces anticancer effects in several types of cancer. We previously demonstrated decreased growth and proliferation, increased levels of DNA damage, and the selective induction of cell death in breast cancer and melanoma cells. Due to these effects, it appears that TRPM2 has a novel role in cancer cells. Further, this novel role appears to involve nuclear function, because our studies, as well as those from other independent groups, demonstrate a nuclear localization of TRPM2 in various types of cancers. Thus, as an emerging therapeutic target, it is important to describe research techniques that can be utilized to analyze TRPM2 function, determine its effects in cancerous and noncancerous cells, and provide molecular biological methods to inhibit or downregulate its function.

Microneedle-Assisted Transdermal Delivery of 2D Bimetallic Metal-Organic Framework Nanosheet-Based Cascade Biocatalysts for Enhanced Catalytic Therapy of Melanoma.

Current conventional treatments for malignant melanoma still face limitations, especially low therapeutic efficacy and serious side effects, and more effective strategies are urgently needed to develop them. Delivering biocatalysts into tumors to efficiently trigger in situ cascade reactions has shown huge potential in producing more therapeutic species or generating stronger tumoricidal effects for augmented tumor therapy. Recently, ultrathin 2D metal-organic framework (MOF) nanosheets have acquired great interest in biocatalysis owing to their large surface areas and abundant accessible active catalytic sites. Herein, an enhanced catalytic therapeutic strategy against melanoma is developed by biocompatible microneedle (MN)-assisted transdermal delivery of a 2D bimetallic MOF nanosheet-based cascade biocatalyst (Cu-TCPP(Fe)@GOD). Profiting from the constructed dissolving MN system, the loaded Cu-TCPP(Fe)@GOD hybrid nanosheets can be accurately delivered into the melanoma sites through skin barriers, and subsequently, trigger the specific cascade catalytic reactions in response to the acidic tumor microenvironment to effectively generate highly toxic hydroxyl radical (• OH) and deplete glucose nutrient for inducing the death of melanoma cells. The ultimate results prove the high melanoma inhibition effect and biosafety of such therapeutic modality, exhibiting a new and promising strategy to conquer malignant melanoma.

Upregulation of CD86 and IL-12 by rhododendrol in THP-1 cells cocultured with melanocytes through ROS and ATP

BackgroundThe tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma. ObjectiveAlthough an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood. MethodsWe hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined. ResultsWhen RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid. ConclusionRD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma.

Stimuli-responsive nanoparticle-nanofiber hybrids for drug delivery and photodynamic therapy

Hybrid nanomaterials possess integrated multi-components to syncretize various properties and functions within a single entity. Owing to this synergistic effect, they promise efficient anti-cancer therapy. In line with this target, we produced stimuli-responsive nanoparticle-nanofiber hybrids (NNHs) via embedding photoresponsive natural melanin nanoparticles (MNPs) within a biocompatible polycaprolactone (PCL) nanofiber matrix. Electrospinning was performed to produce monolithic and core–shell structured NNHs using a single and a coaxial nozzle. The NNHs were upgraded to drug delivery systems by model hydrophilic drug-ampicillin (amp)-loading. The drug release results showed that monolithic PCL meshes displayed a burst release, whereas nanohybrid formation with MNPs improved the release profile toward Fickian diffusion. Core-shell NNH presented a more sustained drug release profile than its MNP-free replica and monolithic NNH because its encapsulating shell layer hindered the diffusion of the drug. The photodynamic therapy accompanied by UV-A-irradiation on monolithic and core–shell NNHs yielded up to 34 % and 37 % malignant melanoma cell death. Moreover, this study proved the potency of MNPs-enhanced NNHs in drug delivery and photodynamic therapy applications. Even so, more efforts should be concerted to unlock unknown features of the NNHs, which have the power to advance emerging areas, including but not limited to material science, biosensing, and theranostics.

484 Melatonin induces melanoma cell death and synergizes with BRAF/MEK inhibitors by altering cell bioenergetics

Melanoma is a leading cause of cancer deaths worldwide. Although targeted therapy and immunotherapy have improved the outcome of patients with metastatic disease, unwanted side effects are a problem. Herein, by using human melanoma cell lines in vitro, we explore that melatonin enhances anti-tumor activity of commonly used BRAF/MEK inhibitors, i.e. vemurafenib (VF) and cobimetinib (CB), respectively. Our results have demonstrated that compared with VF/CB alone, melatonin significantly reduced proliferation read-outs (colony assay, drop assay) and induced of apoptosis (cleaved Casp-3, PARP) in melanoma cells.

Arginase 2 negatively regulates sorafenib-induced cell death by mediating ferroptosis in melanoma.

Ferroptosis, a newly defined and iron-dependent cell death, morphologically and biochemically differs from other cell deaths. Melanoma is a serious type of skin cancer, and the poor efficacy of current therapies causes a major increase in mortality. Sorafenib, a multiple kinase inhibitor, has been evaluated in clinical phase trials of melanoma patients, which shows modest efficacy. Emerging evidence has demonstrated that arginase 2 (Arg2), type 2 of arginase, is elevated in various types of cancers including melanoma. To investigate the role and underlying mechanism of Arg2 in sorafenib-induced ferroptosis in melanoma, reverse transcriptase-quantitative polymerase chain reaction, western blot analysis, adenovirus and lentivirus transduction, and in vivo tumor homograft model experiments were conducted. In this study, we show that sorafenib treatment leads to melanoma cell death and a decrease in Arg2 at both the mRNA and protein levels. Knockdown of Arg2 increases lipid peroxidation, which contributes to ferroptosis, and decreases the phosphorylation of Akt. In contrast, overexpression of Arg2 rescues sorafenib-induced ferroptosis, which is prevented by an Akt inhibitor. In addition, genetic and pharmacological suppression of Arg2 is able to ameliorate the anticancer activity of sorafenib in melanoma cells in vitro and in tumor homograft models. We also show that Arg2 suppresses ferroptosis by activating the Akt/GPX4 signaling pathway, negatively regulating sorafenib-induced cell death in melanoma cells. Our study not only uncovers a novel mechanism of ferroptosis in melanoma but also provides a new strategy for the clinical applications of sorafenib in melanoma treatment.

Quality by design steered development of Niclosamide loaded liposomal thermogel for Melanoma: In vitro and Ex vivo evaluation

Melanoma is the most malignant form of skin cancer across the globe. Conventional therapies are currently ineffective which could be attributed to the rampant chemo-resistance, metastasis, inability to cross the skin barriers and accumulate within the tumor microenvironment. This advent brings in the principles of drug repurposing by repositioning Niclosamide (NIC), an anthelmintic drug for skin cancer. Incorporation into the liposomes facilitated enhanced melanoma cell uptake and apoptosis. Cytotoxicity studies revealed 1.756-fold enhancement in SK-MEL-28 cytotoxicity by NIC-loaded liposomes compared to free drug. Qualitative and quantitative cell internalization indicated greater drug uptake within the melanoma cells illustrating the efficacy of liposomes as efficient carrier systems. Nuclear staining showed blebbing and membrane shrinkage. Elevated ROS levels and apoptosis shown by DCFDA and acridine orange-ethidium bromide staining revealed greater melanoma cell death by liposomes compared to free drug. Incorporating NIC liposomes into the thermogel system restricted the liposomes as a depot onto the upper skin layers. Sustained zero order release up to 48 h with liposomes and 23.58-fold increase in viscosity led to the sol-to-gel transition at 33℃ was observed with liposomal thermogel. Ex vivo gel permeation studies revealed that C-6 loaded liposomes incorporated within the thermogel successfully formed a depot over the upper skin layer for 6 h to prevent transdermal delivery and systemic adverse effects. Thus, it could be concluded that NIC loaded liposomal thermogel system could be an efficacious therapeutic alternative for the management of melanoma.

Lithium Enhances Autophagy and Cell Death in Skin Melanoma: An Ultrastructural and Immunohistochemical Study.

Lithium is an inhibitor of glycogen synthase kinase 3 beta, which is traditionally used in the treatment of bipolar disorders and has antitumor effects. The aim of the current study was to determine if lithium salt causes autophagy and apoptosis in skin melanoma cells to enhance cell death. Light microscopy, transmission electron microscopy, immunohistochemistry, and immunofluorescence were used to study the mechanism of action of lithium carbonate in B16 melanoma cells in vivo. Proliferating cell nuclear antigen immunofluorescence assay revealed that the proliferation of B16 melanoma cells was suppressed by lithium treatment for 7 days. Electron microscopy demonstrated a significant increase in the number of autophagic vacuoles in lithium-treated cells relative to control. In addition, levels of autophagy markers LC3 beta and LAMP1 found in lithium-treated tumor xenografts were higher than levels of these markers in the control tumors. Lithium induced caspase-3 expression and apoptotic cell death in tumor cells. Thus, lithium carbonate is the compound that inhibits cell proliferation and stimulates cell death in melanoma cells through induction of autophagy and apoptosis. Stimulation of autophagy by lithium could contribute to the development of autophagic cell death in tumor cells.

Comprehensive characterisation of immunogenic cell death in melanoma revealing the association with prognosis and tumor immune microenvironment.

Increasing evidence has highlighted the critical functions of immunogenic cell death (ICD) within many tumors. However, the therapeutic possibilities and mechanism of utilizing ICD in melanoma are still not well investigated. Melanoma samples involved in our study were acquired from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. First, pan-cancer analysis of ICD systematically revealed its expression characteristics, prognostic values, mutation information, methylation level, pathway regulation relationship in multiple human cancers. The non-negative matrix factorization clustering was utilized to separate the TCGA-melanoma samples into two subtypes (i.e. C1 and C2) with different prognosis and immune microenvironment based on the expression traits of ICD. Then, LASSO-Cox regression analysis was utilized to determine an ICD-dependent risk signature (ICDRS) based on the differentially expressed genes (DEGs) between the two subtypes. Principal component analysis and t-distributed stochastic neighbor embedding analysis of ICDRS showed that high- and low-risk subpopulations could be clearly distinguished. Survival analysis and ROC curves in the training, internal validation, and external validation cohorts highlighted the accurate prognosis evaluation of ICDRS. The obvious discrepancies of immune microenvironment between the different risk populations might be responsible for the different prognoses of patients with melanoma. These findings revealed the close association of ICD with prognosis and tumor immune microenvironment. More importantly, ICDRS-based immunotherapy response and targeted drug prediction might be beneficial to different risk subpopulations of patients with melanoma. The innotative ICDRS could function as a marker to determine the prognosis and tumor immune microenvironment in melanoma. This will aid in patient classification for individualized melanoma treatment.

NRF2 mediates melanoma addiction to GCDH by modulating apoptotic signalling.

Tumour dependency on specific metabolic signals has been demonstrated and often guided numerous therapeutic approaches. We identify melanoma addiction to the mitochondrial protein glutaryl-CoA dehydrogenase (GCDH), which functions in lysine metabolism and controls protein glutarylation. GCDH knockdown induced cell death programmes in melanoma cells, an activity blocked by inhibition of the upstream lysine catabolism enzyme DHTKD1. The transcription factor NRF2 mediates GCDH-dependent melanoma cell death programmes. Mechanistically, GCDH knockdown induces NRF2 glutarylation, increasing its stability and DNA binding activity, with a concomitant transcriptional upregulation of ATF4, ATF3, DDIT3 and CHAC1, resulting in cell death. In vivo, inducible inactivation of GCDH effectively inhibited melanoma tumour growth. Correspondingly, reduced GCDH expression correlated with improved survival of patients with melanoma. These findings identify melanoma cell addiction to GCDH, limiting apoptotic signalling by controlling NRF2 glutarylation. Inhibiting the GCDH pathway could thus represent a therapeutic approach to treat melanoma.

Melanosomal targeting via caveolin-1 dependent endocytosis mediates ZN(II) phthalocyanine phototoxic action in melanoma cells.

Melanosomes have been considered crucial targets in melanoma treatments. In this study we explored the role of melanosomes in photodynamic therapy (PDT), employing the synthetic Zn(II) phthalocyanine Pc13, a potent photosensitizer that promotes melanoma cell death after irradiation. Phototoxic action is mediated by reactive oxygen species increase. The internalization mechanism of Pc13 and its consequent subcellular localization were evaluated in melanotic B16-F0 cells. Pharmacological inhibitors of dynamin or caveolae, but not of clathrin, decreased Pc13 cellular uptake and phototoxicity. Similar results were obtained when cells over-expressed dominant negative mutants of dynamin-2 and caveolin-1, indicating that Pc13 is internalized by caveolae-mediated endocytosis. Confocal microscopy analysis revealed that Pc13 targets melanosomes and damage of these structures after irradiation was demonstrated by transmission electron microscopy. Treatment of pigmented B16-F0 and WM35 melanoma cells with the melanin synthesis inhibitor phenylthiourea for 48h led to cell depigmentation and enhanced cell death after irradiation, whereas a 3-h period of inhibition did not modify melanin content but produced a marked reduction of Pc13 phototoxicity, together with a decrease of oxidative melanin synthesis intermediates. In contrast, the effect of Pc13 in amelanotic A375 cells was not altered by phenylthiourea treatment. These results provide evidence that melanosomes have a dual role in the efficacy of PDT. While melanin antagonizes the phototoxic action of Pc13, the release of cytotoxic synthetic intermediates to cytosol after irradiation and melanosome damage is conducive to the phototoxic response. Based on these findings, we demonstrate that melanosome-targeted PDT could be an effective approach for melanoma treatment.