Metabolism Of Melanoma Cells Research Articles

UVA-induced metabolic changes in non-malignant skin cells and the potential role of pyruvate as antioxidant

The exposure to UVA (320–400 nm) irradiation is a major threat to human skin concerning photoaging and carcinogenesis. It has been shown that UVA irradiation can induce reactive oxygen species (ROS) and DNA mutations, such as 8-hydroxydeoxyguanosine. Furthermore, UVA induces the expression of photoaging-associated matrix metalloproteases (MMPs), especially of matrix metalloprotease 1 (MMP 1) and matrix metalloprotease 3 (MMP 3). In addition to this, it was recently shown that UVA-induced ROS also increase glucose metabolism of melanoma cells, however, the influence of UVA on the glucose metabolism of non-malignant cells of the human skin has, so far, not been investigated in detail. Here, we investigated the UVA-induced changes in glucose metabolism and the functional relevance of these changes in primary fibroblasts—normal non-malignant cells of the skin. These cells showed an UVA-induced enhanced glucose consumption and lactate production and changes in pyruvate production. As it has been proposed that pyruvate could have antioxidant properties we tested the functional relevance of pyruvate as protective agent against UVA-induced ROS. Our initial experiments support earlier publications, demonstrating that pyruvate treated with H2O2 is non-enzymatically transformed to acetate. Furthermore, we show that this decarboxylation of pyruvate to acetate also occurs upon UVA irradiation. In addition to this, we could show that in fibroblasts pyruvate has antioxidant properties as enhanced levels of pyruvate protect cells from UVA-induced ROS and partially from a DNA mutation by the modified base 8-hydroxydeoxyguanosine. Furthermore, we describe for the first time, that the interaction of UVA with pyruvate is relevant for the regulation of photoaging-associated MMP 1 and MMP 3 expression.Graphical abstract

KIF22 promotes the proliferation and glycolysis of melanoma by activating EGFR/STAT3 signaling

ObjectivesMelanoma is one of the leading causes of cancer death. Kinesin Family member 22 (KIF22) is essential for the invasion of melanoma cells, but the role and mechanism of KIF22 in the proliferation and glycolysis in melanoma remains unknown. MethodsKIF22 expression in melanoma tissues and the relationship between KIF22 high expression and overall survival rate in patients with melanoma were analyzed using the Tnmplot database. KIF22 expression in melanoma cells was examined by western blot. Then, KIF22 was silenced and CCK-8 assay, EDU staining and flow cytometry analysis were adopted for assessing cell proliferation and apoptosis. In addition, the glycolysis metabolism of melanoma cells was reflected by detecting Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR). The expression of proteins related to apoptosis, glycolysis and EGFR/STAT3 signaling was tested by western blot. Subsequently, melanoma cells were treated with EGF or Colivelin to further elucidate the regulatory effect of KIF22 on EGFR/STAT3 signaling. ResultsKIF22 expression was notably upregulated in melanoma tissues and cells, and KIF22 high expression was associated with a poor prognosis. Moreover, KIF22 insufficiency suppressed proliferation and accelerated apoptosis of melanoma cells. Additionally, glycolysis was reduced by KIF22 depletion, evidenced by the decreased ECAR and increased OCR, accompanied by the downregulated expression of HK2, PKM2 and LDHA. Importantly, the impacts of KIF22 depletion on the progression of melanoma were partially attenuated after EGF or Colivelin treatment. ConclusionCollectively, KIF22 knockdown suppressed the proliferation and glycolysis and facilitated the apoptosis of melanoma cells by inactivating EGFR/STAT3 signaling.

Detachment stress mediated bioenergetic switch of malignant melanoma cells into anti-Warburg phenotype.

One of the biological features of cancer cells is their aerobic glycolysis by extensive glucose fermentation to harvest energy, so called Warburg effect. Melanoma is one of the most aggressive human cancers with poor prognosis and high mortality for its high metastatic ability. During the metastatic process, the metastatic tumor cells should survive under detachment stress. However, whether the detachment stress could affect the tumor phenotype is worthy to investigate.We had established the cell model of human melanoma cells under detachment stress, which mimicked circulating melanoma. It had been demonstrated that the detachment stress altered melanoma cell activities, malignancy, and drug sensitivity. In this study, we found that adherent melanoma cells were more sensitive to glucose depletion. Gene expression profiling altered expressions of transporters associated with glucose metabolism. In addition, detachment stress reduced lactate secretion owing to the reduced MCT4 and GLUT1 expressions, the altered glycolytic and respiratory capacities, and the increased superoxide production. Detachment stress also increases the sensitivity of melanoma cells toward the blockade of electron transport chains. Investigation of the change in glucose metabolism of melanoma cells under detachment stress would be critical to provide a novel molecular mechanism to develop potential therapeutics.

The role of autophagy and vesicular trafficking in melanoma development

The role of autophagy and vesicular trafficking in carcinogenesis, including melanoma, is ambiguous: on the one hand, they contribute to the maintenance of intracellular homeostasis and the tumor progression, on the other hand, they can initiate cancer cell death. These processes have a significant impact on the metabolism of melanoma cells and could be associated with metastasis, tumor growth and progression. The review examines the mechanisms of autophagy and vesicular trafficking and presents the modern literature data demonstrating the role of the intracellular transport system in oncogenesis and melanoma development. A literature search was performed in the PubMed database.

Hypoxia and Extracellular Acidification as Drivers of Melanoma Progression and Drug Resistance.

Hypoxia and elevated extracellular acidification are prevalent features of solid tumors and they are often shown to facilitate cancer progression and drug resistance. In this review, we have compiled recent and most relevant research pertaining to the role of hypoxia and acidification in melanoma growth, invasiveness, and response to therapy. Melanoma represents a highly aggressive and heterogeneous type of skin cancer. Currently employed treatments, including BRAF V600E inhibitors and immune therapy, often are not effective due to a rapidly developing drug resistance. A variety of intracellular mechanisms impeding the treatment were discovered. However, the tumor microenvironment encompassing stromal and immune cells, extracellular matrix, and physicochemical conditions such as oxygen level or acidity, may also influence the therapy effectiveness. Hypoxia and acidification are able to reprogram the metabolism of melanoma cells, enhance their survival and invasiveness, as well as promote the immunosuppressive environment. For this reason, these physicochemical features of the melanoma niche and signaling pathways related to them emerge as potential therapeutic targets.

Paraoxonase-2 Silencing Enhances Sensitivity of A375 Melanoma Cells to Treatment with Cisplatin.

Melanoma represents the most aggressive skin cancer, being responsible for the majority of deaths related with these neoplasms. Despite chemotherapy represents a frontline approach for management of the advanced stages of the disease, it displayed poor response rates and short-term efficacy due to melanoma cell resistance. Therefore, the discovery of molecules that can be used for effective targeted therapy of melanoma is crucial. In this study, we evaluated the impact of paraoxonase-2 (PON2) silencing on proliferation, viability, and resistance to treatment of the A375 melanoma cell line with chemotherapeutic drugs dacarbazine (DTIC) and cisplatin (CDDP). Due to the enzymes ability to counteract oxidative stress, we also evaluated the effect of enzyme knockdown on reactive oxygen species (ROS) production in cells treated with CDDP. The data reported clearly demonstrated that PON2 knockdown led to a significant reduction of cell proliferation and viability, as well as to an enhancement of A375 sensitivity to CDDP treatment. Moreover, enzyme downregulation was associated with an increase of ROS production in CDDP-treated cells. Although further analyses will be necessary to understand how PON2 could influence melanoma cell metabolism and phenotype, our results seem to suggest that the enzyme may serve as an interesting molecular target for effective melanoma treatment.

Potential Role of HLA Class I Antigens in the Glycolytic Metabolism and Motility of Melanoma Cells.

Besides playing a crucial role in immune surveillance, human leukocyte antigens (HLA) possess numerous non-immune functions involved in cell communication. In the present study, screening of a panel of HLA class I- and HLA class II-specific monoclonal antibodies (mAbs) for their effects on the metabolism of human melanoma cells showed for the first time that the HLA-B,C-specific mAb B1.23.2 reduced the expression level of key glycolytic enzymes, but did not affect that of mitochondrial respiration effectors. As a result, the metabolism of melanoma cells shifted from a Warburg metabolism to a more oxidative phosphorylation. In addition, the HLA-B,C-specific mAb B1.23.2 downregulated the expression of glutamine transporter and glutaminase enzyme participating in the reduction of tricarboxylic acid cycle. The HLA-B,C-specific mAb B1.23.2-mediated reduction in energy production was associated with a reduction of melanoma cell motility. On the whole, the described results suggest that HLA class I antigens, and in particular the gene products of HLA-B and C loci play a role in the motility of melanoma cells by regulating their metabolism.

Mitochondrial transcription factor A (TFAM) shapes metabolic and invasion gene signatures in melanoma

Mitochondria are central key players in cell metabolism, and mitochondrial DNA (mtDNA) instability has been linked to metabolic changes that contribute to tumorigenesis and to increased expression of pro-tumorigenic genes. Here, we use melanoma cell lines and metastatic melanoma tumors to evaluate the effect of mtDNA alterations and the expression of the mtDNA packaging factor, TFAM, on energetic metabolism and pro-tumorigenic nuclear gene expression changes. We report a positive correlation between mtDNA copy number, glucose consumption, and ATP production in melanoma cell lines. Gene expression analysis reveals a down-regulation of glycolytic enzymes in cell lines and an up-regulation of amino acid metabolism enzymes in melanoma tumors, suggesting that TFAM may shift melanoma fuel utilization from glycolysis towards amino acid metabolism, especially glutamine. Indeed, proliferation assays reveal that TFAM-down melanoma cell lines display a growth arrest in glutamine-free media, emphasizing that these cells rely more on glutamine metabolism than glycolysis. Finally, our data indicate that TFAM correlates to VEGF expression and may contribute to tumorigenesis by triggering a more invasive gene expression signature. Our findings contribute to the understanding of how TFAM affects melanoma cell metabolism, and they provide new insight into the mechanisms by which TFAM and mtDNA copy number influence melanoma tumorigenesis.

UVA, metabolism and melanoma: UVA makes melanoma hungry for metastasis.

Ultraviolet (UV) radiation has a plethora of effects on human tissues. In the UV spectrum, wavelengths above 320nm fall into the UVA range, and for these, it has been shown that they induce reactive oxygen species (ROS), DNA mutations and are capable to induce melanoma in mice. In addition to this, it was recently shown that UVA irradiation and UVA-induced ROS also increase glucose metabolism of melanoma cells. UVA irradiation causes a persistent increase in glucose consumption, accompanied by increased glycolysis, increased lactic acid production and activation of the pentose phosphate pathway. Furthermore, it was shown that the enhanced secretion of lactic acid is important for invasion of melanoma in vitro. The current knowledge of this link between UVA, metabolism and melanoma, possible mechanisms of UVA-induced glucose metabolism and their starting points are discussed in this review with focus on ROS- and UVA-induced cellular stress signalling, DNA damage signalling and DNA repair systems. When looking at the benefits of UVA-induced glucose metabolism, it becomes apparent that there are more advantages of these metabolic changes than one would expect. Besides the role of lactic acid as initiator of protease expression and invasion, its role for immune escape of melanoma cells and the pentose phosphate pathway-derived nicotinamide adenine dinucleotide phosphate (NADPH) as part of a ROS detoxification strategy are discussed.

Gamma irradiation of ocular melanoma and lymphoma cells in the presence of gold nanoparticles: invitro study.

The aim of this work was to determine whether conjugation of cultivated choroidal melanoma and Burkitt's lymphoma cells with gold nanoparticles (GNPs) is beneficial for these series of ocular cancer patients. GNPs are radiosensitizers and can sensitize tumors to radiotherapy.This application has been examined in several tumor types, but not in choroidal melanoma. This study shows the results of in vitro study on the choroidal melanoma and also Burkitt's lymphoma cells in the presence of GNPs during continuous gamma irradiation. Cytotoxicity of GNPs were assessed for five different concentrations then cultured melanoma and Burkitt's lymphoma cells were irradiated with a Gamma source in the presence and absence of NPs. Incubation of melanoma cells with GNP concentrations below 100 μg/ml, accompanied by gamma irradiation, increased cell death (P value = 0.016) . In the absence of irradiation, GNPs at these concentrations did not affect cultured melanoma cell metabolism. Reduced cell viability resulted from a significant increase in absorbed energy by the tumor. Moreover, GNP concentrations higher than 200 μg/ml induced cytotoxicity in melanoma cells. Cytotoxicity assay in GNPs‐loaded Burkitt's lymphoma cells showed a slight decrease in cell viability at 50 μg/ml and clear cytotoxicity at concentrations higher than 100 μg/ml (P value = 0.035). Concentration and proper injection doses of GNPs in sensitive tissues such as the human eye are important variables yet to be determined.This is the first report of choroidal melanoma dosimetry performed in the presence of GNPs and provides valuable insights into future therapeutic approaches. Further in vitro study with more different sizes and concentrations is needed to determine the optimum size and concentration before any clinical research in this regard.

The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells

Reprogramming of cellular metabolism is now considered one of the hallmarks of cancer. Most malignant cells present with altered energy metabolism which is associated with elevated reactive oxygen species (ROS) generation. This is also evident for melanoma, the leading cause of skin cancer related deaths. Altered mechanisms affecting mitochondrial bioenergetics pose attractive targets for novel anticancer therapies. Antimicrobial peptides have been shown to exhibit selective anticancer activities. In this study, the anti-melanoma potential of the antimicrobial peptide, nisin Z, was evaluated in vitro. Nisin Z was shown to induce selective toxicity in melanoma cells compared to non-malignant keratinocytes. Furthermore, nisin Z was shown to negatively affect the energy metabolism (glycolysis and mitochondrial respiration) of melanoma cells, increase reactive oxygen species generation and cause apoptosis. Results also indicate that nisin Z can decrease the invasion and proliferation of melanoma cells demonstrating its potential use against metastasis associated with melanoma. As nisin Z seems to place a considerable extra burden on the energy metabolism of melanoma cells, combination therapies with known anti-melanoma agents may be effective treatment options.

Abstract 3047: PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis

Abstract Circular RNAs are a novel class of non-coding RNAs with functions that remain poorly characterized in normal and pathological conditions. CDR1as is a non-canonical circRNA observed to act as a sponge for miR-7 in brain tissues. Analysis of RNA-seq data of melanocytes and melanoma cell lines and short-term cultures revealed loss of CDR1as expression as a hallmark of melanoma cells. We confirmed silencing of CDR1as in melanoma cells and tissues by RT-qPCR using divergent primers. Clinically, we observed CDR1as loss associated with metastatic progression and poor patient outcomes in a cohort of fresh-frozen melanoma tissue samples. Depletion of CDR1as in melanoma cell lines enhanced invasion in vitro and lung metastasis in vivo, demonstrating functional significance of CDR1as silencing. Surprisingly, CDR1as depletion had no clear effect on miR-7 activity in melanoma cells, and miR-7 inhibition was insufficient to rescue CDR1as silencing-induced invasion. Moreover, GSEA analyses of proteomic profiling of melanoma cells depleted of CDR1as revealed reductions of proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial function, suggesting CDR1as loss may alter metabolism of melanoma cells. Mining of CLIP-Seq data sets and subsequent RIP-PCR revealed direct interactions of CDR1as with the IGF2BP family of proteins and TARDBP, each of which are involved in regulation of mitochondrial function. These data suggest that CDR1as could regulate the subcellular localization and/or function of proteins involved in OXPHOS and mitochondrial respiration. To understand the upstream mechanism driving CDR1as loss in melanoma, we sought to examine the primary transcript from which CDR1as arises. Interestingly, examination of the CDR1as locus revealed an upstream long non-coding RNA, LINC00632, as a plausible primary transcript of CDR1as. CDR1as and LINC00632 expression is strikingly correlated across human tissues and in melanoma cell lines (r>.65). Moreover, depletion of LINC00632 using GapmeRs reduced expression of CDR1as, suggesting they arise from the same precursor transcript. Intriguingly, from data mining of ChIP-Seq datasets, we observed that the PRC2-dependent repressive chromatin mark H3K27me3, is abundant in the genomic locus of LINC00632 in melanoma cells. We validated the presence of H3K27me3 at LINC00632 by ChIP-PCR in melanoma cells lacking CDR1as expression. Strikingly, treatment of such melanoma cell lines with the EZH2 inhibitor, GSK126, induced robust re-expression of both CDR1as and LINC00632, and removal of H3K27me3 at LINC00632. Our data document that CDR1as is epigenetically silenced in melanoma, and its loss promotes melanoma invasion and metastasis. Moreover, CDR1as loss could contribute to metabolic adaptations during metastasis through misregulation of RNA binding proteins. Citation Format: Doug Hanniford, Rana Moubarak, Jochen Imig, Alejandro Ulloa, Beatriz Sanchez Sendra, Alcida Karz, Iman Osman, Ioannis Aifantis, Eva Hernando. PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis [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 3047. doi:10.1158/1538-7445.AM2017-3047

Naringin suppresses the metabolism of A375 cells by inhibiting the phosphorylation of c-Src

Elevation of glycolysis, increase in lactic acid production, and enhancement of mitochondrial biogenesis are all the changes of energy metabolism of melanoma cells. Melanoma cells' metabolism and energy production networks play an important role in cancer proliferation, survival, motility, invasiveness, metastasis, and angiogenesis. Since the Warburg theory was put forward in the 1930s, more researchers focus on finding new ways for effectively eliminating cancer cells by targeting their energy metabolism. In this study, we found naringin has the inhibitory effects on the glucose metabolism of A375 cells, a melanoma cell line, in a concentration-dependent manner. We also found that naringin could significantly reduce the phosphorylation of c-Src. In summary, we demonstrated that naringin inhibits the malignant phenotype of A375 cells by suppressing c-Src and its downstream signaling pathway. More importantly, we provide the novel mechanism that, as a natural inhibitor of c-Src, naringin could be an effective candidate for the treatment of melanoma.

Impact of hyperpigmentation on superoxide flux and melanoma cell metabolism at mitochondrial complex II.

Melanogenesis is a highly conserved process of cytophotoprotection from UV radiation present in many species. Although both mitochondrial function and UV radiation insults are well-documented promoters of increased cellular stress, their individual molecular relationships with skin pigmentation have not been clearly resolved. This study provides evidence for a direct relationship between cellular melanin content, superoxide flux, and mitochondrial function at complex II. Direct and significant correlation between increased pigmentation and complex II turnover was observed in genetically different melanoma cell lines of varied basal pigmentation states (P < 0.01). The same trend was also observed when comparing genetically identical cell cultures with increasing levels of induced pigmentation (P < 0.005). The observation of increased steady-state levels of the catalytic complex II succinate dehydrogenase subunit A alongside hyperpigmentation suggested coregulation of activity and pigment production (P < 0.01). The study also presents novel evidence for a relationship between hyperpigmentation and increased superoxide-generating capacity at complex II. By amperometrically monitoring superoxide flux from differently pigmented FM55 melanocytes and their isolated mitochondria, a dynamic and responsive relationship between pigmentation, complex II function, and intracellular superoxide generation was observed (P < 0.005). The data support hyperpigmentation as a protective antioxidant mechanism in response to complex II-mediated reactive oxygen species generation.

Glutathione efflux associated with a low γ-glutamyl transpeptidase activity in human melanoma cells

The cellular concentration of reduced glutathione (GSH) modulates the sensitivity of human melanoma cells to alkylating drugs in vitro. To investigate whether the membrane-associated enzyme gamma-glutamyl transpeptidase (gamma-GTP) involved in GSH breakdown was expressed in melanoma cells, the enzymatic activity of gamma-GTP as well as the secretion of GSH were measured in human melanoma cells from four different cell lines (Me8, JUSO, GLL19, Swift). All the cells showed low gamma-GTP activities (0-1 mU/mg protein) and released GSH in culture supernatants at significant rates. After incubation for 24 h in growth medium containing 0.1 mmol/L cystine, the levels of GSH in supernatants ranged from 56 to 111 nmol GSH/mg protein. The GSH metabolism of melanoma cells was also evaluated by measuring the levels of the melanogenesis intermediate 5-S-cysteinyldopa under different experimental conditions. The results of these experiments suggest that melanoma cells have a low ability to metabolize the tripeptide GSH, which appears to be responsible for GSH secretion and accumulation in culture supernatants.