bafilomycin-a1 and Melanoma

Melanoma incidence increases every year worldwide and is responsible for 80% of skin cancer deaths. Due to its metastatic potential and resistance to almost any treatments such as chemo, radio, immune and targeted-therapy, the patients still have a poor prognosis, especially at metastatic stage. Considering that, it is crucial to find new therapeutic approaches to overcome melanoma resistance. Here we investigated the effect of cisplatin (CDDP), one of the chemotherapeutic agents used for melanoma treatment, in association with nutritional deprivation in murine melanoma cell lines. Cell death and autophagy were evaluated after the treatment with cisplatin, nutritional deprivation and its association using an in vitro model of murine melanocytes malignant transformation to metastatic melanoma. Our results showed that nutritional deprivation augmented cell death induced by cisplatin in melanoma cells, especially at the metastatic subtype, with slight effects on melanocytes. Mechanistic studies revealed that although autophagy was present at high levels in basal conditions in melanoma cells, was not essential for cell death process that involved mitochondrial damage, reactive oxygen species production and possible glycolysis inhibition. In conclusion, nutritional shortage in combination with chemotherapeutic drugs as cisplatin can be a valuable new therapeutic strategy to overcome melanoma resistance.

Topics: Animals; Apoptosis; Autophagy; Autophagy-Related Protein 7; Cell Line; Cisplatin; Glucose; Macrolides; Melanocytes; Melanoma; Membrane Potentials; Mice; Microscopy, Fluorescence; Microtubule-Associated Proteins; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering

Autophagy can function in a dual role in cancer development and progression: It can be cytoprotective or contribute to cell death. Therefore, determining the contextual role of autophagy between these two opposing effects is important. So far, little is known about the role of autophagy in uveal melanoma. In the present study, we looked to investigate the autophagic process, as well as its effect on cell survival in uveal melanoma cell lines under stressed conditions (starvation). The possible role of autophagy during BRAF inhibition in uveal melanoma was also sought.. Two human uveal melanoma cell lines, OCM1A, which harbors the BRAF mutation V600E and Mel 290, which is BRAF wild type, were studied. Autophagy levels were determined by Western blot assay with/without the addition of autophagic flux inhibitor (bafilomycin A1). Cell proliferation was assessed by an MTT assay.. Starvation triggered autophagy in BRAF V600E-mutant OCM1A cells but not in BRAF wild-type Mel 290 cells. Enhanced autophagy helped the OCM1A cells survive under stressed conditions. The BRAF inhibitor vemurafenib upregulated autophagy through suppression of the PI3K/Akt/mTOR/p70S6 K pathway in BRAF V600E-mutant uveal melanoma cells. Autophagy inhibition impaired the treatment efficacy of vemurafenib in BRAF V600E-mutant uveal melanoma cells.. Our data demonstrate that starvation-trigged autophagy, which is BRAF V600E dependent, promotes cancer cell survival in uveal melanoma. Vemurafenib induces autophagic cell death rather than adaptive cell survival in BRAF V600E-mutant melanoma.

Topics: Autophagy; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Humans; Indoles; Macrolides; Melanoma; Mutation; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Uveal Neoplasms; Vemurafenib

Targeting the sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) signalling axis is emerging as a promising strategy in the treatment of cancer. However, the effect of such an approach on survival of human melanoma cells remains less understood. Here, we show that the sphingosine analogue FTY720 that functionally antagonises S1PRs kills human melanoma cells through a mechanism involving the vacuolar H(+) -ATPase activity. Moreover, we demonstrate that FTY720-triggered cell death is characterized by features of necrosis and is not dependent on receptor-interacting protein kinase 1 or lysosome cathepsins, nor was it associated with the activation of protein phosphatase 2A. Instead, it is mediated by increased production of reactive oxygen species and is antagonized by activation of autophagy. Collectively, these results suggest that FTY720 and its analogues are promising candidates for further development as new therapeutic agents in the treatment of melanoma.

Topics: Adenosine Triphosphate; Autophagy; Cell Death; Cell Line, Tumor; Cell Membrane; Dimethyl Sulfoxide; Fingolimod Hydrochloride; Humans; Macrolides; Melanoma; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Protein Phosphatase 2; Reactive Oxygen Species; Sphingosine; Vacuolar Proton-Translocating ATPases

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a key enzyme in normal development and malignant processes. The regulation of MT1-MMP activity on the cell surface is a complex process involving autocatalytic processing, tissue inhibitor of MMPs (TIMP) binding and constitutive internalization. However, the fate of internalized MT1-MMP is not known. Acidification of intracellular vacuolar compartments is essential for membrane trafficking, protein sorting and degradation. This acidification is controlled by vacuolar H(+)-ATPases, which can be selectively inhibited by bafilomycin-A(1). Here, we treated human tumour cell lines expressing MT1-MMP with bafilomycin-A(1), and analysed its effects on MT1-MMP activity, internalization and processing. We show that the activity of MT1-MMP on the cell surface is constitutively down-regulated through a vacuolar H(+)-ATPase-dependent degradation process. Blockade of this degradation caused the accumulation of TIMP-free active MT1-MMP molecules on the cell surface, although internalization was not affected. As a consequence of this impaired degradation, pro-MMP-2 activation was strongly enhanced. This study demonstrates that the catalytic activity of MT1-MMP on the cell surface is regulated through a vacuolar H(+)-ATPase-dependent degradation process.

Topics: Anti-Bacterial Agents; Enzyme Activation; Enzyme Inhibitors; Humans; Macrolides; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Melanoma; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinase-2; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Vacuolar Proton-Translocating ATPases

In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum. Based on studies described here, we propose that aberrant vacuolar proton ATPase (V-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway. Amelanotic but not melanotic melanoma cells or normal melanocytes display elevated proton export as observed by the acidification of the extracellular medium and their ability to maintain neutral intracellular pH. Tyrosinase activity and transit through the Golgi were restored by either maintaining the melanoma cells in alkaline medium (pH 7.4-7.7) or by restricting glucose uptake. The translocation of tyrosinase out of the endoplasmic reticulum and the induction of cell pigmentation in the presence of the ionophore monensin or the specific V-ATPase inhibitors concanamycin A and bafilomycin A1 supported a role for V-ATPases in this process. Because it was previously shown that V-ATPase activity is increased in solid tumors in response to an acidified environment, the appearance of hypopigmented cells in tyrosinase-positive melanoma tumors may indicate the onset of enhanced glycolysis and extracellular acidification, conditions known to favor metastatic spread and resistance to weak base chemotherapeutic drugs.

Topics: Acids; Anti-Bacterial Agents; Cells, Cultured; Endoplasmic Reticulum; Enzyme Inhibitors; Glucose; Golgi Apparatus; Humans; Hydrogen-Ion Concentration; Macrolides; Melanoma; Monophenol Monooxygenase; Tumor Cells, Cultured; Vacuolar Proton-Translocating ATPases

In this study, we describe the activation of melanogenesis by selective vacuolar type H(+)-ATPase inhibitors (bafilomycin A1 and concanamycin A) in amelanotic human and mouse melanoma cells which express tyrosinase but show no melanogenesis. Addition of the inhibitors activated tyrosinase within 4 h, and by 24 h the cells contained measurable amounts of melanin. These effects were not inhibited by cycloheximide (2 microgram/ml) which is consistent with a post-translational mechanism of activation. Our findings suggest that melanosomal pH could be an important and dynamic factor in the control of melanogenesis in mammalian cells.

Topics: Animals; Anti-Bacterial Agents; Cycloheximide; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Macrolides; Melanins; Melanoma; Melanosomes; Mice; Monophenol Monooxygenase; Protein Processing, Post-Translational; Proton-Translocating ATPases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tumor Cells, Cultured; Vacuolar Proton-Translocating ATPases