Extracellular acidity strengthens mesenchymal stem cells to promote melanoma progression

Mesenchymal stem cells (MSC) participate to tumor stroma development and several evidence suggests that they play a role in facilitating cancer progression. Because melanoma often shows extracellular pH low enough to influence host cell as tumor cell behavior, the aim of this study is to elucidate whether acidity affects cross talk between MSC and melanoma cells to disclose new liaisons promoting melanoma progression, and to offer new therapeutic opportunities. We found that MSC grown in a low pH medium (LpH-MSC) stimulate melanoma xenografts more than MSC grown in a standard pH medium. LpH-MSC express a higher level of TGFβ that is instrumental of epithelial-to-mesenchymal transition (EMT)-like phenotype induction in melanoma cells. LpH-MSC profile also shows a switching to an oxidative phosphorylation metabolism that was accompanied by a forced glycolytic pathway of melanoma cells grown in LpH-MSC-conditioned medium. Metformin, an inhibitor of mitochondrial respiratory chain was able to reconvert oxidative metabolism and abrogate TGFβ expression in LpH-MSC. In addition, esomeprazole, a proton pump inhibitor activated in acidosis, blocked TGFβ expression in LpH-MSC through the downregulation of IkB. Both agents, metformin and esomeprazole, inhibited EMT profile in melanoma cells grown in LpH-MSC medium, and reduced glycolytic markers. Thus, acidosis of tumor microenvironment potentiates the pro-tumoral activity of MSC and orchestrates for a new potential symbiosis, which could be target to limit melanoma progression.     

Metabolic strategies of melanoma cells: Mechanisms,interactions with the tumor microenvironment,and therapeutic implications

Melanomas are metabolically heterogeneous, and they are able to adapt in order to utilize a variety of fuels that facilitate tumor progression and metastasis. The significance of metabolism in melanoma is supported by growing evidence of impact on the efficacy of contemporary therapies for this disease. There are also data to support that the metabolic phenotypes of melanoma cells depend upon contributions from both intrinsic oncogenic pathways and extrinsic factors in the tumor microenvironment. This review summarizes current understanding of the metabolic processes that promote cutaneous melanoma tumorigenesis and progression, the regulation of cancer cell metabolism by the tumor microenvironment, and the impact of metabolic pathways on targeted and immune therapies.     

Rewiring of purine metabolism in response to acidosis stress in glioma stem cells

Glioma stem cells (GSCs) contribute to therapy resistance and poor outcomes for glioma patients. A significant feature of GSCs is their ability to grow in an acidic microenvironment. However, the mechanism underlying the rewiring of their metabolism in low pH remains elusive. Here, using metabolomics and metabolic flux approaches, we cultured GSCs at pH 6.8 and pH 7.4 and found that cells cultured in low pH exhibited increased de novo purine nucleotide biosynthesis activity. The overexpression of glucose-6-phosphate dehydrogenase, encoded by G6PD or H6PD, supports the metabolic dependency of GSCs on nucleotides when cultured under acidic conditions, by enhancing the pentose phosphate pathway (PPP). The high level of reduced glutathione (GSH) under acidic conditions also causes demand for the PPP to provide NADPH. Taken together, upregulation of G6PD/H6PD in the PPP plays an important role in acidic-driven purine metabolic reprogramming and confers a predilection toward glioma progression. Our findings indicate that targeting G6PD/H6PD, which are closely related to glioma patient survival, may serve as a promising therapeutic target for improved glioblastoma therapeutics. An integrated metabolomics and metabolic flux analysis, as well as considering microenvironment and cancer stem cells, provide a precise insight into understanding cancer metabolic reprogramming.     

Ret transgenic mouse model of spontaneous skin melanoma: focus on regulatory T cells

Ret transgenic mouse model of skin malignant melanoma is characterized by the overexpression of the human ret transgene in melanin‐containing cells. Transgenic mice spontaneously develop skin tumors with metastases in lymph nodes, lungs, liver, brain, and the bone marrow. Tumor lesions show typical melanoma morphology and express melanoma‐associated antigens. Although transgenic mice demonstrate an accumulation of melanoma antigen‐specific memory and effector T cells, their anti‐tumor effects could be blocked by highly immunosuppressive leukocytes enriched in the tumor microenvironment and in the periphery. Here, we discuss the role of one of the most potent immunosuppressive subset, regulatory T cells, in the melanoma progression in this model.     

Metformin protects PC12 cells and hippocampal neurons from H2O 2-induced oxidative damage through activation of AMPK pathway

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H₂O₂-induced cell death. Metformin significantly ameliorated cell death due to H₂O₂ insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H₂O₂. Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H₂O₂. Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.     

Loss of XRCC1 confers a metastatic phenotype to melanoma cells and is associated with poor survival in patients with melanoma

Ultraviolet (UV) radiation‐induced DNA damage and genomic instability is one of the leading causes for melanoma. X‐ray repair cross‐complementary protein 1, XRCC1, plays a critically important role in base excision repair pathway. This study was therefore performed to analyze the correlation between XRCC1 expression, melanoma progression, and patient survival. Using a tissue microarray with a total of 119 patients with melanoma, we demonstrate that loss of XRCC1 expression is associated with the progression of disease from dysplastic nevi to primary melanoma and to metastatic melanoma. We found that the loss of XRCC1 was correlated with the progression of melanoma from AJCC stage II to stage III and with worse overall and disease‐specific 5‐yr and 10‐yr survival of patients with melanoma. Furthermore, we also illustrate the inhibitory effect of XRCC1 on melanoma cell invasion and migration, which are the regulatory events in melanoma metastasis.     

Growth factor modulation of melanoma growth stimulatory activity mRNA expression in human malignant melanoma cells correlates with cell growth

This report demonstrates that the expression of melanoma growth stimulatory activity (MGSA) mRNA can be modulated in a positive fashion in the Hs294T human melanoma cell line by PDGF and MGSA. There is close correlation between MGSA expression and the pattern of cell growth in Hs294T cells.     

Adhesion of lactic acid bacteria to caco-2 cells and their effect on cytokine secretion

Cytokines secreted by human enterocytes play a critical role in mucosal and systemic immunity. Intestinal microorganisms can influence this secretion. In the present study, 30 strains of lactic acid bacteria were characterized for their adhesion to Caco-2 cells and their potential to stimulate proinflammatory cytokine secretion by this cell line. The bacteria adhered in a strain-dependent manner to Caco-2 cells. Contact with lactobacilli did not result in the production of IL-6 or IL-8. A slight IL-6 and IL-8 production by a Caco-2 cell was detected after exposure to 8 of the tested Bifidobacterium strains. No correlation was found between adhesion and cytokine induction among the bacteria tested. This indicates that lactic acid bacteria, even those with strong adhesive properties, are not very likely to trigger an inflammatory response in human enterocytes.     

Selectin ligands on human melanoma cells

Twelve established human melanoma lines were screened for surface expression of the carbohydrate antigens Lewis^a (Le^a), sialyl Lewis^a (SLe^a), dimeric sialyl Lewis^a (diSLe^a), sialyl Lewis^X (SLe^X) and dimeric sialyl Lewis^X (diSLe^X). None of the lines expressed SLe^X, but 11/12 were positive for diSLe^X and 7/12 were positive for SLe^a. Although both diSLe^X and SLe^a have been reported to bind to E-selectin, none of the melanoma lines exhibited E-selectin-dependent adhesion to activated human umbilical vein endothelial cells (HUVECs). Three melanoma lines infected with a retroviral vector carrying the cDNA for the human Lewis fucosyltransferase (FucT-III) subsequently expressed SLe^X at their cell surface and exhibited E-selectin-dependent adhesion to activated HUVECs. Treatment of these transduced cells with inhibitors of O-linked or N-linked protein glycosylation significantly inhibited E-selectin-meiiated adhesion, though fluorescence-activated cell sorter analysis indicated no decrease in cell surface expression of SLe^X, SLe^a or diSLe^X. This suggests that the majority of SLe^X/SLe^a-type glycans endogenously procuded by human melanoma cells are not protein-associated and do not mediate E-selectin-dependent adhesion. These results support the hypothesis that E-selectin-dependent adhesion requires presentation of SLe^X-type moieties on appropriate glycoproteins.     

Preparation of a monoclonal antibody to a melanoma growth-stimulatory activity released into serum-free culture medium by Hs0294 malignant melanoma cells

Autostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid-soluble, heat-stable, trypsin-sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse-phase high-performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth-stimulatory activity (MGSA) resides in a 16-Kd moiety, though bioactivity is also associated with 24-26 and less than 14-Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H-thymidine incorporation in serum-free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in serum-free medium in a dose-dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum-free medium or medium containing 0.8% calf serum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse-phase high-performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16-Kd and approximately 13-14-Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the approximately 13-16-Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors.     

MX 2 is a novel regulator of cell cycle in melanoma cells

MX2 protein is a dynamin‐like GTPase2 that has recently been identified as an interferon‐induced restriction factor of HIV‐1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome‐wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context‐dependent.     

Effect of serum withdrawal on the proliferation of B16F10 melanoma cells

B16F10 murine melanoma cell proliferation was inhibited after 48 h in medium with serum in the range 0.1 to 0.5% by volume. Cell viability was mostly retained, whereas cells completely deprived of serum died. Growth-arrested cultures showed serum-dependent suppression of DNA synthesis. The response was typically that of a 'cell cycle freeze', verified by flow cytometric distribution of cells. Consequently, serum deprivation did not lead to synchrony when serum was restored to arrested populations. Furthermore, there was no change in PCNA expression in arrested cells.     

Effects of trace metals on mouse B16 melanoma cells in culture

The effects of fourteen metal ions (As³⁺, As⁵⁺, Cd²⁺, Co²⁺, Cr³⁺, Cr⁶⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Se⁴⁺, V⁵⁺, VO²⁺) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10⁻⁴ M, metal ions such as As³⁺, As⁵⁺, Cd²⁺, Cr⁶⁺, Se⁴⁺, V⁵⁺, VO²⁺, and, to a minor extent, Li⁺, Hg²⁺, and Co²⁺ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg²⁺>Cr⁶⁺=Cd²⁺>As³⁺, As⁵⁺>V⁵⁺, VO²⁺>Se⁴⁺=Ni²⁺=Co²⁺=Li⁺. An increased synthesis of melanin in B16 cells was noted after incubation with Co²⁺, Ni²⁺, Cd²⁺, and Li⁺, whereas Se⁴⁺ had, on the contrary, an inhibiting effect on melanogenesis.     

Metabolic rewiring in cancer cells overexpressing the glucocorticoid-induced leucine zipper protein (GILZ): Activation of mitochondrial oxidative phosphorylation and sensitization to oxidative cell death induced by mitochondrial targeted drugs

Cancer cell metabolism is largely controlled by oncogenic signals and nutrient availability. Here, we highlighted that the glucocorticoid-induced leucine zipper (GILZ), an intracellular protein influencing many signaling pathways, reprograms cancer cell metabolism to promote proliferation. We provided evidence that GILZ overexpression induced a significant increase of mitochondrial oxidative phosphorylation as evidenced by the augmentation in basal respiration, ATP-linked respiration as well as respiratory capacity. Pharmacological inhibition of glucose, glutamine and fatty acid oxidation reduced the activation of GILZ-induced mitochondrial oxidative phosphorylation. At glycolysis level, GILZ-overexpressing cells enhanced the expression of glucose transporters in their plasmatic membrane and showed higher glycolytic reserve. ¹H NMR metabolites quantification showed an up-regulation of amino acid biosynthesis. The GILZ-induced metabolic reprograming is present in various cancer cell lines regardless of their driver mutations status and is associated with higher proliferation rates persisting under metabolic stress conditions. Interestingly, high levels of OXPHOS made GILZ-overexpressing cells vulnerable to cell death induced by mitochondrial pro-oxidants. Altogether, these data indicate that GILZ reprograms cancer metabolism towards mitochondrial OXPHOS and sensitizes cancer cells to mitochondria-targeted drugs with pro-oxidant activities.     

Sestrin2 integrates Akt and mTOR signaling to protect cells against energetic stress-induced death

The phosphoinositide-3 kinase/Akt (PI3K/Akt) pathway has a central role in cancer cell metabolism and proliferation. More importantly, it is one of the cardinal pro-survival pathways mediating resistance to apoptosis. The role of Akt in response to an energetic stress is presently unclear. Here, we show that Sestrin2 (Sesn2), also known as Hi95, a p53 target gene that protects cells against oxidative and genotoxic stresses, participates in the protective role of Akt in response to an energetic stress induced by 2-deoxyglucose (2-DG). Sesn2 is upregulated in response to an energetic stress such as 2-DG and metformin, and mediates the inhibition of mammalian target of rapamycin (mTOR), the major cellular regulator of energy metabolism. The increase of Sesn2 is independent of p53 but requires the anti-apoptotic pathway, PI3K/Akt. Inhibition of Akt, as well as loss of Sesn2, sensitizes cells to 2-DG-induced apoptosis. In addition, the rescue of Sesn2 partially reverses the pro-apoptotic effects of 2-DG. In conclusion, we identify Sesn2 as a new energetic stress sensor, which appears to be protective against energetic stress-induced apoptosis that integrates the pro-survival function of Akt and the negative regulation of mTOR.     

Plasmodium falciparum: effect of time in continuous culture on binding to human endothelial cells and amelanotic melanoma cells

An in vitro correlate of the binding in vivo of Plasmodium falciparum-infected erythrocytes to capillary and venular endothelium, using cultured human endothelial cells and amelanotic melanoma cells, was previously developed. The effects of different times in continuous culture on binding of erythrocytes infected with nine different isolates of P. falciparum is now reported. Four isolates, which bound at the time they were first tested, rapidly lost the ability to bind after 26-43 days in culture. One of these, the Cameroun isolate, tested 12 h after the blood was obtained from the patient, had the highest rate of binding of all isolates (680 infected erythrocytes per 100 melanoma cells). After 37 days in culture, only 18 infected erythrocytes per 100 melanoma cells bound. Three isolates first tested after 30-62 days in culture bound poorly. In contrast, two others, the Vietnam (VI) and Brazil (It), continued to bind during the period of study. The Brazil (It) isolate studied after 43 days in culture bound 505 infected erythrocytes per 100 melanoma cells; its clone ItG2G1 continued to bind equally well after 400 days in culture. The ultrastructural morphology of knobs on the binding and nonbinding infected erythrocytes were indistinguishable. Since evidence from other studies indicates that knobs are necessary for binding to endothelium, it is proposed that some parasites in continuous culture may not express the molecules responsible for binding, although the morphologic knobs are still present.     

Intracellular oxidation of methyl p-coumarate is involved in anti-melanogenic and cytotoxic activities against melanoma cells

Tyrosinase-catalyzed L-tyrosine oxidation is a key step in melanogenesis, and intense melanin formation is often a problem in chemotherapies or food preservation. Methyl p-coumarate is isolated from fresh flower of medicinal plant, Trixis michuacana var longifolia (D. Dow) C., and it suppressed melanogenesis in cultured murine B16-F10 melanoma cells while p-coumaric acid did not show anti-melanogenic activity. Methyl p-coumarate exhibited cytotoxicity with an IC₅₀ of 130 μM (23.2 µg/mL), and p-coumaric acid showed similar activity, but to a lesser extent, suggesting that the anti-melanogenic activity of methyl p-coumarate is at least due to the melanocytotoxicity. This cytotoxicity of methyl p-coumarate was reduced in a dose-dependent manner by ascorbic acid but not with butylated hydroxyanisole. Moreover, methyl 4-methoxycinnamate, which lacks the oxidizable phenolic hydroxyl group, still exhibited comparable cytotoxicity to methyl p-coumarate. In addition, anethole did not show noticeable cytotoxicity, indicating that the enone moiety is an essential element in eliciting melanocytotoxicity. Thus, the enone moiety in methyl p-coumarate is a biologically critical nucleophilic group as a Michael reaction acceptor contributing to the anti-melanogenic activity and cytotoxicity against melanoma cells.