interleukin-8 and Melanoma

Cutaneous melanoma accounts for only about 7% of skin cancers but is causing almost 90% of deaths. Melanoma cells have a distinct repertoire of mutations from other cancers, a high plasticity and degree of mimicry toward vascular phenotype, stemness markers, versatility in evading and suppress host immune control. They exert a significant influence on immune, endothelial and various stromal cells which form tumor microenvironment. The metastatic stage, the leading cause of mortality in this neoplasm, is the outcome of a complex, still poorly understood, cross-talk between tumor and other cell phenotypes. There is accumulating evidence that Interleukin-8 (IL-8) is emblematic for advanced melanomas. This work aimed to present an updated status of IL-8 in melanoma tumor cellular complexity, through a comprehensive analysis including data from other chemokines and neoplasms. The multiple processes and mechanisms surveyed here demonstrate that IL-8 operates following orchestrated programs within signaling webs in melanoma, stromal and vascular cells. Importantly, the yet unknown molecularity regulating IL-8 impact on cells of the immune system could be exploited to overturn tumor fate. The molecular and cellular targets of IL-8 should be brought into the attention of even more intense scientific exploration and valorization in the therapeutical management of melanoma.

Topics: Chemokines; Disease Progression; Humans; Interleukin-8; Melanoma; Prognosis; Tumor Microenvironment

The incidence of melanoma is rising at an alarming rate and we are still awaiting an effective treatment for this malignancy. In its early stage, melanoma can be cured by surgical removal, but once metastasis has occurred there is no effective treatment. Recent findings have suggested multiple functional implications of CXCL8 and its cognate receptors, CXCR1 and CXCR2, in melanoma pathogenesis, thus underscoring their importance as targets for cancer therapy. This review provides an update on the roles of CXCL8 and its receptors in melanoma progression and metastasis.

Topics: Animals; Disease Progression; Humans; Interleukin-8; Melanoma; Neoplasm Metastasis; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Skin Neoplasms

The incidence of melanoma has been steadily increasing over the last 3 decades. Currently, there are several approved treatments for metastatic melanoma, including chemotherapy and biologic therapy as both single treatments and in combination, but none is associated with a significant increase in survival. The chemotherapeutic agent dacarbazine is the standard treatment for metastatic melanoma, with a response rate of 15-20%, although most responses are not sustained. One of the main problems with melanoma treatment is chemotherapeutic resistance. The mechanisms of resistance of melanoma cells to chemotherapy have yet to be elucidated. Following treatment with dacarbazine, melanoma cells activate the extracellular signal-regulated kinase pathway, which results in over-expression and secretion of interleukin (IL)-8 and vascular endothelial growth factor. Melanoma cells utilize this mechanism to escape from the cytotoxic effect of the drug. We have previously reported on the development of fully human neutralizing antibodies against IL-8 (anti-IL-8-monoclonal-antibody [ABX-IL8]). In preclinical studies, ABX-IL8 inhibited tumor growth, angiogenesis, and metastasis of human melanoma in vivo. We propose that combination treatment with dacarbazine and IL-8 will potentiate the cytotoxic effect of the drug. Furthermore, formation of metastasis is a multistep process that includes melanoma cell adhesion to endothelial cells. Melanoma cell adhesion molecule (MUC18) mediates these processes in melanoma and is therefore a good target for eliminating metastasis. We have developed a fully human antibody against MUC18 that has shown promising results in preclinical studies. Since resistance is one of the major obstacles in the treatment of melanoma, we propose that utilization of antibodies against IL-8 or MUC18 alone, or as part of a 'cocktail' in combination with dacarbazine, may be a new treatment modality for metastatic melanoma that overcomes resistance of the disease to chemotherapy and significantly improves survival of patients.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; CD146 Antigen; Dacarbazine; Disease Progression; Drug Resistance, Neoplasm; Humans; Immunotherapy; Interleukin-8; Melanoma; Neoplasm Metastasis; Skin Neoplasms

Metastatic melanoma is associated with high rate of patients' mortality and represents a great challenge for cancer therapies because of its notorious resistance to chemotherapeutic drugs. Considerable efforts have been made over the last 2 decades in pursuit of new treatment modalities and identification of molecular events associated with melanoma progression and development of metastases. The acquisition of the metastatic phenotype is associated with overexpression of the adhesion molecule MCAM/MUC18 and the angiogenic factor IL-8. In this review, we summarize our current knowledge on MCAM/MUC18 and IL-8, their transcriptional regulation, and their role in melanoma growth, angiogenesis and metastasis. Further, we report on the development of new fully human antibodies, anti-MCAM/MUC18 (ABX-MA1) and anti-IL-8 (ABX-IL8), and their effects on tumor growth and metastasis in animal models. Collectively, our studies suggest that ABX-MA1 and ABX-IL8 could serve as new modalities for the treatment of melanoma either alone, or in combination with conventional chemotherapy or other antitumor agents.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; CD146 Antigen; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Interleukin-8; Melanoma

This review article has described briefly studies supporting the concept that IL-8 expression and its regulation by inflammatory cytokines like IL-1 may play an important role in controlling the phenotypes associated with melanoma progression and metastasis. It is clear from the experiments presented here that IL-8 is an important autocrine multifunctional cytokine that modulates melanoma/cell proliferation, migration by induction of extracellular matrix degradation enzymes and induces neovascularization, all of which are critical for melanoma growth and metastasis. In addition, their expression in melanoma tumor specimens suggests an association between IL-8 expression and tumor aggressiveness. Further, inflammatory cytokines produced by either tumor cells or stromal cells may regulate IL-8 expression, which can control melanoma growth and enhance our current knowledge regarding melanoma progression and metastasis. Understanding these events and their significance will allow us to design novel therapeutic approaches for treatment of melanoma.

Topics: Animals; Humans; Interleukin-8; Melanoma; Neoplasm Metastasis

Expression of interleukin-8 (IL-8) by human melanoma cells correlates with their metastatic potential in vivo. Moreover, UVB irradiation of primary cutaneous melanoma induces IL-8 mRNA and protein production and increases both tumor growth and metastasis in nude mice. Although IL-8 has been shown to be an angiogenic factor, the biological consequences of increased IL-8 production by melanoma cells and the role of IL-8 in the metastatic process remains unclear. The purpose of this review is to determine the role of IL-8 in tumor growth and metastasis of human melanoma. Transfection of nonmetastatic and IL-8-negative melanoma cells with the IL-8 gene rendered them highly tumorigenic and increased their metastatic potential in nude mice. The IL-8-transfected cells displayed upregulation of MMP-2 expression and activity and increased invasiveness through Matrigel-coated filters. Activation of MMP-2 by IL-8 can enhance the invasion of host stroma by the tumor cells and increase angiogenesis and, hence, metastasis. In addition to UVB, IL-8 can also be upregulated by hypoxia conditions, suggesting that the environment plays a major role in regulating IL-8 expression and metastasis. The studies summarized in this review suggest that in melanoma, IL-8 may serve as the angiogenic factor distinguishing benign from malignant cells.

Topics: Animals; Cell Hypoxia; Disease Progression; Gelatinases; Humans; Interleukin-8; Matrix Metalloproteinase 2; Melanoma; Metalloendopeptidases; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms, Radiation-Induced; Neovascularization, Pathologic; Skin; Skin Neoplasms; Transfection; Ultraviolet Rays

Topics: Animals; Antigens, Neoplasm; Biomarkers; Cell Adhesion; Cytokines; DNA, Neoplasm; Genes, p53; Humans; Integrins; Interleukin-10; Interleukin-8; Melanoma; Neoplasm Metastasis; Oncogenes; Vaccination

Anti-programmed cell death protein 1 (PD-1) therapy provides long-term clinical benefits to patients with advanced melanoma. The composition of the gut microbiota correlates with anti-PD-1 efficacy in preclinical models and cancer patients. To investigate whether resistance to anti-PD-1 can be overcome by changing the gut microbiota, this clinical trial evaluated the safety and efficacy of responder-derived fecal microbiota transplantation (FMT) together with anti-PD-1 in patients with PD-1-refractory melanoma. This combination was well tolerated, provided clinical benefit in 6 of 15 patients, and induced rapid and durable microbiota perturbation. Responders exhibited increased abundance of taxa that were previously shown to be associated with response to anti-PD-1, increased CD8

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Immunological; CD8-Positive T-Lymphocytes; Drug Resistance, Neoplasm; Fecal Microbiota Transplantation; Gastrointestinal Microbiome; Humans; Interleukin-8; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Melanoma; Myeloid Cells; Programmed Cell Death 1 Receptor; Skin Neoplasms; Tumor Microenvironment

Purpose Interleukin-10 (IL-10) stimulates the expansion and cytotoxicity of tumor-infiltrating CD8+ T cells and inhibits inflammatory CD4+ T cells. Pegylation prolongs the serum concentration of IL-10 without changing the immunologic profile. This phase I study sought to determine the safety and antitumor activity of AM0010. Patients and Methods Patients with selected advanced solid tumors were treated with AM0010 in a dose-escalation study, which was followed by a renal cell cancer (RCC) dose-expansion cohort. AM0010 was self-administered subcutaneously at doses of 1 to 40 μg/kg once per day. Primary end points were safety and tolerability; clinical activity and immune activation were secondary end points. Results In the dose-escalation and -expansion cohorts, 33 and 18 patients, respectively, were treated with daily subcutaneous injection of AM0010. AM0010 was tolerated in a heavily pretreated patient population. Treatment-related adverse events (AEs) included anemia, fatigue, thrombocytopenia, fever, and injection site reactions. Grade 3 to 4 nonhematopoietic treatment-related AEs, including rash (n = 2) and transaminitis (n = 1), were observed in five of 33 patients. Grade 3 to 4 anemia or thrombocytopenia was observed in five patients. Most treatment-related AEs were transient or reversible. AM0010 led to systemic immune activation with elevated immune-stimulatory cytokines and reduced transforming growth factor beta in the serum. Partial responses were observed in one patient with uveal melanoma and four of 15 evaluable patients with RCC treated at 20 μg/kg (overall response rate, 27%). Prolonged stable disease of at least 4 months was observed in four patients, including one with colorectal cancer with disease stabilization for 20 months. Conclusion AM0010 has an acceptable toxicity profile with early evidence of antitumor activity, particularly in RCC. These data support the further evaluation of AM0010 both alone and in combination with other immune therapies and chemotherapies.

Topics: Adult; Aged; Aged, 80 and over; Anemia; Carcinoma, Renal Cell; Cytokines; Drug Eruptions; Exanthema; Fatigue; Female; Fever; Humans; Injections, Subcutaneous; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukin-8; Kidney Neoplasms; Male; Melanoma; Middle Aged; Neoplasms; Polyethylene Glycols; Recombinant Proteins; Thrombocytopenia; Transforming Growth Factor beta; Uveal Neoplasms; Young Adult

Cytokines play a crucial role in the host's immune response. In melanoma patients, cytokine profiles seems to be related to the clinical course and their imbalance could be associated to tumour progression. Thus, we studied a panel of baseline cytokines and their behaviour during treatment in order to verify their correlation with clinical outcomes. Interleukin-6, interleukin-8, interleukin-10, interleukin-12 and soluble receptor of interleukin-2 were evaluated in 90 out of 176 metastatic melanoma patients enrolled in a phase III study comparing chemotherapy and biochemotherapy. We divided patients into three different groups according to their own cytokine levels (low, intermediate and high) and then we correlated these groups with some clinical features. We also monitored the cytokines during the treatment in a subgroup of 37 patients. In univariate analysis, higher values of interleukin-6 (P = 0.005), soluble receptor of interleukin-2 (P = 0.001) and interleukin-12 (P = 0.010) were correlated with a worse survival. Conversely, interleukin-8 was unable to discriminate patients with different prognoses, and interleukin-10 was undetectable in the majority of patients. In multivariate analysis, only soluble receptor of interleukin-2 maintained its independent role in survival. The impact of baseline cytokines on response was insignificant. Regarding the behaviours of cytokines during treatment, the most remarkable aspect was a progressive increase of interleukin-12 and soluble receptor of interleukin-2 in patients with a better survival. In our metastatic melanoma patients, higher basal levels of interleukin-6, interleukin-12 and soluble receptor of interleukin-2 were associated with a worse survival. In contrast, a progressive increase of interleukin-12 and soluble receptor of interleukin-2 was observed during treatment in patients with a better survival.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Cytokines; Dacarbazine; Female; Humans; Immunologic Factors; Interleukin-10; Interleukin-12; Interleukin-2; Interleukin-6; Interleukin-8; Male; Melanoma; Middle Aged; Receptors, Interleukin-6; Skin Neoplasms; Survival Analysis; Survival Rate; Vinblastine

Serum concentrations of angiogenic factors have been reported to correlate with tumour burden and prognosis in metastatic melanoma. The present study was performed to assess the value of angiogenic factors in serum in indicating response or failure to chemotherapy and immunochemotherapy in stage IV melanoma. Thirty-five patients suffering from stage IV melanoma according to the American Joint Committee on Cancer (AJCC) criteria were included in this prospective study. Before and following chemotherapy or immunochemotherapy, serum levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-AB), vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) were measured. Staging examinations following chemotherapy revealed 15 patients with response to therapy (complete response, partial response, stable disease), 14 patients with progressive disease and six patients with mixed response. Patients who responded to therapy showed a significant decrease in the serum level of IL-8 at the time of staging examinations, whereas patients with progressive disease did not. Following chemotherapy, serum concentrations of PDGF-AB had significantly decreased in both patients with response and patients with progressive disease. Comparing the VEGF and bFGF levels of responders and non-responders after a single administration of cytostatics showed significantly lower concentrations in patients with response to therapy. In all patients, a high intra- and inter-individual variability of serum values was observed during application of therapy. It can be concluded that low IL-8 serum levels after chemotherapy indicate response to chemotherapy in stage IV melanoma patients. The persistence of elevated serum levels of VEGF and bFGF following the initial cytostatic administration may help to identify patients resistant to chemotherapy. The distinct variability of serum levels indicates that processes other than tumour angiogenesis also influence the serum concentration of the examined angiogenic factors.

Topics: Adult; Aged; Angiogenic Proteins; Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Dacarbazine; Female; Fibroblast Growth Factor 2; Humans; Interferon-alpha; Interleukin-2; Interleukin-8; Male; Melanoma; Middle Aged; Neoplasm Staging; Platelet-Derived Growth Factor; Prospective Studies; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A; Vindesine

As an adjuvant therapy for patients with high risk of recurrent melanoma, high-dose interferon (IFN)-alpha2b therapy has been shown to have some efficacy. We examined 22 patients with resected melanoma who were treated with repeated injections of recombinant IFN-alpha2b during the treatment. Both angiogenic and immune parameters were measured. White blood cells (WBCs) and lymphocyte numbers, lymphocyte subpopulations, serum concentrations of IFN-alpha and anti-IFN-alpha antibodies, and the serum vascular endothelial growth factor (VEGF), interleukin (IL)-8, and basis fibroblast growth factor (bFGF) concentrations were determined over time in resected, recurrence-free patients with American Joint Committee on Cancer (AJCC) stage III melanoma with one or less (LN+ < or = 1, n = 7) or more than one (LN+ > 1, n = 8) lymph nodes involved, and AJCC stage IV resected disease (n = 7). Follow-up and recurrence-free intervals were longer in stage III (LN+ < or = 1) patients compared with stage IV patients (P < 0.05). The number of WBCs and lymphocytes decreased during the treatment for all patient groups (P < 0.001). In addition, percentages of CD8 and CD20 were higher in stage IV patients than in stage III (LN+ > 1) and stage III (LN+ < or = 1) patients at the beginning of therapy (P < 0.05). A significant increase in the percentage of CD20+ cells, mostly B lymphocytes, was observed in the stage III (LN+ > 1) and stage III (LN+ < or = 1) patients over time but not in stage IV patients (P < 0.001). Low IL-8 and bFGF concentrations at the beginning of therapy were associated with significantly longer recurrence-free survival (P < 0.05). These results warrant a larger trial to determine if the differences observed in patients before treatment can provide prognostic markers in patients receiving IFN-alpha2b therapy.

Topics: Adjuvants, Immunologic; Adult; Aged; Antineoplastic Agents; B-Lymphocytes; Disease-Free Survival; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Growth Substances; Humans; Interferon alpha-2; Interferon-alpha; Interleukin-8; Leukocyte Count; Lymphokines; Male; Melanoma; Middle Aged; Neovascularization, Pathologic; Prognosis; Recombinant Proteins; Skin Neoplasms; T-Lymphocytes; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The systemic side effects of isolated limb perfusion (ILP) with rhTNF alpha and melphalan are characterised by the induction of a systemic inflammatory response syndrome (SIRS). Procalcitonin (PCT), a serum marker of bacterial sepsis, was investigated with respect to its role in SIRS after TNF-ILP. Serum-PCT was analysed in 24 patients (12 male, 12 female), who treated by ILP for regionally metastasized melanoma (n = 8) or locally advanced soft tissue sarcoma (n = 16). Serum samples were analysed pre- and intraoperatively, and at defined intervals after reperfusion of the limb. In addition to PCT, serum IL-6 and IL-8 were analysed in 11 patients. PCT was significantly elevated over baseline after ILP with a maximum between 8 and 36 hours (p < 0.001). Even 96 hours after reperfusion, PCT was still significantly elevated as compared to baseline levels (p = 0.005). There was no correlation to the systemic leakage rate during the perfusion. IL-6 and IL-8 were also significantly increased after ILP (p = 0.001), but the maximum peaks of both cytokines were reached much earlier than for PCT (IL-8 max. at 1 hour and IL-6 max. at 4 hours after reperfusion). Serum procalcitonin is induced as part of the specific SIRS after ILP with rhTNF alpha and melphalan. It may be induced directly by rhTNF alpha or by different cytokines, as serum peaks of IL-6 and IL-8 are reached well before the peak of PCT. Determination of PCT prior to and after ILP with TNF might be useful to assess patients at risk of developing hyperdynamic shock.

Topics: Antineoplastic Combined Chemotherapy Protocols; Calcitonin; Calcitonin Gene-Related Peptide; Chemotherapy, Cancer, Regional Perfusion; Extremities; Female; Humans; Interleukin-6; Interleukin-8; Male; Melanoma; Melphalan; Predictive Value of Tests; Protein Precursors; Sarcoma; Skin Neoplasms; Soft Tissue Neoplasms; Systemic Inflammatory Response Syndrome; Tumor Necrosis Factor-alpha

The use of IL-1 in humans is associated with dose-limiting toxicity which resembles that of TNF-alpha or IL-2. Activation of neutrophils is thought to contribute to the toxicity caused by these two cytokines. We studied the effect of IL-1 in vivo on changes in neutrophil numbers and neutrophil degranulation as well as on the formation of neutrophil agonists, such as complement activation products, and on levels of TNF, IL-6, IL-8, and nitrite/nitrate (as a measure of nitric oxide production). Six patients with metastatic melanoma were treated with 3 ng/kg recombinant human IL-1 beta daily. One hour after the start of the 30-min IL-1 infusion, which caused mild cardiovascular toxicity, plasma levels of IL-6 reached a peak of 25 +/- 9 ng/L (mean +/- SEM), IL-8 reached a peak of 311 +/- 100 ng/L at 2 h, and nitrite/nitrate peaked after 10 h to 89 +/- 27 mumol/L. IL-1 did not induce significant changes in plasma levels of TNF or of the complement activation products C3a and C4b/c. Although IL-1 induced neutrophilia, levels of elastase and lactoferrin did not change. The failure of IL-1 to degranulate neutrophils was confirmed in an ex vivo model with whole blood culture in which doses of up to 100 microgram/L IL-1 beta or IL-1 alpha failed to induce significant elastase or lactoferrin release, whereas TNF, tested as a positive control, was able to do so. These results demonstrate that, unlike TNF, IL-1 does not cause neutrophil degranulation in man, despite its ability to cause neutrophilia and the rapid release of IL-6, IL-8, and nitrite/nitrate.

Topics: Adult; Antineoplastic Agents; Cell Degranulation; Complement Activation; Female; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Leukocyte Count; Male; Melanoma; Middle Aged; Neutrophils; Nitrates; Nitrites

Isolated limb perfusion (ILP) with tumor necrosis factor (TNF), interferon gamma, and melphalan (M) has been reported to result in high response rates for extremity melanoma and sarcoma. We have evaluated the relationship of systemic TNF exposure to induction of several secondary mediators and incidence of systemic toxicity.. Nineteen patients with extremity melanoma (n = 16) or sarcoma (n = 3), underwent 90-minute ILP with TNF-alpha, interferon gamma (0.2 mg), and M (10 to 13 mg/L of limb volume) (TNF/IFN/M) (n = 12), or M alone (n = 7). Continuous intraoperative monitoring (CIM) for systemic leak from the perfusion circuit was performed using radioactive iodine-131 albumin. Cytokine levels in the perfusate and systemic circulation during and after ILP were measured by enzyme-linked immunosorbent assay.. Systemic leaks > or = 1% from the perfusion circuit occurred in six patients who received TNF/IFN/M and in four who received M alone. Hypotension that required vasopressor support occurred in six of six patients with evidence of a leak (> or = 1%) and zero of six patients without a leak (< 1%). These six patients had significantly higher peak systemic TNF levels during and after perfusion than patients without a leak (2.8 and 8.2 ng/mL v 0.7 and 2.0 ng/mL, respectively; P < .05). All patients who received TNF/IFN/M had significantly greater increases in systemic interleukin-6 (IL-6) levels than in patients with M alone (12,395 +/- 10,374 pg/mL v 79.4 +/- 7.2 pg/mL, respectively; P < .001). Intracellular adhesion molecule (ICAM), IL-8, and TNF-R levels were also increased after ILP with TNF/IFN/M.. ILP with TNF/IFN/M can be safely performed, as I131 albumin provides a sensitive measure of systemic leakage from the perfusion circuit. Patients with a measured leak of > or = 1% develop mild and transient postoperative hypotension with significantly higher systemic TNF levels and lower perfusate TNF levels than in patients without leaks.

Topics: Adult; Aged; Aged, 80 and over; Arm; Chemotherapy, Cancer, Regional Perfusion; Cytokines; Female; Histiocytoma, Benign Fibrous; Humans; Interferon-gamma; Interleukin-6; Interleukin-8; Leg; Leiomyosarcoma; Male; Melanoma; Melphalan; Middle Aged; Receptors, Tumor Necrosis Factor; Sarcoma, Ewing; Skin Neoplasms; Tumor Necrosis Factor-alpha

To study the role that interleukin-8 might play in the activation of polymorphonuclear neutrophils during interleukin-2 therapy and the relationship of these phenomena to interleukin-2 induced toxicity.. A cohort study with measurements before and after the administration of interleukin-2.. Medical oncology department of a large teaching hospital.. Fourteen patients with metastatic renal cell carcinoma and 10 with metastatic melanoma being treated in a phase 2 study of the sequential combination of interferon-gamma and interleukin-2.. Plasma levels of tumour necrosis factor-alpha, interleukins-6 and 8 and markers of neutrophil activation (neutrophil elastase and lactoferrin) were measured in patients receiving 5 daily injections of interferon-gamma (100 micrograms/m2/day) followed by 5 days of interleukin-2 (18 x 10(6) IU/m2/day).. Tumour necrosis factor-alpha rose from baseline levels of 32 (range, 12 to 56) to 343 (103 to 787) pg/ml 3 hours after interleukin-2 administration returning to baseline values 21 hours later. Interleukins-6 and -8 rose from baseline levels of 6 (5 to 10) and 75 (35 to 100) to 2151 (152 to 7259) and 1283 (490 to 2500) pg/ml, respectively, at 4 hours after interleukin-2 with both returning to baseline values by 24 hours. Peak levels of neutrophil elastase and lactoferrin, both markers of neutrophil activation, occurred 6 hours after interleukin-2 administration.. These data indicate that following administration of interleukin-2 tumour necrosis factor-alpha is released followed sequentially by rises in interleukins-6 and -8. It is hypothesised that these events result in activation of polymorphonuclear neutrophils. These activated neutrophils may play an important role in initiating endothelial cell damage leading to the haemodynamic toxicity and the capillary leak syndrome which is typically seen following the administration of interleukin-2.

Topics: Adult; Aged; Carcinoma, Renal Cell; Cohort Studies; Female; Humans; Infusions, Intravenous; Interferon-gamma; Interleukin-2; Interleukin-6; Interleukin-8; Kidney Neoplasms; Lactoferrin; Leukocyte Count; Leukocyte Elastase; Male; Melanoma; Middle Aged; Neutrophil Activation; Pancreatic Elastase; Tumor Necrosis Factor-alpha

Melanoma has the highest propensity of all cancers to metastasize to the brain with a large percentage of late-stage patients developing metastases in the central nervous system (CNS). It is well known that metastasis establishment, cell survival, and progression are affected by tumour-host cell interactions where changes in the host cellular compartments likely play an important role. In this context, miRNAs transferred by tumour derived extracellular vesicles (EVs) have previously been shown to create a favourable tumour microenvironment. Here, we show that miR-146a-5p is highly expressed in human melanoma brain metastasis (MBM) EVs, both in MBM cell lines as well as in biopsies, thereby modulating the brain metastatic niche. Mechanistically, miR-146a-5p was transferred to astrocytes via EV delivery and inhibited NUMB in the Notch signalling pathway. This resulted in activation of tumour-promoting cytokines (IL-6, IL-8, MCP-1 and CXCL1). Brain metastases were significantly reduced following miR-146a-5p knockdown. Corroborating these findings, miR-146a-5p inhibition led to a reduction of IL-6, IL-8, MCP-1 and CXCL1 in astrocytes. Following molecular docking analysis, deserpidine was identified as a functional miR-146a-5p inhibitor, both in vitro and in vivo. Our results highlight the pro-metastatic function of miR-146a-5p in EVs and identifies deserpidine for targeted adjuvant treatment.

Topics: Astrocytes; Brain Neoplasms; Extracellular Vesicles; Humans; Interleukin-6; Interleukin-8; Melanoma; MicroRNAs; Molecular Docking Simulation; Tumor Microenvironment

Elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been frequently reported in malignant melanoma suggesting that XIAP renders apoptosis resistance and thereby supports melanoma progression. Independent of its anti-apoptotic function, XIAP mediates cellular inflammatory signalling and promotes immunity against bacterial infection. The pro-inflammatory function of XIAP has not yet been considered in cancer. By providing detailed in vitro analyses, utilising two independent mouse melanoma models and including human melanoma samples, we show here that XIAP is an important mediator of melanoma neutrophil infiltration. Neutrophils represent a major driver of melanoma progression and are increasingly considered as a valuable therapeutic target in solid cancer. Our data reveal that XIAP ubiquitylates RIPK2, involve TAB1/RIPK2 complex and induce the transcriptional up-regulation and secretion of chemokines such as IL8, that are responsible for intra-tumour neutrophil accumulation. Alteration of the XIAP-RIPK2-TAB1 inflammatory axis or the depletion of neutrophils in mice reduced melanoma growth. Our data shed new light on how XIAP contributes to tumour growth and provides important insights for novel XIAP targeting strategies in cancer.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Disease Models, Animal; Humans; Inhibitor of Apoptosis Proteins; Interleukin-8; Melanoma; Mice; Neutrophil Infiltration; Receptor-Interacting Protein Serine-Threonine Kinase 2; Skin Neoplasms; X-Linked Inhibitor of Apoptosis Protein

ALDH1A1 is a cytosolic enzyme upregulated in tumor cells, involved in detoxifying cells from reactive aldehydes and in acquiring resistance to chemotherapeutic drugs. Its expression correlates with poor clinical outcomes in a number of cancers, including melanoma. The present study hypothesized that the increased ALDH1A1 expression and activity upregulated the release of proangiogenic factors from melanoma cells, which regulate angiogenic features in endothelial cells (ECs) through a rearrangement of the Notch pathway.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Endothelial Cells; Interleukin-8; Melanoma; Mice; Neovascularization, Pathologic; Receptors, Notch; Retinal Dehydrogenase; Signal Transduction; Tumor Microenvironment

Early diagnosis of malignant melanoma is critical for effective treatment and reduced patient mortality. However, current clinical and histological variables show limited accuracy in diagnosis. Serum or urine level of 5-S-cysteinyldopa (5-S-CD) is a commonly used melanoma biomarker in Japan owing to its increased sensitivity compared with other melanoma markers. However, its use as a diagnostic marker has shown some limitations. Therefore, here we examined the combination of 5-S-CD with melanoma inhibitory activity, which showed sensitivity in detecting melanoma comparable with that of 5-S-CD, and interleukin-8, a cytokine linked with melanoma progression, in a cohort of Japanese patients with melanoma. Our results revealed that the triple combination of 5-S-CD, melanoma inhibitory activity, and interleukin-8 showed high diagnostic accuracy in detecting melanoma compared with each of the individual factors. Importantly, the triple marker showed specificity and utility in detecting early-stage melanoma. Our results suggest the utility of the triple marker as a diagnostic biomarker for melanoma patients.

Topics: Biomarkers, Tumor; Cysteinyldopa; Cytokines; Humans; Interleukin-8; Melanoma; Melanoma, Cutaneous Malignant; Skin Neoplasms

Topics: Aged; Animals; Carcinogenesis; Cell Line, Tumor; Female; Humans; Interleukin-8; Keratins, Hair-Specific; Keratins, Type II; Male; Melanoma; Mice; Mice, Inbred BALB C; Middle Aged; Skin Neoplasms

Melanoma is one of the most aggressive forms of human cancer and its incidence has significantly increased worldwide over the last decades. This neoplasia has been characterized by the release of a wide variety of soluble factors, which could stimulate tumor cell proliferation and survival in an autocrine and paracrine manner. Consequently, we sought to evaluate the pattern of soluble factors produced by pre-metastatic and metastatic melanoma established cultures, and to determine whether these factors can be detected in the autologous serum of malignant melanoma patients. Our results showed that both melanoma cultures had a common profile of 27 soluble factors mainly characterized by the high expression of VEGF-A, IL-6, MCP-1, IL-8, and SDF-1. In addition, when we compared supernatants, we observed significant differences in VEGF-A, BDNF, FGF-2, and NGF-β concentrations. As we found in melanoma cultures, serum samples also had their specific production pattern composed by 21 soluble factors. Surprisingly, PDGF-BB and EGF were only found in serum, whereas IL-2, IL-4, IL-8, IL31, FGF2, and GRO-α were only expressed in the supernatant. Significant differences in PDGF-BB, MIP-1β, HGF, PIGF-1, BDNF, EGF, Eotaxin, and IP-10 were also found after comparing autologous serum with healthy controls. According to this, no correlation was found between culture supernatants and autologous serum samples, which suggests that some factors may act locally, and others systemically. Nonetheless, after validation of our results in an independent cohort of patients, we concluded that PDGF-BB, VEGF-A, and IP-10 serum levels could be used to monitor different melanoma stages.

Topics: Autocrine Communication; Becaplermin; Cell Proliferation; Chemokine CCL2; Chemokine CXCL10; Chemokine CXCL12; Cytokines; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Interleukin-8; Male; Melanoma; Neoplasm Proteins; Paracrine Communication; Vascular Endothelial Growth Factor A

IL-8 (CXCL-8) is a chemoattractant factor for myeloid leukocytes, that is produced in large quantities by many solid tumors. Levels of IL-8, which can act upon a variety of immune and nonimmune cells, can tell us a lot about tumors, including their size (positive association) and how likely they are to respond to immunotherapy (negative association). This is because the IL-8 produced by tumors can promote angiogenesis, recruit immunosuppressive cells like neutrophils and myeloid-derived suppressor cells (MDSCs), and stimulate epithelial-to-mesenchymal transition, which is a precursor to metastasis. In a pooled analysis of several clinical trials in kidney cancer, melanoma, and lung cancer, it was found that patients with higher baseline concentrations of IL-8 in the blood experienced worse outcomes and lower overall survival after being treated with immunotherapy. Currently, the field that relates IL-8 to immunotherapy is leading to numerous and promising clinical trials that combine the inhibition of IL-8 with existing immunotherapeutic therapies. For this reason, multiple constructs based on IL-8 agonists are being developed clinically by the pharmaceutical and biotech industries.

Topics: Humans; Immunotherapy; Interleukin-8; Melanoma; Myeloid-Derived Suppressor Cells

Melanoma has the highest mortality rate of all skin tumors, and metastases are the major cause of death from it. The molecular mechanism leading to melanoma metastasis is currently unclear.. With the goal of revealing the underlying mechanism, three data sets with accession numbers GSE8401, GSE46517 and GSE7956 were downloaded from the Gene Expression Omnibus (GEO) database. After identifying the differentially expressed gene (DEG) of primary melanoma and metastatic melanoma, three kinds of analyses were performed, namely functional annotation, protein-protein interaction (PPI) network and module construction, and co-expression and drug-gene interaction prediction analysis.. A total of 41 up-regulated genes and 79 down-regulated genes was selected for subsequent analyses. Results of pathway enrichment analysis showed that extracellular matrix organization and proteoglycans in cancer are closely related to melanoma metastasis. In addition, seven pivotal genes were identified from PPI network, including CXCL8, THBS1, COL3A1, TIMP3, KIT, DCN, and IGFBP5, which have all been verified in the TCGA database and clinical specimens, but only CXCL8, THBS1 and KIT had significant differences in expression.. To conclude, CXCL8, THBS1 and KIT may be the hub genes in the metastasis of melanoma and thus may be regarded as therapeutic targets in the future.

Topics: Carcinogenesis; Computational Biology; Databases, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Male; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Protein Interaction Maps; Proto-Oncogene Proteins c-kit; Thrombospondin 1

Skin is constantly exposed to UVR, the most critical risk factor for melanoma development. Hyaluronan is abundant in the epidermal extracellular matrix and may undergo degradation by UVR. It is hypothesized that an intact hyaluronan coat around the cells protects against various agents including UVR, whereas hyaluronan fragments promote inflammation and tumorigenesis. We investigated whether hyaluronan contributes to the UVB-induced inflammatory responses in primary melanocytes. A single dose of UVB suppressed hyaluronan secretion and the expression of hyaluronan synthases HAS2 and HAS3, the hyaluronan receptor CD44, and the hyaluronidase HYAL2, as well as induced the expression of inflammatory mediators IL6, IL8, CXCL1, and CXCL10. Silencing HAS2 and CD44 partly inhibited the inflammatory response, suggesting that hyaluronan coat is involved in the process. UVB alone caused little changes in the coat, but its removal with hyaluronidase during the recovery from UVB exposure dramatically enhanced the surge of these inflammatory mediators via TLR4, p38, and NF-κB. Interestingly, exogenous hyaluronan fragments did not reproduce the inflammatory effects of hyaluronidase. We hypothesize that the hyaluronan coat on melanocytes is a sensor of tissue injury. Combined with UVB exposure, repeated injuries to the hyaluronan coat could maintain a sustained inflammatory state associated with melanomagenesis.

Topics: Carcinogenesis; Cells, Cultured; Chemokine CXCL1; Chemokine CXCL10; Epidermis; Extracellular Matrix; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Interleukin-6; Interleukin-8; Melanocytes; Melanoma; Primary Cell Culture; Signal Transduction; Skin Neoplasms; Toll-Like Receptor 4; Ultraviolet Rays

The protein bcl-xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl-xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild-type embryos, bcl-xL-overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino-mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl-xL-overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA-seq public databases of human melanoma biopsies revealed that bcl-xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co-expression of bcl-xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl-xL-induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl-xL-expressing melanoma.

Topics: Animals; Animals, Genetically Modified; bcl-X Protein; Cell Line, Tumor; Heterografts; Humans; Interleukin-8; Melanoma; Neovascularization, Pathologic; Recombinant Proteins; Tumor Microenvironment; Zebrafish

UVA radiation, which accounts for about 95% of the solar spectrum, contributes to and may be the etiological factor of skin cancers of which malignant melanoma is the most aggressive. UVA causes oxidative stress in various types of cells in the skin, keratinocyte, melanocytes, and fibroblasts, which is responsible for its cytotoxic effect. Here we used a transwell system to explore how the responses of melanoma cells to a low dose of UVA (20kJ/m

Topics: Apoptosis; Bystander Effect; Cell Line; Cell Survival; Coculture Techniques; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Melanoma; Membrane Potential, Mitochondrial; Reactive Nitrogen Species; Reactive Oxygen Species; Skin Neoplasms; Ultraviolet Rays

Project High-tech Omics-based Patient Evaluation (HOPE), including comprehensive whole-exome sequencing (WES) and gene expression profiling (GEP) using freshly resected tumor specimens, has been in progress since its implementation in 2014. Among a total of 1,685 cancer patients, 13 melanoma patients were registered in the HOPE Project and were characterized using multi-omics analyses. Among the 13 melanoma patients, 4 were deceased, and 9 were alive. The mean overall survival (OS) and relapse‑free survival (RFS) times of the melanoma patients were 16.9 and 14.7 months, respectively. Previously, we developed an immune response‑associated gene list, which consisted of 164 genes in Project HOPE, for evaluating the immunological status. In the present study, the association of immune response‑associated gene expression with immunological parameters, such as programmed death-ligand 1 (PD-L1) and CD8 expression levels, single nucleotide variant (SNV) number, and Vogelstein driver gene mutation number, was investigated. With respect to PD-L1 expression, both immuno-suppression and immuno-stimulation-related genes were upregulated in PD-L1-positive melanomas. In contrast, regarding Vogelstein driver mutations, several T-cell activation-related genes were significantly downregulated in the high driver gene mutation group. In addition, many T-cell activation-related genes were upregulated in the CD8-positive melanomas. The correlation of immune response-associated gene expression with the survival time of the melanoma patients was investigated. Eight specific genes were commonly identified as genes that were significantly correlated for both the overall OS and RFS time, which could be possible prognostic factors for melanoma patients. These results revealed that an immune response-associated gene panel could be an informative tool for evaluating the immunological status prior to clinical immunotherapy in the upcoming era of genomic cancer medicine.

Topics: Adult; Aged; Aged, 80 and over; Asian People; B7-H1 Antigen; Biomarkers, Tumor; Female; Follow-Up Studies; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Male; Melanoma; Middle Aged; Mutation; Neoplasm Recurrence, Local; Prognosis; Survival Rate

Paclitaxel (PTX) is a potent anti-cancer drug commonly used for the treatment of advanced breast cancer (BCA) and melanoma. Toll-like receptor 4 (TLR4) promotes the production of pro-inflammatory cytokines associated with cancer chemoresistance. This study aims to explore the effect of TLR4 in PTX resistance in triple-negative BCA and advanced melanoma and the effect of compound A (CpdA) to attenuate this resistance.. BCA and melanoma cell lines were checked for the response to PTX by cytotoxic assay. The response to PTX of TLR4-transient knockdown cells by siRNA transfection was evaluated compared to the control cells. Levels of pro-inflammatory cytokines, IL-6 and IL-8, and anti-apoptotic protein, XIAP were measured by real-time PCR whereas the secreted IL-8 was quantitated by ELISA in TLR4-transient knockdown cancer cells with or without CpdA treatment. The apoptotic cells after adding PTX alone or in combination with CpdA were detected by caspase-3/7 assay.. PTX could markedly induce TLR4 expression in both MDA-MB-231 BCA and MDA-MB-435 melanoma cell lines having a basal level of TLR4 whereas no significant induction in TLR4-transient knockdown cells occurred. The siTLR4-treated BCA cells revealed more dead cells after PTX treatment than that of mock control cells. IL-6, IL-8 and XIAP showed increased expressions in PTX-treated cells and this over-production effect was inhibited in TLR4-transient knockdown cells. Apoptotic cells were detected higher when PTX and CpdA were combined than PTX treatment alone. Isobologram exhibited the synergistic effect of CpdA and PTX. CpdA could significantly decrease expressions of IL-6, XIAP and IL-8, as well as excreted IL-8 levels together with reduced cancer viability after PTX treatment.. The acquired TLR4-mediated PTX resistance in BCA and melanoma is explained partly by the paracrine effect of IL-6 and IL-8 released into the tumor microenvironment and over-production of anti-apoptotic protein, XIAP, in BCA cells and importantly CpdA could reduce this effect and sensitize PTX-induced apoptosis in a synergistic manner. In conclusion, the possible impact of TLR4-dependent signaling pathway in PTX resistance in BCA and melanoma is proposed and using PTX in combination with CpdA may attenuate TLR4-mediated PTX resistance in the treatment of the patients.

Topics: Acetates; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Interleukin-6; Interleukin-8; Melanoma; Paclitaxel; Signal Transduction; Toll-Like Receptor 4; Tumor Microenvironment; Tyramine

The goal of this study was to assess the relationship between the severity of obstructive sleep apnoea (OSA) and the levels of carcinogenesis- and tumour growth-related biomarkers in patients with cutaneous melanoma.This multicentre observational study included patients who were newly diagnosed with melanoma. The patients were classified as non-OSA (apnoea-hypopnoea index (AHI) 0-5 events·h

Topics: Adult; Aged; Biomarkers, Tumor; Carcinogenesis; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hypoxia; Intercellular Adhesion Molecule-1; Interleukin-8; Male; Melanoma; Middle Aged; S100 Calcium Binding Protein beta Subunit; Skin Neoplasms; Sleep Apnea, Obstructive; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

Genistein, a natural isoflavone found in soybean products, is considered as a powerful anti-cancer agent, although the involved mechanisms are not fully understood. There is a growing body of evidence that, among the genes inhibited by genistein and responsible for cell cycle progression, invasion, metastasis, and angiogenesis, IL-8 occupies a relevant place. On the other hand, it is equally well documented that IL-8 is upregulated by prostaglandin E2 (PGE

Topics: Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Cell Proliferation; Dinoprostone; Dose-Response Relationship, Drug; Female; Genistein; Humans; Interleukin-8; Male; Melanoma; Receptors, Prostaglandin E, EP3 Subtype; Tumor Cells, Cultured

Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition. Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis. We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions. This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling. We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability. Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent. These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies.

Topics: Antigens, Neoplasm; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Interleukin-8; Male; Melanoma; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; RNA Stability; RNA-Binding Proteins

Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance. Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance. Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance. In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1β and fibroblast-derived CXCR2 ligands. Fibroblasts require IL-1β to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth. In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors. Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors. We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatment.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Chemokine CXCL1; Fibroblasts; Humans; Inflammation; Interleukin-1; Interleukin-1beta; Interleukin-8; Ligands; Macrophages; MAP Kinase Signaling System; Melanoma; Mice, Knockout; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; Receptors, Interleukin-1; Receptors, Interleukin-8B; Skin Neoplasms; Stromal Cells

Surrogate biomarkers of efficacy are needed for anti-PD1/PD-L1 therapy, given the existence of delayed responses and pseudo-progressions. We evaluated changes in serum IL-8 levels as a biomarker of response to anti-PD-1 blockade in melanoma and non-small-cell lung cancer (NSCLC) patients.. Metastatic melanoma and NSCLC patients treated with nivolumab or pembrolizumab alone or nivolumab plus ipilimumab were studied. Serum was collected at baseline; at 2-4 weeks after the first dose; and at the time-points of response evaluation. Serum IL-8 levels were determined by sandwich ELISA. Changes in serum IL-8 levels were compared with the Wilcoxon test and their strength of association with response was assessed with the Mann-Whitney test. Accuracy of changes in IL-8 levels to predict response was estimated using receiver operation characteristics curves.. Twenty-nine melanoma patients treated with nivolumab or pembrolizumab were studied. In responding patients, serum IL-8 levels significantly decreased between baseline and best response (P <0.001), and significantly increased upon progression (P =  0.004). In non-responders, IL-8 levels significantly increased between baseline and progression (P =  0.013). Early changes in serum IL-8 levels (2-4 weeks after treatment initiation) were strongly associated with response (P <0.001). These observations were validated in 19 NSCLC patients treated with nivolumab or pembrolizumab (P =  0.001), and in 15 melanoma patients treated with nivolumab plus ipilimumab (P <0.001). Early decreases in serum IL-8 levels were associated with longer overall survival in melanoma (P =  0.001) and NSCLC (P =  0.015) patients. Serum IL-8 levels also correctly reflected true response in three cancer patients presenting pseudoprogression.. Changes in serum IL-8 levels could be used to monitor and predict clinical benefit from immune checkpoint blockade in melanoma and NSCLC patients.

Topics: Adult; Antineoplastic Agents, Immunological; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Lung Neoplasms; Male; Melanoma; Middle Aged; Skin Neoplasms; Survival Analysis

Topics: Animals; Antimalarials; Antineoplastic Agents; Autophagosomes; Autophagy; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Chloroquine; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Melanoma; Mice; NF-kappa B; Sequestosome-1 Protein; Signal Transduction

Among the most important traditional medicinal fungi,

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Humans; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Mice; Mice, Inbred C57BL; Reishi; Triple Negative Breast Neoplasms

Nuclear factor of activated T cell (NFAT1, NFATC2) is a transcription factor that binds and positively regulates IL2 expression during T-cell activation. NFAT1 has important roles in both innate and adaptive immune responses, but its involvement in cancer is not completely understood. We previously demonstrated that NFAT1 contributes to melanoma growth and metastasis by regulating the autotaxin gene (Enpp2). Here, we report a strong correlation between NFAT1 expression and metastatic potential in melanoma cell lines and tumor specimens. To elucidate the mechanisms underlying NFAT1 overexpression during melanoma progression, we conducted a microarray on a highly metastatic melanoma cell line in which NFAT1 expression was stably silenced. We identified and validated two downstream targets of NFAT1, IL8, and MMP3. Accordingly, NFAT1 depletion in metastatic melanoma cell lines was associated with reduced IL8 and MMP3 expression, whereas NFAT1 overexpression in a weakly metastatic cell line induced expression of these targets. Restoration of NFAT1 expression recovered IL8 and MMP3 expression levels back to baseline, indicating that both are direct targets of NFAT1. Moreover, in vivo studies demonstrated that NFAT1 and MMP3 promoted melanoma tumor growth and lung metastasis. Collectively, our findings assign a new role for NFAT1 in melanoma progression, underscoring the multifaceted functions that immunomodulatory factors may acquire in an unpredictable tumor microenvironment. Cancer Res; 76(11); 3145-55. ©2016 AACR.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Proliferation; Female; Humans; Immunoenzyme Techniques; Interleukin-8; Lung Neoplasms; Matrix Metalloproteinase 3; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; NFATC Transcription Factors; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Tumour microenvironment plays a critical role in cell invasion and metastasis. To investigate the role of cancer-associated fibroblasts (CAFs) in melanoma cell invasiveness, we used 3D spheroid invasion assay. The effect of conditioned media from normal fibroblasts and CAFs cultivated alone or co-cultivated with melanoma cells on BLM or A2058 melanoma spheroid invasion was analysed. We found that conditioned media from CAFs and CAFs co-cultured with melanoma cells, especially, promote invasion and migration, without significant effect on melanoma cell proliferation. We further analysed the expression of pro-invasive cytokines IL-8 and IL-6 in media and found that melanoma cells are dominant producers of IL-8 and fibroblasts are dominant producers of IL-6 in 2D monocultures, while co-cultivation of CAFs with melanoma cells induces production/secretion of IL-6 and IL-8 into the media. The analyses of IL-6 levels in 3D cultures and human melanoma samples, however, revealed that at least in some cases IL-6 is also produced directly by melanoma cells. Analysis of the role of IL-6 and IL-8 in CAF-induced melanoma invasion, using neutralising antibodies, revealed that simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness. In summary, these experiments indicate the important role of CAFs and IL-8 and IL-6 cytokines in melanoma cell invasiveness.

Topics: Cancer-Associated Fibroblasts; Cell Movement; Cell Proliferation; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Melanoma; Neoplasm Invasiveness

CD34 promotes melanoma cell motility, negative regulation of cellular response to hypoxia and positive regulation of vasculogenesis. Interleukin 8 (IL-8) is responsible for angiogenic response in endothelial cells, increases proliferation, metastasis, and survival of melanoma cells. The aim of our study was evaluation of relationship between CD34 immunoexpression and IL-8 serum concentrations in melanoma patients. The study was conducted on patients with melanoma that were divided in: Clark II (17 patients - 19.3%), Clark III (33 patients - 37.5%), Clark IV (22 patients - 25%), and Clark V (16 patients - 18.2%) levels. CD34 expression was absent for Clark II melanomas, and positive for Clark III (6.1%), Clark IV (40.9%), and Clark V (56.2%). The CD34 immune-mark was highly positive only for Clark IV and V levels. Interleukin 8 (IL-8) had high values (above 15 pg/mL) for all patients with melanoma (58.9% - Clark II; 87.8% - Clark III; 90.9% - Clark IV and 93.7% - Clark V). We have found a strong and statistically significant correlation between CD34 and IL-8 for Clark IV (r = 0.54, P < 0.05) and Clark V (r = 0.72, P < 0.05) melanomas. CD34 and IL-8 are associated with poor prognosis of melanoma, metastasis, and neoangiogenesis.

Topics: Adolescent; Adult; Antigens, CD34; Biomarkers, Tumor; Enzyme Activation; Female; Humans; Immunohistochemistry; Interleukin-8; L-Lactate Dehydrogenase; Male; Melanoma; Middle Aged; Neovascularization, Pathologic; Young Adult

The BRAF(V600E) mutation confers constitutive kinase activity and accounts for >90% of BRAF mutations in melanoma. This genetic alteration is a current therapeutic target; however, the antitumorigenic effects of the BRAF(V600E) inhibitor vemurafenib are short-lived and the majority of patients present tumor relapse in a short period after treatment. Characterization of vemurafenib resistance has been essential to the efficacy of next generation therapeutic strategies. Herein, we found that acute BRAF inhibition induced a decrease in active MMP-2, MT1-MMP and MMP-9, but did not modulate the metalloproteinase inhibitors TIMP-2 or RECK in naïve melanoma cells. In vemurafenib-resistant melanoma cells, we observed a lower growth rate and an increase in EGFR phosphorylation followed by the recovery of active MMP-2 expression, a mediator of cancer metastasis. Furthermore, we found a different profile of MMP inhibitor expression, characterized by TIMP-2 downregulation and RECK upregulation. In a 3D spheroid model, the invasion index of vemurafenib-resistant melanoma cells was more evident than in its non-resistant counterpart. We confirmed this pattern in a matrigel invasion assay and demonstrated that use of a matrix metalloproteinase inhibitor reduced the invasion of vemurafenib resistant melanoma cells but not drug naïve cells. Moreover, we did not observe a delimited group of cells invading the dermis in vemurafenib-resistant melanoma cells present in a reconstructed skin model. The same MMP-2 and RECK upregulation profile was found in this 3D skin model containing vemurafenib-resistant melanoma cells. Acute vemurafenib treatment induces the disorganization of collagen fibers and consequently, extracellular matrix remodeling, with this pattern observed even after the acquisition of resistance. Altogether, our data suggest that resistance to vemurafenib induces significant changes in the tumor microenvironment mainly by MMP-2 upregulation, with a corresponding increase in cell invasiveness.

Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; GPI-Linked Proteins; Humans; Indoles; Interleukin-8; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Neoplasm Invasiveness; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Tissue Inhibitor of Metalloproteinase-2; Tumor Microenvironment; Up-Regulation; Vemurafenib

Owing to activation of several resistance-mediating pathways including NF-κB signaling, metastasized melanoma is almost universally resistant against chemotherapy. Given that blocking of NF-κB either by proteasome-, pan-IKK- or selective IKKβ-inhibitors may increase the susceptibility of melanoma cells to chemotherapy, we have assessed the role of the second kinase within the IKK complex, IKKα. While expression of IKKα and overall activation of NF-κB were heterogeneous, the IKKα-specific p100/p52 processing was detected in a small subset of melanomas (1/9 primary and 1/12 metastatic melanomas) as well as in 1/8 melanoma cell lines. Down-modulation of IKKα by siRNA resulted in diminution of doxorubicin-induced NF-κB activation, constitutive and TNFα-stimulated expression of CXCL8 and ICAM-1, and cell migration. In contrast, overexpression of IKKα in melanoma cells did not significantly affect progression-related functions. Thus, IKKα may be a worthwhile target only in selected individualized therapies but not in general melanoma therapy.

Topics: Cell Line, Tumor; Cell Movement; Disease Progression; Gene Expression Regulation; Gene Knockdown Techniques; Humans; I-kappa B Kinase; Intercellular Adhesion Molecule-1; Interleukin-8; Melanoma; NF-kappa B p52 Subunit; Precision Medicine; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor-alpha

Metastasis of melanoma cells during the recurrence or the late stage of melanoma has been characterized as the dissemination of tumor cells under anchorage independency. The secreted interleukin-8 (IL-8) and its conical receptors from melanoma cells have been associated with melanoma malignancy. However, their correlations with melanoma cells under anchorage independency were unclear. Suspension of adherent melanoma cells generated the suspended melanoma cell model of anoikis resistance. The in-vivo xenograft experiment, in-vitro cell proliferation/migration assay, microarray, and bioinformatics analysis were used to compare the malignancy and gene expression profiling in adherent and suspended melanoma cells. PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and kinase inhibition assay were adapted to validate the expression and regulation of IL-8 and CXCR1/2. Suspended melanoma cells were anoikis resistant and showed elevated malignancy in vivo and in vitro. Gene expression profiling of adherent and suspended melanoma cells showed extensive alteration associated with cell survival/death, cell signaling, and regulation of gene expression. Microarray and bioinformatics analysis on gene set enrichment analysis further showed elevated IL-8 expression in suspended melanoma cells. The upregulation of IL-8 and the effect on chemotaxis were mediated by MEK/ERK activation upon cell suspension. Change in JNK phosphorylation induced CXCR1 downregulation under cell suspension, but upregulation by cell reattachment. We suggest the possible roles of elevated IL-8 secretion and change in CXCR expression contributing toward elevated melanoma malignancy upon reattachment from cell suspension. We show that the suspension of melanoma cells is critical in promoting melanoma malignancy in vivo and in vitro.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemotaxis; Computational Biology; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-8; MAP Kinase Kinase 4; Melanoma; Neoplasm Metastasis; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Receptors, Interleukin-8A; Skin Neoplasms

Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).. Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.. Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.. We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

Topics: Adult; Aged; Cell Communication; Cell Differentiation; Cell Line, Tumor; Chemokine CXCL1; Epidermal Cells; Epidermis; Female; Fibroblast Growth Factor 2; Gene Expression Profiling; Humans; Interleukin-8; Keratin-10; Keratin-14; Keratinocytes; Male; Melanocytes; Melanoma; Middle Aged; Neoplasm Metastasis; S100 Proteins; Vascular Endothelial Growth Factor A

Patients with early stage, radial growth phase (RGP) melanoma have a 97% survival rate; however, when the melanoma progresses to the invasive vertical growth phase (VGP), survival rates decrease to 15%. The targets of many clinical trials are the known genetic and molecular mechanisms involved in melanoma progression, with the most common oncogenic mutation being the BRAFV600E. However, less than half of melanomas harbor this mutation, and consequently, do not respond to the current BRAF targeted treatments. It is therefore critical to elucidate alternative mechanisms regulating melanoma progression. Increased expression of the chemokine receptor, CXCR3, on melanoma cells is correlated with increased metastasis and poor patient outcomes, suggesting a role for CXCR3 in the RGP to VGP transition. We found that endogenous CXCR3 can be induced in two RGP cell lines, BOWES (BRAFWT) and WM35 (BRAFV600E), with in vitro environmental stress and nutrient deprivation. Signaling via induced endogenous CXCR3 is linked with IL-8 expression in BOWES cells. Ectopic overexpression of CXCR3 in BOWES cells leads to increased ligand-mediated phERK, cellular migration, and IL-8 expression in vitro, and to increased tumorigenesis and lymph node metastasis in vivo. Our results demonstrate that, in BRAFWT melanomas, CXCR3 signaling mediates significant increases in IL-8 expression, suggesting that CXCR3 expression and signaling may represent a transformative event that drives the progression of BRAFWT melanomas.. Expression of CXCR3 on BRAFWT melanoma cells may be a mediator of melanoma progression.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Humans; Interleukin-8; Male; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Proto-Oncogene Proteins B-raf; Receptors, CXCR3; Signal Transduction

FKBP51 is overexpressed in melanoma and impacts tumour cell properties. However, its comprehensive role in melanoma pathogenesis and underlying mechanism(s) remain elusive.. FKBP51 was stably silenced in aggressive melanoma cell lines and its effect examined in vitro and in mouse model. Histological/immunohistochemical analyses were performed to confirm metastasis, angiogenesis and neutrophil infiltration. Gene expression was analyzed by qRT-PCR, immunoblot and/or ELISA. NF-κB transcriptional activity and promoter binding were monitored by luciferase-based promoter-reporter and ChIP assays, respectively. Interleukin (IL)-8 inhibition was achieved by gene silencing or neutralising-antibody treatment.. FKBP51 silencing reduced melanoma growth, metastasis, angiogenesis and neutrophil infiltration and led to IL-8 downregulation through NF-κB suppression in cell lines and tumour xenografts. IL-8 inhibition drastically decreased growth, migration and invasiveness of FKPB51-overexpressing cells; whereas its treatment partially restored the suppressed phenotypes of FKBP51-silenced melanoma cells. Interleukin-8 depletion in conditioned medium (CM) of FKBP51-overexpressing melanoma cells inhibited endothelial cell proliferation and capillary-like structure formation, whereas its treatment promoted these effects in endothelial cells cultured in CM of FKBP51-silenced melanoma cells.. FKBP51 promotes melanoma growth, metastasis and angiogenesis, and IL-8 plays a key role in these processes. Thus, targeting of FKBP51 or its upstream or downstream regulatory pathways could lead to effective therapeutic strategies against melanoma.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Melanoma; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; Promoter Regions, Genetic; Tacrolimus Binding Proteins

The aim of the present study was to evaluate the usefulness of four interleukins (IL-2, IL-6, IL-8 and IL-10) for melanoma detection and correlate these interleukins with sentinel node metastasis positivity.. A group of 236 persons was assessed: 175 patients with melanomas and 61 healthy persons. Melanoma patients were divided to four groups according to Breslow score. We determined IL-2, IL-6, IL-8 and IL-10 in each plasma sample. Interleukin plasma levels were assayed using a Human Cytokine Milliplex Map kit. Measurements were performed using the Bio-Plex MAGPIX Multiplex Reader. Plasma samples were collected prior to surgery or any other form of treatment. All melanoma diagnoses were histologically verified.. We compared interleukin plasma levels in the healthy group and plasma levels in each Breslow score stage. In the first Breslow score stage, IL-2 (p<0.0001), IL-6 (p=0.0004) and IL-10 (p<0.0001) were positive. In the second Breslow score, stage IL-2 (p<0.0001), IL-6 (p<0.0001), IL-8 (p=0.0017) and IL-10 (p<0.0001) were positive. By comparing the group of positive and negative sentinel node metastasis, we observed a statistically significant difference in two interleukins: The median of IL-2 levels in the negative group was 5.88 pg/ml compared to 32.57 pg/ml in the positive group (p=0.0005). The median of IL-6 levels in the negative group was 4.80 pg/ml compared to 32.02 pg/ml in the positive group (p=0.0048).. Interleukins IL-2, IL-6 and IL-10 are promising biomarkers of early-stage melanoma. IL-2 and IL-6 appear to be prognostic biomarkers.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Early Detection of Cancer; Female; Humans; Interleukin-10; Interleukin-2; Interleukin-6; Interleukin-8; Lymphatic Metastasis; Male; Melanoma; Middle Aged; Neoplasm Staging; Prognosis; Sentinel Lymph Node Biopsy

There are several circulatory biomarkers that are involved in forecasting the clinical outcome of cutaneous melanoma. Serum/plasma vitamin D status is one of the markers intensively studied in this type of cutaneous cancer. The combination of validated serum biomarkers (like LDH) with new biomarkers such as IL-8, angiogenic factor, and vitamin D is still at the dawn of research. Hence, we are aiming to establish the predictive power of inflammatory biomarkers, such as IL-8, and metabolic ones, such as vitamin D. These candidate biomarkers are intended to aid classical biomarkers, such as LDH, in the prognosis of cutaneous melanoma.. Serum vitamin D and IL-8 were quantified in melanoma patients and in matching healthy controls.. Median serum vitamin D concentrations were significantly lower (p = 0.003) in melanoma patients as compared to healthy control subjects, while around 65% of the investigated patients have proven a severe circulatory deficiency of this vitamin. IL-8 was found increased (p = 0.001) in melanoma patients as compared to controls.. Upregulation of proangiogenic factors associated with vitamin D deficiency can prove to be potent future biomarkers candidates, enhancing the predictive power of classical LDH.

Topics: Adolescent; Adult; Biomarkers; Calcifediol; Cohort Studies; Disease Progression; Female; Humans; Inflammation; Interleukin-8; L-Lactate Dehydrogenase; Male; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Neovascularization, Pathologic; Prognosis; Skin Neoplasms; Up-Regulation; Vitamin D Deficiency; Young Adult

Patients diagnosed with melanoma brain metastases have few treatment options and poor prognosis, and improved treatment strategies for these patients require detailed understanding of the underlying pathobiology. In this investigation we studied the vascularity and invasiveness of artificial brain metastases established from four human melanoma cell lines.. A-07, D-12, R-18, and U-25 cells transfected with GFP were injected intracerebrally and intra-arterially in nude mice. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or by histological examination. Expression and secretion of factors involved in angiogenesis and invasion were assessed by quantitative PCR, ELISA, and immunohistochemistry.. The melanoma cells grew preferentially in the meninges and ventricles after intracerebral and intra-arterial injection. Intertumor heterogeneity in the aggressiveness of meningeal tumors reflected differences in angiogenic activity and expression of vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL-8). In contrast, growth and invasion of the brain parenchyma relied primarily on vascular co-option. The cell lines showed different patterns of invasion from meninges to the scull and from meninges to the brain parenchyma, and these differences were associated with differences in expression of the matrix metalloproteinases MMP-2 and MMP-9. Furthermore, the melanoma cells produced multiple brain lesions after intracerebral implantation by using the meningeal linings of the brain as transport routes.. The melanoma cell lines showed different growth patterns in the brain, and these differences were associated with differences in expression of the angiogenic factors VEGF-A and IL-8 and the matrix metalloproteinases MMP-2 and MMP-9.

Topics: Animals; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Humans; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Optical Imaging; Radiography; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Melanoma cells facilitate endothelial gap formation, the first step during tumor transendothelial migration, which is mediated by both adhesion and endogenously produced chemokines (in particular, interleukin-8 (IL-8)). Tetraspanins are localized to the cell surface in cancer and participate in various functions including invasion of tissues mediated by secretion of cytokines and matrix metalloproteinases. However, little is known about the role of CD82 tetraspanins in malignant melanomas during cancer cell invasion. In this study, we investigated the functional importance of CD82 expression in melanoma-mediated gap formation by using cDNAs to induce CD82 expression in highly invasive melanoma cell lines. Results showed that CD82 expression inhibited melanoma cell-induced gap formation, melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inducible expression of CD82 in highly metastatic melanoma cells significantly increased p21 expression upon binding of Duffy antigen receptor group (DARC), inducing tumor cell senescence and interrupting IL-8-mediated vascular endothelial (VE)-cadherin disassembly. Taken together, these studies provide a rationale for using drug therapies that restore CD82 expression and inhibit IL-8 production to inhibit late-stage melanoma cell extravasation and subsequent metastasis development.

Topics: Adult; Aged; Animals; Duffy Blood-Group System; Endothelial Cells; Female; Gene Expression; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Kangai-1 Protein; Lung Neoplasms; Male; Melanoma; Mice; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Protein Binding; Receptors, Cell Surface

Malignant melanoma cells are known to have altered expressions of growth factors as compared with normal melanocytes. Thrombomodulin (TM) is a thrombin receptor on endothelial cells that converts thrombin from a procoagulant to an anticoagulant enzyme. TM expression is downregulated in tumor cells, and this phenomenon correlates with tumor cell invasiveness and a poor prognosis in patients with cancer. In this study, we evaluated TM expression in two human melanoma cell lines that are known to have either low (WM35) or high (A375) aggressive phenotypes. Analysis by quantitative real-time PCR (qPCR) showed that the mRNA expression of TM is modestly (WM35) or dramatically (A375) downregulated in melanoma cells, as compared with human primary melanocytes. TM expression levels inversely correlated with in-vitro migration properties of tumor cells. In addition, interleukin-8 expression also correlated with the degree of aggressiveness, as indicated by high expression levels of this cytokine in A375 cells. Overexpression of TM in A375 cells by transient transfection reversed their aggressive phenotype and dramatically decreased interleukin-8 expression by these cells. Taken together, these results suggest that downregulation of TM plays a crucial role in melanocyte transformation and melanoma progression.

Topics: Adenomatous Polyposis Coli Protein; Antigens, CD; Cell Line, Tumor; Cell Movement; Disease Progression; Down-Regulation; Endothelial Protein C Receptor; Humans; Interleukin-8; Melanocytes; Melanoma; Neoplasm Invasiveness; Receptors, Cell Surface; RNA, Messenger; Thrombin; Thrombomodulin; Thromboplastin; Transfection

Glutamate-triggered signal transduction is thought to contribute widely to cancer pathogenesis. In melanoma, overexpression of the metabotropic glutamate receptor (GRM)-1 occurs frequently and its ectopic expression in melanocytes is sufficient for neoplastic transformation. Clinical evaluation of the GRM1 signaling inhibitor riluzole in patients with advanced melanoma has demonstrated tumor regressions that are associated with a suppression of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathways. Together, these results prompted us to investigate the downstream consequences of GRM1 signaling and its disruption in more detail. We found that melanoma cells with enhanced GRM1 expression generated larger tumors in vivo marked by more abundant blood vessels. Media conditioned by these cells in vitro contained relatively higher concentrations of interleukin-8 and VEGF due to GRM1-mediated activation of the AKT-mTOR-HIF1 pathway. In clinical specimens from patients receiving riluzole, we confirmed an inhibition of MAPK and PI3K/AKT activation in posttreatment as compared with pretreatment tumor specimens, which exhibited a decreased density of blood vessels. Together, our results demonstrate that GRM1 activation triggers proangiogenic signaling in melanoma, offering a mechanistic rationale to design treatment strategies for the most suitable combinatorial use of GRM1 inhibitors in patients.

Topics: Angiogenesis Inhibitors; Animals; Cell Movement; Heterocyclic Compounds, 3-Ring; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Inhibitor of Apoptosis Proteins; Interleukin-8; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Receptors, Metabotropic Glutamate; Riluzole; Signal Transduction; Sirolimus; Skin Neoplasms; Survivin; Tumor Burden; Vascular Endothelial Growth Factor A

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population characterized by immunosuppressive activity. Elevated levels of MDSC in peripheral blood are found in inflammatory diseases as well as in malignant tumors where they are supposed to be major contributors to mechanisms of tumor-associated tolerance. We investigated the frequency and function of MDSC in peripheral blood of melanoma patients and observed an accumulation of CD11b(+) CD33(+) CD14(+) HLA-DR(low) MDSC in all stages of disease (I-IV), including early stage I patients. Disease progression and enhanced tumor burden did not result in a further increase in frequencies or change in phenotype of MDSC. By investigation of specific MDSC-associated cytokines in patients' sera, we found an accumulation of IL-8 in all stages of disease. T-cell proliferation assays revealed that MDSC critically contribute to suppressed antigen-specific T-cell reactivity and thus might explain the frequently observed transient effects of immunotherapeutic strategies in melanoma patients.

Topics: Case-Control Studies; CD11b Antigen; Cell Proliferation; Cells, Cultured; Coculture Techniques; Disease Progression; HLA-DR Antigens; Humans; Interleukin-8; Leukocytes, Mononuclear; Lipopolysaccharide Receptors; Lymphocyte Activation; Lymphocyte Count; Melanoma; Myeloid Cells; Neoplasm Staging; Receptors, Antigen, T-Cell; Sialic Acid Binding Ig-like Lectin 3; Skin Neoplasms; T-Lymphocytes, Regulatory; Tetanus Toxoid; Tumor Burden; Tumor Escape

Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF-ERK (extracellular signal-regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)-ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor-resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

Topics: Animals; Blotting, Western; Cell Shape; Dimethyl Sulfoxide; Drug Resistance, Neoplasm; Enzyme-Linked Immunosorbent Assay; Humans; Indoles; Interleukin-8; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Skin Neoplasms; Statistics, Nonparametric; Sulfonamides

Imiquimod (IMQ) is a synthetic Toll-like receptor (TLR7/8) ligand that can trigger antiviral and antitumor activities. Despite evidence of potent therapeutic effects, the clinical use of IMQ in melanoma is impeded by incomplete understanding of its mechanisms of action. Mice and humans differ in many aspects of immunity, including TLR7 expression patterns, thus impeding the use of mouse models in translating discoveries into clinical applications. In this article, we investigated the mechanisms behind IMQ effects in vivo in a human context of melanoma and immunity using an innovative melanoma-bearing humanized mouse model. In this model, IMQ strongly inhibited melanoma tumor development through prompt mobilization of plasmacytoid dendritic cells and by triggering their cytotoxic functions, and through upregulation of expression of type 1 IFN response genes. IMQ also drastically impeded tumor vascularization by inducing the downregulation of angiogenic factors vascular endothelial growth factor, angiogenin, IL-8, and fibroblast growth factor. Our results revealed the short- and long-term multifactorial effects of IMQ converging toward inhibition of melanoma development. By providing a better understanding of the mechanisms of action of IMQ in melanoma, our study opens the way for its further clinical use in the treatment of metastatic melanoma.

Topics: Administration, Topical; Aminoquinolines; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Dendritic Cells; Disease Models, Animal; Down-Regulation; Fibroblast Growth Factors; Humans; Imiquimod; Interleukin-8; Melanoma; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Middle Aged; Neovascularization, Pathologic; Ribonuclease, Pancreatic; Skin Neoplasms; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The transcription factor NF-kappaB (NF-kB) is a key regulator of cytokine and chemokine production in melanoma and is responsible for symptoms such as anorexia, fatigue, and weight loss. In addition, NF-kB is believed to contribute to progression of the disease by upregulation of cell cycle and anti-apoptotic genes and to contribute to resistance against targeted therapies and immunotherapy. In this study, we have examined the ability of the bromodomain and extra-terminal (BET) protein inhibitor I-BET151 to inhibit NF-kB in melanoma cells. We show that I-BET151 is a potent, selective inhibitor of a number of NF-kB target genes involved in induction of inflammation and cell cycle regulation and downregulates production of cytokines such as IL-6 and IL-8. SiRNA studies indicate that BRD2 is the main BET protein involved in regulation of NF-kB and that I-BET151 caused transcriptional downregulation of the NF-kB subunit p105/p50. These results suggest that BET inhibitors may have an important role in treatment of melanoma where activation of NF-kB may have a key pathogenic role.

Topics: Animals; Apoptosis; Autocrine Communication; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chemokines; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Interleukin-6; Interleukin-8; Melanoma; Mice; NF-kappa B; Protein Serine-Threonine Kinases; Skin Neoplasms; Transcription Factors

The drug efflux transporter ABCB5 identifies cancer stem-like cells (CSC) in diverse human malignancies, where its expression is associated with clinical disease progression and tumor recurrence. ABCB5 confers therapeutic resistance, but other functions in tumorigenesis independent of drug efflux have not been described that might help explain why it is so broadly overexpressed in human cancer. Here we show that in melanoma-initiating cells, ABCB5 controls IL1β secretion, which serves to maintain slow cycling, chemoresistant cells through an IL1β/IL8/CXCR1 cytokine signaling circuit. This CSC maintenance circuit involved reciprocal paracrine interactions with ABCB5-negative cancer cell populations. ABCB5 blockade induced cellular differentiation, reversed resistance to multiple chemotherapeutic agents, and impaired tumor growth in vivo. Together, our results defined a novel function for ABCB5 in CSC maintenance and tumor growth.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Gene Expression; Heterografts; Humans; Interleukin-1beta; Interleukin-8; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Microarray Analysis; Neoplastic Stem Cells; Receptors, Interleukin-8A; Signal Transduction; Transfection

Spreading of melanoma is associated with efficient extravasation of circulating tumor cells from the vascular system into distant target organs. This process is accompanied and supported by proinflammatory and procoagulatory conditions. In this study, we analysed the ability of human melanoma cell lines to activate endothelial cells (ECs) in vitro. Some melanoma cells, that is, MV3, were shown to trigger an prompt calcium-flux-dependent, procoagulatory endothelial response that was accompanied by luminal release of ultra-large von Willebrand factor (ULVWF) fibres that were immobilized to the endothelial surface layer. In contrast to MV3-derived supernatant, prolonged treatment of ECs with WM9-derived supernatant mediated a pronounced activation of nuclear factor kappa B (NFκB). NFκB activation in ECs was dependent on both IL-1α and IL-1β secreted from melanoma cells. Melanoma-derived IL-1 mediated an upregulation of proinflammatory cytokines IL-6 and IL-8, the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and the procoagulatory tissue factor (TF) in ECs. Our data show that melanoma cells activate ECs either directly and within seconds or by an IL-1-mediated NFκB activation. Both pathways of EC activation convert the regular repressive function of ECs on inflammation and coagulation to a proinflammatory and procoagulatory surface that supports tumor progression.

Topics: Calcium Signaling; Capillary Permeability; Cell Line, Tumor; Cytokines; Disease Progression; Endothelium, Vascular; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-6; Interleukin-8; Melanoma; Models, Biological; NF-kappa B; Phenotype; Thromboplastin; Up-Regulation; Vascular Cell Adhesion Molecule-1; von Willebrand Factor

During melanoma cell extravasation through the vascular endothelium, melanoma cells interact with endothelial cells through secretion of cytokines and by adhesion between proteins displayed on opposing cell surfaces. How these tumor cell associated signals together regulate the dynamics of intracellular signaling pathways within endothelial cells leading to endothelial cell-cell junction disruption is not well understood. Here, we used a combination of experimental and computational approaches to examine the individual and combined effects of activation of the vascular cell adhesion molecule (VCAM)-1, interleukin (IL)-8, and IL-1β signaling pathways on the integrity of vascular junctions. Our simulations predict a multifaceted interplay of signaling resulting from individual activation of VCAM-1, IL-8 and IL-1β pathways that is neither synergistic nor additive compared to all inputs turned on simultaneously. Furthermore, we show that the levels of phosphorylated proteins associated with actinomyosin contractility and junction disassembly peak prior to those related to actin remodeling. The results of this work provide insight into the dynamics of tumor-mediated endothelial junction disassembly and suggest that targeting proteins downstream of several interaction pathways may be the most effective therapeutic approach to reduce melanoma extravasation.

Topics: Actins; Antigens, CD; Cadherins; Cell Line, Tumor; Coculture Techniques; Cytoskeleton; Endothelial Cells; Gene Knockout Techniques; Human Umbilical Vein Endothelial Cells; Humans; Intercellular Junctions; Interleukin-1beta; Interleukin-8; Melanoma; Models, Biological; Myosin-Light-Chain Kinase; Phosphorylation; Signal Transduction; Vascular Cell Adhesion Molecule-1

Melanoma is a multifactorial disease with a strong genetic component and known risk factors such as excessive ultraviolet exposure, intermittent sunburns and fair skin type. The prognosis is poor if diagnosis is delayed, in spite of recent treatment advances. Evidence is mounting that the incidence of melanoma is higher in the immunosuppressed and individuals with highly stressful occupations. We present a case series of individuals diagnosed with multiple cutaneous melanomas over a few months to 1 year. All had encountered psychological stressors in their lives, and the melanomas were diagnosed briefly after encountering these stressors. No known causes of immunosuppression were detected to explain the sporadic occurrence of melanomas in these individuals. There is evidence in the current literature that stress can lead to immune disregulation, predisposing an individual to various disease states including melanoma. Stress hormones such as norepinephrine have been shown to cause upregulation of cytokines such as Interleukin 6 and 8, which are proangiogenic and support tumour progression. Coupled with genetic and environmental factors, stress appears to play a role in melanoma formation and progression. Large prospective studies are required to study the link between stress and melanoma and gain further insight into the etiology of melanoma.

Topics: Anxiety; Dysplastic Nevus Syndrome; Female; Humans; Interleukin-6; Interleukin-8; Male; Melanoma; Middle Aged; Risk Factors; Skin Neoplasms; Stress, Psychological; Vascular Endothelial Growth Factor A

Dacarbazine induces a clinical response only in 15% of melanoma patients. New treatment strategies may involve combinations of drugs with different modes of action to target the tumor heterogeneity. We aimed to determine whether the combined treatment of heterogeneous melanoma cell populations in vitro with the alkylating agent dacarbazine and the nuclear factor-κB inhibitor parthenolide could be more effective than either drug alone. A panel of melanoma cell lines, including highly heterogeneous populations derived from surgical specimens, was treated with dacarbazine and parthenolide. The effect of drugs on the viable cell number was examined using an acid phosphatase activity assay, and the combination effect was determined by median-effect analysis. Cell death and cell-cycle arrest were assessed by flow cytometry. Gene expression was measured by real-time PCR and changes in the protein levels were evaluated by western blotting. Secretion of vascular endothelial growth factor and interleukin-8 was determined using an enzyme-linked immunosorbent assay. The self-renewing capacity was assessed using a clonogenic assay. Dacarbazine was less effective in heterogeneous melanoma populations than in the A375 cell line. Parthenolide and dacarbazine synergistically reduced the viable cell numbers. Both drugs induced cell-cycle arrest and apoptotic cell death. Importantly, parthenolide abrogated the baseline and dacarbazine-induced vascular endothelial growth factor secretion from melanoma cells in heterogeneous populations, whereas interleukin-8 secretion was not significantly affected by either drug. Parthenolide eradicated melanoma cells with self-renewing capacity also in cultures simultaneously treated with dacarbazine. The combination of parthenolide and dacarbazine might be considered as a new therapeutic modality against metastatic melanoma.

Topics: Antineoplastic Agents, Alkylating; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dacarbazine; Dose-Response Relationship, Drug; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Interleukin-8; Melanoma; NF-kappa B; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sesquiterpenes; Skin Neoplasms; Vascular Endothelial Growth Factor A

Introduction Uveal melanoma (UM) is a highly vascularised tumour generally treated with radiotherapy (RT). A recent preclinical study from our group [1] demonstrated that RT-associated anti-angiogenic therapy has more than additive effects on cell growth, by modulating vascular endothelial growth factor (VEGF) levels. The pro-angiogenic interleukin-8 (IL-8) is highly expressed in both tumour and endothelial cells and is associated with resistance to VEGF-targeted therapies in various tumour types. The aim of this study is to investigate IL-8 release in response to the anti-angiogenic drug bevacizumab (AV) and RT given alone and in combination. Material and methods The human ocular melanoma cells (OCM-1) and human umbilical vein endothelial cells (HUVEC) were grown in transwell plates. AV was administered at a 2,500 μg/ml dose and cells were irradiated with a 6 Gy dose. IL-8 concentrations were determined by ELISA assay. Protein expression was detected by western blot. Results AV alone or in combination with RT reduces VEGF levels in both cell lines when co-cultured; unexpectedly, RT alone did not increase VEGF levels. In transwell plate AV alone lowered IL-8 secretion in both cell lines. This inhibitory effect was reduced when co-cultured cells are treated with AV + RT, suggesting that RT-induced VEGF may reactivate IL-8 secretion, enhancing an alternative pathway to sustain tumour angiogenesis. Conclusions These data indicate that the UM microenvironment, beside VEGF, can activate IL-8 signalling as an alternative pro-angiogenic pathway.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Bevacizumab; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Combined Modality Therapy; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Melanoma; Neovascularization, Pathologic; Receptors, Interleukin-8B; Uveal Neoplasms; Vascular Endothelial Growth Factor A

Ingenol mebutate is the active ingredient in Picato® a new drug for the treatment of actinic keratosis. A number of derivatives related to ingenol mebutate were prepared by chemical synthesis from ingenol with the purpose of investigating the SAR and potency in assays relating to pro-inflammatory effects (induction of PMN oxidative burst and keratinocyte cytokine release), the potential of cell death induction, as well as the chemical stability. By modifications of the ingenol scaffold several prerequisites for activity were identified. The chemical stability of the compounds could be linked to an acyl migration mechanism. We were able to find analogues of ingenol mebutate with comparable in vitro properties. Some key features for potent and more stable ingenol derivatives have been identified.

Topics: Antineoplastic Agents; Apoptosis; Cell Line; Diterpenes; Humans; Interleukin-8; Keratinocytes; Keratosis, Actinic; Leukocytes, Mononuclear; Melanoma; Oxidative Stress; Reactive Oxygen Species; Skin Neoplasms; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Here, we explored the effects of the novel class II-specific histone deacetylase inhibitors (HDACis) MC1568 and MC1575 on interleukin-8 (IL-8) expression and cell proliferation in cutaneous melanoma cell line GR-M and uveal melanoma cell line OCM-3 upon stimulation with phorbol 12-myristate 13-acetate (PMA). We found that PMA upregulated IL-8 transcription via the AP-1 binding site and identified c-Jun as the transcription factor involved in this eventS. MC1568 and MC1575 inhibited IL-8 levels and cell proliferation in either unstimulated or PMA-stimulated melanoma cells. They acted by suppressing (i) c-Jun binding to the IL-8 promoter, (ii) recruitment of histones 3 and 4, RNA polymerase II and TFIIB to the c-Jun promoter, and (iii) c-Jun expression. Our findings provide new insights into mechanisms underlying anti-tumoral activities of class II-specific HDACis in human melanoma and suggest that they may constitute a novel therapeutic strategy for improving the treatment of this cancer.

Topics: Acetylation; Cell Line, Tumor; Cell Proliferation; DNA-Directed RNA Polymerases; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Interleukin-8; Melanoma; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-jun; Pyrroles; RNA, Messenger; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factor TFIIB; Transcription, Genetic; Uveal Neoplasms

Recent studies sight β-adrenergic receptor (AR) antagonists as novel therapeutic agents for melanoma, as they may reduce disease progression. Here within, we evaluated the expression of β-ARs in a series of human cutaneous melanocytic lesions, and studied the effect of their endogenous agonists, norepinephrine (NE) and epinephrine (E), on primary and metastatic human melanoma cell lines. Using immunohistochemistry, we found that both β1- and β2-ARs are expressed in tissues from benign melanocytic naevi, atypical naevi and malignant melanomas and that expression was significantly higher in malignant tumours. Melanoma cell lines (human A375 primary melanoma cell line and human Hs29-4T metastatic melanoma cell lines) also expressed β1- and β2-ARs by measuring transcripts and proteins. NE or E increased metalloprotease-dependent motility, released interleukin-6 and 8 (IL-6, IL-8) and vascular endothelial growth factor (VEGF). These effects of catecholamines were inhibited by the unselective β-AR antagonist propranolol. The role of soluble factors elicited by catecholamines seemed pleiotropic as VEGF synergized with NE increased melanoma invasiveness through 3D barriers, while IL-6 participated in stromal fibroblast activation towards a myofibroblastic phenotype. Our results indicate that NE and E produce in vitro via β-ARs activation a number of biological responses that may exert a pro-tumorigenic effect in melanoma cell lines. The observation that β-ARs are upregulated in malignant melanoma tissues support the hypothesis that circulating catecholamines NE and E, by activating their receptors, favour melanoma progression in vivo.

Topics: Adult; Blotting, Western; Cell Line, Tumor; Cytokines; DNA Primers; Epinephrine; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Male; Melanoma; Metalloproteases; Middle Aged; Norepinephrine; Real-Time Polymerase Chain Reaction; Receptors, Adrenergic, beta; Skin Neoplasms; Vascular Endothelial Growth Factor A

Recent studies have indicated an important role of proteinases and proteinase-activated receptors (PARs) in tumorigenesis. Although a role for PARs has been described in various skin tumors including melanoma, the underlying cellular mechanisms have not been understood. Recent studies have suggested PAR(1) as a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. Moreover, changes in the expression patterns of PAR(1) and PAR(2) correlate with skin cancer progression, and PAR(1) is overexpressed in melanoma. Therefore, we sought to elucidate the putative role of PAR(1)- and PAR(2)-mediated signal transduction pathways during melanoma progression. Activation of both PAR(1) and PAR(2) led to rapid phosphorylation of protein kinase D1 (PKD1) in cultured WM9 melanoma cells. PKD1 is known to be involved in cell migration, integrin regulation, and intracellular vesicle transport. Downregulation of PKD1 by siRNA resulted in diminished proliferation, decreased αvβ3 integrin regulation, and secretion of pro-angiogenic chemokine IL-8 in WM9 cells. In conclusion, our results show that PAR(1) and PAR(2) are involved in WM9 cell proliferation and secretion of IL-8 by activation of PKD1. Inactivation of the PKD1 pathway may be beneficial for the inhibition of PAR-induced melanoma proliferation and for maintenance of the inflammatory tumor environment.

Topics: Cell Line, Tumor; Cell Shape; Enzyme Activation; Humans; Integrin alphaVbeta3; Interleukin-8; Melanoma; Phosphorylation; Receptor, PAR-1; Receptor, PAR-2; Skin Neoplasms; TRPP Cation Channels

The treatment results of patients with locoregional melanoma are still not satisfactory - up to 50% of patients experience recurrence and (or) disease dissemination. The aim of the study was to assess the prognostic value of clinical factors and serum cytokines of patients with cutaneous melanoma in locoregional stage.. 149 patients at stage I-III according AJCC treated between 2007-2010 were included. Pre-surgery serum levels of VEGF IL-8 and sTNF-R1 were analyzed by ELISA method in 74 melanoma patients and 50 healthy controls. The median follow-up time was 16 months (range: 1-81 months).. The most important factors influencing the disease-free survival (DFS) are: staging system according to AJCC) (p < 0.001), regional nodal stage (pN) (p < 0.001), primary tumor (Breslow) thickness (pT) (p = 0.013) and melanoma ulceration (p = 0.004). The serum levels of selected cytokines were significantly higher in melanoma patients than in healthy volunteers (VEGF, p < 0.001; sTNF-R1, p < 0.001; IL-8, p = 0.001). There were no significant relationships between level of cytokine, recurrence or clinical/pathological parameters.. The AJCC staging system gives the most accurate insight into prognosis of melanoma patients at locoregional stage after primary therapy. Cytokine serum profile in melanoma patients at locoregional stage has limited value for predicting tumor burden and treatment outcomes.

Topics: Adult; Aged; Biomarkers, Tumor; Disease-Free Survival; Female; Humans; Interleukin-8; Lymphatic Metastasis; Male; Melanoma; Middle Aged; Neoplasm Staging; Prognosis; Skin Neoplasms; TNF Receptor-Associated Factor 1; Vascular Endothelial Growth Factor A

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.

Topics: Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Interleukin-8; Lymphangiogenesis; Lymphatic Metastasis; Lymphatic Vessels; Lysophospholipids; Melanoma; NF-kappa B; Receptors, Lysophosphatidic Acid; RNA, Small Interfering; Sentinel Lymph Node Biopsy; Signal Transduction; Up-Regulation

Side population (SP) cells are identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and it represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a substrate of ABCB1, both in vitro and in vivo. However, melanoma SP cells were also resistant to temozolomide, which is not a substrate for ABC transporters, through IL-8 upregulation. In addition, gene profiling studies identified three signaling pathways (NF-κB, α6-β4-integrin, and IL-1) as differentially upregulated in melanoma SP cells, and there was a significant increase of PCDHB11 and decrease of FUK and TBX2 in these cells. Therefore, we provide evidence that SP is an enriched source of chemoresistant cells in human melanomas, and suggest that the selected genes and signaling pathways of SP cells may be a potential target for effective melanoma therapies. To our knowledge, this is a previously unreported study to isolate SP cells from melanoma patients and to investigate the gene expression profiling of these cells.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Coloring Agents; Dacarbazine; Drug Resistance, Neoplasm; Female; Humans; In Vitro Techniques; Interleukin-8; Melanoma; Mice; Mice, Nude; Paclitaxel; Side-Population Cells; Skin Neoplasms; Temozolomide; Up-Regulation; Xenograft Model Antitumor Assays

Combination therapy of paclitaxel (taxol) with natural anti-tumor agents that are capable of inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Our previous study showed that icariside II (IS), derived from Herba Epimedii, inhibited the proliferation of melanoma cells in vivo and in vitro through the regulation of apoptosis. In this report, the combination effects of paclitaxel and IS were investigated in human melanoma A375 cells. As compared to the treatment with paclitaxel alone, the co-administration of IS and paclitaxel resulted in an enhancement of apoptosis as revealed by WST-8 and PI assays. Meanwhile, Western blot analysis showed that the co-administration of IS and paclitaxel resulted in increases of cleaved caspase-3, one of the terminal pro-apoptotic proteins. In melanoma, IL-8 and VEGF are positively correlated with disease stage and a high probability of progression. We demonstrated that treatment of A375 cells with IS in combination with paclitaxel resulted in a significant decrease in the production of IL-8 and VEGF, compared with paclitaxel alone. Recent studies suggest that TLR4-MyD88-ERK signaling may be a novel target for reversing chemoresistance to paclitaxel. Our flow cytometry and Western blot data showed that paclitaxel activated TLR4-MyD88-ERK signaling and that IS treatment could effectively inhibit this paclitaxel-induced activation of TLR4-MyD88-ERK signaling. In conclusion, this study demonstrated for the first time that IS could potentiate paclitaxel-induced apoptosis in melanoma cells. These effects were mediated, at least in part, by inhibiting the activation of the TLR4 signal transduction pathways. These findings support further preclinical evaluation of IS as a new potential anti-tumor agent.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Line, Tumor; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Flavonoids; Flow Cytometry; Humans; Interleukin-8; Melanoma; Paclitaxel; Signal Transduction; Toll-Like Receptor 4; Vascular Endothelial Growth Factor A

CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed, and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that overexpression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared with vector control-transfected cells. We also observed increased clonogenic ability, growth, and invasive potential of CXCL-8 overexpressing cells in vitro. Knockdown of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis.

Topics: Animals; Carcinogenesis; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Interleukin-8; Melanoma; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Stromal Cells

The switch of human melanoma cell phenotype from non to highly tumorigenic and metastatic is triggered by the increase of procathepsin L secretion, which modifies the tumour microenvironment. The aim of the present study was to identify components involved in the regulation of procathepsin L secretion in melanoma cells. We focused on Rab family members, i.e. Rab3A, Rab4A, Rab4B, Rab5A, Rab8A, Rab11A, Rab27A and Rab33A, which are involved in distinct regulatory pathways. From analysis of mRNA and protein expression of these Rab components and their knockdown by specific siRNAs (small interfering RNAs) it emerged that Rab4A protein is involved in the regulation of procathepsin L secretion. This result was strengthened as procathepsin L secretion was either inhibited by expression of a Rab4A dominant-negative mutant or increased by overexpression of the wild-type Rab4A. Rab4A regulation: (i) discriminates between procathepsin L secretion and expression of intracellular cathepsin L forms; (ii) did not modify other Rab proteins and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression, or IL-8 (interleukin-8) and MMP-2 (matrix metalloproteinase-2) secretion; and (iii) was still efficient during unglycosylated procathepsin L secretion. Thus down- or up-regulation of Rab4A expression or Rab4A function triggered inhibition or increase of procathepsin L secretion respectively. Furthermore, Rab4A regulation, by modifying procathepsin L secretion, switches the tumorigenic phenotype of human melanoma cells in nude mice.

Topics: Animals; Cathepsin L; Enzyme Precursors; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Interleukin-8; Matrix Metalloproteinase 2; Melanoma; Mice; Mice, Nude; rab4 GTP-Binding Proteins; RNA, Small Interfering; Tumor Cells, Cultured; Up-Regulation

Most melanoma cells are characterized by the V600E mutation in B-Raf kinase. This mutation leads to increased expression of interleukin (CXCL8), which plays a key role in cell growth and angiogenesis. Thus CXCL8 appears to be an interesting therapeutic target. Hence, we performed vaccination of mice with GST-CXCL8, which results in a reduced incidence of syngenic B16 melanoma cell xenograft tumors. We next addressed the molecular mechanisms responsible for aberrant CXCL8 expression in melanoma. The CXCL8 mRNA contains multiples AU-rich sequences (AREs) that modulate mRNA stability through the binding of tristetraprolin (TTP). Melanoma cell lines express very low TTP levels. We therefore hypothesized that the very low endogenous levels of TTP present in different melanoma cell lines might be responsible for the relative stability of CXCL8 mRNAs. We show that TTP is actively degraded by the proteasome and that extracellular-regulated kinase inhibition results in TTP accumulation. Conditional expression of TTP in A375 melanoma cells leads to CXCL8 mRNA destabilization via its 3' untranslated regions (3'-UTR), and TTP overexpression reduces its production. In contrast, downregulation of TTP by short hairpin RNA results in upregulation of CXCL8 mRNA. Maintaining high TTP levels in melanoma cells decreases cell proliferation and autophagy and induces apoptosis. Sorafenib, a therapeutic agent targeting Raf kinases, decreases CXCL8 expression in melanoma cells through reexpression of TTP. We conclude that loss of TTP represents a key event in the establishment of melanomas through constitutive expression of CXCL8, which constitutes a potent therapeutic target.

Topics: Animals; Antibodies; Antineoplastic Agents; Apoptosis; Autophagy; Benzamides; Benzenesulfonates; Cell Line, Tumor; Cell Proliferation; Chemokine CXCL1; Chemokine CXCL5; Dichlororibofuranosylbenzimidazole; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Genes, Reporter; Half-Life; Humans; Immunotherapy, Active; Interleukin-8; Leupeptins; MAP Kinase Kinase Kinases; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Niacinamide; Phenylurea Compounds; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins; Pyridines; Receptors, Interleukin-8B; RNA Stability; RNA, Messenger; RNA, Small Interfering; Sorafenib; Transfection; Tristetraprolin; Tumor Cells, Cultured; Vaccination

Melanoma accounts for only a small portion of skin cancer but it is associated with high mortality. Melanoma serum biomarkers that may aid early diagnosis or guide therapy are needed clinically. However, studies of serum biomarkers have often been hampered by the serum interference that causes false readouts in immunological tests. Here we show that, after using a special buffer to eliminate the serum interference, IL-8 and cathepsin B levels were significantly elevated in melanoma patients (p < 0.05). More importantly, the combination of IL-8 and cathepsin B were also studied as a prognosis marker for melanoma mortality. Our study provides a novel approach to examine serum biomarkers.

Topics: Aged; Biomarkers, Tumor; Breast Neoplasms; Cathepsin B; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Male; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Staging; Survival Analysis

The coincidence of skin tumors and elevated mast cell (MC) numbers has been known for many years. However, it has remained controversial whether, in this context, MCs promote or inhibit tumor growth. Addressing this problem, different melanoma and squamous cell carcinoma cell lines were co-cultivated with primary, dermal MC for 24 h and gene or protein expression of cytokines tumor necrosis factor (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) estimated. Co-culture with MCs led to an increase in IL-8 gene expression and IL-8 protein release from melanoma cells and IL-6 and IL-8 gene expression and protein release from squamous cell carcinoma cells, respectively. Moreover induction of IL-6 and IL-8 was primarily regulated by MC-derived TNF-α. Our data suggest an interplay between MCs and tumor cells, which results in altered cytokine release and may, thus, have an impact on tumor growth, invasion and neovascularisation.

Topics: Antibodies; Carcinoma, Squamous Cell; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Gene Expression; Histamine; Histamine H1 Antagonists; Histamine H2 Antagonists; Humans; Interleukin-6; Interleukin-8; Mast Cells; Melanoma; Skin Neoplasms; Tumor Necrosis Factor-alpha

Many melanoma cells continuously produce interleukin-8 (IL-8). The involvement of signal transducer and activator of transcription 3 (STAT3) in the constant production of IL-8 in melanoma cells was examined. The level of IL-8 production correlated well with that of the phosphorylated (activated) STAT3 in six human melanoma cell lines. Introduction of the constitutively activated form of STAT3 (STAT3-C) into WM35 melanoma cells, that show low levels of IL-8 and phosphorylated STAT3, enhanced IL-8 production. Knockdown of STAT3 suppressed IL-8 production in WM1205Lu cells that contain a high level of IL-8 accompanied by STAT3 phosphorylation. Introduction of STAT3-C markedly increased the luciferase activity in WM1205Lu cells transfected with reporter vectors linked to the 5'-flanking region of the IL-8 gene from -546 to +44 base pair (bp) and from -272 to +44 bp, but not in cells expressing reporter plasmids from -133 to +44 bp and from -98 to +44 bp. These results indicate that the upregulation of IL-8 production is caused by constitutive STAT3 activation at the level of gene transcription in melanoma cells.

Topics: Adenoviridae; Cell Line, Tumor; Humans; Interleukin-8; Luciferases; Melanoma; Phosphorylation; Plasmids; Skin Neoplasms; STAT3 Transcription Factor; Transcription, Genetic; Transfection; Up-Regulation

To complete the metastatic journey, cancer cells have to disseminate through the circulation and extravasate to distal organs. However, the extravasation process, by which tumor cells leave a blood vessel and invade the surrounding tissue from the microcirculation, remains poorly understood at the molecular level. In this study, tumor cell adhesion to the endothelium (EC) and subsequent extravasation were investigated under various flow conditions. Results have shown polymorphonuclear neutrophils (PMNs) facilitate melanoma cell adhesion to the EC and subsequent extravasation by a shear-rate dependent mechanism. Melanoma cell-PMN interactions are mediated by the binding between intercellular adhesion molecule-1 (ICAM-1) on melanoma cells and beta2 integrins on PMNs. In addition, the fluid convection affects the extent of activation of beta2 integrins on PMNs by endogenously secreted interleukin 8 (IL-8) within the tumor microenvironment. Results also indicate that shear rate affects the binding kinetics between PMNs and melanoma cells, which may contribute to the shear-rate dependence of melanoma extravasation in a shear flow when mediated by PMNs.

Topics: Animals; Biomechanical Phenomena; CD18 Antigens; Cell Adhesion; Cell Line, Tumor; Coculture Techniques; Endothelial Cells; Humans; Interleukin-8; L Cells; Leukocyte Rolling; Leukocytes; Lymphocyte Function-Associated Antigen-1; Macrophage-1 Antigen; Melanoma; Mice; Neoplasm Metastasis; Neutrophils; Rheology; Signal Transduction

It is unknown why only a minority of circulating tumor cells trapped in lung capillaries form metastases and involvement of immune cells remains uncertain. A novel model has been developed in this study showing that neutrophils regulate lung metastasis development through physical interaction and anchoring of circulating tumor cells to endothelium. Human melanoma cells were i.v. injected into nude mice leading to the entrapment of many cancer cells; however, 24 hours later, very few remained in the lungs. In contrast, injection of human neutrophils an hour after tumor cell injection increased cancer cell retention by approximately 3-fold. Entrapped melanoma cells produced and secreted high levels of a cytokine called interleukin-8 (IL-8), attracting neutrophils and increasing tethering beta(2) integrin expression by 75% to 100%. Intercellular adhesion molecule-1 on melanoma cells and beta(2) integrin on neutrophils interacted, promoting anchoring to vascular endothelium. Decreasing IL-8 secretion from melanoma cells lowered extracellular levels by 20% to 50%, decreased beta(2) integrin on neutrophils by approximately 50%, and reduced neutrophil-mediated extravasation by 25% to 60%, resulting in approximately 50% fewer melanoma cells being tethered to endothelium and retained in lungs. Thus, transendothelial migration and lung metastasis development decreased by approximately 50%, showing that targeting IL-8 in melanoma cells has the potential to decrease metastasis development by disrupting interaction with neutrophils.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Coculture Techniques; Female; Humans; Interleukin-8; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Neoplastic Cells, Circulating; Neutrophils; RNA, Small Interfering; Skin Neoplasms

Analysis of cytokine and chemokine production by tumor cell lines including five lung cancers, a malignant mesothelioma, and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma, and the malignant melanoma. We investigated the migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3(+) CD4 regulatory T cells (Tregs) in migrated T cells to both IL-6- and IL-8-producing tumors. Marked induction of CXCR1 expression on Foxp3(+) CD4 Tregs by IL-6 followed by IL-8-mediated migration appeared to be responsible for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8R expression by IL-6 is one of the mechanisms for tumor escape.

Topics: CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cell Movement; Forkhead Transcription Factors; Humans; Interleukin-6; Interleukin-8; Lung Neoplasms; Melanoma; Mesothelioma; Receptors, Interleukin-8A; T-Lymphocytes, Regulatory; Tumor Cells, Cultured; Tumor Escape

Melanoma is one of the most therapy-resistant cancers. Activating mutations in BRAF and NRAS are the source of extracellular signal regulated protein kinase (ERK) pathway activation. We show that melanoma cell lines, originating in different metastatic sites, with BRAF or NRAS mutations, in addition to active mitogen activated protein kinase (MAPK)-ERK, also have highly activated stress activated protein kinase (SAPK)-p38. This is in direct contrast to carcinoma cells in which the activity of the two kinases appears to be mutually exclusive; high level of p38 activity inhibits, through a negative feedback, ERK activity and prevents tumorigenesis. Melanomas are insensitive to ERK inhibition by p38 and utilize p38-signaling pathway for migration and growth in vivo. We found a positive functional loop linking the high ERK activity to surface expression of alphaVbeta3-integrin. This integrin, by interacting with vitronectin, induces p38 activity and increases IL-8 production, enhancing cell migration. Because the negative loop from p38 to ERK is lost, the two kinases can remain simultaneously activated. Inhibition of ERK and p38 activities is more effective in blocking in vivo growth than inhibition of each kinase individually. Future therapies might have to consider targeting of both pathways.

Topics: Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; Genes, ras; Humans; Integrin alphaVbeta3; Interleukin-8; Melanoma; Mutation; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins B-raf; Tumor Cells, Cultured

Studies suggest that stress can be a co-factor for the initiation and progression of cancer. The catecholamine stress hormone, norepinephrine (NE), may influence tumor progression by modulating the expression of factors implicated in angiogenesis and metastasis. The goal of this study was to examine the influence of NE on the expression of VEGF, IL-8, and IL-6 by the human melanoma cell lines, C8161, 1174MEL, and Me18105. Cells were treated with NE and levels of VEGF, IL-8, and IL-6 were measured using ELISA and real-time PCR. The expression of beta-adrenergic receptors (beta-ARs) mRNA and protein were also assessed. Finally, immunohistochemistry was utilized to examine the presence of beta1- and beta2-AR in primary and metastatic human melanoma biopsies. We show that NE treatment upregulated production of VEGF, IL-8, and IL-6 in C8161 cells and to a lesser extent 1174MEL and Me18105 cells. The upregulation was associated with induced gene expression. The effect on C8161 cells was mediated by both beta1- and beta2-ARs. Furthermore, 18 of 20 melanoma biopsies examined expressed beta2-AR while 14 of 20 melanoma biopsies expressed beta1-AR. Our data support the hypothesis that NE can stimulate the aggressive potential of melanoma tumor cells, in part, by inducing the production VEGF, IL-8, and IL-6. This line of research further suggests that interventions targeting components of the activated sympathetic-adrenal medullary (SAM) axis, or the utilization of beta-AR blocking agents, may represent new strategies for slowing down the progression of malignant disease and improving cancer patients' quality of life.

Topics: Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-16; Interleukin-8; Melanoma; Norepinephrine; Polymerase Chain Reaction; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; RNA, Messenger; Stress, Physiological; Up-Regulation; Vascular Endothelial Growth Factor A

Redox dysregulation in cancer cells represents a chemical vulnerability that can be targeted by pro-oxidant redox intervention. Dietary constituents that contain an electrophilic Michael acceptor pharmacophore may therefore display promising chemopreventive and chemotherapeutic anti-cancer activity. Here, we demonstrate that the cinnamon-derived dietary Michael acceptor trans-cinnamic aldehyde (CA) impairs melanoma cell proliferation and tumor growth. Feasibility of therapeutic intervention using high doses of CA (120 mg/kg, po, daily, 10 days) was demonstrated in a human A375 melanoma SCID mouse xenograft model. Low-micromolar concentrations (IC(50)< 10 microM) of CA, but not closely related CA derivatives devoid of Michael acceptor activity, suppressed proliferation of human metastatic melanoma cell lines (A375, G361, LOX) with G1 cell-cycle arrest, elevated intracellular ROS, and impaired invasiveness. Expression array analysis revealed that CA induced an oxidative stress response in A375 cells, up-regulating heme oxygenase 1, sulfiredoxin 1 homolog, thioredoxin reductase 1, and other genes, including the cell-cycle regulator and stress-responsive tumor suppressor gene cyclin-dependent kinase inhibitor 1A, a key mediator of G1-phase arrest. CA, but not Michael-inactive derivatives, inhibited NF-kappaB transcriptional activity and TNFalpha-induced IL-8 production in A375 cells. These findings support a previously unrecognized role of CA as a dietary Michael acceptor with potential anti-cancer activity.

Topics: Acrolein; Animals; Cell Line, Tumor; Cell Proliferation; Cinnamomum zeylanicum; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase; Gene Expression Profiling; Heme Oxygenase-1; Humans; Interleukin-8; Melanoma; Mice; Mice, SCID; Microarray Analysis; Neoplasm Invasiveness; Neoplasm Transplantation; NF-kappa B; Oxidative Stress; Oxidoreductases Acting on Sulfur Group Donors; Plant Growth Regulators; Signal Transduction; Thioredoxin Reductase 1; Tumor Necrosis Factor-alpha

Crucial steps in tumor growth and metastasis are proliferation, survival, and neovascularization. Previously, we have shown that receptors for CXCL-8, CXCR1, and CXCR2 are expressed on endothelial cells and CXCR2 has been shown to be a putative receptor for angiogenic chemokines. In this report, we examined whether tumor angiogenesis and growth of CXCL-8-expressing human melanoma cells are regulated in vivo by a host CXCR2-dependent mechanism. We generated mCXCR2(-/-), mCXCR2(+/-), and wild-type nude mice following crosses between BALB/c mice heterozygous for nude(+/-) and heterozygous for mCXCR2(+/-). We observed a significant inhibition of human melanoma tumor growth and experimental lung metastasis in mCXCR2(-/-) mice as compared with wild-type nude mice. Inhibition in tumor growth and metastasis was associated with a decrease in melanoma cell proliferation, survival, inflammatory response, and angiogenesis. Together, these studies show the importance of host CXCR2-dependent CXCL-8-mediated angiogenesis in the regulation of melanoma growth and metastasis.

Topics: Animals; Cell Growth Processes; Cell Line, Tumor; Female; Humans; Interleukin-8; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Neovascularization, Pathologic; Receptors, Interleukin-8B

In view of several studies highlighting an observation of an erythematous eruption in the vicinity of or distant from the lesion in melanoma patients (The Brenner sign), this study sought to assess whether this phenomenon might be related to the blood level of cytokines IL-6 and IL-8.. Sera specimens obtained from 27 patients with melanoma, of which 15 had erythematous eruptions and 12 did not, were studied by immunohistochemistry for the expression of IL-6 and IL-8.. IL-6 was detected in all melanoma patients in both groups. The mean level of IL-6 in the redness group (2.41 pg/L) was significantly higher than in the group without redness (1.25 pg/L). IL-8 was detected in all 27 melanoma patients in the two groups. The serum level was less than 5 pg/L in only 1 patient (6.7%) in the redness group, and in 6 patients (50%) in the group without redness, a statistically significant difference.. The Brenner sign appears to reflect a more advanced disease and herald a poor prognosis according to its correlation with the IL-8 and IL-6 blood level. However, in view of the biphasic effect of IL-8 level on tumor progression, and IL-6's ability to inhibit early stage melanoma, redness in melanoma patients could be a sign of a better prognosis of the melanoma.

Topics: Erythema; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Melanoma

We examined the autocrine/paracrine role of interleukin-8 (CXCL8) and the functional significance of CXCL8 receptors, CXCR1 and CXCR2, in human malignant melanoma proliferation, migration, invasion and angiogenesis. We found that a panel of seven cell lines, even though at different extent, secreted CXCL8 protein, and expressed CXCR1 and CXCR2 independently from the CXCL8 expression, but depending on the oxygen level. In fact, hypoxic exposure increases the expression of CXCR1 and CXCR2. The cell proliferation of both M20 and A375SM lines, expressing similar levels of both CXCR1 and CXCR2 but secreting low and high amounts of CXCL8, respectively, was significantly enhanced by CXCL8 exposure and reduced by CXCL8, CXCR1 and CXCR2 neutralising antibodies, indicating the autocrine/paracrine role of CXCL8 in melanoma cell proliferation. Moreover, an increased invasion and migration in response to CXCL8 was observed in several cell lines, and a further enhancement evidenced under hypoxic conditions. A CXCL8-dependent in vivo vessel formation, evaluated through a matrigel assay, was also demonstrated. Furthermore, when neutralising antibodies against CXCR1 or CXCR2 were used, only the involvement of CXCR2, but not CXCR1 was observed on cell migration and invasion, while both receptors played a role in angiogenesis. In summary, our data demonstrate that CXCL8 induces cell proliferation and angiogenesis through both receptors and that CXCR2 plays an important role in regulating the CXCL8-mediated invasive and migratory behaviour of human melanoma cells. Thus, blocking the CXCL8 signalling axis promises an improvement for the therapy of cancer and, in particular, of metastatic melanoma.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Interleukin-8; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Neovascularization, Pathologic; Receptors, Interleukin-8A; Receptors, Interleukin-8B

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for migration of tumor cells out of the vascular system to establish metastases. We found that neutrophils (PMN) increased melanoma cell extravasation. Endogenous IL-8 liberated from melanoma cells or from PMN induced by melanoma cells contributed to PMN-facilitated melanoma cell arrest on the EC in the microcirculation. Functional blocking of IL-8 receptors on PMN or neutralizing soluble IL-8 in the tumor circulation decreased the level of CD11b/CD18 up-regulation on PMN and subsequently reduced melanoma cell extravasation. We also found that targeting mutant V600EB-Raf interrupted melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. B-Raf encodes a RAS-regulated kinase that mediates cell growth and malignant transformation kinase pathway activation. Results showed that inhibition of V600EB-Raf reduced IL-8 secretion from melanoma cells and reduced the capacity of IL-8 production from the tumor microenvironment involving PMN. Furthermore, reduction in intercellular adhesion molecule-1 (ICAM-1) expression on melanoma cells was found after V600EB-Raf knockdown. These results provide new evidence for the complex role of secreted chemokine and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the EC, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Coculture Techniques; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-8; Leukocytes; Lung Neoplasms; Melanoma; Mice; Neoplasm Metastasis; RNA, Small Interfering

Cancer patients undergoing treatment with systemic cancer chemotherapy drugs often have abnormal growth factor and cytokine profiles. Thus, serum levels of interleukin-8 (IL-8) are elevated in patients with malignant melanoma. In addition to IL-8, aggressive melanoma cells secrete, through its transcriptional regulator hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), which promotes angiogenesis and metastasis of human cancerous cells. Whether these responses are related to adenosine, a ubiquitous mediator expressed at high concentrations in cancer and implicated in numerous inflammatory processes, is not known and is the focus of this study. We have examined whether the DNA-damaging agents etoposide (VP-16) and doxorubicin can affect IL-8, VEGF, and HIF-1 expressions in human melanoma cancer cells. In particular, we have investigated whether these responses are related to the modulation of the adenosine receptor subtypes, namely, A(1), A(2A), A(2B), and A(3). We have demonstrated that A(2B) receptor blockade can impair IL-8 production, whereas blocking A(3) receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with VP-16 and doxorubicin. This understanding may present the possibility of using adenosine antagonists to reduce chemotherapy-induced inflammatory cytokine production and to improve the ability of chemotherapeutic drugs to block angiogenesis. Consequently, we conclude that adenosine receptor modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively.

Topics: Blotting, Western; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Enzyme-Linked Immunosorbent Assay; Etoposide; Flow Cytometry; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; MAP Kinase Signaling System; Melanoma; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptor, Adenosine A2B; Receptor, Adenosine A3; Receptors, Purinergic P1; RNA Interference; Vascular Endothelial Growth Factor A

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glucose-6-Phosphate Isomerase; Humans; Interleukin-8; Melanoma; Models, Biological; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Skin Neoplasms

Galectin-3 (Gal-3) is a beta-galactoside-binding protein that is involved in cancer progression and metastasis. Using a progressive human melanoma tissue microarray, we previously demonstrated that melanocytes accumulate Gal-3 during the progression from benign to dysplastic nevi to melanoma and further to metastatic melanoma. Herein, we show that silencing of Gal-3 expression with small hairpin RNA results in a loss of tumorigenic and metastatic potential of melanoma cells. In vitro, Gal-3 silencing resulted in loss of tumor cell invasiveness and capacity to form tube-like structures on collagen ("vasculogenic mimicry"). cDNA microarray analysis after Gal-3 silencing revealed that Gal-3 regulates the expression of multiple genes, including endothelial cell markers that appear to be aberrantly expressed in highly aggressive melanoma cells, causing melanoma cell plasticity. These genes included vascular endothelial-cadherin, which plays a pivotal role in vasculogenic mimicry, as well as interleukin-8, fibronectin-1, endothelial differentiation sphingolipid G-protein receptor-1, and matrix metalloproteinase-2. Chromatin immunoprecipitation assays and promoter analyses revealed that Gal-3 silencing resulted in a decrease of vascular endothelial-cadherin and interleukin-8 promoter activities due to enhanced recruitment of transcription factor early growth response-1. Moreover, transient overexpression of early growth response-1 in C8161-c9 cells resulted in a loss of vascular endothelial-cadherin and interleukin-8 promoter activities and protein expression. Thus, Gal-3 plays an essential role during the acquisition of vasculogenic mimicry and angiogenic properties associated with melanoma progression.

Topics: Animals; Antigens, CD; Apoptosis; Cadherins; Cell Line; Cell Proliferation; Female; Galectin 3; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Interleukin-8; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Microvessels; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis

Interleukin-8 (IL-8) is an important mediator in neutrophil-mediated acute inflammation but has also a wide range of actions on various cells types. We demonstrated that infection of melanoma cells and fibroblasts with cell-associated varicella-zoster virus (VZV) and infection of a T cell line with cell-free VZV resulted in an induction of IL-8 secretion in vitro. The inhibition of the VZV replication with a drug interfering with its DNA replication had no effect on the IL-8 release. Since the IL-8 promoter contains binding sites for NF-kappaB and AP-1, melanoma cells and the T cell line were treated with inhibitors of NF-kappaB, JNK/SAPK or p38/MAPK prior to infection. In melanoma cells, the JNK/SAPK pathway was shown to be important for the IL-8 secretion during the VZV replication, whereas in the T cell line, not only the JNK/SAPK but also the p38/MAPK pathways were required for IL-8 secretion. The neutralisation of the IL-8 bioactivity had no significant consequence on the VZV replication, suggesting that IL-8 acts neither as a proviral nor as an antiviral cytokine during the VZV replication in vitro.

Topics: Cell Line, Tumor; Fibroblasts; Herpesviridae Infections; Herpesvirus 3, Human; Humans; Interleukin-1beta; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Melanoma; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; T-Lymphocytes; Transcription, Genetic; Tumor Necrosis Factor-alpha; Virus Replication

Small fragments of the extracellular matrix component hyaluronic acid (sHA) are typically produced at sites of inflammation and tissue injury and have been shown to be associated with tumor invasiveness and metastasis. Here we report that exposure of human melanoma cells to sHA leads to nuclear factor kB (NFk-B) activation followed by enhanced expression of matrix metalloprotease (MMP) 2 and interleukin (IL)-8, factors that can contribute to melanoma progression. At the receptor level, we found that Toll-like receptor (TLR) 4 is involved in this signalling pathway, similar to the case in dendritic and endothelial cells. Specifically, we found that melanoma cells expressed TLR4 on their surface in vivo and in vitro, and using specific siRNA, we could clearly demonstrate the functional importance of TLR4 in sHA-triggered activation of IL-8 expression in melanoma cells. Furthermore, we also found that sHA treatment enhanced the motility of melanoma cells, an effect that could again be blocked by TLR4-specific siRNA. Together, our results suggest that sHA in melanoma might promote tumor invasiveness by inducing MMP- and cytokine-expression, in part in a TLR4-dependent manner, providing new insights into the relationship between cancer and innate immunity.

Topics: Adjuvants, Immunologic; Cell Line, Tumor; Cell Movement; Cell Nucleus; Disease Progression; Humans; Hyaluronic Acid; Immunity, Innate; Interleukin-8; Matrix Metalloproteinase 2; Melanoma; NF-kappa B; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Toll-Like Receptor 4; Up-Regulation

B-RAF mutations have been identified in the majority of melanoma and a large fraction of colorectal and papillary thyroid carcinoma. Drug discovery efforts targeting mutated B-RAF have yielded several interesting molecules, and currently, three compounds are undergoing clinical evaluation. Inhibition of B-RAF in animal models leads to a slowing of tumor growth and, in some cases, tumor reduction. Described within is a novel series of diaryl imidazoles with potent, single-digit nanomolar, anti-B-RAF activity. One compound from this series has been detailed here and has been shown to block B-RAF(V600E)-dependent extracellular signal-regulated kinase 1/2 phosphorylation in SK-MEL-28 melanoma cells as well as soft agar colony formation and proliferation. Importantly, interleukin-8 (IL-8) was identified by quantitative real-time PCR and ELISA as a product of the elevated mitogen-activated protein kinase signaling in these cells. Plasma concentrations of IL-8 in mice bearing melanoma xenografts were significantly reduced following exposure to B-RAF inhibitors. Taken together, these data suggest that IL-8 could serve as a tractable clinical biomarker.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Humans; Imidazoles; Interleukin-8; Melanoma; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; RNA, Messenger; Transplantation, Heterologous

Inflammation facilitates tumor progression including metastasis. Interleukin-8 (IL-8) is a chemokine that regulates polymorphonuclear neutrophil (PMN) mobilization and activity and we hypothesize that this cytokine influences tumor behavior. We have demonstrated that IL-8 is crucial for PMN-mediated melanoma extravasation under flow conditions. In addition, IL-8 is up-regulated in PMNs upon co-culturing with melanoma cells. Melanoma cells induce IkappaB-alpha degradation in PMNs indicating that NF-kappaB signaling is active in PMNs. Furthermore, the production of IL-8 in PMNs is NF-kappaB dependent. We have further identified that interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) from PMN-melanoma co-cultures synergistically contribute to IkappaB-alpha degradation and IL-8 synthesis in PMNs. Taken together, these findings show that melanoma cells induce PMNs to secrete IL-8 through activation of NF-kappaB and suggest a model in which this interaction promotes a microenvironment that is favorable for metastasis.

Topics: Cell Communication; Cell Line, Tumor; Cell Movement; Cells, Cultured; Cytokines; Humans; Inflammation; Interleukin-8; Melanoma; Neutrophils; NF-kappa B; Transfection

Previous reports have shown that cellular functions could be influenced by visual light (400-700 nm). Recent evidence indicates that cellular proliferation could be triggered by the interaction of a helium-neon laser (He-Ne laser, 632.8 nm) with the mitochondrial photoacceptor-cytochrome c oxidase. Our previous studies demonstrated that He-Ne irradiation induced an increase in cell proliferation, but not migration, in the melanoma cell line A2058 cell. The aim of this study was to investigate the underlying mechanisms involved in photostimulatory effects induced by an He-Ne laser. Using the A2058 cell as a model for cell proliferation, the photobiologic effects induced by an He-Ne laser were studied. He-Ne irradiation immediately induced an increase in mitochondrial membrane potential (delta psi(mt)), ATP, and cAMP via enhanced cytochrome c oxidase activity and promoted phosphorylation of Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) expressions. He-Ne irradiation-induced A2058 cell proliferation was significantly abrogated by the addition of delta psi(mt) and JNK inhibitors. Moreover, treatment with an He-Ne laser resulted in delayed effects on IL-8 and transforming growth factor-beta1 release from A2058 cells. These results suggest that He-Ne irradiation elicits photostimulatory effects in mitochondria processes, which involve JNK/AP-1 activation and enhanced growth factor release, and ultimately lead to A2058 cell proliferation.

Topics: Adenosine Triphosphate; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Electron Transport Complex IV; Helium; Humans; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Ki-67 Antigen; Lasers; Melanoma; Membrane Potentials; Mitochondria; Neon; Phosphorylation; Transcription Factor AP-1; Transforming Growth Factor beta1

Monocytes differentiate into dendritic cells (DC) in response to GM-CSF combined with other cytokines including IL-4 and IL-15. Here, we show that IL15-DC are efficient in priming naive CD8+ T cells to differentiate into melanoma antigen-specific cytotoxic T lymphocytes (CTL). While both melanoma peptide-pulsed IL15-DC and IL4-DC expand high-precursor frequency MART-1-specific CD8+ T cells after two stimulations in vitro, IL15-DC require much lower peptide concentration for priming. IL15-DC are more efficient in expanding gp100-specific CD8+ T cells and can expand CD8+ T cells specific for Tyrosinase and MAGE-3. CTL primed by IL15-DC are superior in their function as demonstrated by (i) higher IFN-gamma secretion, (ii) higher expression of Granzyme B and Perforin, and (iii) higher killing of allogeneic melanoma cell lines, most particularly the HLA-A*0201+ Sk-Mel-24 melanoma cells that are resistant to killing by CD8+ T cells primed with IL4-DC. Supernatants of the sonicated cells demonstrate unique expression of IL-1, IL-8 and IL-15. Therefore, membrane-bound IL-15 might contribute to enhanced priming by IL15-DC. Thus, IL-15 induces myeloid DC that are efficient in priming and maturation of melanoma antigen-specific CTL.

Topics: Antigens, CD; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Cytotoxicity Tests, Immunologic; Dendritic Cells; Granzymes; Humans; Interferon-gamma; Interleukin-1; Interleukin-15; Interleukin-4; Interleukin-8; K562 Cells; Leukocyte Common Antigens; Lipopolysaccharides; Lymphocytes; Melanoma; Monocytes; Monophenol Monooxygenase; Neoplasm Proteins; Receptors, CCR7; Receptors, Chemokine; T-Lymphocytes, Cytotoxic

Ultraviolet radiation (UVR) is known to be involved in the initiation and progression of malignant melanoma. Many studies have focused on the initiation of melanoma, but less is known about the effect of UVR on established tumor cells. Here, we show that after ultraviolet-B (UVB) irradiation, melanoma cells (MM) are able to secrete autocrine factors that enhance their motility. Time-lapse videomicroscopy of UVB irradiated (15 or 30 mJ/cm(2)) MM showed an initial decrease in MM cell motility one hour after irradiation, with subsequent increase 24 h after UV-B treatment. Conditioned media harvested from MM 24 h following UV-B irradiation specifically enhanced the motility of un-irradiated MM, suggesting that a newly synthesized soluble factor released by UVB MM is involved. As interleukin 8 (IL-8) is known to be up-regulated by different cell types after UV-B irradiation, we investigated IL-8 expression after UVB exposure. Quantitative RT-PCR and ELISA demonstrated an induction of IL-8 in MM by UVB (15 or 30 mJ/cm(2)), and addition of recombinant IL-8 to cell cultures enhanced cell motility to a similar degree than UVB. Importantly, blocking IL-8 activity by a neutralizing anti IL-8 antibody inhibited the up-regulation of MM motility after UVB treatment. We conclude that UVB enhances MM motility and that this effect is mediated at least in part by IL-8 released by MM in an autocrine fashion. Our findings are consistent with the hypothesis that UVB is not only involved in the initiation of melanoma, but may also be important for some aspects of tumor progression.

Topics: Actin Cytoskeleton; Actins; Antibodies; Autocrine Communication; Cell Line, Tumor; Cell Movement; Gene Expression; Humans; Interleukin-8; Melanoma; RNA, Messenger; Skin Neoplasms; Solubility; Ultraviolet Rays

Endotoxin/lipopolysacccharide (LPS) is a potent inflammatory stimulus, which acts on tumour infiltrating leukocytes by eliciting a wide range of factors promoting invasion and metastasis. Less known is the effect of LPS directly on tumour cells. In this study, we analysed whether tumour cell lines from different origin (melanoma, ovarian carcinoma, neuroblastoma) are responsive to LPS in vitro. Results showed that only melanoma cells significantly up-regulated the production of IL-8 and cell adhesion, when triggered with LPS. These effects were associated with the constitutive expression of TLR-4 mRNA in these cells and the expression on the cell membrane of the complete LPS-binding receptor.

Topics: Cell Adhesion; Cell Proliferation; Escherichia coli; Female; Humans; Interleukin-8; Lipopolysaccharides; Melanoma; Neuroblastoma; Ovarian Neoplasms; RNA, Messenger; Toll-Like Receptor 4; Tumor Cells, Cultured

We examined the expression of CXCL8 (interleukin-8), its receptors, CXCR1 and CXCR2, and vessel density in human melanoma by immunohistochemical analysis of tumors from different Clark levels, depths, and thicknesses. Expression of CXCL8 and CXCR2 was lower in Clark level I and II specimens than in level III through V specimens and metastases. CXCR1 expression was observed ubiquitously in the majority of human melanoma tumor specimens irrespective of disease state, with the highest intensity in Clark level III specimens. We observed a significant difference in CXCL8 and CXCR2 expression between thin (0.75 mm) melanomas and between thin and metastatic lesions. Positive correlations were observed between Clark level and CXCL8 or CXCR2 and between thickness and CXCR2 expression. We found no correlation between vessel density and Clark level or thickness. Our data suggest that expression of CXCL8 and CXCR2 contributes to aggressive growth and metastasis in human malignant melanoma. Consistent with the transition from radial to vertical growth phase melanoma, expression of CXCL8 and its receptor, CXCR2, may be key in the switch to an aggressive, more metastatic phenotype.

Topics: Disease Progression; Humans; Immunohistochemistry; Interleukin-8; Melanoma; Neovascularization, Pathologic; Receptors, Interleukin-8A; Receptors, Interleukin-8B

It has been determined previously that polymorphonuclear leukocytes, or PMNs, can facilitate melanoma cell extravasation through the endothelium under shear conditions. The interactions between melanoma cells and PMNs are mediated by the beta2-integrins expressed by PMNs and intercellular adhesion molecules (ICAM-1) expressed on melanoma cells. In this study, the kinetics of these interactions was studied using a parallel plate flow chamber. The dissociation rates were calculated under low force conditions for ICAM-1 interactions with both beta2-integrins, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), together and separately by using functional blocking antibodies on PMNs. The kinetics of PMNs stimulated with IL-8 was also determined. It was concluded that the small number of constitutively expressed active beta2-integrins on PMNs are sufficient to bind to ICAM-1 expressed on melanoma cells and that the intrinsic dissociation rate for these adhesion molecules appear to be more dependent on what method is used to determine them than on what cells express them.

Topics: Adult; CD18 Antigens; Cell Adhesion; Cell Line, Tumor; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Kinetics; Lymphocyte Function-Associated Antigen-1; Macrophage-1 Antigen; Melanoma; Neutrophils; Skin Neoplasms

Previously, we found polymorphonuclear neutrophils (PMNs) increased melanoma cell extravasation under flow conditions (Intl J Cancer 106: 713-722, 2003). In this study, we characterized the effect of hydrodynamic shear on PMN-facilitated melanoma extravasation using a novel flow-migration assay. The effect of shear stress and shear rate on PMN-facilitated melanoma extravasation was studied by increasing the medium viscosity with dextran to increase shear stress independently of shear rate. Under fixed shear rate conditions, melanoma cell extravasation did not change significantly. In contrast, the extravasation level increased at a fixed shear stress but with a decreasing shear rate. PMN-melanoma aggregation and adhesion to the endothelium via beta(2)-integrin/intracellular adhesion molecule-1 (ICAM-1) interactions were also studied. Lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, whereas Mac-1 (CD11b/CD18) affected prolonged PMN-melanoma aggregation. Blockage of E-selectin or ICAM-1 on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. We have found PMN-melanoma adhesion is correlated with the inverse of shear rate, whereas the PMN-endothelial adhesion correlated with shear stress. Interleukin-8 (IL-8) also influenced PMN-melanoma cell adhesion. Functional blocking of the PMN IL-8 receptors, CXCR1 and CXCR2, decreased the level of Mac-1 upregulation on PMNs while in contact with melanoma cells and reduced melanoma extravasation. We have found PMN-facilitated melanoma adhesion to be a complex multistep process that is regulated by both microfluid mechanics and biology.

Topics: Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Endothelium, Vascular; Flow Cytometry; Humans; Interleukin-8; Melanoma; Neoplasm Metastasis; Neutrophils; Stress, Mechanical; Tumor Cells, Cultured

Omega-3 polyunsaturated fatty acids (n-3 PUFA) inhibit ultraviolet B (UVB)-induced inflammation and other inflammatory states, in vivo. We examined whether this may be mediated by modulation of interleukin (IL)-8, a chemokine pivotal to skin inflammation induced by UVB, in epidermal and dermal cells. We also explored the ability of n-3 PUFA to protect against tumor necrosis factor (TNF)-alpha induction of IL-8, and assessed relative potencies of the principal dietary n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Pre-supplementation, both HaCaT keratinocyte and CCD922SK fibroblast cell lines showed dose-responses for UVB-induced IL-8 release (p<0.001), assessed 48 h post-irradiation. Cells were supplemented with > or =90% purified EPA, DHA, oleic acid (OA) or vehicle control, for 4.5 d. EPA and DHA supplements were bioavailable to keratinocytes and fibroblasts. In keratinocytes, EPA and DHA were shown to reduce basal secretion of IL-8 by 66% and 63%, respectively (p<0.05), and UVB-induced levels by 66% and 65% at 48 h after 100 mJ per cm2, respectively, (p<0.01). A similar pattern occurred in fibroblasts, whereas OA had no influence on IL-8 release in either cell line. In addition, TNF-alpha-induced IL-8 secretion by keratinocytes was reduced by 54% and 42%, respectively, by EPA and DHA (p<0.001). Hence both n-3 PUFA inhibit production of UVB- and TNF-alpha-induced IL-8 in skin cells; this may be important in the photoprotective and other anti-inflammatory effects conferred by these agents.

Topics: Adult; Cell Line, Tumor; Dermatitis; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Acids, Omega-3; Female; Fibroblasts; Humans; Interleukin-8; Keratinocytes; Male; Melanoma; Middle Aged; Radiation Dosage; Skin Neoplasms; Tumor Necrosis Factor-alpha; Ultraviolet Rays

Cancer patients with recurrent local disease after radiation therapy have increased probability of developing regional and distant metastases. The mechanisms behind this observation were studied in the present work by using D-12 and R-18 human melanoma xenografts growing in preirradiated beds in BALB/c-nu/nu mice as preclinical models of recurrent primary tumors in humans. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Based on earlier studies, we hypothesized that metastasis was governed primarily by the proangiogenic factor interleukin-8 (IL-8) in D-12 tumors and by the invasive growth-promoting receptor urokinase-type plasminogen activator receptor (uPAR) in R-18 tumors. Pimonidazole was used as a hypoxia marker, and hypoxia, microvascular hotspots, and the expression of IL-8 and uPAR were studied by immunohistochemistry. The metastatic frequency was significantly higher in tumors in preirradiated beds than in control tumors in unirradiated beds, and it increased with the preirradiation dose. D-12 tumors showed increased fraction of hypoxic cells, increased fraction of IL-8-positive cells, and increased density of microvascular hotspots in preirradiated beds, and R-18 tumors showed increased fraction of hypoxic cells and increased fraction of uPAR-positive cells in preirradiated beds. Strong correlations were found between these parameters and metastatic frequency. IL-8 was up-regulated in hypoxic regions of D-12 tumors, and uPAR was up-regulated in hypoxic regions of R-18 tumors. Daily treatment with anti-IL-8 antibody (D-12) or anti-uPAR antibody (R-18) suppressed metastasis significantly. Our preclinical study suggests that primary tumors recurring after inadequate radiation therapy may show increased metastatic propensity because of increased fraction of hypoxic cells and hypoxia-induced up-regulation of metastasis-promoting gene products. Two possible mechanisms were identified: hypoxia may enhance metastasis by inducing neoangiogenesis facilitating hematogenous spread and by promoting invasive growth facilitating lymphogenous spread. The aggressive behavior of postirradiation local recurrences suggests that they should be subjected to curative treatment as early as possible to prevent further metastatic dissemination. Moreover, the possibility that patients with a high probability of developing local recurrences after radiation therapy may benefit from postirradiation treatment with antiangiogenic and/or

Topics: Animals; Cell Growth Processes; Cell Hypoxia; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Lung Neoplasms; Lymphatic Metastasis; Melanoma; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Transplantation, Heterologous; Up-Regulation

Interleukin (IL)-8 and transforming growth factor (TGF)-beta1 are proangiogenic factors overexpressed in advanced human melanoma. We investigated the effects of TGF-beta1 on IL-8 expression in the well-characterized A375 human melanoma system. We demonstrated by enzyme-linked immunoassay and Northern blot analysis that TGF-beta1 selectively induced IL-8 expression, at both protein and mRNA levels, in highly metastatic A375SM cells but not cells of their poorly metastatic parental line A375P. Transient transfection with luciferase reporter gene constructs revealed that TGF-beta1 activated IL-8 promoter activity in A375SM cells but not A375P cells. Studies with progressive 5' deletion constructs and site-specific mutations demonstrated that a construct containing -133 to +44 of the 5'-flanking sequence was necessary and sufficient for maximal TGF-beta1-induced transcription response and that TGF-beta1-induced activation of IL-8 promoter depended on AP-1 (-126 to -120 bp), NF-kappaB (-94 to -71 bp), and C/EBP-like factor NF-IL6 (-94 to -81 bp) in this region. Interestingly, both A375P and A375SM cells expressed type I and type II TGF-beta receptors and TGF-beta1 induced the nuclear translocation of Smad3 protein in both A375P and A375SM cells. Moreover, both A375P and A375SM cells were susceptible to TGF-beta1-induced growth inhibition. Our data thus demonstrated that TGF-beta1 selectively induced IL-8 expression in highly metastatic A375SM melanoma cells. This TGF-beta1-induced IL-8 expression could be an amplification cascade responsible for overexpression of IL-8 in human melanoma and one of potential mechanisms by which TGF-beta1 promotes angiogenesis, growth, and metastasis of human melanoma.

Topics: Cell Line, Tumor; Fluorescent Antibody Technique; Humans; Interleukin-8; Melanoma; Transforming Growth Factor beta; Transforming Growth Factor beta1

Nuclear factor-kappaB (NF-kappaB) plays a central role in cell survival and proliferation in human melanoma; therefore, the authors explored the possibility of exploiting NF-kappaB for melanoma treatment by using curcumin, an agent with known, potent, NF-kappaB-inhibitory activity and little toxicity in humans.. Three melanoma cell lines (C32, G-361, and WM 266-4), all of which had B-raf mutations, were treated with curcumin, and the authors assessed its effects on viability ((3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide assay) and apoptosis (flow-cytometric analysis of annexin V/propidium iodide-stained cells). Curcumin-treated cells also were examined for NF-kappaB binding activity (electrophoretic mobility shift assay) and for the activity of its upstream regulator, IkappaB kinase (IKK) (immune complex kinase assay). In addition, relevant signaling, as reflected by B-Raf kinase activity (kinase cascade assay), and steady-state levels of activated, downstream effectors, as reflected by mitogen-activated signal-regulated protein kinase (MEK), extracellular signal-regulated protein kinase (ERK), and Akt phosphorylation levels (immunoblots), were assessed.. Curcumin treatment decreased cell viability of all 3 cell lines in a dose-dependent manner (50% inhibitory concentration = 6.1-7.7 microM) and induced apoptosis. NF-kappaB and IKK were active constitutively in all melanoma cell lines examined, and curcumin, under apoptosis-inducing conditions, down-regulated NF-kappaB and IKK activities. However, curcumin did not inhibit the activities of B-Raf, MEK, or ERK, and Akt phosphorylation was enhanced. Furthermore, in the presence of curcumin, the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate] no longer suppressed Akt phosphorylation.. Curcumin has potent antiproliferative and proapoptotic effects in melanoma cells. These effects were associated with the suppression of NF-kappaB and IKK activities but were independent of the B-Raf/MEK/ERK and Akt pathways.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Curcumin; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Humans; I-kappa B Kinase; Interleukin-8; Melanoma; NF-kappa B; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Signal Transduction

The endothelin pathway plays a critical role in melanoma tumor progression by a variety of mechanisms that enhance tumor cell growth, invasion, and metastasis. Here, we investigate the effect of this pathway on CXC chemokine expression in human melanoma cells and melanocytes. As determined by ELISA, endothelin-1 (ET-1) induces CXCL1 and CXCL8 secretion in three human melanoma cell lines in a concentration-dependent fashion. These responses are mediated by the endothelin-B receptor and are sustained over a 40 h time course. ET-1 does not induce CXCL1 secretion in primary human melanocytes but ET-3, an endothelin isoform, induces a low level of CXCL1 secretion in certain cultures. Neither ET-1 nor ET-3 induces secretion of CXCL8 in primary human melanocytes; thus, this response may be specific for melanocytic cells that have undergone malignant transformation. We have previously demonstrated that ET-1 induces changes in the expression of adhesion molecules in melanoma cells such that invasion and metastasis are favored. This study demonstrates that ET-1 additionally induces secretion of CXC chemokines critical for melanoma metastasis and tumor progression.

Topics: Cell Line, Tumor; Chemokine CXCL1; Chemokines, CXC; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-3; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Melanocytes; Melanoma; Receptor, Endothelin B; Skin Neoplasms

Previous studies have shown that neutrophils (PMNs) facilitate melanoma cell extravasation [M.J. Slattery, C. Dong, Neutrophils influence melanoma adhesion and migration under flow conditions, Intl. J. Cancer 106 (2003) 713-722] Little is known, however, about the specific interactions between PMNs, melanoma and the endothelium (EC) or the molecular mechanism involved under flow conditions. The aim of this study is to investigate a "two-step adhesion" hypothesis that involves initial PMN tethering on the EC and subsequent melanoma cells being captured by tethered PMNs. Different effects of hydrodynamic shear stress and shear rate were analyzed using a parallel-plate flow chamber. Results indicate a novel finding that PMN-facilitated melanoma cell arrest on the EC is modulated by shear rate, which is inversely-proportional to cell-cell contact time, rather than by the shear stress, which is proportional to the force exerted on formed bonds. Beta2 integrins/ICAM-1 adhesion mechanisms were examined and the results indicate LFA-1 and Mac-1 cooperate to mediate the PMN-EC-melanoma interactions under shear conditions. In addition, endogenously produced IL-8 contributes to PMN-facilitated melanoma arrest on the EC through the CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) on PMN. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the EC.

Topics: Cell Adhesion; Cell Adhesion Molecules; Culture Media; Dextrans; Endothelium; Humans; Interleukin-8; Melanoma; Neutrophils; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Shear Strength; Stress, Mechanical; Tumor Cells, Cultured

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for the migration of tumor cells out of the vascular system to establish metastases. Innate immune system processes can potentially promote tumor progression through inflammation dependant mechanisms. White blood cells, neutrophils (PMN) in particular, are being studied to better understand how the host immune system affects cancer cell adhesion and subsequent migration and metastasis. Melanoma cell interaction with the EC is distinct from PMN-EC adhesion in the circulation. We found PMN increased melanoma cell extravasation, which involved initial PMN tethering on the EC, subsequent PMN capture of melanoma cells and maintaining close proximity to the EC. LFA-1 (CD11a/CD18 integrin) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, while Mac-1 (CD11b/CD18 integrin) affected prolonged PMN-melanoma aggregation. Blocking E-selectin or ICAM-1 (intercellular adhesion molecule) on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. Results indicated a novel finding that PMN-facilitated melanoma cell arrest on the EC could be modulated by endogenously produced interleukin-8 (IL-8). Functional blocking of the IL-8 receptors (CXCR1 and CXCR2) on PMN, or neutralizing soluble IL-8 in cell suspensions, significantly decreased the level of Mac-1 up-regulation on PMN while communicating with melanoma cells and reduced melanoma extravasation. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines, and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the endothelium in the microcirculation, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.

Topics: Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cell Movement; Endothelial Cells; Humans; Interleukin-8; Melanoma; Neutrophils; Shear Strength; Tumor Cells, Cultured

Topics: Cell Line, Tumor; Dacarbazine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Humans; Interleukin-8; Melanoma; Mitogen-Activated Protein Kinases; NF-kappa B; Transcription Factor AP-1; Transcription, Genetic; Transfection; Up-Regulation; Vascular Endothelial Growth Factor A

In recent years, the incidence of cutaneous melanoma has increased more than that of any other cancer. Dacarbazine is considered the gold standard for treatment, having a response rate of 15% to 20%, but most responses are not sustained. Previously, we have shown that short exposure of primary cutaneous melanoma cells to dacarbazine resulted in the upregulation of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). The purpose of the present study was to determine how long-term exposure of melanoma cells to dacarbazine would affect their tumorigenic and metastatic potential in vivo.. The primary cutaneous melanoma cell lines SB2 and MeWo were repeatedly exposed in vitro to increasing concentrations of dacarbazine, and dacarbazine-resistant cell lines SB2-D and MeWo-D were selected and examined for their ability to grow and metastasize in nude mice.. The dacarbazine-resistant cell lines SB2-D and MeWo-D exhibited increased tumor growth and metastatic behavior in vivo. This increase could be explained by the activation of RAF, MEK, and ERK, which led to the upregulation of IL-8 and VEGF. More IL-8, VEGF, matrix metalloproteinase-2 (MMP-2), and microvessel density (CD-31) were found in tumors produced by SB2-D and MeWo-D in vivo than in those produced by their parental counterparts. No mutations were observed in BRAF.. Our results have significant clinical implications. Treatment of melanoma patients with dacarbazine could select for a more aggressive melanoma phenotype. We propose that combination treatment with anti-VEGF/IL-8 or MEK inhibitors may potentiate the therapeutic effects of dacarbazine.

Topics: Animals; Antineoplastic Agents, Alkylating; Cell Line, Tumor; Dacarbazine; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Melanoma; Mice; Mice, Nude; Skin Neoplasms; Up-Regulation; Vascular Endothelial Growth Factor A

IL-8 is a strong chemoattractant for neutrophils, and it is constitutively produced by many tumors, including human melanomas. To determine the biologic importance of IL-8 for melanoma cells from primary and metastatic lesions, we transduced selected cell lines constitutively producing low levels of IL-8 with IL-8 cDNA using a replication-deficient adenoviral vector. Nontumorigenic SBcl2 primary melanoma cells formed tumors when transduced with increasing plaque-forming units of IL-8 per cell. However, at high IL-8 transduction levels (100 ng/ml/10(5) cells in 48 hr), tumor growth was impaired due to massive neutrophil infiltration. A similar biphasic response was observed in WM115 primary melanomas, which are tumorigenic but not metastatic. Depletion of neutrophils with an antibody that blocks the accumulation of granulocytes at the site of inflammation enabled transduced primary melanomas secreting high levels of IL-8 to survive and grow. In contrast, highly tumorigenic and metastatic 451Lu cells showed marked increases in tumor growth and number of metastatic foci in the lungs depending on the expression levels of IL-8. Cytotoxicity assays with isolated neutrophils confirmed the preferential killing of primary over metastatic melanoma cells. SBcl2 cells stimulated by IL-8 to form tumors in immunodeficient mice were induced to produce VEGF, suggesting that the angiogenic response is enhanced due to increased growth factor production. Our results demonstrate that nontumorigenic primary melanomas depend on IL-8 stimulation in vivo for growth and that tumor growth depends on the level of neutrophil infiltration. Metastatic melanomas proliferate in vivo independently of infiltrating neutrophils.

Topics: Adenoviridae; Animals; Chemotaxis, Leukocyte; Cytotoxicity, Immunologic; Disease Progression; Endothelial Growth Factors; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lymphokines; Melanoma; Mice; Mice, Nude; Neutrophils; Skin Neoplasms; Transfection; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Dysregulated activation of Ras or its downstream effectors such as mitogen-activated protein kinase kinase and ERK has been shown to play a critical role in tumorigenesis of many cancer types. However, in melanoma, activating mutations in Ras are rarely observed and are limited to N-Ras in UV-exposed cells. In this study, we identify constitutively activated ERK in almost all melanoma cell lines and in tumor tissues tested, which is in contrast to normal melanocytes and several early stage radial growth phase melanoma lines where ERK can be activated by serum or growth factors. Constitutive activation of ERK is preceded by phosphorylation of mitogen-activated protein kinase kinase and c-RAF. In all of the melanoma cell lines tested, Ras is constitutively activated without underlying mutations. On the contrary, activating mutations in the kinase domain of BRAF are present in the majority of the cell lines tested. Furthermore, ERK activation can be partially inhibited from the cell surface using inhibitors of fibroblast growth factor and hepatocyte growth factor but not interleukin 8 signaling pathways. These data suggest that melanoma growth, invasion, and metastasis are attributable to constitutively activated ERK apparently mediated by excessive growth factors through autocrine mechanisms and BRAF kinase activation.

Topics: Enzyme Activation; Fibroblast Growth Factors; Growth Substances; Hepatocyte Growth Factor; Humans; Interleukin-8; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; Tumor Cells, Cultured

There is increasing evidence that both cell adhesion molecules and soluble factors are involved in tumor metastasis. We have found that endothelial cells secrete chemoattractants that can induce melanoma cell chemotaxis. Protein separation on an ion-exchange column shows the association of IL-8 with fractions that contain the chemoattractant activity. This activity is completely lost from the conditioned medium after immunoprecipitation with anti-IL-8 antibodies, indicating that IL-8 is the major melanoma chemoattractant secreted by endothelial cells. IL-877, the predominant endothelial IL-8 isoform that contains 77 amino acids, is found to be twice as potent as the more common 72-amino acid isoform IL-872. Antibody inhibition studies indicate that the chemotactic response of melanoma cells is mediated by the CXC-chemokine receptor CXCR1 and not by the more promiscuous CXCR2. When stimulated by tumor necrosis factor alpha, the nonresponsive WM35 melanoma cells synthesize a higher level of CXCR1 and become chemotactic toward interleukin (IL)-8. Pretreatment of cells with pertussis toxin nullifies their chemotactic response, suggesting the involvement of G proteins. Antibodies against either IL-8 or CXCR1 inhibit melanoma transendothelial migration in a coculture assay by 30%. These results are consistent with a role for IL-8-induced chemotaxis in the transendothelial migration of melanoma cells.

Topics: Cells, Cultured; Chemotactic Factors; Chemotaxis; Coculture Techniques; Culture Media, Conditioned; Endothelium; Humans; Interleukin-8; Melanoma; Models, Biological; Receptors, Interleukin-8A; Tumor Cells, Cultured

The incidence of cutaneous malignant melanoma in the United States has increased more than any other cancer in recent years. Chemotherapy for metastatic melanoma is disappointing, there being anecdotal cases of complete remission. Dacarbazine (DTIC) is considered the gold standard for treatment, having a response rate of 15-20%, but most responses are not sustained. The mechanisms for the increased chemotherapeutic resistance of melanoma are unclear. The objective of this study was to determine the mechanisms by which melanoma cells escape the cytotoxic effect of DTIC. Here, we show that DTIC induced interleukin (IL)-8 and vascular endothelial growth factor (VEGF) protein overexpression and secretion via transcriptional up-regulation in the two melanoma cell lines SB-2 and MeWo. Luciferase activity driven by the IL-8 and VEGF promoters was up-regulated by 1.5-2- and 1.6-3.5-fold, respectively, in the SB2 and MeWo melanoma cell lines. The mitogen-activated protein kinase signal transduction pathway seemed to regulate at least partially the activation of IL-8, whereas it was not involved in VEGF promoter regulation. Electrophoretic mobility shift analysis analyses have revealed an increase in binding activity of activator protein 1 (c-Jun) and nuclear factor-kappaB after DTIC treatment for both melanoma cell lines. Metastatic melanoma cell lines secreting high levels of IL-8 and VEGF were more resistant to DTIC than early primary melanomas secreting low levels of the cytokines. In addition, transfection of the primary cutaneous melanoma SB-2 cells with the IL-8 gene rendered them resistant to the cytotoxic effect of the drug, whereas the addition of IL-8-neutralizing antibody to metastatic melanoma cells lowered their sensitivity to DTIC. Taken together, our data demonstrate that DTIC can cause melanoma cells to secrete IL-8 and VEGF, which might render them resistant to the cytotoxic effects of the drug. We propose that combination treatment with anti-VEGF/IL-8 agents may potentiate the therapeutic effects of DTIC.

Topics: Cell Line, Tumor; Dacarbazine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Humans; Interleukin-8; Melanoma; Mitogen-Activated Protein Kinases; NF-kappa B; Transcription Factor AP-1; Transcription, Genetic; Transfection; Up-Regulation; Vascular Endothelial Growth Factor A

Anti-angiogenic agents regulate tumor growth by inhibiting endothelial cell proliferation and invasion. Carboxyamido-triazole (CAI), an inhibitor of non-voltage-operated calcium entry and calcium influx-mediated pathways, has angiogenesis and invasion inhibitory activity. We hypothesized that CAI may express its anti-angiogenic effects through negative regulation of pro-angiogenic cytokine production and/or function. In vivo, orally administered CAI prevented A2058 human melanoma xenograft growth and concomitantly resulted in a marked reduction in circulating vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In vitro, A2058 cell secretion of VEGF was inhibited by CAI treatment under limiting micronutrient conditions that approximate the tumor microenvironment, media restriction, and acidification to pH 6.8 (P=0.0003 and P=0.0006, respectively). VEGF and HIF-1alpha message and protein were also reduced by CAI treatment. Oral CAI treatment reduced vascular ingrowth in vivo into VEGF-containing Matrigel plugs. Commensurate with those findings, human umbilical vein endothelial cell (HUVEC) migration towards VEGF was reduced below background by exposure to CAI in the migration chamber (P<0.0001). An 88% reduction in circulating IL-8 concentration was measured in CAI-treated animals. However, IL-8 protein secretion and gene expression were increased by CAI treatment in culture (P< or =0.01), where CAI caused a dose-dependent acidification of the culture milieu (P< or =0.005). This paradox suggests that IL-8 production in vitro may be more sensitive to ambient pH than cytosolic calcium. These observations suggest that CAI inhibition of tumor cell VEGF production and endothelial cell response to VEGF results in disruption of signaling between the tumor and its microenvironment, causing a net anti-angiogenic effect.

Topics: Animals; Antineoplastic Agents; Cell Movement; Cell Transplantation; Culture Media; Endothelial Growth Factors; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hydrogen-Ion Concentration; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lymphokines; Melanoma; Mice; Neovascularization, Pathologic; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transplantation, Heterologous; Triazoles; Tumor Cells, Cultured; Umbilical Veins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

In the present study, we examined the autocrine/paracrine role of IL-8 in melanoma growth and metastasis by analyzing the expression and functional significance of IL-8 receptors, CXCR1 and CXCR2 in human malignant melanoma cells with different metastatic potential. CXCR1 and CXCR2 mRNA and protein levels were analyzed by reverse trannscriptase-based polymerase chain reaction, immunohistochemistry, immunoprecipitation, flow cytometry and ligand binding assay in melanoma cells in vitro and xenografted in nude mice. Melanoma cells constitutively expressed CXCR1 and CXCR2 mRNA and protein. Highly metastatic A375SM cells expressed higher levels of CXCR1 and CXCR2 mRNA and protein in vitro and in vivo as compared to low metastatic A375P and non-metastatic SBC-2 melanoma cells. Treatment of SBC-2 and A375P cells with exogenously added recombinant IL-8 significantly enhanced their proliferation and invasive potential. Further neutralizing antibodies to CXCR1 and CXCR2 inhibited proliferation and invasive potential of unstimulated and IL-8-stimulated A375P cells. In summary, the data suggest that constitutive expression of CXCR1 and CXCR2 play an important role regulating the IL-8-mediated metastatic phenotype in human malignant melanoma cells.

Topics: Animals; Cell Division; Humans; Interleukin-8; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Receptors, Interleukin-8A; Receptors, Interleukin-8B

Successive events of growth factor-induced autocrine and paracrine activation promote tumor growth and metastasis. Insulin-like growth factor-I (IGF-I) stimulates melanoma cells to grow, survive, and migrate. Interleukin-8 (IL-8) is produced by melanoma cells and has been correlated with melanoma metastasis, but the biological functions of this cytokine have not been elucidated. We show here that IGF-I-induced migration of melanoma cells could be inhibited by neutralizing antibody against IL-8. IGF-I overexpression induced IL-8 production in melanoma cells, especially in biologically early melanomas by accelerating its transcription rate via activation of mitogen-activated protein kinase pathway. IGF-I treatment phosphorylated c-Jun and stimulated the binding of AP-1 but not NF-kappaB to the IL-8 promoter. These data identify IL-8 as a new target of IGF-I in melanoma and suggest that some of the biological functions of IGF-I are mediated by IL-8.

Topics: Antibodies, Monoclonal; Blotting, Northern; Blotting, Western; Cell Movement; Cell Nucleus; DNA Primers; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Humans; In Vitro Techniques; Insulin; Insulin-Like Growth Factor I; Interleukin-8; Melanoma; RNA, Messenger; Tumor Cells, Cultured

The aim of this study was to investigate whether tumour hypoxia and/or vascular hot spots promote the development of metastatic disease. The D-12 human melanoma xenograft line was used as a tumour model. Hypoxia and vascular hot spots were detected by immunohistochemistry using pimonidazole as a hypoxia marker and anti-CD31 antibody to visualize endothelial cells. Vascular hot spots were found to be induced in hypoxic foci, owing to hypoxia-induced up-regulation of angiogenesis stimulatory factors. This effect was mediated by interleukin 8 and possibly also by vascular endothelial growth factor. Interleukin 8 positive foci showed a high degree of co-localization with hypoxic foci, as revealed by immunohistochemistry. The incidence of spontaneous pulmonary metastases was associated with the density of hypoxic foci, the density of interleukin 8 positive foci and the density of vascular hot spots in the primary tumour. Treatment with neutralizing antibody against interleukin 8 and/or vascular endothelial growth factor resulted in hypoxia-induced necrosis rather than hypoxia-induced vascular hot spots and inhibited metastasis. Our study suggests a cause-effect relationship between hypoxia and metastasis in cancer and hence an elevated probability of metastatic disease in patients having primary tumours characterized by high densities of hypoxic foci and vascular hot spots.

Topics: Animals; Endothelial Growth Factors; Female; Humans; Hypoxia; Immunohistochemistry; Interleukin-8; Lung Neoplasms; Lymphokines; Melanoma; Mice; Mice, Inbred BALB C; Microcirculation; Neoplasm Metastasis; Regional Blood Flow; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

In the present study, we demonstrate that upregulation of interleukin-1beta(IL-1beta)-mediated and tumor necrosis factor-alpha (TNF-alpha)-mediated IL-8 expression in human malignant melanoma cells is modulated by the activation of nuclear factor-kappaB (NF-kappaB). Addition of capsaicin (8-methyl-N-vanillyl-6-nonenamide), a known inhibitor of NF-kappaB, resulted in the inhibition of constitutive as well as IL-1beta-induced and TNF-alpha-induced IL-8 expression in melanoma cells. The inhibition of IL-8 expression was dependent on the concentration of capsaicin and duration of treatment. Further, electrophoretic mobility shift assay (EMSA) of nuclear extracts from melanoma cells showed a constitutive activation of NF-kappaB and activated protein 1 (AP-1), which was upregulated following treatment with IL-1beta. Treatment of melanoma cells with capsaicin inhibited activation of constitutive and IL-1beta-induced NF-kappaB, but not AP-1, leading to inhibition of IL-8 expression. Further, downregulation of IL-8 expression in capsaicin-treated melanoma cells resulted in inhibition of in vitro cell proliferation. These results demonstrate that constitutive and induced NF-kappaB activation regulates IL-8 expression in melanoma cells. Downregulation of constitutive and induced NF-kappaB activation in malignant melanoma cells leads to inhibition of IL-8 production and in vitro cell proliferation.

Topics: Capsaicin; Cell Division; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Interleukin-8; Kinetics; Melanoma; NF-kappa B; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

Interleukin-8 (IL-8) has recently been shown to contribute to human melanoma progression by functioning as a mitogenic and angiogenic factor. In the present study, we investigated whether targeting IL-8 by a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy to control angiogenesis, growth, and metastasis of melanoma. The human melanoma cells A375SM (high IL-8 producer) and TXM-13 (intermediate IL-8 producer) were injected subcutaneously into nude mice, which were then treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks). Tumor growth of both melanomas in ABX-IL8-treated mice was significantly inhibited when compared with control IgG-treated animals. ABX-IL8 treatment also suppressed experimental metastasis when the melanoma cells were injected intravenously. IL-8 blockade by ABX-IL8 significantly inhibited the promoter activity and the collagenase activity of matrix metalloproteinase-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane in vitro. In vivo, ABX-IL8 treatment resulted in decreased expression of matrix metalloproteinase-2, and decreased vascularization (angiogenesis) of tumors concomitant with increased apoptosis of tumor cells. Moreover, in an in vitro vessel formation assay, ABX-IL8 directly interfered with the tubule formation by human umbilical vein endothelial cells. Taken together, these results point to the potential utility of ABX-IL8 as a modality to treat melanoma and other solid tumors either alone or in combination with conventional chemotherapy or other anti-tumor agents.

Topics: Animals; Antibodies; Cell Division; Down-Regulation; Humans; Interleukin-8; Matrix Metalloproteinase Inhibitors; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Neovascularization, Pathologic; Tumor Cells, Cultured

To determine the predictive value of the angiogenic serum factors angiogenin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8) for the prognosis of patients with malignant melanoma.. Angiogenin, VEGF, bFGF, and IL-8 were measured in sera of 125 melanoma patients with different stages of disease and with or without current therapy including interferon alfa and different cytostatics in comparison with 30 healthy controls using enzyme-linked immunosorbent assay.. Serum levels of angiogenin, VEGF, bFGF, and IL-8 were significantly increased in melanoma patients compared with healthy controls. Elevated serum concentrations of VEGF, bFGF, and IL-8 were associated with advanced disease stages and tumor burden. Cytostatic therapy of patients was accompanied by increased serum levels of angiogenin, bFGF, and IL-8. As shown by univariate analysis, elevated serum levels of VEGF (P = .0001 and .0036), bFGF (P < .00005 and < .00005), and IL-8 (P < .00005 and < .00005) were strongly correlated with a poor overall and progression-free survival, respectively. Multivariate analysis revealed stage of disease (P = .0238), tumor burden (P = .0347), VEGF (P = .0036), bFGF (P = .0252), and IL-8 (P = .0447) as independent predictive factors of overall survival. Tumor burden (P = .0081), VEGF (P = .0245), and IL-8 (P = .0089) were found as independent predictive factors of progression-free survival.. Our data suggest that the angiogenic serum factors VEGF, bFGF, and IL-8 are useful predictive markers for overall and progression-free survival in melanoma patients.

Topics: Analysis of Variance; Angiogenesis Inducing Agents; Biomarkers; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Growth Substances; Humans; Interleukin-8; Lymphokines; Male; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Ribonuclease, Pancreatic; Survival Analysis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The heterotrimeric lymphotoxin alpha(1)beta(2) (LTalpha(1)beta(2)) complex and LIGHT, a new member of the tumor necrosis factor (TNF) superfamily, have been identified as membrane-anchored ligands for the LTbeta receptor (LTbetaR), a member of the TNF receptor (TNFR) superfamily. Although some of the biologic activities of this receptor have been described using either soluble LTalpha(1)beta(2) as a ligand or agonistic monoclonal antibodies (mAb), very little is known about the signaling of LIGHT via the LTbetaR. To gain more insight into the biologic functions of LIGHT, we generated a recombinant soluble form of human LIGHT (rsHuLIGHT). We demonstrate here that this rsHuLIGHT is capable of binding to the LTbetaR. Interestingly, receptor-mediated ligand precipitation analysis revealed that rsHuLIGHT bound only to human LTbetaR but not to mouse LTbetaR, indicating a species-specific receptor ligand interaction. Activation of A375 human melanoma cells by rsHuLIGHT induced an increased secretion of interleukin-8 (IL-8). Furthermore, rsHuLIGHT caused growth arrest of A375 cells even in the absence of interferon-gamma (IFN-gamma).

Topics: Animals; COS Cells; Growth Inhibitors; Humans; Immunoglobulin Fc Fragments; Interleukin-8; Ligands; Lymphotoxin-alpha; Melanoma; Membrane Proteins; Mice; Protein Binding; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Member 14; Receptors, Virus; Recombinant Fusion Proteins; Solubility; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor Ligand Superfamily Member 14; Tumor Necrosis Factor-alpha; Up-Regulation

We studied the effects of 17beta-estradiol, progesterone, and dihydrotestosterone on in vitro growth of human metastatic melanoma. Each sex hormone inhibited the growth of melanoma receptor-dependently; 17beta-estradiol inhibited 3H-thymidine uptake of estrogen receptor-positive WM266-4 and NM26, but not that of the receptor-negative HS15. Progesterone inhibited 3H-thymidine uptake of progesterone receptor-positive WM266-4 and HS15, but not that of the receptor-negative NM26. Dihydrotestosterone inhibited 3H-thymidine uptake of androgen receptor-positive HS15 and NM26, but not that of the receptor-negative WM266-4. The growth inhibition by each hormone was counteracted by the respective hormone receptor antagonist. The combination of more than two hormones neither gave additive nor synergistic growth inhibition. The growth inhibition by each sex hormone was counteracted by interleukin-8 but not by the other growth factors. Each sex hormone reduced the constitutive interleukin-8 secretion and mRNA levels in the respective receptor-positive melanoma but not in the receptor-negative melanoma. Transient transfection showed that each sex hormone inhibited the constitutive chloramphenicol acetyltransferase expression driven by interleukin-8 promoter in the respective receptor-positive melanoma but not in the receptor-negative melanoma. Transfection with a series of 5'-deleted interleukin-8 promoter/chloramphenicol acetyltransferase reporter constructs demonstrated that the sequences between -98 and -63 bp on interleukin-8 promoter may be involved in the transcriptional repression. These data suggest that 17beta-estradiol, progesterone, and dihydrotestosterone suppress the growth of melanoma by inhibiting interleukin-8 production in a receptor-dependent manner.

Topics: Antibodies; Cell Division; Chloramphenicol O-Acetyltransferase; Dihydrotestosterone; Estradiol; Female; Gene Expression Regulation, Neoplastic; Gonadal Steroid Hormones; Humans; Interleukin-8; Lymph Node Excision; Male; Melanoma; Progesterone; Promoter Regions, Genetic; Skin Neoplasms; Transcription, Genetic; Transfection; Tumor Cells, Cultured

We studied the effects of various gangliosides on in vitro growth of human metastatic melanoma WM266-4. GD1b, GT1b, and GQ1b inhibited 3H-thymidine uptake and growth rate of WM266-4 whereas the other gangliosides were ineffective. The growth inhibition by GD1b, GT1b, and GQ1b was counteracted by interleukin-8 but not by the other growth factors. The growth inhibition by gangliosides was not detected in the presence of anti-interleukin-8 antibody. GD1b, GT1b, and GQ1b reduced the constitutive interleukin-8 secretion and mRNA levels in WM266-4. Transient transfection showed that GD1b, GT1b, and GQ1b inhibited the constitutive chloramphenicol acetyltransferase expression driven by interleukin-8 promoter in WM266-4. Transfection with a series of 5'-deleted mutants demonstrated that the sequences between -98 and -62 bp on interleukin-8 promoter may be involved in the transcriptional repression by these gangliosides. Cyclic AMP analog dibutyryl cAMP counteracted GD1b, GT1b, and GQ1b-induced inhibition of interleukin-8 production at the levels of protein secretion, mRNA expression, and promoter activity. GD1b, GT1b, and GQ1b reduced cAMP level and protein kinase A activity in WM266-4. These gangliosides suppressed adenylate cyclase activity without altering that of cyclic nucleotide phosphodiesterase in WM266-4. The data indicate that GD1b, GT1b, and GQ1b may suppress the growth of melanoma by inhibiting interleukin-8 production via the inhibition of adenylate cyclase.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclase Inhibitors; Cell Division; Chloramphenicol O-Acetyltransferase; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Female; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Melanoma; Promoter Regions, Genetic; RNA, Messenger; Skin Neoplasms; Transcription, Genetic; Tumor Cells, Cultured

We examined whether macrophage infiltration is associated with angiogenesis in cutaneous melanoma. The numbers of macrophages and microvessels increased significantly with increasing depth of tumor and with tumor angiogenesis. Macrophage infiltration thus appeared to provide a useful diagnostic marker for the progression of cutaneous melanoma. We further examined whether human melanoma cells produce angiogenic factors in response to macrophage-derived cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 alpha (IL-1alpha). Treatment of melanoma cells with TNFalpha and IL-1alpha in vitro enhanced the production of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and of basic fibroblast growth factor (bFGF) to a lesser degree, in human melanoma cells. Lipopolysaccharide (LPS)-activated human monocytes enhanced production of IL-8, VEGF, TNF alpha, as well as IL-1alpha, but not bFGF. Co-culture of human monocytes and human melanoma cells was also found to significantly enhance production of IL-8 and VEGF in the absence and presence of LPS, compared with either monocytes or melanoma cells alone. The production of IL-8 and VEGF from co-cultured melanoma cells and LPS-activated monocytes was blocked when anti-TNF-alpha antibody or anti-IL-1alpha antibody was co-administrated. This is direct evidence that production of the potent angiogenic factors IL-8 and VEGF from melanoma cells is up-regulated through TNFalpha and/or IL-1alpha secreted by activated monocytes/macrophages, influencing both tumor growth and angiogenesis in melanomas.

Topics: Adult; Aged; Aged, 80 and over; Angiogenesis Inducing Agents; Cell Communication; Cell Movement; Endothelial Growth Factors; Humans; Interleukin-1; Interleukin-8; Lymphokines; Macrophages; Melanoma; Middle Aged; Monocytes; Neoplasm Staging; Neovascularization, Pathologic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Isolated, hyperthermic limb perfusion (ILP) with recombinant human tumor necrosis factor-alpha (rhTNF-alpha) and melphalan is a highly effective treatment for locoregional metastases of malignant melanoma and for advanced soft tissue sarcoma of the limb. The major systemic side effects are characterized by the induction of a systemic inflammatory response syndrome (SIRS). Procalcitonin (PCT), a serum marker of bacterial sepsis, was investigated with respect to its role in SIRS after ILP.. University surgical oncology division with an integrated eight-bed intensive care unit.. Thirty-seven patients were treated by ILP with rhTNF-alpha and melphalan (n = 26) or with cytostatics alone (n = 11) for soft tissue sarcoma or malignant melanoma.. The course of serum PCT, interleukin (IL)-6, and IL-8 was analyzed intra- and postoperatively. Hemodynamic variables including heart rate, mean arterial pressure, cardiac index, pulmonary arterial pressure, pulmonary capillary occlusion pressure, and pulmonary and systemic vascular resistance were recorded in parallel.. PCT was significantly elevated over baseline after ILP with a maximum between 8 hrs (peak level 16.0+/-18.8 (SD) ng/mL) and 36 hrs (13.8+/-15.7 ng/mL) (p < .001). The increase in serum PCT was significantly more pronounced after ILP with rhTNF-alpha/melphalan than after ILP with cytostatics alone (p < .001). IL-6 and IL-8 were also significantly increased after ILP (p = .001), reaching peak concentrations at 1 hr and 4 hrs postoperatively. Significant changes in heart rate, mean arterial pressure, cardiac index, and systemic vascular resistance were observed during and after ILP; however, PCT levels could not be correlated to these variables. Pulmonary arterial pressure, pulmonary capillary occlusion pressure, and pulmonary vascular resistance showed no significant changes.. Serum procalcitonin is induced as part of the SIRS after ILP with rhTNF-alpha/melphalan. It may be induced directly by rhTNF-alpha or other cytokines, because serum peaks of IL-6 and IL-8 precede the peak of PCT. Because there is no correlation between serum levels of PCT and hemodynamic variables, this marker cannot be applied to assess the severity of SIRS reaction after ILP.

Topics: Adult; Aged; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Calcitonin; Calcitonin Gene-Related Peptide; Cardiovascular Physiological Phenomena; Chemotherapy, Cancer, Regional Perfusion; Cisplatin; Extremities; Female; Glycoproteins; Humans; Interleukin-6; Interleukin-8; Male; Melanoma; Melphalan; Middle Aged; Protein Precursors; Recombinant Proteins; Sarcoma; Time Factors; Tumor Necrosis Factor-alpha

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro- and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. In most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor.

Topics: Angiostatins; Animals; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Humans; Interleukin-8; Lymphokines; Melanoma; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neovascularization, Pathologic; Peptide Fragments; Plasminogen; Platelet-Derived Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The purpose of this study was to determine the role of nuclear factor (NF)-kappaB/relA activity in the induction of angiogenesis and production of metastasis by human melanoma cells. Highly metastatic melanoma variant cells expressed high levels of constitutive NF-kappaB activity. Transfection of highly metastatic human melanoma variant cells with a dominant-negative mutant inhibitor of nuclear factor-kappaB alpha (Ikappabeta alpha) expression vector (Ikappabeta alphaM) decreased the level of constitutive NF-kappaB activity, inhibited s.c. tumor growth, and prevented lung metastasis in nude mice. Furthermore, the slow-growing s.c. tumors formed by the IkappaB alphaM-transfected cells exhibited a decrease in microvessel density (angiogenesis), which correlated with a decrease in the level of interleukin-8 expression. Collectively, these results demonstrate that NF-kappaB/reLA activity significantly contributes to tumorigenicity, angiogenesis, and metastasis of human melanoma cells implanted in nude mice.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Division; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Female; Genes, Dominant; Humans; I-kappa B Proteins; Immunohistochemistry; Interleukin-8; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Microcirculation; Mutation; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; NF-kappa B; Platelet Endothelial Cell Adhesion Molecule-1; Promoter Regions, Genetic; Time Factors; Transfection; Tumor Cells, Cultured

The purpose of this study was to determine whether constitutive NF-kappaB activity regulates the expression level of interleukin-8 (IL-8) in metastatic human melanoma cells. Cultures of metastatic human A375 melanoma cells expressed higher levels of IL-8 mRNA and protein than nonmetastatic A375 human melanoma cells. No discernible differences in IL-8 half-life were found between metastatic and nonmetastatic cells, but cells that overexpressed IL-8 had a higher transcription rate and increased IL-8 promoter activity. Analysis of the IL-8 promoter using deletion mutants revealed that the region within -133 was essential for constitutive IL-8 promoter activity and that mutation of NF-kappaB binding sites eliminated the constitutive IL-8 promoter activity. The activation of constitutive IL-8 transcription directly correlated with the level of constitutive NF-kappaB activity. Transfection of melanoma cells with a dominant-negative mutant IkappaBalpha expression vector (pLXSN-IkappaBalphaM) significantly decreased the level of constitutive NF-kappaB activity and expression of IL-8, demonstrating that constitutive NF-kappaB/relA activities contribute to overexpression of IL-8 in highly metastatic human melanoma cells.

Topics: Base Sequence; Blotting, Northern; Blotting, Western; DNA-Binding Proteins; Down-Regulation; Electrophoresis, Agar Gel; Enzyme-Linked Immunosorbent Assay; Humans; I-kappa B Proteins; Interleukin-8; Luciferases; Lung Neoplasms; Melanoma; Mutation; NF-kappa B; NF-KappaB Inhibitor alpha; RNA, Messenger; Transfection; Tumor Cells, Cultured

Angiogenesis is a significant prognostic factor in melanoma, but the angiogenic factors controlling the neovascularization are not well defined. The purpose of this study was to investigate whether the angiogenesis and metastasis of melanoma are promoted by vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), platelet-derived endothelial cell growth factor (PD-ECGF), and/or basic fibroblast growth factor (bFGF). Cells from human melanoma lines (A-07, D-12, R-18, and U-25) transplanted to BALB/c nu/nu mice were used as tumor models. Expression of angiogenic factors was studied by ELISA, Western blotting, and immunohistochemistry. Angiogenesis was assessed by using an intradermal angiogenesis assay. Lung colonization and spontaneous lung metastasis were determined after i.v. and intradermal inoculation of tumor cells, respectively. The specific roles of VEGF, IL-8, PD-ECGF, and bFGF in tumor angiogenesis, lung colonization, and spontaneous metastasis were assessed in mice treated with neutralizing antibody. The melanoma lines expressed multiple angiogenic factors, and each line showed a unique expression pattern. Multiple angiogenic factors promoted angiogenesis in the most angiogenic melanoma lines, whereas angiogenesis in the least angiogenic melanoma lines was possibly promoted solely by VEGF. Tumor growth, lung colonization, and spontaneous metastasis were controlled by the rate of angiogenesis and hence by the angiogenic factors promoting the angiogenesis. Lung colonization and spontaneous metastasis in A-07 were inhibited by treatment with neutralizing antibody against VEGF, IL-8, PD-ECGF, or bFGF. Each of these angiogenic factors may promote metastasis in melanoma, because inhibition of one of them could not be compensated for by the others. Our observations suggest that efficient antiangiogenic treatment of melanoma may require identification and blocking of common functional features of several angiogenic factors.

Topics: Animals; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Growth Substances; Humans; Immunohistochemistry; Interleukin-8; Lymphokines; Melanoma; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neovascularization, Pathologic; Thymidine Phosphorylase; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Antisense Elements (Genetics); Humans; In Situ Hybridization; Interleukin-8; Melanoma; Reproducibility of Results; Research Design; RNA, Messenger; Sensitivity and Specificity

Cultures of retinal explants have been established as a useful tool to investigate effects of pathogenic agents in vitro. We used such cultures as a model to study the effects of choroidal melanoma on retinal organisation and function.. Rabbit retinal explants were co-cultured with human choroidal melanoma cells, or exposed to supernatants from choroidal melanoma cell cultures, for various periods from 1 day to 10 days. The retinal explants were then studied by histology and immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin. The release of the pro-inflammatory interleukins IL-6 and IL-8 into the media was measured by enzyme-linked immunosorbent assay.. Both in the co-cultures and after treatment with choroidal melanoma cell supernatants for more than 1 week, the layered structure of the retinae became disorganised. Retinal glial (Müller) cells displayed gliosis as indicated by increased GFAP immunoreactivity and decreased immunoreactivity for vimentin. Additionally, the secretion of cytokines, particularly of IL-8, was significantly modulated. The retinal explants produced much less IL-8 than the melanoma cells in separate cultures but increased their IL-8 release significantly after a few days' exposure to melanoma cell-conditioned medium.. The results show that in cases of choroidal melanoma, the well-known morphological and inflammatory alterations of the retina are accompanied by glial cell reactivity and up-regulated retinal cytokine secretion, and may be caused by soluble factors secreted and induced by the melanoma.

Topics: Animals; Choroid Neoplasms; Coculture Techniques; Culture Media; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Humans; Interleukin-6; Interleukin-8; Melanoma; Neuroglia; Organ Culture Techniques; Rabbits; Retina; Up-Regulation; Vimentin

Modulation of tumour cell growth by tumour-infiltrating leucocytes is of high importance for the biological behaviour of malignant neoplasms. In melanoma, tumour-associated macrophages (TAM) and tumour-infiltrating lymphocytes (TIL) are of particular interest as inhibitors or enhancers of cell growth. Recruitment of leucocytes from the peripheral blood into the tumour site is mediated predominantly by chemotaxins, particularly by the group of chemokines. The aim of this study was to identify peptides released by human melanoma cells with monocyte chemotactic properties. To assure the presence of biologically active mediators, biochemical purification and biological characterization of peptides was based on a detection system dependent on bioactive, monocyte chemotactic activity in vitro. Cell culture supernatants of melanoma cells were fractioned by heparin-sepharose followed by preparative reversed-phase HPLC steps to enrich monocyte chemotactic activity in one single band on a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel. These purified fractions were shown to react with RANTES-specific antibodies in an enzyme-linked immunosorbent assay (ELISA) as well as in Western blot analysis. Amino acid sequencing of the N-terminal protein fragment confirmed 100% homology to the RANTES protein. Further analysis showed that four out of eight melanoma cell lines constitutively expressed and secreted the beta-chemokine RANTES as detected by ELISA. The amount of RANTES protein secreted (up to 50 ng ml(-1)) was about 5-50 times higher than interleukin 8 (IL-8), determined in the same supernatant samples. Tumour necrosis factor alpha, (TNF-alpha), not, however, IL-2, interferon-gamma (IFN-gamma), or (alpha-melanocyte-stimulating hormone (alpha-MSH) was able to up-regulate RANTES and interleukin 8 secretion. Furthermore, higher levels of RANTES secretion in vitro were associated with increased tumour formation upon s.c. injection of six human melanoma cell lines in nude mice. Our data provide evidence that a subset of melanoma cells express mRNA and secrete RANTES protein which may be partly responsible for the recruitment of monocytes, T-cells and dendritic cells into the tumours. However, transplantation experiments in nude mice suggest that effects of RANTES may also benefit tumour progression. Further studies are needed to dissect the underlying mechanisms.

Topics: Animals; Blotting, Western; Chemokine CCL5; Chemotactic Factors; Chemotaxis, Leukocyte; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Immunity, Cellular; Interleukin-8; Male; Melanoma; Mice; Mice, Nude; Tumor Cells, Cultured

Growth-related oncogene-alpha (GROalpha) was first described as an autocrine mitogen and growth factor for melanoma cells. More recent studies show that GROalpha, interleukin-8 (IL-8) and other members of the alpha-chemokine superfamily are also angiogenic. Therefore, we sought to determine if inhibitors of the alpha-chemokine receptor would be effective in inhibiting the tumour growth and pulmonary metastasis of human melanoma cells. We determined that melanocytes and 12 human melanoma cell lines produce both GROalpha and IL-8. The proliferation of A375SM, a highly metastatic cell line, and C8161-C were significantly increased by human recombinant GROalpha and inhibited by anti-human GROalpha monoclonal antibody. Antileukinate, a potent inhibitor of alpha-chemokine receptor binding, inhibited the binding of GROalpha to its receptors in melanocytes and all 12 melanoma cell lines tested. Antileukinate also suppressed proliferation of A375SM and C8161-C cells in a dose-dependent manner, and the suppression was not due to cytotoxic effects. Furthermore, continuous administration of antileukinate inhibited the tumour growth and pulmonary metastasis of A375SM cells in athymic BALB/c nude mice. These findings suggest that antileukinate inhibits the growth of melanoma cells by preventing GROalpha from binding to its receptors. This suggests a possible use of alpha-chemokine receptor inhibitors such as antileukinate in the treatment of malignant melanoma.

Topics: Animals; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Dose-Response Relationship, Drug; Dose-Response Relationship, Immunologic; Female; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lung Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Peptides; Time Factors; Tumor Cells, Cultured

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.

Topics: Animals; Carcinogens; CD4-Positive T-Lymphocytes; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Interleukin-17; Interleukin-6; Interleukin-8; Male; Melanoma; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Transfection; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Besides its proinflammatory properties, interleukin-8 (IL-8) has been suggested as an important promoter for melanoma growth. To study the role of IL-8 in melanoma biology, we determined the in vivo expression of IL-8 mRNA by in situ hybridization in primary melanoma lesions and metastases. High levels of melanoma cell-associated IL-8-specific transcripts were exclusively detected in close vicinity of necrotic/hypoxic areas of melanoma metastases, whereas both in primary melanomas and in non-necrotic metastases IL-8 expression was low or absent. To analyze further the up-regulation of IL-8 mRNA expression in necrotic/hypoxic tumor areas, human melanoma cell lines of different aggressiveness exposed to severe hypoxic stress (anoxia) were used as an in vitro model. Anoxia induced IL-8 mRNA and protein expression in the highly aggressive/metastatic cell lines MV3 and BLM but not in the low aggressive cell lines IF6 and 530. As shown by IL-8 promoter-dependent reporter gene analysis and mRNA stability assays, elevated mRNA levels in melanoma cells were due to both enhanced transcriptional activation and enhanced IL-8 mRNA stability. Interestingly, transcriptional activation was abolished by mutations in the AP-1 and the NF-kappaB-like binding motifs, indicating that both sites are critical for IL-8 induction. Concomitantly, anoxia induced an enhanced binding activity of AP-1 and NF-kappaB transcription factors only in the highly aggressive cells. From our in vitro and in vivo data we suggest that anoxia-induced regulation of IL-8 might be a characteristic feature of aggressive tumor cells, thus indicating that IL-8 might play a critical role for tumor progression in human malignant melanoma.

Topics: Antigens, CD; Cell Hypoxia; Gene Expression; Genes, Reporter; Humans; Immunohistochemistry; In Situ Hybridization; Interleukin-8; Melanoma; Necrosis; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Promoter Regions, Genetic; Receptors, Chemokine; Receptors, Interleukin; Receptors, Interleukin-8A; Receptors, Interleukin-8B; RNA, Messenger; Skin Neoplasms; Transcription Factor AP-1; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation

It was recently demonstrated that interleukin-8 (IL-8) is produced by cultured melanoma cells and acts as an essential autocrine growth factor. Earlier studies from our laboratory demonstrated a direct correlation between IL-8 expression in human variant cell lines and their metastatic potential in nude mice. In the present study we examined the expression of IL-8 in human malignant melanomas using immunohistochemistry to correlate IL-8 levels with disease stage. None of the radial growth phase (RGP) tumours (melanoma in situ) expressed IL-8. In contrast, 50% of the vertical growth phase (VGP) tumours (invasive melanoma), which have a high risk of metastasis, showed IL-8 immunoreactivity. Further, all the metastatic lesions analysed showed intense staining for IL-8; the levels were higher than those observed in the primary skin tumours. In summary, the data suggest an association between the expression of IL-8 and the metastatic phenotype.

Topics: Biomarkers, Tumor; Disease Progression; Humans; Immunohistochemistry; Interleukin-8; Lymphatic Metastasis; Melanoma; Phenotype; Skin Neoplasms; Soft Tissue Neoplasms

Dissemination of uveal melanomas is almost exclusively haematogenous, making angiogenesis of the tumour a prerequisite for the formation of metastases. Uveal melanomas must employ strategies to evade the immune system in order to escape immune surveillance. We therefore determined the expression of the following angiogenic and immunosuppressive factors in seven human uveal melanoma cell lines using reverse transcriptase-polymerase chain reaction (RT-PCR): secreted interleukin-1 receptor antagonist (sIL-1ra), interleukin (IL)-6, IL-8, IL-10, transforming growth factor (TGF)-alpha, TGFbeta, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and angiopoietin-2. In addition, the secretion of sIL-1ra, IL-6, IL-8, IL-10, TGFbeta and VEGF was assayed by enzyme linked immunosorbent assay (ELISA). The potential of uveal melanoma cell lines to convert plasminogen to angiostatin was tested in an in vitro assay. All the factors except angiopoietin-1 were determined in one or more cell lines using RT-PCR, although these results were not necessarily confirmed by ELISA. Expression of VEGF and angiopoietin-2 was found in all seven cell lines. Production of angiostatin was observed in one cell line. All seven cell lines examined expressed angiogenic factors and most cell lines expressed immunosuppressive factors. The expression of VEGF and angiopoietin-2 in combination with a lack of angiopoietin-1 expression suggest high vascular remodelling capacity and could be of great relevance for the metastatic potential of uveal melanoma.

Topics: Angiopoietin-1; Angiopoietin-2; Angiostatins; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Growth Substances; Humans; Immunosuppressive Agents; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Interleukin-6; Interleukin-8; Melanoma; Membrane Glycoproteins; Neovascularization, Pathologic; Peptide Fragments; Plasminogen; Protein Biosynthesis; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factors; Tumor Cells, Cultured; Uveal Neoplasms

Previous in vitro studies have demonstrated that endogenously produced human interleukin-8 (IL-8) can act as an important growth factor for human melanoma cells in vitro. The present study, has investigated whether IL-8 mRNA expression in primary melanomas may be of prognostic relevance with regard to melanoma progression and metastatic spread. In order to evaluate the clinical significance of IL-8 mRNA expression of melanoma cells in vivo, 59 melanocytic tissue specimens (37 primary melanomas and 22 melanocytic naevi) were studied using a semiquantitative in situ hybridization technique. Significant mRNA expression of IL-8 was found in 59 per cent (22/37) of melanomas. In 19 per cent (7/37) of the malignant melanomas, additional hybridization signals were noted within keratinocytes of the overlying epidermis. In contrast, paralesional normal-appearing epidermis and melanocytes in non-malignant lesions (melanocytic naevi) showed no IL-8 mRNA. Analysis of the relationship between IL-8 expression and clinico-histopathological features showed a significant association between IL-8 mRNA expression and the histological melanoma subtype (IL-8 mRNA: 14/19 in superficial spreading melanoma versus 4/12 in nodular melanoma, p< 0.05). Furthermore, IL-8 expression in primary tumours could be correlated with the patients' clinical course, with time to progression being significantly reduced in primary tumours expressing IL-8 in either the tumour cells or keratinocytes of the overlying epidermis. These results demonstrate for the first time that IL-8 expression, as detected by in situ hybridization in primary tumours, may serve as a significant prognostic factor for tumour progression in human malignant melanoma.

Topics: Adult; Aged; Chi-Square Distribution; Disease-Free Survival; Female; Gene Expression; Humans; In Situ Hybridization; Interleukin-8; Male; Melanoma; Middle Aged; Neoplasm Proteins; RNA, Messenger; Skin Neoplasms

Here, we report the molecular regulation of interleukin (IL)-8 expression in human melanoma cells. The inflammatory cytokines IL-1beta and tumor necrosis factor-alpha (TNF-alpha) up-regulated IL-8 expression, in a time- and concentration-dependent manner, in three metastatic melanoma variants, SBC-2 (nonmetastatic), A375P (low metastatic), and A375SM (high metastatic), by increased transcription of the IL-8 gene, leading to increased levels of IL-8 mRNA and protein production. Furthermore, we report that IFN-alpha and IFN-beta did not inhibit steady-state IL-8 production. However, IFN-alpha and IFN-beta inhibited IL-1beta or TNF-alpha-mediated up-regulation of IL-8 mRNA. In addition, IFN-beta demonstrated a more potent inhibitory effect at a lower concentration than did IFN-alpha. Both pretreatment and simultaneous treatment of melanoma cells with IFN-alpha or IFN-beta inhibited the IL-1beta and TNF-alpha up-regulation of IL-8 mRNA levels. This inhibition was at the transcriptional levels and was unaffected by a protein synthesis inhibitor, suggesting that this did not require de novo protein synthesis. Further, modulation of IL-8 levels by IL-1beta, alone or in combination with IFN-beta, affected the proliferation of melanoma cells. In summary, our data suggest that the up-regulation of IL-8 expression in melanoma cells is regulated at the transcriptional level and is rapidly and specifically inhibited by IFN-alpha or IFN-beta, independent of de novo protein synthesis, perhaps due to a transient modification of a preexisting factor(s).

Topics: Animals; Cell Division; Gene Expression Regulation, Neoplastic; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Interferon-beta; Interleukin-1; Interleukin-8; Melanoma; Mice; Mice, Nude; RNA, Messenger; Stimulation, Chemical; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

Melanomas produce multiple cytokines which may influence their growth in vivo. Experimental evidence suggests that granulocyte macrophage-colony stimulating factor (GM-CSF) can induce a potent anti-melanoma response. whereas interleukin-8 (IL-8) may act as a growth factor in human melanoma. Little is currently known regarding the production of these cytokines by human melanoma in vivo. In this study we tested the hypothesis that endogenous production of GM-CSF and IL-8 can be correlated with the depth of human malignant melanoma surgical specimens. We examined 45 melanocytic human tissue samples consisting of 27 primary cutaneous melanomas, 9 metastatic melanomas, and 9 dysplastic nevi for in vivo GM-CSF and IL-8 production using immunohistochemistry. The majority of thin melanomas (< or = 0.76 mm) stained highly positive for GM-CSF with little or no staining for IL-8 whereas the medium (>0.76- < or = 4.0 mm) and thick (>4.0 mm) melanoma specimens showed little or no staining for GM-CSF and significant amounts of IL-8 staining. Metastatic melanoma as well as dysplastic nevi specimens had little or no GM-CSF and IL-8 staining. These results support the hypothesis that endogenous melanoma cytokines such as GM-CSF and IL-8 with opposing effects on tumor progression play an important role in melanoma growth and regulation.

Topics: Biomarkers, Tumor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-8; Melanoma; Neoplasm Invasiveness; Skin Neoplasms

IL-8 is expressed by activated and neoplastic astrocytes and enhances the survival of hippocampal neurons in vitro. Since mRNA encoding chemokine receptors have been demonstrated in brain, the expression of chemokine receptors by specific cell types in anatomic regions of the central nervous system (CNS) was investigated. Archival tissues from various regions of the CNS were stained with specific mAbs to the Duffy Ag/receptor for chemokines, a promiscuous receptor that binds selected chemokines; the specific receptor for IL-8 (CXCR1); and the receptor (CXCR2) shared by IL-8 and melanoma growth stimulatory activity. The Duffy Ag/receptor for chemokines was expressed exclusively by Purkinje cells in the cerebellum. Chemokine binding and radioligand cross-linking confirmed the presence of a high affinity, promiscuous chemokine receptor in the cerebellum. Although CXCR1 was not expressed in the CNS, CXCR2 was expressed at high levels by subsets of projection neurons in diverse regions of the brain and spinal cord, including the hippocampus, dentate nucleus, pontine nuclei, locus coeruleus, and paraventricular nucleus, and in the anterior horn, interomediolateral cell column, and Clarke's column of the spinal cord. Fibers that express CXCR2 included those in the superior cerebellar peduncle and the substantia gelatinosa. Immunohistochemical analysis of the involved brain tissues from patients with Alzheimer's disease revealed expression of CXCR2 in the neuritic portion of plaques surrounding deposits of amyloid. These data suggest that chemokines may play a role in reactive processes in normal neuronal function and neurodegenerative disorders.

Topics: Antigens, CD; Antigens, Protozoan; Brain; Carrier Proteins; Cell Division; Chemokines; Duffy Blood-Group System; Humans; Immunohistochemistry; Interleukin-8; Melanoma; Neurons; Protozoan Proteins; Receptors, Cell Surface; Receptors, Cytokine; Receptors, Interleukin; Receptors, Interleukin-8A

We reported earlier that IL-1 inhibits the growth of human melanoma cells (A375-6), and that these cells become resistant to IL-1 after prolonged periods of culture. The resistant cells constitutively produce IL-alpha and IL-6 with IL-6 production was induced by endogenous IL-1 in an autocrine manner. The cells are also resistant to IL-6 anti-proliferative effects. In the present study, we show that the resistant clones exhibited up-regulated expression of intercellular-adhesion molecule 1 (ICAM-1) and vitronectin receptor (integrin alpha(v)beta3) when compared with the IL-1-sensitive clone, A375-6. Moreover, these IL-1-resistant clones exhibited many other metastatic characteristics, such as expression of IL-8 mRNA, production of matrix metalloproteinases (MMP-2 and MMP-9), and augmented invasion activity. However, contrary to our expectations, the IL-1-resistant cells did not exhibit experimental metastasis in a nude-mouse model, similarly to the IL-1-sensitive parental A375-6 cell line. In contrast, the highly metastatic clone A375-SM exhibited alpha(v)beta3 expression at a level comparable to that of the IL-1-resistant cells, but expressed low or no ICAM-1, metalloproteinase and displayed little in vitro invasion activity. These results show that the metastatic characteristics of IL-1-resistant cells are not sufficient to produce metastasis in vivo and suggest that these resistant clones may provide a good model system for characterizing the molecular mechanisms of metastasis.

Topics: Animals; Cell Division; Cell Line; Collagenases; Drug Resistance, Neoplasm; Flow Cytometry; Gelatinases; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Metalloendopeptidases; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Messenger; Skin Neoplasms; Transcription, Genetic; Transplantation, Heterologous; Tumor Cells, Cultured

Expression of interleukin-8 (IL-8) by human melanoma cells correlates with their metastatic potential. Moreover, UV-B irradiation of primary cutaneous melanoma cells induces IL-8 mRNA and protein production and increases both tumor growth and metastasis in nude mice. Although IL-8 has been shown to be an angiogenic factor, the biological consequences of increased IL-8 production by melanoma cells and the role of IL-8 in the metastatic process remains unclear. The purpose of this study was to determine the role of IL-8 in tumor growth and metastasis of human melanoma cells. Nonmetastatic SB-2 melanoma cells with negligible levels of IL-8 were transfected with IL-8 cDNA and subsequently analyzed for changes in their tumorigenic and metastatic potential. Enforced expression of IL-8 rendered the melanoma cells highly tumorigenic and increased their metastatic potential as compared with parental and control transfected cells. The IL-8-transfected cells displayed up-regulation in M(r) 72,000 collagenase type IV (MMP-2) mRNA and collagenase activity and increased invasiveness through Matrigel-coated filters. Moreover, when the MMP-2 promoter was linked upstream of the chloramphenicol acetyltransferase (CAT) reporter gene, CAT activity was up-regulated in IL-8 but not in control transfected cells, suggesting that IL-8 is involved in MMP-2 gene transcription. Activation of type IV collagenase by IL-8 can enhance the invasion of host stroma by the tumor cells and increase angiogenesis and, hence, metastasis.

Topics: Animals; Chloramphenicol O-Acetyltransferase; Gelatinases; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Melanoma; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; RNA, Messenger; Skin Neoplasms; Transfection; Tumor Cells, Cultured; Up-Regulation

Three human MGSA/GRO genes encode 3 highly related chemokines, MGSA/GRO alpha, -beta and -gamma. All 3 MGSA/GRO proteins bind to the same receptors, but with differing affinities, and stimulate a number of biological responses including chemotaxis, angiogenesis, and growth regulation. We have previously demonstrated that MGSA/GRO alpha can be isolated from culture medium conditioned by malignant melanoma cells and that continuous secretion of MGSA/GRO alpha contributes to the transformation of immortalized murine melanocytes. The present study was designed to determine whether MGSA/GRO beta or -gamma have similar effects on melanocyte tumorigenicity. Stable Melan-a clones expressing either human MGSA/GRO beta or -gamma exhibited enhanced ability to form large colonies in soft agar and tumors in nude mice. The clones expressing the MGSA/GRO beta or -gamma transgene formed tumors within 2 months after injection; the tumors were highly pigmented and expressed immunoreactive MGSA/GRO beta or -gamma protein. Furthermore, when conditioned medium from Melan-a clones expressing MGSA/GRO alpha, -beta or -gamma transgenes were examined for the ability to induce angiogenesis in the rat cornea, strong angiogenic responses were observed. This angiogenic response was blocked by antibodies to the respective MGSA/GRO protein, but not by normal rabbit serum. By contrast, angiogenic responses were observed in only 2 of 12 corneal implants (17%) containing medium conditioned by Melan-a clones expressing the neomycin resistance marker alone.

Topics: Amino Acid Sequence; Animals; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Melanoma; Mice; Mice, Nude; Molecular Sequence Data; Neovascularization, Pathologic; Rabbits; Rats; Rats, Inbred F344; Receptors, Interleukin; Receptors, Interleukin-8B; Transgenes; Tumor Cells, Cultured

The glucocorticoid dexamethasone inhibited the production of the rat cytokine-induced neutrophil chemoattractant CINC/gro, a counterpart of human melanoma growth-stimulating activity that belongs to the interleukin-8 (IL-8) family, in the normal rat kidney epithelial cell line NRK-52E stimulated with interleukin-1 beta (IL-1 beta), lipopolysaccharide, or tumor necrosis factor alpha. The accumulation of CINC/gro mRNA induced by these activators was also decreased comparably by dexamethasone. A nuclear run-on assay revealed that dexamethasone decreased the IL-1 beta-induced transcription of the CINC/gro gene. The half-life of CINC/gro mRNA transcripts did not change significantly after exposure to dexamethasone, suggesting that this glucocorticoid acts mainly at the transcriptional level. Transfection with luciferase expression vectors containing 5'-deleted and mutated CINC/gro gene sequences demonstrated that the 5'-flanking region containing the NF-kappa B binding site is involved in the IL-1 beta- and dexamethasone-induced activation and repression of the CINC/gro gene expression, respectively. Furthermore, a tandem repeat of the NF-kappa B sequence in the CINC/gro gene conferred the inducibility by IL-1 beta and suppression of luciferase activity by dexamethasone. In an electrophoretic mobility shift assay, dexamethasone diminished the IL-1 beta-induced formation of NF-kappa B complexes, which consisted of p65 and p50. Western blotting revealed that dexamethasone inhibited the IL-1 beta-induced translocation of p65 from the cytoplasm into the nucleus, while the nuclear level of NF-kappa B p50 remained almost unchanged. In addition, the degradation of I kappa B-alpha induced by IL-1 beta was not inhibited by dexamethasone. These results indicated that the suppression of the CINC/gro gene transcription by glucocorticoid occurs through the impairment of NF-kappa B activation, possibly by interference with the translocation of NF-kappa B p65 from the cytoplasm into the nucleus, thereby suppressing transactivation of the CINC/gro gene.

Topics: Animals; Base Sequence; Blotting, Northern; Cell Division; Cell Line; Cell Nucleus; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Cytokines; Dexamethasone; Gene Expression; Growth Inhibitors; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1; Interleukin-8; Kidney; Kinetics; Luciferases; Melanoma; Molecular Sequence Data; NF-kappa B; Oligonucleotide Probes; Rats; Recombinant Fusion Proteins; Recombinant Proteins; RNA, Messenger; Suppression, Genetic; Transcription, Genetic; Transfection; Tumor Necrosis Factor-alpha

Incidence and mortality of human malignant melanoma has risen rapidly over recent decades. Although the notorious resistance to treatment is characteristic for metastatic malignant melanoma, only a few experimental models have been established to study the metastatic cascade or to test new alternative treatment modalities. Thus, new human models are wanted. Here, we describe the metastatic behaviour of seven human melanoma cell lines derived from two primary cutaneous melanomas (WM 98-1, WM 1341) and five metastases established from liver (UKRV-Mel-4), skin (M7, M13), pleural effusion (UKRV-Mel-2) and lymph node (MV3). All cell lines were analysed for their capacity to grow in nude mice after s.c. and i.v. administration. M13 cells developed liver metastases spontaneously after s.c. injection, and subsequent passages of M13 and M7 melanoma cells caused liver metastases after i.v. injection, whereas MV3 and WM98-1 gave rise to lung metastases, using the same inoculation route. In contrast, WM 1341, UKRV-Mel-2 and UKRV-Mel-4 grew only very slowly in nude mice after s.c. injection and did not cause any metastases after i.v. or s.c. administration. The pattern of metastases or growth kinetics did not correlate with the interleukin 8 or tumour necrosis factor secretion of cell lines. Adhesion molecules and growth factor receptor expression on the cell lines differed widely, as determined by flow cytometry, with the low metastatic cell lines (UKRV-Mel-2, UKRV-Mel-4 and WM 1341) demonstrating a marked reduction in VLA-1 and VLA-5 expression compared with the metastatic lines (M7, M13, MV3 and WM 98-1). Expression of pigment-related proteins such as tyrosinase, TRP-1, TRP-2, Melan-A/MART-1, gp100, MAGE1 or MAGE-3 was not associated with growth and metastatic characteristics of the melanoma cell lines analysed. In conclusion, the established human melanoma cell lines exhibited diverse growth behaviour in nude mice in congruence with some early established prognostic markers such as VLA-1 and VLA-5. The xenografts provide good models for further study of metastatic processes as well as for evaluation of alternative treatment modalities including new pharmaceutical drugs and gene therapeutic targeting using tissue-specific gene regulatory elements for gene targeting.

Topics: Animals; Antigens, Neoplasm; Base Sequence; Cell Adhesion Molecules; Cell Division; Female; Flow Cytometry; HLA Antigens; Humans; Interleukin-8; Male; Melanoma; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Transplantation; Phenotype; Pigments, Biological; Skin Neoplasms; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In the past, interleukin-8 (IL-8) could be demonstrated within keratinocytes in normal epidermis and inflammatory skin diseases, like psoriasis and eczema. Using monoclonal antibodies, the distribution of IL-8 immunoreactivity was inversely related to the density of inflammatory infiltrate. Other in vitro observations indicated IL-8 to be a growth factor for keratinocytes. These results prompted an immunohistochemical examination of IL-8 immunoreactivity in malignant and semimalignant epithelial tumours of human skin. Whereas IL-8 could not be detected within the transformed cells of epithelial tumours or melanoma, some tumour cells within well differentiated squamous cell carcinoma and Bowen's disease showed IL-8 immunoreactivity. Thus, loss of IL-8 immunoreactivity can be a sign of malignant transformation. This indicates an important role in growth regulation as well as terminal differentiation of human keratinocytes.

Topics: Bowen's Disease; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Humans; Immunohistochemistry; Interleukin-8; Keratinocytes; Melanoma; Skin; Skin Neoplasms

In the present study we evaluated the haematological and immunological changes in 4 patients with advanced melanoma and 6 patients with advanced renal cell carcinoma treated with subcutaneous interleukin (IL)-2 and interferon (IFN)-alfa-2b. Serum samples taken before and during six weeks' courses of IL-2 plus IFN-alfa were assayed for the presence of IL-2, soluble IL-2-receptor (sIL-2R), soluble intercellular adhesion molecule-1 (sICAM-1), IL-6 and IL-8. In addition, whole blood counts were taken. Eosinophilia occurred in all patients, lymphocytosis in 8 patients. The higher maximum level of IL-2 during treatment seemed to be connected to longer survival: it was a median of 578 pg/ml in the patients with a median survival of 7 months, and 1025 pg/ml in the patients who survived a median of 15 months. Conversely, an increase in sIL-2R was an unfavourable sign: it was a median of 8-fold and 3-fold in the patients with a median survival of 7 and 16 months, respectively. During treatment, sICAM-1 levels paralleled with those of sIL-2R. There was major intraindividual and interindividual variation in serum IL-6 and IL-8 levels with no distinctive kinetic pattern. Thus, no definite conclusions could be drawn. However, it seems worthwhile to measure IL-2, sIL-2R and sICAM-1 during immunotherapy; their prognostic value should be further evaluated in a larger patient population.

Topics: Adult; Aged; Blood Cell Count; Carcinoma, Renal Cell; Drug Therapy, Combination; Female; Humans; Injections, Subcutaneous; Intercellular Adhesion Molecule-1; Interferon-alpha; Interleukin-2; Interleukin-6; Interleukin-8; Kidney Neoplasms; Male; Melanoma; Middle Aged; Receptors, Interleukin-2; Skin Neoplasms

Interleukin-8 (IL-8) is a cytokine that is thought to promote melanoma tumour progression. We evaluated and adapted a non-radioactive, reverse transcriptase-polymerase chain reaction method for semiquantitative analysis of IL-8 mRNA expression. Using this technique we studied the regulation of IL-8 levels in the melanoma cell line Colo 38. Seeding of melanoma cells into culture dishes resulted in a significant increase of IL-8 expression, which could be attributed to adherence. A pronounced increase of IL-8 mRNA expression and protein production was induced by tumour necrosis factor-alpha (TNF alpha). Interferon-gamma (IFN gamma) partially inhibited TNF alpha-induced IL-8 secretion, whereas no influence on IL-8 mRNA levels was detected. The inhibitory affect of IFN gamma on melanoma cells is in contrast to its stimulatory effect on melanocytes.

Topics: Antineoplastic Agents; Humans; Interferon-gamma; Interleukin-8; Melanoma; Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The constitutive expression of interleukin-8 (IL-8) by human melanoma cells correlates with their metastatic potential. The exposure of human melanoma cells to the inflammatory cytokines IL-1 beta or tumor necrosis factor-alpha (TNF-alpha) upregulated IL-8 expression in a time-dependent and concentration-dependent manner. This enhanced expression of IL-8 was inhibited by cycloheximide or actinomycin-D. Treatment of melanoma cells with interferon (IFN) alpha, beta, or gamma did not affect the constitutive expression of IL-8, but IFN-alpha and IFN-beta blocked the upregulation of IL-8 expression in cells treated with IL-1 beta or TNF-alpha subsequent to or simultaneously with the IFN. These data suggest that the expression of IL-8 in human melanoma cells can be upregulated by inflammatory cytokines and that IFN-alpha and IFN-beta can counterregulate this stimulation.

Topics: Animals; Cycloheximide; Dactinomycin; Female; Gene Expression Regulation, Neoplastic; Humans; Interferon alpha-2; Interferon-alpha; Interferon-beta; Interferon-gamma; Interleukin-1; Interleukin-10; Interleukin-8; Lung Neoplasms; Lymphatic Metastasis; Melanoma; Mice; Mice, Nude; Middle Aged; Neoplasm Proteins; Nucleic Acid Synthesis Inhibitors; Protein Synthesis Inhibitors; Recombinant Proteins; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Induction of interleukin-8 (IL-8) by IL-1 or tumor necrosis factor (TNF), and repression by interferons or glucocorticoids have been shown to involve sequences between nucleotides -94 and -71 of the 5'-flanking region, and the transcription factors NF-IL-6 and NF-kappaB. The A3 cell line was derived from the human melanoma cell line G-361 by stable transfection with part of the IL-8 promoter (nucleotides -101 to +40 from transcription start) fused to the luciferase coding region. These regulatory sequences were sufficient for transcriptional activation by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, IL-1beta, or TNF-alpha. Simultaneous treatment of A3 cells with ATRA and TNF-alpha resulted in a dose- and time-dependent synergistic increase in luciferase expression and IL-8 mRNA levels. Transient transfections of the parental cell line demonstrated that the NF-kappaB binding site is essential for this synergistic transactivation. Electrophoretic mobility shift assays with nuclear extracts of A3 cells showed that stimulation with ATRA and TNF-alpha for more than 16 h resulted in enhanced NF-kappaB binding compared to that induced by TNF-alpha alone. The simultaneous treatment with ATRA and TNF-alpha also resulted in changes in the composition of NF-kappaB complexes bound to the IL-8 NF-kappaB site, preventing the formation of two TNF-alpha-inducible binding activities. We suggest that these complexes consist of repressive factors which, when removed, allow enhanced binding of NF-kappaB to its cognate site.

Topics: Drug Synergism; Humans; Inflammation Mediators; Interleukin-8; Melanoma; NF-kappa B; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

The objective of the present study was to investigate the effects of isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan in patients with cancer on, first, plasma levels of cytokines, second, systemic monocyte and T-lymphocyte distribution and, third, the ability of mononuclear cells to produce cytokines upon stimulation in vitro. Six patients undergoing an ILP were entered into the study (group 1). In addition, patients undergoing a major surgical operation (group 2) minor operation (group 3) as well as healthy volunteers (group 4) were included as control groups. Sensitive enzyme-linked immunosorbent assays (ELISAs) were used to measure TNF-alpha and interleukin-6 (IL-6) plasma levels at various time points during and after operation. Furthermore, the percentage of monocytes and T lymphocytes was determined in all studied groups using a FACScan. In addition, cytokine production upon stimulation with lipopolysaccharide (LPS) and a combination of anti-CD3/anti-CD28 monoclonal antibodies in whole-blood cultures was investigated. Increased plasma levels of TNF-alpha and IL-6 in patients undergoing ILP was observed, but only IL-6 appeared to be increased in patients treated with a major operation. No significant fluctuations were found in the other groups studied. Concerning the number of monocytes, a significant decrease was observed only in patients treated with ILP. Furthermore, a decreased production of TNF-alpha, IL-6 and IL-8 upon various types of stimulation in vitro was found in those patients, but also after a major operation. In conclusion, the results of the present study show increased plasma levels of cytokines in patients treated with ILP and major operation. Furthermore, a decrease in numbers of monocytes in the circulation and the ability of mononuclear cells to produce cytokines in vitro may be induced by administration of TNF-alpha in ILP. Although similar results were found in patients treated with major operation, the underlying mechanisms of this phenomenon remain to be elucidated.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; CD28 Antigens; CD3 Complex; Chemotherapy, Cancer, Regional Perfusion; Female; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Male; Melanoma; Middle Aged; Monocytes; Sarcoma; T-Lymphocytes; Time Factors; Tumor Necrosis Factor-alpha

UV radiation has been shown to play a role in the initiation of human cutaneous melanoma, but its role in the development of malignant melanoma to the metastatic state is not very well defined. Although previous studies have concentrated on the effect of UV-B on the host immune response, the effect of UV-B on the tumor cells was not elucidated. Here we show that UV-B can induce interleukin 8 (IL-8) mRNA and protein secretion in human cutaneous melanoma with negligible expression of IL-8. UV-B-induced IL-8 was constitutively expressed 60 days after irradiation in tumors implanted in mice. Induction of IL-8 was UV-B dose dependent and blocked by cyclohexamide, indicating that de novo protein synthesis is required for its expression. The UV-irradiated cells demonstrated enhanced tumorigenicity and metastatic potential in nude mice. The increase in tumorigenicity and metastatic ability could be explained by the increase in Mr 72,000 type IV collagenase activity and angiogenesis attributed to the induction of IL-8 after irradiation. The acquisition of the metastatic phenotype induced by UV-B could not be attributed to abnormalities in the p53 or MTS-1 (p16INK4) genes. To the best of our knowledge, this is the first report to show that UV-B can increase the aggressiveness of human cutaneous melanoma for growth and metastasis.

Topics: Animals; Carrier Proteins; Collagenases; Cyclin-Dependent Kinase Inhibitor p16; Cycloheximide; Dose-Response Relationship, Radiation; Gene Expression Regulation, Neoplastic; Genes, p53; In Vitro Techniques; Interleukin-8; Lung Neoplasms; Male; Matrix Metalloproteinase 9; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; RNA, Messenger; Skin Neoplasms; Tumor Cells, Cultured; Ultraviolet Rays

We investigated the role of nitric oxide (NO) in the expression of interleukin-8 (IL-8) in the human melanoma cell line, G361. Three NO donors, 3-morpholinosydnonimine hydrochloride (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-L-glutathione (SNOG), all caused an increase in both IL-8 protein secretion and promoter activity. Truncation of the promoter showed that 101 bp of the 5' flanking region proximal to the transcription start site are sufficient for the response to NO. Furthermore, mutation of the NF-kappa B and NF-IL-6 binding sites led to a significant decrease in NO-stimulated promoter activity. The nitric oxide synthase inhibitor, NG-amino-L-homoarginine (NAHA), inhibited TNF-alpha-stimulated IL-8 promoter activity by 60%. Addition of excess L- but not D-arginine partially reversed the NAHA-mediated inhibition. These results demonstrate that NO is an endogenous regulator of IL-8 production in G361 melanoma cells.

Topics: Base Sequence; Binding Sites; CCAAT-Enhancer-Binding Proteins; Cell Line; DNA Primers; DNA-Binding Proteins; Gene Expression; Glutathione; Humans; Interleukin-8; Melanoma; Molecular Sequence Data; Molsidomine; Mutagenesis, Site-Directed; NF-kappa B; Nitric Oxide; Nitroso Compounds; Nuclear Proteins; Penicillamine; Platelet Aggregation Inhibitors; Polymerase Chain Reaction; Promoter Regions, Genetic; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Vasodilator Agents

It was recently demonstrated that IL-8 is produced by melanoma cell lines and acts as an essential autocrine growth factor. We studied the constitutive production of IL-8 by melanoma cell lines and the serum concentrations in patients with metastatic melanoma. All of 10 melanoma cell lines investigated constitutively produced IL-8 (mean 315 +/- 58 pg/10(5) cells per 24 h. IL-8 was detectable (mean 159 +/- 13.1 pg/ml) in the serum of 21 out of 56 patients by an enzyme-linked immunosorbent assay (ELISA; detection limit < 100 pg/ml). There was a significant correlation with tumour load, whereas no correlation with metastatic sites was found. No increased IL-8 levels were seen in healthy controls or patients with metastatic renal cell carcinoma. These results suggest that IL-8 is constitutively produced by melanoma cells in vivo.

Topics: Cytokines; Enzyme-Linked Immunosorbent Assay; Growth Substances; Humans; Interferon-alpha; Interferon-gamma; Interleukin-8; Melanoma; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The in vitro expression level of interleukin-8 (IL-8) correlates with the metastatic potential of human melanoma cells. The purpose of this study was to determine whether the expression level of IL-8 in human melanoma cells is influenced by the organ microenvironment. A375P cells, a low metastatic human melanoma, and A375SM cells, a highly metastatic variant, were injected into the subcutis (s.c.), spleen (to produce liver metastases), and lateral tail vein (to produce lung metastases) of athymic nude mice. Northern blot and immunohistochemical analyses determined that s.c. tumors, lung lesions, and liver lesions expressed high, intermediate, and low IL-8, mRNA, and protein, respectively. This differential regulation of IL-8 was not due to the size or density of the lesions or to selection of subpopulations of cells. We based this conclusion on the results of three experiments: (a) melanoma cell lines established in culture from in vivo-growing tumors exhibited similar levels of IL-8 mRNA transcripts; (b) in a crossover experiment, the level of IL-8 mRNA was always high in A375 tumors reestablished in the skin and low in the tumors reestablished in the liver, regardless of whether the melanoma cells had been first harvested from s.c. or liver tumors; and (c) A375 melanoma cells cocultured with human keratinocytes produced high levels of IL-8 protein, whereas A375 cells cocultured with highly differentiated human hepatoma cells produced decreased levels. When A375P cells were then incubated with cytokines associated with keratinocytes (IL-1 and interferon beta) or hepatocytes (transforming growth factor alpha or beta), IL-1 enhanced the production of IL-8 protein, whereas TGF-beta decreased its production. These data show that IL-8 expression in melanoma cells is modulated by local host factors.

Topics: Adaptation, Psychological; Animals; Cell Communication; Cell Division; Female; Humans; Interleukin-1; Interleukin-8; Keratinocytes; Liver Neoplasms, Experimental; Lung; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Organ Specificity; RNA, Messenger; Skin Neoplasms; Skin Physiological Phenomena; Transforming Growth Factor beta

Melanoma growth stimulatory activity (MGSA)/growth regulated (GRO) and interleukin-8 (IL-8) are highly related chemokines that have a causal role in melanoma progression. Expression of these chemokines is similar in that both require the NF-kappa B element and additional regions such as the CAAT/enhancer binding protein (C/EBP) element of the IL-8 promoter. The constitutive and cytokine IL-1-induced promoter activity of the chemokine MGSA/GRO alpha in normal retinal pigment epithelial and the Hs294T melanoma cells is partially regulated through the NF-kappa B element, which binds both NF-kappa B p50 and RelA (NF-kappa B p65) homodimers and heterodimers. Mutational analysis of the MGSA/GRO alpha promoter reveals that, in addition to the NF-kappa B element, the immediate upstream region (IUR) is necessary for basal expression in retinal pigment epithelial and Hs294T cells. Gel mobility shift and UV cross-linking analyses demonstrate that several constitutive DNA binding proteins interact with the IUR. Although this region has sequence similarity to the several transcription factor elements including C/EBP, the IUR includes sequences that have no similarity to previously identified enhancer regions. Furthermore, RelA transactivates through either the NF-kappa B element or the IUR, suggesting a putative interaction between NF-kappa B and this novel complex.

Topics: Animals; Base Sequence; CCAAT-Enhancer-Binding Proteins; Cell Line; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Cross-Linking Reagents; Cytokines; DNA Mutational Analysis; DNA-Binding Proteins; Gene Expression; Growth Inhibitors; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Melanoma; Molecular Sequence Data; NF-kappa B; Nuclear Proteins; Pigment Epithelium of Eye; Promoter Regions, Genetic; Sequence Homology, Nucleic Acid; Transcription Factor RelA; Transcription Factors; Tumor Cells, Cultured; Ultraviolet Rays

FCE 27266, 2,2,'-(carbonyl-bis(imino-N-methyl-4,2-pyrrole carbonyl-imino¿N-methyl-4,2-pyrrole¿carbonylimino])-bis- (1,5-naphthalene) disulfonic acid, is a noncytotoxic compound able to complex bFGF, PDGF beta, IL-8, VEGF and IL-1 beta and to inhibit the binding to their receptors. A single intravenous treatment 48 h prior to intravenous injection with tumor cells was associated with 60% inhibition of lung metastasis from B16F10 murine melanoma and 82% inhibition of liver metastasis from M5076 murine reticulosarcoma. Marginal inhibition was observed in the latter model, administering the drug 24 h after tumor cell injection. Efficacy was maintained in athymic mice, with 95 and 100% inhibition of lung metastasis from B16F10 melanoma and A375 human melanoma. The antimetastatic activity was confirmed in two models of spontaneous metastasis: in Lewis lung carcinoma implanted intramuscularly, daily intraperitoneal treatment from day 1 to 17 was associated with 77% inhibition of lung metastasis; on M5076 reticulosarcoma implanted intramuscularly, daily intraperitoneal treatment from day 1 to 14 prior to amputation of the tumor was associated with significant inhibition of liver metastasis (79%); conversely, daily intraperitoneal treatment from day 15 to 28 starting 1 day after amputation was marginally effective. The administered doses did not inhibit the growth of the primary tumor in both models. It is concluded that FCE 27266 is a novel, promising molecule, with significant efficacy on lung and liver metastases of murine and human origin; its mode of action is still under study and is probably exerted through inhibition of growth factors and cytokines influencing the different steps of angiogenesis and metastasis.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Binding, Competitive; Cell Line; Distamycins; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; HL-60 Cells; Humans; Interleukin-1; Interleukin-8; Kinetics; Liver Neoplasms; Lung Neoplasms; Lymphokines; Lymphoma, Large B-Cell, Diffuse; Male; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Neoplasm Metastasis; Platelet-Derived Growth Factor; Receptors, Growth Factor; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

We correlated the steady state transcription and protein secretion of interleukin 8 (IL-8) in 13 different human melanoma cell lines with their ability to grow and produce metastasis in nude mice. Highly metastatic cells expressed higher steady state levels of IL-8 mRNA transcripts than did low metastatic cells. In situ mRNA hybridization analyses confirmed the pattern of mRNA expression on a cellular level. Increased mRNA expression directly correlated with secretion of IL-8 protein as determined by enzyme-linked immunosorbent assay. Recombinant IL-8 stimulated the proliferation of low metastatic A375P cells in a dose-dependent manner, a stimulation that was abrogated by the use of a polyclonal antibody against IL-8. The data suggest that IL-8 can be an autocrine growth factor for human melanoma cells and that IL-8 is involved in melanoma metastasis.

Topics: Animals; Base Sequence; Blotting, Northern; Cell Division; Gene Expression; Humans; In Situ Hybridization; Interleukin-8; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; RNA, Messenger; Stimulation, Chemical; Transcription, Genetic

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.

Topics: Base Sequence; Cell Line; Cell Membrane; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Cloning, Molecular; DNA Primers; Female; Growth Substances; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Keratinocytes; Kidney; Kinetics; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Open Reading Frames; Phosphorylation; Placenta; Polymerase Chain Reaction; Pregnancy; Receptors, Interleukin; Receptors, Interleukin-8A; RNA, Messenger; Transfection; Tumor Cells, Cultured

We screened a panel of 8 primary and 21 metastatic melanoma cell lines for constitutive secretion of cytokines. Melanomas expressed bioactivity for TGF-beta (8/25 lines) and IFN (7/12), but not IL-2. Immunoassays detected IL-1 alpha (4/25), IL-1 beta (12/25), IL-6 (13/29), IL-8 (29/29), TGF-beta 2 (5/12) and GM-CSF (11/29), but not IL-3, IL-4, TNF-alpha, or IFN-gamma. There was no preferential association of cytokine production with cells cultured from primary versus metastatic disease, and only IL-8 was produced by all lines tested. These data demonstrate that cultured melanomas produce a variety of cytokines which are potentially capable of influencing tumor growth in vivo.

Topics: Biological Assay; Culture Media, Conditioned; Cytokines; Enzyme-Linked Immunosorbent Assay; Humans; Interferons; Interleukin-8; Interleukins; Melanoma; Neoplasm Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Monocyte chemotactic and activating factor (MCAF) is an important mediator of monocyte recruitment to sites of chronic inflammation and neoplasia. In the present study, we determined whether MCAF can also enhance the activation of tumoricidal capacity of monocytes. Human monocytes incubated with MCAF and subthreshold concentrations of lipopolysaccharide (LPS) exhibited synergistic tumoricidal activity against allogeneic A375 melanoma cells, irrespective of their metastatic potential. The sequence of MCAF and LPS treatment was crucial. Monocytes treated first with MCAF for 4 h and then with LPS for 18 h were highly cytotoxic to the melanoma cells, whereas monocytes first treated with LPS and then with MCAF were not. Treatment of monocytes with MCAF and LPS also significantly increased production of tumor necrosis factor. These data suggest that like interferon-gamma, MCAF can prime human monocytes to respond to LPS. Interleukin-8, a chemokine for neutrophils, did not enhance the monocytes' LPS-triggered tumoricidal response. Collectively, these data show that MCAF can influence the recruitment and tumoricidal activation of blood monocytes. Therefore, MCAF may be an important mediator of tumor regression.

Topics: Chemokine CCL2; Chemotactic Factors; Cytokines; Cytotoxicity, Immunologic; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Humans; Interferon-gamma; Interleukin-8; Lipopolysaccharides; Lymphatic Metastasis; Lymphocyte Activation; Lymphocytes; Melanoma; Recombinant Proteins; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Normal melanocytes require a number of exogenous growth factors in contrast to most metastatic malignant melanomas. This investigation demonstrates that endogenously produced human IL-8 can act as an important growth factor for human melanoma cells. In the present study, six out of eight human melanoma cell lines tested secrete IL-8 protein into the culture supernatant. In two of these IL-8-secreting melanoma cell lines, SK-MEL 13 and SK-MEL 23, we have determined the IL-8 requirement for their proliferative capacity. These melanoma cell lines produced significant amounts of bioactive IL-8 as measured by the ELISA technique. Secretion of human IL-8 was inducible by IL-1 and by PMA. Human IL-8-specific mRNA was already detected in unstimulated melanoma cells. In addition, human IL-8-R mRNA could be detected for the first time in human melanoma cells. Exposure of the two melanoma cell lines in vitro to antisense oligonucleotides targeted against two different sites of human IL-8 mRNA-inhibited cell proliferation, colony formation in soft agar, and secretion of IL-8 protein into culture supernatant in a dose dependent fashion. Effects were reversible either by removal of the oligomers or by addition of exogenous IL-8 protein. In contrast, exposure to IL-8 sense probes or oligonucleotides in sense or antisense orientation specific for IL-7, TGF-alpha, TGF-beta, and MGSA had no such effect. A monospecific immune serum and two IL-8-specific mAb were also capable of inhibiting melanoma cell proliferation in the same manner. These results provide strong evidence for an autocrine IL-8 synthesis and for an IL-8-dependent proliferation in a subgroup of human melanomas. Furthermore, they suggest that IL-8 may play a role not only in immunomodulation but also in melanoma progression and metastatic spread.

Topics: Base Sequence; Cell Division; Humans; Interleukin-8; Melanoma; Molecular Sequence Data; Oligonucleotides, Antisense; Receptors, Immunologic; Receptors, Interleukin-8A; RNA, Messenger; Tumor Cells, Cultured

Two cDNAs coding for distinct interleukin 8 (IL-8) receptors, IL-8R1 [Murphy and Tiffany (1991) Science 253, 1280-1283] and IL-8R2 [Holmes, Lee, Kuang, Rice and Wood (1991) Science 253, 1278-1280] have been reported, and biochemical studies on human neutrophils have revealed two proteins (p70 and p44) that bind IL-8 with high affinity [Moser, Schumacher, von Tscharner, Clark-Lewis and Baggiolini (1991), J. Biol. Chem. 266, 10666-10671]. We have cloned the cDNA coding for IL-8R1 from a library of differentiated HL-60 cells. Transfection of this cDNA into Jurkat cells resulted in the expression of high-affinity binding for IL-8 and two related cytokines, GRO alpha and neutrophil-activating peptide 2 (Kd 0.5-1.0 nM). Northern-blot analysis with the IL-8R1 cDNA as probe revealed abundant expression of transcripts of different size in human neutrophils and low-level expression of a single RNA species in HL-60 cells differentiated with dimethyl sulphoxide and retinoic acid. Because of the extensive nucleotide sequence similarity of the cDNAs for IL-8R1 and IL-8R2, the reverse-transcription PCR method was used for analysis of RNA expression in myeloid and lymphoid cells, 19 cell lines established from human primary melanomas or metastases, two melanocyte and one fibroblast cell lines. IL-8R1 mRNA transcripts were expressed at high levels in neutrophils, and to a lesser extent in blood monocytes and the myeloid cell lines, HL-60 and AML 193, but were not found in THP-1 cells, lymphocytes and Jurkat cells. IL-8R2 mRNA transcripts, by contrast, were found in all blood cells and related cell lines, as well as in all melanoma, melanocyte and fibroblast cell lines tested. As for IL-8R1, IL-8R2 mRNA expression was highest in neutrophils. These results suggest that IL-8R1 and IL-8R2 may both be involved in neutrophil activation by IL-8 and related cytokines, and presumably correspond to p70 and p44, the receptors that were identified biochemically. Possible IL-8 functions on lymphocytes and melanoma cells, e.g. chemotaxis and proliferation, must be independent of IL-8R1 and may be mediated by IL-8R2.

Topics: Base Sequence; Cells, Cultured; Cloning, Molecular; DNA; Humans; Interleukin-8; Lymphocytes; Melanoma; Molecular Sequence Data; Oligodeoxyribonucleotides; Phagocytes; Receptors, Immunologic; Receptors, Interleukin-8A; RNA, Messenger; Tumor Cells, Cultured

Human melanoma growth stimulating activity (MGSA) is a mitogenic factor first identified in the conditioned media of human melanoma cells. Structurally, MGSA belongs to a superfamily of proteins that includes interleukin-8 (IL-8) and platelet factor 4. These proteins are involved in inflammatory processes, and an understanding of their mechanism of action should provide insight into their pathophysiology. In this study, we report the high level expression of recombinant human MGSA in Escherichia coli. The structure was confirmed by mass spectrometry and NH2-terminal amino acid sequencing. Receptor binding studies were carried out in a human melanoma cell line, Hs294T, and in U937 cells. Direct binding experiments with 125I-MGSA in Hs294T cells have allowed us to identify a novel MGSA receptor in these cells, with a KD of 3.9-4.25 nM and approximately 52,960-67,758 binding sites/cell. These MGSA-binding sites were specific and could not be displaced by unlabeled IL-8. The MGSA receptor in these cells is biologically active, and the addition of ligand induces cellular proliferation in a dose-dependent manner. In U937 cells, unlabeled IL-8 and MGSA were able to completely displace radiolabeled IL-8. Scatchard analysis of the displacement binding data was consistent with binding to a single class of binding sites, and the calculated KD values were 2.4 +/- 0.6 nM for IL-8 and 3.2 +/- 0.80 nM for MGSA. Treatment of U937 cells with IL-8 or MGSA produced a rapid increase in Ca2+ flux; however, subsequent incubation with either ligand failed to produce any further Ca2+ flux. The IL-8 receptor in U937 cells was covalently labeled with 125I-IL-8 to reveal a protein with a molecular mass of 69 kDa.

Topics: Amino Acid Sequence; Binding Sites; Blotting, Western; Calcium; Cell Division; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Kinetics; Mass Spectrometry; Melanoma; Molecular Weight; Neoplasm Proteins; Peptide Fragments; Plasmids; Receptors, Cell Surface; Receptors, Cytokine; Recombinant Proteins; Tumor Cells, Cultured

The objective of this study was 1) to investigate the in vivo production of IL-8 in patients undergoing IL-2 immunotherapy and 2) to study the influence of IL-1Ra, soluble TNF receptor p75 (TNFsRp75), and a TNFsRp75-Fc fusion protein on IL-2-induced IL-8 production in vitro. Circulating IL-8 was assessed both in plasma and erythrocyte lysates prepared from patients undergoing IL-2 immunotherapy. IL-8 was detectable in the plasma within 2-4 h after the first IL-2 infusion, reached a peak level after 4 h, and declined rapidly to undetectable within 8 h. Erythrocyte-bound IL-8 was also detected within 4 h of the first IL-2 dose, but levels were higher than those measured in plasma and remained elevated long after the plasma levels had become undetectable. On day 4 of therapy, the increases in both plasma and the erythrocyte-lysate IL-8 levels induced by an IL-2 injection were less pronounced than on day 1. Although IL-1Ra and TNFsRp75-Fc individually had only a modest suppressive effect on IL-2-induced IL-8 production by PBMC in vitro, the combination of IL-1Ra and TNFsRp75-Fc markedly down-regulated IL-2-induced IL-8 synthesis and steady-state mRNA levels. TNFsRp75 had no effect on IL-2-induced IL-8 synthesis. Our studies suggest that the transient detection of IL-8 in plasma early in the course of IL-2 treatment is due to erythrocyte sequestration and that suppressed synthesis, due in part to high levels of circulating IL-1 and TNF antagonists, may play a role later in the course of treatment.

Topics: Calcium; Carcinoma, Renal Cell; Erythrocytes; Gene Expression; Humans; Immunotherapy; Interleukin 1 Receptor Antagonist Protein; Interleukin-2; Interleukin-8; Leukocytes, Mononuclear; Lymphocyte Activation; Melanoma; Receptors, Cell Surface; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; RNA, Messenger; Sialoglycoproteins

Melanoma growth-stimulatory activity (MGSA)/GRO is well characterized as a potent neutrophil chemoattractant. In the present study, we have demonstrated that MGSA induced a dose-dependent decrease in the expression of interstitial collagens by rheumatoid synovial fibroblasts. The decrease was observed over a dose range of 0.6-6.0 nM MGSA. This effect was specific, as MGSA had no demonstrable effect on the expression of collagen-degrading metalloproteinases, nor did it affect the collagenase inhibitor, tissue inhibitor of metalloproteinases. It also had no effect on the proliferation rate of these fibroblasts, unlike its mitogenic effect on melanoma cells. The ability to inhibit collagen expression was also demonstrated by another member of the C-X-C branch of the platelet factor 4 superfamily, interleukin-8 (IL-8), but not by RANTES, MIP-1 alpha, or MIP-1 beta, which belong to the C-C branch. Steady-state levels of expression of MGSA and IL-8 transcripts in normal adult tissues were dissimilar, suggesting that expression may be an important level at which the activity of these cytokines is regulated. Direct binding experiments with 125I-MGSA on synovial fibroblasts have allowed us to identify an MGSA receptor with a KD of 10.1 nM and approximately 75,000 binding sites/fibroblast. 125I-MGSA binding was specific and could not be displaced by unlabeled IL-8. These results suggest that MGSA, as well as IL-8, may play a role other than that of neutrophil chemo-attractant and more specifically, may be important in the regulation of collagen turnover.

Topics: Arthritis, Rheumatoid; Base Sequence; Blotting, Northern; Cell Division; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Collagen; Cytokines; Fibroblasts; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Kinetics; Melanoma; Multigene Family; Synovial Membrane; Transcription, Genetic; Tumor Cells, Cultured

The capacity of human melanocytes and melanoma cells to produce IL-8 and monocyte chemotactic and activating factor (MCAF) was investigated. Melanocytes expressed mRNA for IL-8 and MCAF, when stimulated with either IL-1 alpha or TNF alpha, but not when stimulated with IL-6, IFN gamma, or LPS alone. IL-8 and MCAF could be induced in a dose-dependent fashion with doses as low as 0.1 ng/ml TNF alpha and 0.5 ng/ml IL-1 alpha. IL-8 and MCAF mRNA were rapidly expressed and peaked between 2 and 4 h for IL-8 and between 4 and 8 h for MCAF. This correlated well with the accumulation of IL-8 antigen as measured by a radioimmunoassay. Supernatants from melanocyte cultures stimulated with either IL-1 alpha or TNF alpha and separated on a heparin-Sepharose column became positive for neutrophil and monocyte chemotactic activity in a dose- and time-dependent fashion. When IFN gamma was added to melanocyte cultures stimulated with suboptimal doses of TNF alpha there was a synergistic increase in secreted IL-8 protein and monocyte chemotactic activity. These data provide further evidence for the possible role of melanocytes in the initiation of an inflammatory reaction. Three different malignant melanoma cell lines stimulated with either TNF alpha or IL-1 alpha expressed IL-8 mRNA, but not mRNA for MCAF. The IL-8 mRNA signal corresponded well with the amount of secreted IL-8 protein. These data suggest that IL-8 and MCAF may play a role in growth regulation and spreading of melanomas.

Topics: Antigens; Chemokine CCL2; Chemotactic Factors; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Melanocytes; Melanoma; RNA, Messenger; Skin Neoplasms; Tumor Cells, Cultured

Natural or recombinant neutrophil activating cytokine (IL-8) induced migration across polycarbonate filters of human A 2058 melanoma cells. Anti-IL-8 antibodies blocked IL-8 induced melanoma cell migration. Checkerboard experiments revealed a gradient-dependent response of A2058 melanoma cells to IL-8. Filters exposed to IL-8 and washed supported melanoma cell migration, thus implying a haptotactic component in the response. The homologous polypeptide platelet factor 4 was inactive. The observation that IL-8 affects melanoma cells emphasizes the need for a comprehensive analysis of the spectrum of action of platelet factor 4-related peptides. The effect of the inflammatory cytokine IL-8 on melanoma cells may be relevant to augmented secondary localization of tumors at sites of inflammation.

Topics: Chemotactic Factors; Chemotaxis; Humans; Interleukin-8; Interleukins; Melanoma; Platelet Factor 4; Recombinant Proteins; Tumor Cells, Cultured

Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cell Division; Cell Line; Chemokine CXCL1; Chemokines, CXC; Chemotaxis, Leukocyte; Cricetinae; Cricetulus; DNA; Fibroblasts; Genetic Vectors; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Neutrophils; Recombinant Fusion Proteins; Tumor Cells, Cultured