Tryptophan protects human melanoma cells against gamma-interferon and tumour necrosis factor-alpha: a unifying mechanism of action.

The sensitivity and resistance of six human melanoma cell lines to gamma-interferon (gamma-IFN) and tumour necrosis factor-alpha (TNF-alpha) have been examined. Amelanotic cell lines were more sensitive to gamma-IFN and TNF-alpha than melanotic cells. The cytotoxicity of gamma-IFN and TNF-alpha could be reversed in all cells by the addition of L- or D-tryptophan to the culture medium. Melanoma cells resistant to gamma-IFN excrete calcium activated neutral protease (CANP) and as a consequence, make L-tryptophan available by the hydrolysis of serum proteins in the culture medium. Resistance to gamma-IFN could be reversed by the addition of specific CANP inhibitor, whereas gamma-IFN-sensitive strains became more resistant with the addition of CANP to the culture medium. It has been confirmed that gamma-IFN induces indoleamine 2,3-dioxygenase in melanoma cells. This enzyme utilizes the superoxide anion (O2-) as a substrate for the oxidation of either L- or D-tryptophan to N-formylkynurenic acid leading to cell death. The induction of this degradative pathway for L-tryptophan kills cells by starvation of this essential and relatively scarce amino acid. TNF-alpha induces manganese-containing superoxide dismutase (MnSOD) which also uses O2- to produce cytotoxic concentrations of hydrogen peroxide. Therefore, it can be concluded that the cytotoxicity of both gamma-IFN and TNF-alpha depends on the availability of L-tryptophan as the substrate for the removal of O2- via indoleamine 2,3-dioxygenase.     

Serum values of tumour necrosis factor-alpha and of soluble tumour necrosis factor-R55 in melanoma patients

Tumour necrosis factor-alpha (TNFalpha) is a cytokine with a variety of biological activities, including an effect on tumour growth. The soluble TNF receptor TNF-R55 (sTNF-R55) inhibits TNFalpha functioning. Serum values of TNFalpha and TNF-R55 have been observed in patients with different cancers. To determine the serum values of TNFalpha and its soluble receptors and to investigate the prognostic value of sTNF-R55, we studied the sera of 68 patients with metastatic melanoma, 109 patients with no recurrent disease after surgical removal of melanoma, and 69 healthy controls. At least four different monoclonal antibodies against human TNFalpha and human TNF-R55, respectively, were prepared to obtain sensitive and reliable sandwich enzyme-linked immunosorbent assays. We found that in patients with metastatic melanoma the serum values of sTNF-R55 were significantly higher (2.41 ng/ml; range 0.02 23.0 ng/ml; P < 0.05) than in the melanoma patients without recurrence (0.54 ng/ml; range 0.02-6.25 ng/ml) and healthy controls (0.5 ng/ml; range 0.02 5.0 ng/ml). The sTNF-R55 concentrations increased as the disease progressed. Patients with metastatic melanoma also had significantly higher concentrations of TNFalpha (3.34 ng/ml; range 0.03-30.0 ng/ml; P<0.05) than patients without recurrence (1.24 ng/ml; range 0.02 23.0 ng/ml). Patients with metastatic melanoma, a high sTNF-R55 concentration, a low TNFalpha concentration and a low TNF:sTNF-R55 ratio had the worst prognosis. Low values of sTNF-R55 (<0.6 ng/ml) were associated with favourable response to chemotherapy (P = 0.007). According to our findings, patients with metastatic melanoma have higher values of sTNF-R55 than the controls and melanoma patients without recurrence. sTNF-R55 values higher than 0.6 ng/ml and a TNF:sTNF-R55 ratio lower than 1.5 are unfavourable prognostic factors for response to chemotherapy.     

Azelaic acid as a competitive inhibitor of thioredoxin reductase in human melanoma cells

Azelaic acid has been shown to inhibit thioredoxin reductase (TR) at the surface of guinea pig and human skin, on cultures of human keratinocytes, melanocytes, melanoma cells, murine melanoma cells (Cloudman S91), and on purified enzymes from Escherichia coli, rat liver, and human melanoma. Human melanoma cells are more resistant to inhibition by azelaic acid than murine melanoma or human melanocytes. Kinetic studies with pure TRs indicate that azelaic acid is a reversible competitive inhibitor. Fluorescence spectroscopy has been used to show that azelaic acid does not interfere with electron transfer from NADPH to FAD on TR. However, azelaic acid does inhibit electron transfer from the dithiolate active site of this enzyme. Inhibition by azelaic acid is pH-dependent, requiring the dissociation of both carboxylate groups, and also the dissociation of the active site dithiol groups. Binding studies with [14C]azelaic acid at different pHs, indicate that inhibition is first due to the formation of a thioester on the active thiolate groups followed by transacylation of a basic amino acid residue in the active site. A comparative study of TR inhibition by C6, C9, C10 and C12 saturated dicarboxylic acids was also determined on guinea pig skin in vivo. These homologous dicarboxylic acids gave greater inhibition with increasing size (i.e. mol wt.).     

Radiation-induced tumour necrosis factor-alpha expression: clinical application of transcriptional and physical targeting of gene therapy

Promising data are emerging on a new anticancer agent, Ad.EGR-TNF, an adenoviral vector, which contains radio-inducible DNA sequences from the early growth response (EGR1) gene promoter and cDNA for the gene encoding human tumour necrosis factor-alpha. Ad.EGR-TNF combines the well-documented broad-spectrum anticancer activity of TNFalpha with the proven clinical usefulness of radiotherapy. Systemic delivery of the TNFalpha protein has had limited success clinically because of severe dose-limiting toxic effects. This limitation has been overcome by the use of a gene delivery approach, combined with a radiation-inducible promoter to express the TNFalpha protein in the irradiated tumour tissue. Preclinical and early phase I clinical testing indicates that effective concentrations of TNFalpha can be delivered to the tumour site without significant systemic exposure or toxic effects. The combination of radiation and TNFalpha gene delivery has produced striking antitumour effects in model systems in animals. In the clinical setting, potent anticancer activity has been observed with a high rate of complete and partial objective tumour responses. A novel mechanism of destruction of the tumour vasculature seems to be central to this distinct antitumour activity. This review summarises the rationale, mechanistic basis, preclinical data, and preliminary clinical findings for this new treatment model.     

Analysis of gene expression profiles in melanoma cells with acquired resistance against antineoplastic drugs

Resistance to various antineoplastic agents is common in the clinical management of malignant melanoma. The biological mechanisms conferring these different drug-resistant phenotypes are still unclear. To identify potential factors mediating drug resistance to melanoma cells, the mRNA expression profiles of the parental drug-sensitive human melanoma cell line MeWo and four derived drug-resistant sublines with acquired resistance against four commonly used drugs for melanoma treatment (cisplatin, etoposide, fotemustine and vindesine) were analysed. We investigated cDNA arrays with 43,000 cDNA clones ( approximately 30,000 unique genes) to study the expression patterns of these cell lines. We were able to simultaneously extract new candidate genes associated with drug resistance in malignant melanoma and to correlate the present findings with previously described resistance-associated genes. Using hierarchical clustering and analysing the overlap of genes with altered expression, we detected similarities between the expression signatures related to cisplatin and fotemustine resistance. The resistance against vindesine required a minimal set of changes in gene expression relative to the parental MeWo cell line. Our study provides new data that may be used to obtain further insight into the resistance characteristics of malignant melanoma.     

Isolated limb perfusion for melanoma patients--a review of its indications and the role of tumour necrosis factor-alpha.

AIMS: The treatment of melanoma in-transit metastases (IT-mets) can vary widely and is dependant on the size and the number of the lesions. When multiple, large lesions exist, isolated limb perfusion (ILP) has established itself as an attractive treatment option with high response rates. METHODS: Review on the various methods of treatment of melanoma in-transit metastases, with a focus on isolated limb perfusion. A Medline based literature search was performed for articles relating to this topic. Additional original papers were obtained from citations in those identified by the initial search. Indications and results are discussed and the extra value of tumour necrosis factor (TNF) is evaluated. RESULTS: ILP with Melphalan results in complete response rates of 40-82% and showed to be 54% in a large retrospective meta-analysis. The addition of TNF can improve these completes response rates (59-85%) and although no data from randomized controlled trials are available, it seems of particular value in large, bulky lesions or in patients with recurrent disease after previous ILP. CONCLUSIONS: TNF-based ILP has earned a permanent place in the treatment of patients with melanoma IT-mets. In patients with a high tumour burden, TNF-based ILP is the most efficacious procedure to obtain local control and achieve limb salvage.     

Tumour necrosis factor-alpha expression in tumour islets confers a survival advantage in non-small cell lung cancer

Background  

We have previously demonstrated that tumour islet infiltration by macrophages is associated with extended survival (ES) in NSCLC. We therefore hypothesised that patients with improved survival would have high tumour islet expression of chemokine receptors known to be associated with favourable prognosis in cancer. This study investigated chemokine receptor expression in the tumour islets and stroma in NSCLC.     

Inhibition of the tumour necrosis factor-alpha autocrine loop enhances the sensitivity of multiple myeloma cells to anticancer drugs

Several autocrine soluble factors, including macrophage inflammatory protein-1α and tumour necrosis factor-alpha (TNF-α), promote the survival and growth of multiple myeloma (MM) cells. We hypothesised that inhibition of the TNF-α autocrine loop may enhance the cytotoxic effect of anticancer drugs in MM cell lines. In the present study, a TNF-α-neutralizing antibody suppressed cell proliferation and enhanced the cytotoxic effect of anticancer drugs on MM cells. In addition, combination treatment with the TNF-α-neutralizing antibody and the chemotherapy agent melphalan inhibited nuclear factor κB (NF-κB) p65 nuclear translocation and mammalian target of rapamycin (mTOR) activation and upregulated the expression of Bax and Bim. Treatment of ARH-77 cells with the NF-κB inhibitor dimethyl fumarate or the mTOR inhibitor rapamycin suppressed NF-κB p65 nuclear translocation and enhanced the cytotoxic effect of melphalan. Furthermore, infliximab, a monoclonal antibody against TNF-α, also enhanced the cytotoxic effect of anticancer drugs in ARH-77 cells. These results indicated that TNF-α-neutralizing antibodies or infliximab enhanced the cytotoxic effect of anticancer drugs by suppressing the TNF receptor/mTOR/NF-κB pathways. The inhibition of TNF-α may thus provide a new therapeutic approach to control tumour progression and bone destruction in MM patients.     

Preferential activity of wild-type and mutant tumor necrosis factor-alpha against tumor-derived endothelial-like cells.

Tumor-derived endothelial-like cells (tEC) were prepared by culturing human umbilical vein endothelial cells (HUVEC) in the presence of HT1080 human fibrosarcoma-conditioned medium. tEC showed higher permeability and less cell-adhesion activity than normal HUVEC (nEC). Tumor necrosis factor-alpha (TNF) is known to have tumor-vasculature disrupting activity. tEC showed higher cytotoxicity to recombinant human TNF (rhTNF) than nEC, and was not observed using HUVEC cultured with WI38 human diploid cell-conditioned medium as a medium-control. These results demonstrate that tEC acquire physiological properties of tumor-associated vasculature, and may be a useful model system for the study of the mechanisms of TNF antitumor action. The TNF-mutant RGD-V29 (code No. F4614), which has an inserted 4Arg-Gly-Asp sequence and an 29Arg-->Val replacement, was found to induce greater preferential destruction of tEC compared to rhTNF. When the preferential activities were evaluated in terms of 30% cytotoxicity (IC30) ratio (nEC/tEC), the ratio was 460 for RGD-V29 compared to 4.2 for rhTNF. RGD-V29 also exhibited cell-adhesive function and bound preferentially to the p55 TNF-receptor. Both these properties of RGD-V29 contributed to the tEC selective cytotoxicity, indicating that the RGD ligands and selective p55 receptor binding on the cells, although uncharacterized, are involved in tEC targeting. Therefore, the TNF mutant RGD-V29 may show greater selectivity toward tumor vasculature than wild-type TNF.     

Thioredoxin reductase, a redox-active selenoprotein, is secreted by normal and neoplastic cells: presence in human plasma

Thioredoxin (Trx) and Trx reductase (TrxR) are redox-active proteins that participate in multiple cellular events, including growth promotion, apoptosis, and cytoprotection. Studies on overexpression of Trx and TrxR in human cancers have indicated a role of these proteins in tumor development. In this study, we analyzed the expression of TrxR in peripheral blood cells, tumor-transformed leukemia, and melanoma cells and found, in addition to abundant plasma membrane localization, that TrxR was released from these cells. Secretory cells were observed at the single cell level using a sensitive enzyme-linked immunospot assay. The release was inducible, and physiological stimulation of human monocytes by IFN-gamma, lipopolysaccharide, and interleukin 1alpha significantly increased the number of TrxR-secreting cells (P = 0.004). Secretion of TrxR followed the classical Golgi pathway, and it was confirmed by metabolic labeling using [35S]methionine and [35S]cysteine. TrxR was also detected for the first time in fresh healthy blood donor plasma (n = 21; median concentration, 18.0 ng/ml), with biological activity as determined by insulin reduction assay. These results highlight the role of extracellular Trx and TrxR during inflammation and tumor progression. Released Trx, with its active site motif containing amino acids Cys-X-X-Cys, was recently shown to have chemoattractant properties beside its previously described antioxidant and cocytokine activities. Regeneration of oxidized Trx requires available TrxR outside the cell, the presence and induction of which is described in this paper for normal and transformed cells.     

Resistance of human melanoma cells against the cytotoxic and complement-enhancing activities of doxorubicin

The anticancer agent doxorubicin has two different effects on human SK-MEL-170 melanoma cells: the well known direct cytotoxicity and a marked enhancement of their susceptibility to killing by the R24 monoclonal anti-GD3) ganglioside antibody and human complement. This complement-enhancing effect of doxorubicin is also present after covalent immobilization onto glycerol-coated glass beads preventing cellular uptake of the drug (M. Panneerselvam, R. Bredehorst, and C-W. Vogel, Proc. Natl. Acad. Sci. USA, 83: 9144-9148, 1986). In order to investigate the effect of doxorubicin resistance on the complement-enhancing activity of the drug, we have established a doxorubicin-resistant SK-MEL-170 subline. The development of drug resistance in these melanoma cells was associated with multiple phenotypical changes including an increased expression of at least four high molecular weight plasma membrane proteins or glycoproteins with molecular weights of approximately 220,000, 180,000, 150,000, and 130,000, respectively. The drug-resistant cells accumulated doxorubicin at approximately 2-fold lower amounts in accordance with a 2-fold higher efflux of doxorubicin from these cells. The basal complement susceptibility of the doxorubicin-resistant cells was reduced by more than 60% presumably as a result of the observed reduced expression of GD3 sites and decreased binding of C3b. Most importantly, the doxorubicin-resistant cells were also resistant against the complement-enhancing effect of the free and immobilized drug. The two doxorubicin resistance phenomena, the resistance to the cytotoxic and to the complement-enhancing activities of the drug, seem to be fundamentally different: (a) to achieve comparable cytotoxic and complement-enhancing effects on drug-sensitive and -resistant SK-MEL-170 cells, the drug-resistant cells required 178-fold higher concentrations of free doxorubicin for the cytotoxic effect but only 5-fold higher amounts of the free drug and 12-fold higher amounts of the immobilized drug for the complement-enhancing effect; (b) resistance against the cytotoxic activity of doxorubicin is associated with reduced intracellular drug accumulation, while the data obtained with immobilized doxorubicin indicate that resistance to the complement-enhancing activity of the drug is independent of cellular drug uptake. These data suggest that different mechanisms are responsible for the two resistance phenomena in doxorubicin-resistant melanoma cells.     

退变性腰椎侧凸TNF-α基因多态性和蛋白表达水平的相关研究

目的研究探讨TNF-α基因单核苷酸多态性和血清中TNF-α水平与退变性腰椎侧凸的相关性。方法选取2009年10月至2011年12月于本院脊柱外科门诊及病房收治的60例退变性腰椎侧凸患者作为研究对象,均行腰椎正侧位X线片及腰椎MRI检查,选取同时期于该院进行健康体检者60例作为正常对照组,两组研究对象均系无血缘关系的北方汉族个体,在性别、年龄、体重指数与病例组相匹配。应用聚合酶链反应-限制性内切酶长度多态性（PCR—RFLP）技术对两组研究对象DNA标本TNF—α基因启动子区特异性片段进行扩增及对扩增的特异性片段用NcoI进行酶切,用酶联免疫吸附测定技术（ELISA）。对两组研究对象血清TNF—α蛋白水平进行检测。利用Adobe Photoshop 6．0软件,测量病例组MRI图像中顶椎间盘及其上下椎间盘内髓核与脑脊液T2加权像的相对信号强度及正位X线片上的Cobb角。结果两组研究对象各基因型频率分布符合Hardy—Weinberg遗传平衡定律,研究对象具有代表性。两组基因型频率差异无统计学意义（P〉0．05）。G／G基因型频率与非G／G基因型频率（C／A、A／A）及等位基因G、A频率两组比较差异均有统计学意义（P〈0．05）。相对危险度比较提示,非G／G基因型比G／G基因型患病的危险性增大,A等位基因携带者比非A等位基因携带者患病的危险性增加。细胞因子TNF-α血清浓度两组之间差异有统计学意义（P〈0．05）,且病例组显著高于对照组。TNF-α血清浓度与髓核相对信号强度呈负相关,与Cobb’s角呈正相关。将病例组根据椎间盘退变等级分组,各组之间差异有统计学意义（P〈0．05）。结论退变性腰椎侧凸患者血清中TNF-α浓度比正常人群血清水平明显增高,血清中TNF-α浓度与侧凸角呈正相关,与退变性腰椎侧凸椎间盘髓核信号强度呈负相关,血清TNF-α浓度越高,椎间盘退变程度越重,TNF—α是退变性腰椎侧凸发生发展的危险因素。     

Interleukin-1beta and tumour necrosis factor-alpha promote the transformation of human immortalised mesothelial cells by erionite

Asbestos fails to induce the transformation of human mesothelial cells in vitro although it has been known as a potential carcinogen to human mesothelial cells. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) are major cytokines released by macrophages after inhalation of asbestos. These cytokines can regulate mesothelial cell proliferation both in vitro and in vivo. In the present study, we used the growth in soft agar as an index of transformation and investigated the role of IL-1beta and TNF-alpha during the process of human mesothelial cell carcinogenesis. Both IL-1beta and TNF-alpha were demonstrated to enhance erionite-induced transformation of the immortalised, non-tumorigenic human mesothelial cell line (MeT-5A) in vitro. The MeT-5A cells could only be transformed when the cells were exposed to a combination of cytokines and erionite, or at least two cytokines together without erionite, for at least 4 months in vitro. The findings presented here suggest that IL-1beta and TNF-alpha play a significant role in the pathogenesis of mesothelioma, and that it might be desirable to block or inhibit cytokine secretion in high risk populations to prevent mesothelioma.     

Circulating tumour cells as tumour biomarkers in melanoma: detection methods and clinical relevance

Circulating tumour cells (CTCs) are cells of solid tumour origin detectable in the peripheral blood. Their occurrence is considered a prerequisite step for establishing distant metastases. Metastatic melanoma was the first malignancy in which CTCs were detected and numerous studies have been published on CTC detection in melanoma at various stages of disease. In spite of this, there is no general consensus as to the clinical utility of CTCs in melanoma, largely due to conflicting results from heterogeneous studies and discrepancies in methods of detection between studies. In this review, we examine the possible clinical significance of CTCs in cutaneous, mucosal and ocular melanoma, focusing on detection methods and prognostic value of CTC detection.