Interleukin 10 production by human melanoma

Interleukin 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these 35 samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.     

Interleukin 4 inhibits growth and induces apoptosis in human breast cancer cells

Interleukin-4 (IL-4) is a pleiotropic cytokine produced by mast cells and T lymphocytes that promotes proliferation and immunoglobulin class-switching in B cells. IL-4 receptors (IL-4Rs) are also expressed by nonhematopoietic cells as well as some tumor cells. Unlike its mitogenic effect on B cells, IL-4 inhibits the growth of some cancer cells in vitro. In this study, we show that IL-4R is expressed by breast and ovarian cancer cell lines. Furthermore, anchorage-dependent and -independent growth of breast cancer cell lines MCF-7 and MDA-MB-231 is inhibited by IL-4 treatment, and this effect requires IL-4R. Interestingly, IL-4 only inhibited proliferating breast cancer cells and had no effect on basal, unstimulated growth. We therefore characterized the effect of IL-4 on breast cancer cell growth stimulated by either estradiol or insulin-like growth factor I (IGF-I). In both anchorage-dependent and -independent growth assays, IL-4 inhibited estradiol-stimulated growth. The antiestrogen effect of IL-4 was not due to IL-4 interference with the estrogen receptor, because IL-4 did not interfere with estrogen receptor-mediated reporter gene transactivation. In contrast, IL-4 had no effect on IGF-I-stimulated proliferation. Because IGF-I is known to inhibit programmed cell death, we examined apoptosis as a possible mechanism of IL-4 action. We established that IL-4 induced apoptosis in breast cancer cells by five independent criteria: (a) morphological indicators including pyknotic nuclei and cytoplasmic condensation; (b) DNA fragmentation; (c) the formation of DNA laddering; (d) the cleavage of poly(ADP-ribose) polymerase; and (e) the presence of cells with sub-G1 DNA content. IL-4 increased the percentage of apoptotic cells in MCF-7 and MDA-MB-231 cells 6.0- and 6.7-fold over that of the control, respectively. Finally, the addition of IGF-I reversed IL-4-induced apoptosis, suggesting that the mechanism of IL-4-induced growth inhibition in human breast cancer cells is the induction of programmed cell death.     

ND-2001 suppresses lung metastasis of human renal cancer cells in athymic mice

Explants of highly metastatic human renal cell carcinoma SN12Cpm6 cells in athymic mice were treated with sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam; ND-2001). ND-2001 (50 micrograms/ml) caused 78% inhibition of lung metastasis of SN12Cpm6 cells (two of five animals remaining metastasis free). The in vitro tumor cell invasion assay showed that ND-2001 (100 micrograms/ml) suppressed the invasive activity of SN12Cpm6 cells to Matrigel matrix at an inhibition rate of 72%. These results suggest that ND-2001 may be a new anti-metastatic drug against human cancer cells.     

Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth

Previously, we found a positive correlation between the expression of platelet-type 12-lipoxygenase (12-LOX) and the progression of human prostate adenocarcinoma (PCa; Gao et al., Urology, 46: 227-237, 1995). To determine the role of 12-LOX in PCa progression, we generated stable 12-LOX-transfected PC3 cells, which synthesize high levels of 12-LOX protein and 12(S)-hydroxyeicosatetraenoic acid metabolite. In vitro, 12-LOX-transfected PC3 cells demonstrated a proliferation rate similar to neo controls. However, following s.c. injection into athymic nude mice, 12-LOX-transfected PC3 cells formed larger tumors than did the controls. Decreased necrosis and increased vascularization were observed in the tumors from 12-LOX-transfected PC3 cells. Both endothelial cell migration and Matrigel implantation assays indicate that 12-LOX-transfected PC3 cells were more angiogenic than their neo controls. These data indicate that 12-LOX stimulates human PCa tumor growth by a novel angiogenic mechanism.     

Positive feedback and angiogenesis in tumor growth control

In vivo tumor growth data from experiments performed in our laboratory suggest that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are angiogenic signals emerging from an up-regulated genetic message in the proliferating rim of a solid tumor in response to tumor-wide hypoxia. If these signals are generated in response to unfavorable environmental conditions, i.e. a decrease in oxygen tension, then the tumor may play an active role in manipulating its own environment. We have idealized this type of adaptive behavior in our mathematical model via a parameter which represents the carrying capacity of the host for the tumor. If that model parameter is held constant, then environmental control is limited to tumor shape and mitogenic signal processing. However, if we assume that the response of the local stroma to these signals is an increase in the host's ability to support an ever larger tumor, then our models describe a positive feedback control system. In this paper, we generalize our previous results to a model including a carrying capacity which depends on the size of the proliferating compartment in the tumor. Specific functional forms for the carrying capacity are discussed. Stability criteria of the system and steady state conditions for these candidate functions are analyzed. The dynamics needed to generate stable tumor growth, including countervailing negative feedback signals, are discussed in detail with respect to both their mathematical and biological properties.     

Molecular mechanisms controlling human melanoma progression and metastasis

Progression from normal melanocyte to metastatic melanoma is a multistep, multigenic process. While relatively easy to conceptualize, the stages of melanoma progression are not necessarily discrete, nor are they unambiguously defined at the morphologic or molecular levels. The ability to stage the lesions more precisely would afford clinicians a greater confidence when designing therapeutic strategies. Unfortunately, a molecular definition of cells at each stage does not yet exist. Toward that end, the purpose of this review is twofold: (1) to highlight recent advances in our understanding of the molecular genetics of human cutaneous melanoma predisposition, and (2) to construct a molecular model of melanoma progression toward metastasis.     

Prostaglandin E1 suppresses tumor necrosis factor-alpha and interleukin-10 production by lipopolysaccharides-stimulated mononuclear cells

Previous studies have shown that the intravenous administration of yohimbine, an alpha 2 antagonist, increases norepinephrine turnover and has related anxiogenic effects in humans. We herein report that yohimbine also increases plasma neuropeptide Y (NPY) in healthy human subjects. This finding is consistent with previous reports in animals, but contrasts with a previously reported study in humans. NPY is a 36 amino acid peptide neurotransmitter located in sympathetic and nonsympathetic nerve fibers, as well as in brain structures such as the locus coeruleus, where it is colocalized with norepinephrine. NPY has been shown to inhibit locus coeruleus neuronal firing, decrease norepinephrine release, and increase postsynaptic noradrenergic signal transduction. When administered centrally, NPY also has anxiolytic properties. This study therefore suggests that yohimbine challenge may be useful in assessing NPY and noradrenergic system interactions in neuropsychiatric disorders such as panic disorder or post traumatic stress disorder in which noradrenergic system dysfunction has been observed.     

P-selectin deficiency attenuates tumor growth and metastasis

Selectins are adhesion receptors that normally recognize certain vascular mucin-type glycoproteins bearing the carbohydrate structure sialyl-Lewisx. The clinical prognosis and metastatic progression of many epithelial carcinomas has been correlated independently with production of tumor mucins and with enhanced expression of sialyl-Lewisx. Metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. We provide a link between these observations by showing that P-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucin-producing carcinoma. P-selectin-deficient mice showed significantly slower growth of subcutaneously implanted human colon carcinoma cells and generated fewer lung metastases from intravenously injected cells. Three potential pathophysiological mechanisms are demonstrated: first, intravenously injected tumor cells home to the lungs of P-selectin deficient mice at a lower rate; second, P-selectin-deficient mouse platelets fail to adhere to tumor cell-surface mucins; and third, tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in P-selectin-deficient animals.     

Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: a novel mechanism of tumor angiogenesis

Intussusceptive microvascular growth refers to vascular network formation by insertion of interstitial tissue columns, called tissue pillars or posts, into the vascular lumen and subsequent growth of these columns, resulting in partitioning of the vessel lumen. While intussusception has been reported in normal developing organs, its existence in solid tumors has not been previously documented. By observing the growth of the human colon adenocarcinoma (LS174T) in vivo for a period of 6 weeks, we demonstrate that intussusception is an important mechanism of tumor angiogenesis. At the leading edge of the tumor, vascular growth was found to occur by both intussusception and endothelial sprouting. In the stabilized regions, intussusception led to network remodeling and occlusion of vascular segments. The formation of some tissue pillars appears to depend on intravascular blood-flow patterns or changes in intravascular shear stress. The rapid vascular remodeling by intussusception could possibly contribute to intermittent blood flow in tumors.     

Interleukin 6 is an autocrine growth factor for normal human pleural mesothelial cells

Interleukin 6 (IL-6) is a multifunctional inflammatory cytokine whose abnormal production has been implicated in a variety of diseases. Our previous study demonstrated that exudative pleural effusions contain a large amount of IL-6, and the levels of IL-6 in pleural effusion have diagnostic and pathophysiologic values. Although IL-6 is produced by a variety of cells, the origin of IL-6 in pleural effusion has not been determined clearly. We hypothesized that pleural mesothelial cells (PMCs) are an important source of IL-6 in pleural diseases. In this study, we tried to demonstrate whether PMCs could produce IL-6 and to characterize the modulation of its production. PMCs were established from patients with nonmalignant pleural effusion. Immunoreactive IL-6 could be detected in cultured supernatants of all PMCs from five patients, and all IL-6 detected in the supernatants were biologically active. IL-6 production was augmented by the addition of interleukin 1 alpha (IL-1 alpha) in a dose-dependent manner and suppressed by dexamethasone. Expression of IL-6 mRNA was spontaneously observed and was increased by IL-1 alpha. PMCs also expressed mRNA for IL-6 receptors gp80 and gpl30. Spontaneous cell growth and DNA synthesis of PMCs were inhibited by the addition of a neutralizing anti-IL-6 monoclonal antibody and were promoted by the addition of IL-6 to the culture. These results suggest that IL-6 is an autocrine growth factor for PMCs.     

Acute alcohol intoxication suppresses natural killer cell activity and promotes tumor metastasis

Alcohol consumption is associated with increased morbidity and mortality related to infectious diseases and malignancy (1-5), although immune mediation of these relationships is controversial. Specifically, the activity of natural killer (NK) cells, which are involved in the resistance to infections and metastasis, can be suppressed in the presence of ethanol in vitro. However, acute consumption or infusion of ethanol in vivo exerts no effects on NK activity assessed in vitro thereafter. Therefore, we have developed and used a method to study the effects of ethanol on NK activity in living rats by using an NK-sensitive metastatic process and selective depletion of NK cells in vivo. Acute ethanol intoxication caused a marked suppression of NK activity in vivo and a tenfold increase in the number of MADB106 tumor metastases. Ethanol had no effect in rats selectively depleted of NK cells or when an NK-insensitive tumor (C4047) was used. These findings suggest that even acute ethanol intoxication markedly suppresses NK activity in the living organism. This suppression may underlie some aspects of the association between alcoholism, infectious disease and malignancies.     

Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer

Apolipoprotein[a] phenotyping is a critically important method to explore the role of kringle-4 repeat number as a modulator of lipoprotein[a]-associated cardiovascular risk. The availability of a kringle-4 number-based reference standard is therefore necessary for a reliable and generally accepted classification of apo[a] phenotypes. We propose here a battery of recombinant apo[a] isoforms that may be used as the reference standard in various gel systems. Five plasmids encoding for r-apo[a] containing a known number (n = 9, 13, 17, 25, 33) of plasminogen-like kringle-4 copies were constructed, and transfected into the human embryonic kidney cell line 293. The electrophoretic mobility of the recombinant apo[a] isoforms expressed by these cells in a hollow-fiber bioreactor was determined after reduction by SDS-gel (agarose, acrylamide or a mixture of both) electrophoresis and immunoblotting using an antibody specific for human apo[a]. The equation of the linear relationship between log r-apo[a] kringle number and relative migration was used to determine the isoform size of apo[a] in normal human plasma. A very good correlation (r = 0.97) was found with the genotype (pulsed-field gel eletrophoresis of kpnI-digested restriction fragments of genomic DNA) and among electrophoretic methods. The proposed recombinant standard offers the possibility to identify apo[a] isoforms within a large range of molecular sizes, 9 to 33 kringle-4 copies, using simple electrophoretic techniques and a nomenclature based on its molecular structure, i.e., the number of kringle-4 repeats.-Anglés-Cano, E., S. Loyau, G. Cardoso-Salda?a, R. Couderc, and P. Gillery. A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping.     

Saikosaponin b2 induces differentiation without growth inhibition in cultured B16 melanoma cells

Some patients of ischemic heart disease have low uptake in 123I-labeled beta methyl-iodophenyl penta-decanoic acid (BMIPP) SPECT in spite of normal uptake in thallium-201 (Tl) SPECT. To investigate their clinical significance, we performed both T1 and BMIPP myocardial SPECT in 26 cases with stable angina (n = 16) and unstable angina (n = 10), and compared with clinical backgrounds electrocardiogram (ECG) and left ventriculography (LVG). In 11 patients of them, the uptake of BMIPP was moderately reduced. We divided 26 cases into two groups according to uptake of BMIPP (normal/reduced). The two groups had no differences in length of angina attack and duration of disease, but they had a significant difference in the abnormality of either ECG or LVG. Three to six months after PTCA, we examined LVG in 18 cases, 12 of 16 cases with the abnormality of LVG showed the improvement of wall motion. We concluded the reduced uptake of BMIPP with normal uptake of Tl was related to more severe ischemia in cases with unstable angina.     

Targeting the tumor vasculature: inhibition of tumor growth by a vascular endothelial growth factor-toxin conjugate

Tumor-derived vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) plays an important role in neovascularization and the development of tumor stroma. Furthermore, VEGF receptors are over-expressed in the endothelial cells of tumor vasculature and almost non-detectable in the vascular endothelium of adjoining normal tissues. The differential expression of receptor offers a selective advantage for targeting cytotoxic toxin polypeptides. We have prepared a vascular targeting reagent by chemically linking recombinant VEGF to a truncated form of diphtheria toxin. The VEGF-toxin conjugate was selectively toxic to endothelial cell lines and inhibited experimental neovascularization of the chick chorioallantoic membrane. In the present study, we examined the effects of VEGF-toxin conjugate on solid tumor growth. Athymic nude mice with established subcutaneous tumors were treated with daily intraperitoneal injections of the VEGF-toxin conjugate or free toxin. When compared with control animals treated with the toxin polypeptide alone, the conjugate-treated animals displayed a significant inhibition of tumor growth. Histological analysis of tumors from conjugate-treated animals revealed hemorrhagic necrosis consistent with a vascular-mediated injury. In contrast, highly vascularized normal tissues from conjugate-treated animals demonstrated no evidence of hemorrhage or tissue injury. The conjugate was well tolerated without apparent toxicities. Our results illustrate the anti-tumor activity of a VEGF-toxin conjugate selectively targeting the tumor neovasculature.     

Anti-cell death activity promotes pulmonary metastasis of melanoma cells

Bcl-2 inhibits apoptosis from a variety of stimuli, and a Bcl-2-binding protein BAG-1 also functions in protection from apoptosis in concert with Bcl-2. Here, we provide evidence that prolonged cell survival introduced by overexpression of Bcl-2 or BAG-1 proteins strongly promotes experimental pulmonary metastasis of melanoma B16-BL6 cells. In murine melanoma cell line B16-BL6, gene transfer-mediated expression of the Bcl-2 or BAG-1 led to prolonged cell survival against serum-starved apoptosis in vitro. The Bcl-2-expressing B16 cells, B16-Bcl-2 and the BAG-1-expressing B16 cells, B16-BAG-1 strongly enhanced pulmonary metastasis in allogenic BALB/c nude mice and whole lung weights were increased by 2.4-fold and 1.4-fold, respectively, compared with control transfectants, suggesting that Bcl-2 is a stronger positive modulator of metastasis. When the viable B16-Bcl-2 and control transfectants were injected subcutaneously into BALB/c nude mice, the colony numbers of pulmonary metastasis of the B16-Bcl-2 transfectant increased by 5.6-fold compared with the control transfectants. These enhanced metastatic potentials in the B16-Bcl-2 and the B16-BAG-1 transfectants were well correlated with anti-cell death activity against serum-starvation and enhanced cell viability on limiting dilution. Analysis of the transfectants however revealed that their growth rates, invasive ability and cell motility were not significantly altered by overexpression of either Bcl-2 or BAG-1 proteins. Taken together, these studies demonstrate that prolonged cell survival is a crucial factor to promote metastasis of melanoma, thereby contributing to tumor progression.     

Inhibition of angiogenesis by antitumor antibiotic C1027 and its effect on tumor metastasis

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na+/H+ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes.