Regulation of murine interleukin-10 production by dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA), one of the predominant androgens secreted by the adrenal cortex, is a potential immunologic regulator. In this report, the effect of DHEA on interleukin-10 (IL-10) production was studied in vivo. Mice were injected s.c. with DHEA or DHEA-sulfate (DHEAS) ranging from 50 microg to 500 microg/g body weight. The serum was collected, and the spleen cells were isolated 48 h after treatment. Results indicate that treatment with DHEA or DHEAS significantly increases the serum level of IL-10. The spleen cells isolated from the DHEA-treated or DHEAS-treated mice also showed an increase in IL-10 secretion and mRNA expression after the cells were activated by concanavalin A (ConA). The maximal dose of DHEA for inducing IL-10 production was 250 microg/g body weight. As IL-10 is a potent differentiation factor of B lymphocytes, the possible role of DHEA in regulation of immunoglobulin (Ig) production was studied in vivo. Results indicated a significant increase in both serum level of Ig (IgG, IgM, IgA) and Ig secretion by spleen cells after the mice were treated with DHEA or DHEAS. Mice injected with both DHEA (250 microg/g body weight) and anti-IL-10 antibody (0.5 mg/g body weight) showed a significantly reduced DHEA-mediated increase in Ig production. Thus, DHEA might affect the function of B lymphocytes via stimulating IL-10 production.     

Notch ligand Delta-like 1 promotes the metastasis of melanoma by enhancing tumor adhesion

Notch signaling plays a vital role in tumorigenicity and tumor progression by regulating proliferation, invasion, and the tumor microenvironment. Previous research by our group indicated that Notch ligand Delta-like 1 (Dll1) is involved in angiogenesis in melanoma, and we noticed that it took a longer time to trypsinize Dll1-expressing B16 melanoma cells than the control cells. In this article, we extended our study to investigate the effects of Dll1 on tumor cell adhesion and metastasis. Dll1 overexpression activated Notch signaling in B16 tumor cells and significantly enhanced the adhering capacity of B16 tumor cells both in vitro and in vivo. B16-Dll1 cells also had a higher metastatic potential than their counterpart in the mouse model of lung metastasis. Along with increased Dll1 expression, N-cadherin, but not E-cadherin, was upregulated in B16-Dll1 cells. These data suggested that Notch ligand Dll1 may enhance the adhesion and metastasis of melanoma cells by upregulation of N-cadherin.     

Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis,tumor growth,and metastasis of human melanoma

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.     

Inhibition of tumor growth by histoincompatible cells expressing interleukin-2.

Murine tumor cells engineered to express IL-2 have been shown to be rejected by the syngeneic host, which is then protected against a subsequent tumorigenic challenge. To assess whether IL-2 has to be produced by the tumor cells themselves, or whether its local delivery would be sufficient to promote such beneficial effects, the syngeneic tumor cells were co-inoculated with allogeneic or xenogeneic cells secreting IL-2, selected after gene transfection. In several murine systems, it was observed that this is an efficient approach for controlling the growth of the syngeneic tumor. However, animals which rejected the tumor were not protected against a subsequent challenge. Several lines of evidence indicate that NK cells play a major role in tumor rejection induced by the IL-2 expressing histoincompatible vector cells. Thus, while local delivery of IL-2 in the vicinity of a tumor might not be sufficient to promote a systemic long-term specific antitumor immune response, it can control the growth of the primary syngeneic tumor. These experiments demonstrate the feasibility of using genetically engineered histoincompatible cells (which are rejected by the host's immune system) as a transient delivery system in vivo.     

Topic 10. Angiogenesis and metastasis growth factors

Sixth International Congress of the Metastasis Research SocietyPosters

Topic 10. Angiogenesis and metastasis growth factors     

重组人白细胞介素-10诱导瘤细胞凋亡

目的 研究重组人白细胞介素-10诱导瘤细胞凋亡的作用。方法利用基因工程技术制备的rhIL-10,分别作用于体外培养的ScaBer、NCIS446、U937瘤细胞,观察瘤细胞的形态改变,MTT法检测rhIL-10对瘤细胞增殖的抑制作用,电泳观察rhIL-10诱导瘤细胞凋亡的情况,TUNEL法检测瘤细胞凋亡百分率。结果电泳出现典型的DNA“lader”,ScaBe,、NCIS446、U937三种瘤细胞的凋亡率为48．5％、45．6％、47．1％,IC50分别为0．043、0．052、0．058mg／mL。结论rhIL-10具有显著抑制瘤细胞生长活性、诱导瘤细胞凋亡的作用。     

Regulation of interleukin-10 and interleukin-22 expression in T helper cells

Interleukin (IL)-10 and IL-22 are crucial regulators of inflammation during immune responses. While IL-10 functions to prevent excessive inflammation by acting on immune cells, IL-22 elicits innate responses from tissue epithelia and promotes wound healing. Although T helper (Th) cells are a major source for both cytokines, IL-10 and IL-22 are rarely co-expressed at high levels in the same cells. Here we discuss a number of common aspects as well as crucial differences in the molecular regulation of both cytokines that might explain their broad, but distinct expression among Th cell subsets.     

V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis

Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma, which exists as four different splice variants. The presence of versican in the extracellular matrix plays a role in tumor cell growth, adhesion and migration, which could be altered by altering the ratio between versican isoforms. We have previously shown that overexpression of the V3 isoform of versican in human melanoma cell lines markedly reduces cell growth in vitro and in vivo, since V3-overexpressing (LV3SN) cultured cells as well as primary tumors arising from these cells grow slower than their vector-only counterparts (LXSN). In the present work, we have extended these observations to demonstrate that the delayed cell growth is due to multiple events since differences in proliferative index as well as in apoptosis are observed in LV3SN cells and tumors compared to LXSN. For example, LV3SN melanoma cells exhibit delayed activation of MAPK in response to EGF, we have also characterized further the primary tumors originated in nude mice from V3-transduced melanoma cells to determine if other events affect the V3 tumor phenotype. For example, hyaluronan content of LV3SN tumors was higher than in LXSN tumors, whereas other related matrix components and vascularization were unaffected. Furthermore, lung metastasis in nude mice occurred only in animals carrying LV3SN tumors, indicating a dual role for this molecule, both as an inhibitor of tumor growth and a metastasis inductor.     

Inhibitory effects of roxithromycin on tumor angiogenesis, growth and metastasis of mouse B16 melanoma cells

We examined the effects of roxithromycin, a 14-membered ring macrolide antibiotic, on tumor angiogenesis, tumor growth and metastasis of mouse B16BL6 melanoma cells. The inhibitory effect of roxithromycin on angiogenesis using mouse dorsal air sac model was dose-dependent, and 100 mg/kg of roxithromycin administered intraperitoneally twice a day reduced the dense capillary network area to about 20% of the control. Administration of roxithromycin histologically reduced the development of microvessels and mononuclear cell infiltration. In vivo tumor growth studies demonstrated that intraperitoneal administration of roxithromycin at 20 mg/kg/day and 50 mg/kg/day reduced tumor size of B16BL6 melanoma to about 56% and 33% (experiment 1), 71% and 48% (experiment 2) of that in the respective controls. Roxithromycin also significantly inhibited pulmonary metastasis of B16BL6 cells in a spontaneous system. The inhibitory activities of roxithromycin on angiogenesis, tumor growth and metastasis were compared with those of a potent angiogenesis inhibitor, TNP-470. These data demonstrated that roxithromycin has potent antiangiogenic and antitumor effects and might have possible therapeutic applications.     

Contextual regulation of inflammation: a duet by transforming growth factor-beta and interleukin-10

Transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) are regulatory cytokines with pleiotropic roles in the immune system. The prominent function of TGF-beta is to maintain T cell tolerance to self or innocuous environmental antigens via its direct effects on the differentiation and homeostasis of effector and regulatory T cells. A critical route for the regulation of T cells by TGF-beta is via activation of a T cell-produced latent form of TGF-beta1 by dendritic cell-expressed avbeta8 integrin. IL-10 operates primarily as a feedback inhibitor of exuberant T cell responses to microbial antigens. T cells are also the principal producers of IL-10, the expression of which is regulated by IL-27, IL-6, and TGF-beta. The collective activity of TGF-beta and IL-10 ensures a controlled inflammatory response specifically targeting pathogens without evoking excessive immunopathology to self-tissues.     

Regulation of parasite antigen-driven immune responses by interleukin-10 (IL-10) and IL-12 in lymphatic filariasis.

We investigated the mechanisms by which interleukin-10 (IL-10) regulates antigen-specific hyporesponsiveness in asymptomatic microfilaremic (MF) individuals. Peripheral blood mononuclear cells from MF individuals (n = 11) were stimulated in vitro with Brugia malayi antigen (BMA) or mycobacterial purified protein derivative (PPD) in the presence of neutralizing anti-IL-10 or isotype control monoclonal antibodies. As expected, BMA stimulated little or no gamma interferon (IFN-gamma) secretion in MF individuals, whereas PPD stimulated IFN-gamma in all but one. Neutralization of endogenous BMA-driven IL-10 secretion led to augmentation of IFN-gamma in seven of nine MF individuals (1.5- to 10-fold) and did so in a BMA-specific manner (PPD-driven IFN-gamma was augmented in only two of eight MF individuals and only 1.5- to 2-fold), indicating that IL-10 downregulates type 1 responses in these individuals. Type 2 responses (IL-5 secretion) were unaffected by the IL-10 blockade. To assess whether IL-12 could reverse the type 1 downregulation observed, the effect of recombinant human IL-12 (rhIL-12) on BMA-driven IL-5 and IFN-gamma production was also evaluated. rhIL-12 augmented both BMA- and PPD-driven IFN-gamma production 5- to 10-fold in six of nine MF individuals. These data demonstrate that IL-10 downregulates BMA-driven type 1 responses and that IL-12 can overcome downregulation of Th1 responses associated with MF but does so in a non-antigen-specific manner.     

Host immunity to mycoplasma antigens introduced into B16 melanoma cells: Effect on tumor growth rate and metastasis

Immunization of syngeneic C57BL/6 mice with X-irradiated Bl6 melanoma cells was previously shown to elicit antibodies specific to viral antigens on the melanoma cells. When immunized mice were challenged with viable subcutaneous transplants of B16 melanoma cells that formed non-metastatic tumors in normal mice, tumors failed to develop in some mice, but there was a high incidence of lung metastasis in mice with progressively growing tumors. To determine whether protective immunity and/or enhanced metastasis were the consequences of immune responses specific for inherent tumor-associated viral antigens, non-metastatic B16 melanoma cells were deliberately infected withMycoplasma arginini. The result was incorporation of perpetuating antigens that elicited, in mycoplasma-immunized mice, humoral and cell-mediated immune responses to infected (B16-M⁺) but not uninfected (B16-M^–) cells. When mycoplasma-immunized mice were challenged with B16-M⁺ and B-16M^– subcutaneous transplants, only B16-M⁺ tumors were rendered slower-growing and appreciably more metastatic. By contrast, in mice immunized against uninfected B16 melanoma cells, both B16-M⁺ and B16-M^– tumors grew more slowly, and metastasized to a greater extent, than corresponding tumors in unimmunized mice. Enhanced metastasis was not experimentally separable from reduced tumor growth rate and was not simply the consequence of a longer period of tumor growth. Evidence suggests that host immunity does not directly promote metastasis, but that reduced tumor growth rates resulting from protective immunity are more conducive to successful dissemination of metastases.     

Production of human tumor necrosis factor alpha, interleukin-6, and interleukin-10 is induced by lactic acid bacteria.

To investigate the role of cytokines in interactions between lactic acid bacteria and the immune system, we measured production of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 from human peripheral blood mononuclear cells after stimulation with live or glutaraldehyde-fixed bacteria. Production of tumor necrosis factor alpha, IL-6, and, in some cases, IL-10 was induced in amounts even greater than those obtained with lipopolysaccharide as a stimulant. Our results suggest that lactic acid bacteria can stimulate nonspecific immunity.     

The inhibition of tumor growth and metastasis by self-assembled nanofibers of taxol

Molecular hydrogels have big potential for local delivery and sustained release of therapeutic agents. In this paper, we reported on a molecular hydrogel mainly formed by the widely used anti-cancer drug of taxol. The hydrogel was formed by an ester bond hydrolysis process from a taxol derivative (Taxol-SA-GSSG, 1) and could be administrated into solid tumors to dramatically hinder their growths and prevent their metastasis. Besides the improved anti-cancer effect compared to the clinically used intravenous (i.v.) injection of Taxol(?), the concentration of taxol in blood was low due to the local administration of taxol hydrogels, which greatly enhanced the dosage tolerance of mice to taxol and might reduce side effects of taxol during chemotherapy. Our observations suggested that the hydrogel mainly composed of taxol would have great potential for its practical applications.     

Increased melanoma growth and metastasis spreading in mice overexpressing placenta growth factor

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family, plays an important role in adult pathological angiogenesis. To further investigate PlGF functions in tumor growth and metastasis formation, we used transgenic mice overexpressing PlGF in the skin under the control of the keratin 14 promoter. These animals showed a hypervascularized phenotype of the skin and increased levels of circulating PlGF with respect to their wild-type littermates. Transgenic mice and controls were inoculated intradermally with B16-BL6 melanoma cells. The tumor growth rate was fivefold increased in transgenic animals compared to wild-type mice, in the presence of a similar percentage of tumor necrotic tissue. Tumor vessel area was increased in transgenic mice as compared to controls. Augmented mobilization of endothelial and hematopoietic stem cells from the bone marrow was observed in transgenic animals, possibly contributing to tumor vascularization. The number and size of pulmonary metastases were significantly higher in transgenic mice compared to wild-type littermates. Finally, PlGF promoted tumor cell invasion of the extracellular matrix and increased the activity of selected matrix metalloproteinases. These findings indicate that PlGF, in addition to enhancing tumor angiogenesis and favoring tumor growth, may directly influence melanoma dissemination.