Lorea Mendoza

Clinical and experimental approaches to the pathophysiology of interleukin-18 in cancer progression

SummaryInterleukin-18 (IL-18, interferon [IFN]-gamma-inducing factor) is a proinflammatory cytokine converted to a biologically active molecule by interleukin (IL)-1beta converting enzyme (caspase-1). A wide range of normal and cancer cell types can produce and respond to IL-18 through a specific receptor (IL-18R) belonging to the toll-like receptor family. The activity of IL-18 is regulated by IL-18-binding protein (IL-18bp), a secreted protein possessing the ability to neutralize IL-18 and whose blood level is affected by renal function and is induced by IFNgamma. IL-18 plays a central role in inflammation and immune response, contributing to the pathogenesis and pathophysiology of infectious and inflammatory diseases. Because immune-stimulating effects of IL-18 have antineoplastic properties, IL-18 has been proposed as a novel adjuvant therapy against cancer. However, IL-18 increases in the blood of the majority of cancer patients and has been associated with disease progression and, in some cancer types, with metastatic recurrence risk and poor clinical outcome and survival. Under experimental conditions, cancer cells can also escape immune recognition, increase their adherence to the microvascular wall and even induce production of angiogenic and tumor growth-stimulating factors via IL-18-dependent mechanism. This is particularly visible in melanoma cells. Thus, the role of IL-18 in cancer progression and metastasis remains controversial. This review examines the clinical correlations and biological effects of IL-18 during cancer development and highlights recent experimental insights into prometastatic and proangiogenic effects of IL-18 and the use of IL-18bp against cancer progression.

Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis.

BackgroundThe recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear.MethodsThe proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 μm in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence.ResultsSpheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells.Conclusion3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.

Mannose receptor-mediated endothelial cell activation contributes to B16 melanoma cell adhesion and metastasis in liver

The role of mannose receptors from hepatic sinusoidal endothelium (HSE) in liver colonization by B16 melanoma (B16M) cells was studied. The expression of high mannose‐type oligosaccharides on the surface of B16M cells was enhanced by in vitro treatment with 1‐deoximannojirimycin (1‐DMM). There was a significant (P < 0.01) enhancement of hepatic metastasis when B16M cells were 1‐DMM‐treated before being intrasplenically injected into C57BL/6J mice. Intraperitoneal administration of 5 mg/kg recombinant human interleukin‐1 receptor antagonist (rHuIL‐1Ra) inhibited the 1‐DMM‐induced enhancement of metastasis. Expression of high mannose‐type oligosaccharides on the surface of 1‐DMM‐treated B16M cells and their in vitro adhesion to the HSE was significantly correlated (R = 0.82). The addition of either 100 μg/ml mannan or paraformaldehyde (PFA)‐fixed 1‐DMM‐treated B16M cells to cultured HSE for a period of 12 h significantly (P < 0.01) increased the release of IL‐1β from the HSE compared to that liberated by the HSE incubated with either basal medium or PFA‐fixed untreated B16M cells. The same HSE treatments also significantly (P < 0.01) increased the degree of adhesion of other B16M cells to HSE, being abrogated by anti‐mouse vascular cell adhesion molecule‐1 (VCAM‐1) antibodies. The conditioned media from HSE cultures, activated by PFA‐fixed, 1‐DMM‐treated B16M cells significantly (P < 0.01) increased B16M cell proliferation when compared to conditioned media from HSE cultures incubated with PFA‐fixed, untreated B16M cells. Thus, 1‐DMM treatment of B16M cells enhanced the development of hepatic metastasis by IL‐1‐dependent mechanisms. The mechanism is consistent with in vitro mannose receptor‐mediated melanoma cell attachment to the HSE, which subsequently upregulates IL‐1β release, VCAM‐1‐dependent adherence, and melanoma growth factor(s) release by HSE. J. Cell. Physiol. 174:322–330, 1998.

Resveratrol prevents inflammation-dependent hepatic melanoma metastasis by inhibiting the secretion and effects of interleukin-18

BackgroundImplantation and growth of metastatic cancer cells at distant organs is promoted by inflammation-dependent mechanisms. A hepatic melanoma metastasis model where a majority of metastases are generated via interleukin-18-dependent mechanisms was used to test whether anti-inflammatory properties of resveratrol can interfere with mechanisms of metastasis.MethodsTwo experimental treatment schedules were used: 1) Mice received one daily oral dose of 1 mg/kg resveratrol after cancer cell injection and the metastasis number and volume were determined on day 12. 2) Mice received one daily oral dose of 1 mg/kg resveratrol along the 5 days prior to the injection of cancer cells and both interleukin-18 (IL-18) concentration in the hepatic blood and microvascular retention of luciferase-transfected B16M cells were determined on the 18th hour. In vitro, primary cultured hepatic sinusoidal endothelial cells were treated with B16M-conditioned medium to mimic their in vivo activation by tumor-derived factors and the effect of resveratrol on IL-18 secretion, on vascular cell adhesion molecule-1 (VCAM-1) expression and on tumor cell adhesion were studied. The effect of resveratrol on melanoma cell activation by IL-18 was also studied.ResultsResveratrol remarkably inhibited hepatic retention and metastatic growth of melanoma cells by 50% and 75%, respectively. The mechanism involved IL-18 blockade at three levels: First, resveratrol prevented IL-18 augmentation in the blood of melanoma cell-infiltrated livers. Second, resveratrol inhibited IL-18-dependent expression of VCAM-1 by tumor-activated hepatic sinusoidal endothelium, preventing melanoma cell adhesion to the microvasculature. Third, resveratrol inhibited adhesion- and proliferation-stimulating effects of IL-18 on metastatic melanoma cells through hydrogen peroxide-dependent nuclear factor-kappaB translocation blockade on these cells.ConclusionsThese results demonstrate multiple sites for therapeutic intervention using resveratrol within the prometastatic microenvironment generated by tumor-induced hepatic IL-18, and suggest a remarkable effect of resveratrol in the prevention of inflammation-dependent melanoma metastasis in the liver.

Inhibition of Cytokine-Induced Microvascular Arrest of Tumor Cells by Recombinant Endostatin Prevents Experimental Hepatic Melanoma Metastasis

We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor α (TNF-α) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-α and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-α, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 μg/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF.

Effect of Asymptomatic Natural Infections due to Common Mouse Pathogens on the Metastatic Progression of B16 Murine Melanoma in C57BL/6 Mice

To investigate whether the presence of infections in C57BL/6 mice influences the metastatic ability of B16 melanoma (B16M) cells, we compared the susceptibility to metastasis development of pathogen-free mice with that of mice from a colony endemically infected with several mouse pathogens. We found that, compared to seronegative controls, mice that were seropositive at least to Mouse Hepatitis Virus (MHV) and Mycoplasma pulmonis: (i) exhibited a higher interindividual variability in all the parameters quantifying metastatic progression; (ii) had elevated serum levels of proinflammatory cytokines both before and at the end of the experiment; (iii) were more susceptible to hepatic metastasis. Interestingly, final levels of tumor necrosis factor (TNF)-α and interleukin (IL)-18 correlated with the extent of hepatic colonization by the melanoma cells. To confirm the metastasis-enhancing effect of MHV and M. pulmonis we measured the ability of B16M cells to metastasize in pathogen-free animals housed for increasing time-intervals in the vicinity of MHV+ animals. Notably, susceptibility to metastasis was lower in animals seronegative to MHV than in MHV+ mice, whereas the latter were less susceptible to metastasis than MHV+M. pulmonis+ mice. Seropositive animals had increased levels of TNF-α and IL-18 suggesting that MHV and M. pulmonis enhance the metastatic ability of melanoma cells by inducing the release of proinflammatory cytokines. While our results highlight the importance of using pathogen-free animals in metastasis studies, they emphasize the need for a comprehensive health monitoring of the mice used in such studies, particularly in case of using facilities lacking appropriate containment measures.

Design, Synthesis, and Functional Evaluation of Leukocyte Function Associated Antigen-1 Antagonists in Early and Late Stages of Cancer Development

The integrin leukocyte function associated antigen 1 (LFA-1) binds the intercellular adhesion molecule 1 (ICAM-1) by its α(L)-chain inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report the structure-based design, small-scale synthesis, and biological activity evaluation of a novel family of LFA-1 antagonists. The design led to the synthesis of a family of highly substituted homochiral pyrrolidines with antiproliferative and antimetastatic activity in a murine model of colon carcinoma, as well as potent antiadhesive properties in several cancer cell lines in the low micromolar range. NMR analysis of their binding to the isolated I-domain shows that they bind to the I-domain allosteric site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

BackgroundHuman melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown.MethodsHerein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied.ResultsMice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNFα and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion- and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFα induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1.ConclusionsWe demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion- and proliferation-stimulating effects of TNFα, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing melanoma cells. These data suggest COX-2 neutralization as a potential anti-metastatic therapy in melanoma patients at high risk of systemic and bone dissemination due to intercurrent infectious and inflammatory diseases.

Proangiogenic Implications of Hepatic Stellate Cell Transdifferentiation into Myofibroblasts Induced by Tumor Microenvironment

Hepatic stellate cells are perisinusoidal fibroblasts that transdifferentiate into myofibroblasts in response to paracrine factors released from cancer cells and cancer-activated endothelial cells. Tumor-associated myofibroblasts exhibit contractility, proliferation, production of extracellular matrix molecules and metalloproteases. They secrete soluble factors inducing proinflammatory and immune suppressant effects. Myofibroblasts are present in avascular micrometastasis prior to endothelial cell recruitment, and act as supporting stroma for tumor neoangiogenesis. In replacement-type cancer growth, the reticular arrangement of tumor-associated myofibroblasts provides a sinusoidal-type angiogenic pattern. In pushing-type cancer growth, fibrous tract-forming myofibroblasts support a portal-type angiogenic pattern. Additionally, tumor-activated myofibroblasts support cancer development via paracrine release of tumor invasion and proliferation-stimulating factors. In summary, this information suggests that the ability of cancer cells to activate hepatic stellate cells and to collaborate with myofibroblasts along the metastatic process may represent a key phenotypic property of liver-colonizing cancer cells. On the other hand, experimental anti-tumor and anti-angiogenic agents have inhibited intrametastatic recruitment and proangiogenic activities of hepatic myofibroblasts, suggesting that targeting tumorigenic effects of these cells may contribute to hepatic metastasis inhibition.

Abstract C46: Design, synthesis, and functional evaluation of leukocyte-function associated antigen-1 (LFA-1) antagonists in early and late stages of cancer development.

The integrin leukocyte function associated antigen 1 (LFA-1) binds the intercellular adhesion molecule 1 (ICAM-1) by its αL-chain inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report the structure-based design, small-scale synthesis, and biological activity evaluation of a novel family of LFA-1 antagonists. The design led to the synthesis of a family of highly substituted homochiral pyrrolidines with antiproliferative and antimetastatic activity in a murine model of colon carcinoma, as well as potent antiadhesive properties in several cancer cell lines in the low micromolar range. NMR analysis of their binding to the isolated I-domain shows that they bind to the I-domain allosteric site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled.