Clarisa Salado

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.

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.

Biological evaluation of morin and its new oxovanadium(IV) complex as antio-xidant and specific anti-cancer agents

It is known that flavonoids possess, among others, antioxidant and antitumoral properties that depend on their molecular structure. The central objective if this study was to investigate the potential antioxidant and antiproliferative properties of the flavonol morin and its new oxovanadium(IV) complex (VOmor) that was synthesized in order to modify the morin chemical structure. Two osteoblast (UMR106 and MC3T3E1), two breast tumor (T47D and SKBR3) and breast epithelial cell lines in culture were used for the antitumoral determinations. Additionally, a comparative study of their antioxidant capacities using different radicals (DPPH, ABTS(+), OH, O2(-), ROO) was performed. Selected mechanisms of action were studied using the breast cancer cell lines. Results obtained show that morin and its complex behaved as good antioxidant agents for some of the radicals and that the complexation improved the behavior with respect to OH and O2(-) radicals being morin more effective as ROO scavenger. A considerable variation in sensitivity was observed in the breast cancer cells but non-specificity was found for the treatment of osteosarcoma. Moreover, the compounds did not affect the normal proliferation of the breast epithelial mammal cells. The mechanistic studies demonstrated that the complex did not generate reactive oxygen species in the cells (confirming the in vitro studies) and did not produce any damage of DNA. The plasmatic membrane was observed to be damaged only in the SKBR3 cell line. In contrast, the perturbation of the mitochondrial membrane potential and the activation of caspase 3/7 for the breast tumor cells revealed an apoptotic cell death process. All these results collectively suggested that VOmor complex could serve as promising pharmacologically active substance against breast cancer treatment.

Bovine serum albumin binding, antioxidant and anticancer properties of an oxidovanadium(IV) complex with luteolin

Chemotherapy using metal coordination compounds for cancer treatment is the work of the ongoing research. Continuing our research on the improvement of the anticancer activity of natural flavonoids by metal complexation, a coordination compound of the natural antioxidant flavone luteolin (lut) and the oxidovanadium(IV) cation has been synthesized and characterized. Using different physicochemical measurements some structural aspects of [VO(lut)(H2O)2]Na·3H2O (VOlut) were determined. The metal coordinated to two cis-deprotonated oxygen atoms (ArO(-)) of the ligand and two H2O molecules. Magnetic measurements in solid state indicated the presence of an effective exchange pathway between adjacent vanadium ions. VOlut improved the antioxidant capacity of luteolin only against hydroxyl radical. The antitumoral effects were evaluated on MDAMB231 breast cancer and A549 lung cancer cell lines. VOlut exhibited higher viability inhibition (IC50=17 μM) than the ligand on MDAMB231 cells but they have the same behavior on A549 cells (ca. IC50=60 μM). At least oxidative stress processes were active during cancer cell-killing. When metals chelated through the carbonyl group and one adjacent OH group of the flavonoid an effective improvement of the biological properties has been observed. In VOlut the different coordination may be the cause of the small improvement of some of the tested properties of the flavonoid. Luteolin and VOlut could be distributed and transported in vivo. Luteolin interacted in the microenvironment of the tryptophan group of the serum binding protein, BSA, by means of electrostatic forces and its complex bind the protein by H bonding and van der Waals interactions.

Insights into the mechanisms underlying the antitumor activity of an oxidovanadium(IV) compound with the antioxidant naringenin. Albumin binding studies

Naringenin, a natural antioxidant present in grapefruit, oranges and the skin of tomatoes showed low antioxidant properties among other flavonoids due to its structural characteristics. Since many flavonoids were shown to have cell-killing and antioxidant activities, naringenin was investigated herein. In parallel with its antioxidant activities the flavonoid showed very low cytotoxicity at concentrations up to 100 μM against lung (A549) and breast (SKBr3 and MDAMB231) cancer cell lines. Furthermore, a newly-synthesized and characterized complex of naringenin and oxidovanadium(IV) ([V(IV)O(nar)2] · 2H2O, VOnar, with weak ferromagnetic coupling) was also studied. As a result, VOnar acted as a better compound on cell-killing and antioxidant activities (in vitro) than naringenin. The anti-proliferative effect of VOnar was accompanied by reactive oxygen species (ROS) generation, cell membrane and DNA damages, cell cycle arrest, caspase 3/7 activation and mitochondrial potential reduction. The higher parameters observed for the MDAMB231 cell line have been related to its low glutathione (GSH) content. The assays of the interaction of bovine serum albumin (BSA) with the complex showed the affinity of protein toward it and that there is only one binding site on the BSA molecule. However, metal complexation decreased the binding affinity to BSA of naringenin probably due to a steric hindrance of the complex.

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.

Specific antitumor activities of natural and oxovanadium(IV) complexed flavonoids in human breast cancer cells

The structure–activity relationships of the natural flavonoids quercetin, chrysin and silibinin have revealed that they meet the key structural requirements for killing tumor cells. When the structures of these three flavonoids are modified by complexation using the oxovanadium(IV) cation their cytotoxic properties in human breast cancer cell lines are enhanced. Breast epithelial cells are used to determine the selectivity of these compounds. The mechanisms of action of the flavonoids and the oxovanadium(IV) cation in the MDA-MB231 cell line seem to be different to the apoptotic mechanisms of cell death exerted by the oxovanadium(IV) complexes. These results showed the mechanisms of the antitumoral effect of these complexes, making them promising compounds for cancer treatment.

Antioxidant, anticancer activities and mechanistic studies of the flavone glycoside diosmin and its oxidovanadium(IV) complex. Interactions with bovine serum albumin

The natural antioxidant flavonoid diosmin, found in citric fruits, showed low antioxidant properties among other flavonoids due to its structural characteristics and low cytotoxicity against lung (A549) and breast (T47D, SKBR3 and MDAMB231) cancer cell lines. The anticancer behavior has been improved by the metal complex generated with the flavonoid and the oxidovanadium(IV) ion. This new complex, [VO(dios)(OH)3]Na5·6H2O (VOdios), has been synthesized and characterized both in solid and solution states. The interaction of the metal ion through the sugar moiety of diosmin precluded the improvement of the antioxidant effects. However, the cell-killing effects tested in human lung A549 and breast T47D, SKBR3 and MDAMB231 cancer cell lines, were enhanced by complexation. The anti-proliferative effects on the human lung cancer cell line were accompanied by cellular ROS generation and an increase in cytoplasm condensation. The breast cancer cell lines did not produce caspase3/7 activation, mitochondrial potential reduction and ROS generation. Therefore, a non-apoptotic form of cell death in a caspase- and oxidative stress-independent manner has been proposed. The protein binding ability has been monitored by the quenching of tryptophan emission in the presence of the compounds using bovine serum albumin (BSA) as a model protein. Both compounds could be distributed and transported in vivo and the complex displayed stronger binding affinity and higher contributions to the hydrogen bond and van der Waals forces.