Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism.

Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models.

Effect of fractalkine-Fc delivery in experimental lung metastasis using DNA/704 nanospheres.

The lung is one target organ to which solid tumors frequently metastasize. Given the systemic adverse effects of currently available treatments, developing effective strategies of drug/gene delivery directly to the lungs is therefore needed. Aerosol delivery is a non-invasive gene transfer approach to target the airways. Here, we sought to evaluate the potential to deliver a fractalkine (FKN)-encoding plasmid formulated with the tetrafunctional amphiphilic block copolymer 704 through aerosolization in two models of pulmonary metastases. FKN is a chemokine recently described as a good candidate to stimulate a strong antitumor immune response in various forms of cancers. Here, we have assessed the effect of single and repeated aerosolizations of FKN-encoding plasmid formulated with 704 on the development of experimental lung metastases of mouse colon carcinoma and osteosarcoma. For this purpose, we have designed FKN-Fc sequences encoding an optimized version of the chemokine. Repeated intratracheal administrations of 704/FKN-Fc markedly inhibited growth of experimental lung metastases of CT-26 and K7M2 cells. Our results showed that tetrafunctional amphiphilic block copolymer 704 is a highly efficient synthetic vector for mediating local and safe gene transfer into the lung. In addition, FKN-Fc gene therapy of pulmonary nodules may provide a promising immunotherapeutic approach.

Tissue-specific differential antitumour effect of molecular forms of fractalkine in a mouse model of metastatic colon cancer.

BACKGROUND AND AIMS: Fractalkine, a chemokine that presents as both a secreted and a membrane-anchored form, has been described as having tumour-suppressive activities in standard subcutaneous models. Here, we investigate the antitumour effect of fractalkine, in its three molecular forms, in two orthotopic models of metastatic colon cancer (liver and lung) and in the standard subcutaneous model. METHODS: We have developed models of skin tumours, liver and pulmonary metastasis and compared the extent of tumour development between C26 colon cancer cells expressing either the native, the soluble, the membrane-bound fractalkine or none. RESULTS: The native fractalkine exhibits the strongest antitumour effect, reducing the tumour size by 93% in the skin and by 99% in the orthotopic models (p<0.0001). Its overall effect results from a critical balance between the activity of the secreted and the membrane-bound forms, balance that is itself dependent on the target tissue. In the skin, both molecular variants reduce tumour development by 66% (p<0.01). In contrast, the liver and lung metastases are only significantly reduced by the soluble form (by 96%, p<0.002) whereas the membrane-bound variant exerts a barely significant effect in the liver (p = 0.049) and promotes tumour growth in the lungs. Moreover, we show a significant difference in the contribution of the infiltrating leukocytes to the tumour-suppressive activity of fractalkine between the standard and the orthotopic models. CONCLUSIONS: Fractalkine expression by C26 tumour cells drastically reduces their metastatic potential in the two physiological target organs. Both molecular forms contribute to its antitumour potential but exhibit differential effects on tumour development depending on the target tissue.

Signal transduction involved in MCP-1-mediated monocytic transendothelial migration.

Monocyte chemoattractant protein-1 (MCP-1) is a major chemoattractant for monocytes and T lymphocytes. The MonoMac6 cell line was used to examine MCP-1 receptor-mediated signal transduction events in relation to MCP-1-mediated monocytic transendothelial migration. MCP-1 stimulates, with distinct time courses, extracellular signal-related kinases (ERK1 and ERK2) and stress-activated protein kinases (SAPK1/JNK1 and SAPK2/p38). SAPK1/JNK1 activation was blocked by piceatannol, indicating that it is regulated by Syk kinase, whereas SAPK2/p38 activation was inhibited by PP2, revealing an upstream regulation by Src-like kinases. In contrast, ERK activation was insensitive to PP2 and piceatannol. Pertussis toxin, a blocker of Go/Gi proteins, abrogated MCP-1-induced ERK activation, but was without any effect on SAPK1/JNK1 and SAPK2/p38 activation. These results underscore the major implication of Go/Gi proteins and nonreceptor tyrosine kinases in the early MCP-1 signaling. Furthermore, MCP-1-mediated chemotaxis and transendothelial migration were significantly diminished by a high concentration of SB202190, a broad SAPK inhibitor, or by SB203580, a specific inhibitor of SAPK2/p38, and abolished by pertussis toxin treatment. Altogether, these data suggest that coordinated action of distinct signal pathways is required to produce a full response to MCP-1 in terms of monocytic locomotion.

Src-regulated extracellular signal-related kinase and Syk-regulated c-Jun N-terminal kinase pathways act in conjunction to induce IL-1 synthesis in response to microtubule disruption in HL60 cells.

A microtubule reorganization is often observed during cellular contacts that are associated to IL-1 production. Here, we show that in HL60 cells, vincristine, a microtubule-disrupting agent that induces a strong production of IL-1, triggers the activation of both extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK-1). While ERK activation is rapid and transient, peaking at 10 min, the JNK1 activation is delayed and more sustained reaching a maximum at 2 h. ERK activation was blocked by CP 118556, indicating it is regulated by a Src-like kinase, while JNK1 was inhibited by piceatannol, revealing an upstream regulation by Syk. Each kind of the nonreceptor tyrosine kinase blockers efficiently inhibits the vincristine-induced IL-1 production and diminishes the level of IL-1 transcripts, indicating that the ERK and JNK pathways act coordinately to elicit the transcription of the IL-1 gene. Furthermore, we found that pertussis toxin, a blocker of Go/Gi proteins, abrogated the vincristine-induced activation of both Src and Syk. Our data support a model where the status of microtubule polymerization influences the activity of Go or Gi proteins that control, in turn, two independent Src/ERK and Syk/JNK1 cascades that are both necessary to sustain IL-1 synthesis.

Signal transduction pathways involved in soluble fractalkine-induced monocytic cell adhesion.

Fractalkine displays features that distinguishes it from the other chemokines. In particular, besides its chemoattractant action it promotes, under physiologic flow, the rapid capture and the firm adhesion of a subset of leukocytes or intervenes in the neuron/microglia interaction. This study verified that indeed the human monocytic MonoMac6 cell line adheres to fibronectin-coated filters in response to soluble fractalkine (s-FKN). s-FKN stimulates, with distinct time courses, extracellular signal-related kinases (ERK1 and ERK2) and stress-activated protein kinases (SAPK1/JNK1 and SAPK2/p38). Both p60 Src and p72 Syk were activated under s-FKN stimulation with a rapid kinetic profile compatible with a downstream regulation on the mitogen-activated protein kinase (MAPK) congeners. The use of specific tyrosine kinase inhibitors revealed that the ERK pathway is strictly controlled by Syk, whereas c-Src up-regulated the downstream SAPK2/p38. In contrast, the SAPK1/JNK1 pathway was not regulated by any of these nonreceptor tyrosine kinases. The s-FKN-mediated increased adherence of MonoMac6 cells was partially inhibited by SB202190, a broad SAPKs inhibitor, PD98059, an MEK inhibitor, LY294002, a phosphatidyl inositol 3-kinase inhibitor, and a pertussis toxin-sensitive G protein. These data highlight that the integration of a complex array of signal transduction pathways is necessary to complete the full s-FNK-dependent adherence of human monocytic cells to fibronectin. (Blood. 2001;97:2031-2037)

Protein kinase activation by warm and cold hypoxia- reoxygenation in primary-cultured rat hepatocytes-JNK(1)/SAPK(1) involvement in apoptosis.

Ischemia-reperfusion procedures induced severe hepatic damages owing to different processes related to hypoxia and reoxygenation (H/R) phases, including the consecutive oxygen free radical (OFR) release. Stress-activated protein kinases (SAPKs) could be activated by extracellular stimuli. The aim of this study was to show whether H/R stress conditions could stimulate these kinases, and especially c-jun-N-terminal kinase (JNK(1)/SAPK(1)), to reveal a potential role of JNK(1)/SAPK(1) in the control of hepatocyte apoptosis. Primary cultured rat hepatocytes, isolated from other liver cells and blood flow, were subjected to warm and cold hypoxia-reoxygenation phases mimicking surgical and transplant conditions. The activation status of SAPKs was evaluated by immunoprecipitation or Western-blotting experiments, whereas apoptosis was assessed by measuring caspase activation and internucleosomal DNA fragmentation in vitro and by TUNEL reaction, in vivo. Hypoxia, and especially hypoxia-reoxygenation, significantly increased JNK(1)/SAPK(1) activation in cultured hepatocytes. Either in warm or cold conditions, OFR scavengers (N-Acetylcystein, Di-Phenyleneiodonium, Deferoxamine) decreased this stimulation. Warm ischemia-reperfusion also led to JNK activation. Hypoxia and especially hypoxia-reoxygenation induced programmed cell death in vivo and in vitro. This last phenomenon was inhibited when hepatocytes were treated with SB 202190, which was described as a potent inhibitor of p38 and JNK activities. Altogether, these results confirmed that JNK(1)/SAPK(1) was activated during the hypoxia-reoxygenation process, and that this activity participated in the onset of the apoptosis program.