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Chemokines are small basic proteins belonging to the family of cytokines and characterized by a chemotactic activity. Their main function is the regulation of migration of leucocytes and other cells (endothelial cells, fibroblasts, epithelial cells etc.) in both physiologic and pathologic conditions. Over the last few years other properties of chemokines have been recognized: it has been shown that they have active roles in several pathologic processes, including cancer. A number of studies confirmed that transformed cells secrete chemokines, which are able to recall leucocytes. This leukocyte infltration does not necessarily represent the immune response of the host: on the contrary, it could favour growth, invasion and metastasization of the cancer. In this work we studied the role of Interleukin-8 (IL-8/CXCL-8) and Monocyte Chemotactic Protein-1 (MCP-Protein-1/CCL2) in the growth of human lung cancer cells. IL-8 is an important chemotactic factor for neutrophils and it shows angiogenetic activity. MCP-1 is the main responsible for the recruiting of monocytes in solid tumours.
In our experiments we used three different lines of lung cancer (A549, Calu-6, e Calu-1). We demonstrated the capacity of all the cancer lines to produce IL-8 and MCP-1 by ELISA. Flow cytometric analysis showed
the presence of receptors for both chemokines. On the basis of these results, we postulated an autocrine effect of the chemokines on the cancer cellsColorimetric tests (XTT and MTT) were performed to study the effect of IL-8 and MCP-1 on cell proliferation, both upon incubation with increasing concentrations of exogenous chemokines, and after antibody mediated neutralization of endogenous chemokines. We then evluated the induction of apoptosis with Annexin V/PI staining and FACS analysis after neutralization of endogenous IL-8 and MCP-1.
Our results show, for the first time, the capacity of these lung cancer cells to produce the chemokynes and express their receptors (CXCR1 e CCR2, respectively). This suggests the possibility of an autocrine effect of chemokines in the context of lung cancer. This concept is supported by the dose-dependent response of the cell lines to increasing concentrations of MCP-1 and IL-8. Moreover, the neutralization of these chemokynes with antibodies leads to a significant reduction in tumor cell proliferation. Preliminary data, obtained with the A549 line, suggest that neutralization of endogen MCP-1 and IL-8 induces apoptosis in the tumor cells.
In conclusion, the three tumor cell lines produce and release soluble MCP-1 and IL-8 in the culture medium. They express their respective
receptors, CCR2 e CXCR1, on the cellular surface. This led to the hypotesis of an autocrine effect of the chemokines on the tumor cells. The results of the cellular vitality tests show that MCP-1 and IL-8 has the ability to favour cell proliferation. This ability could be partly linked to the fact that they can inhibit apoptosis in our experimental conditions. Further test are needed to identify the effect of the chemokines in the cell-death pathways and, in general, in the various phases of the cell cycle.
