PHARMACOLOGICAL CHARACTERIZATION OF THE SIGNAL TRANSDUCTION PATHWAYS MODULATED BY A3 RECEPTORS IN CANCER CELLS: POSSIBLE TARGETS FOR THERAPEUTIC INTERVENTION.

Adenosine is an ubiquitous autacoid that modulates a variety of cellular functions through occupancy of four cell surface G-protein-coupled receptors, named A1, A2A, A2B and A3. In particular, adenosine was found to exert its effects on cell proliferation, clone formation ability, UV resistance, and cell death mainly through the A3 subtype, which is highly expressed in tumor cells. Adenosine also plays a role in the promotion of angiogenesis. In this thesis I have characterized the signal transduction pathways modulated in hypoxia by A3 adenosine receptors in two different human tumor models: the colon cancer HT29 and the melanoma A375 cell lines. I have performed the study in hypoxia, present in most solid tumors, which shifts the cellular phenotype toward an increase in adenosine. Furthermore, hypoxic tumor cells are resistant to conventional chemiotherapy and radiotherapy. I have investigated the modulation of hypoxia-inducible factor-1 (HIF-1) through adenosine via its receptor subtypes. HIF-1 is a transcription factor that functions as a master regulator of oxygen homeostasis. HIF-1 is a heterodimer composed of an inducibly expressed HIF-1α subunit and a constitutively expressed HIF-1β one. In normoxia, HIF-1α is rapidly degraded by the ubiquitin proteasome system, whereas exposure to hypoxic conditions prevents its degradation. I have studied the signaling pathways modulated by A3 receptors which involved Akt, MEK, and p38 MAPK. I have demonstrated that adenosine increased in hypoxic colon carcinoma cells HIF-1α and vascular endothelial growth factor (VEGF) through the A3 receptor stimulation. Furthermore, the stimulation of A2B receptor increased interleukin-8 (IL-8) expression. Pretreatment of cells with caffeine, which is a methylxanthine antagonist of adenosine receptors, significantly reduced adenosine-induced VEGF promoter activity and VEGF and IL-8 expression. The kinases have a key role in A3 receptor ability to enhance HIF-1α and VEGF protein expression. Moreover, Akt, ERK 1/2, and p38 MAPK are required for the IL-8 expression increase induced by A2B receptor activation. Then, I have examined the modulation of IL-8, VEGF and HIF-1 by the DNA-damaging agents etoposide and doxorubicin, and I have analyzed the influence of the adenosinergic signaling on the chemotherapeutic drug effects. I have demonstrated that A2B receptor blockade can impair IL-8 production, whereas blocking A3 receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with etoposide and doxorubicin. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. Exposure of melanoma cells to the chemotherapeutic drugs resulted in the increase of p38, Akt and ERK1/2 phosphorylation levels. Moreover, etoposide and doxorubicin strongly inhibited HIF-1α protein expression. The A2B receptor antagonist MRE 2029F20 attenuated the increase in p38, Akt and ERK1/2 phosphorylation levels induced by the chemotherapeutic drugs, and when used alone it reduced p38, Akt and ERK1/2 phosphorylation basal levels. The A3 receptor antagonist MRE 3008F20 alone reduced HIF-1α protein, and in combination with the chemotherapeutic drugs further decreased HIF-1α protein accumulation. Therefore, the results of this thesis provide evidence of how human tumor growth may be influenced through the adenosinergic system and how the adenosine receptors modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively.