Annalisa Benini

Caffeine inhibits adenosine-induced accumulation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and interleukin-8 expression in hypoxic human colon cancer cells.

Frequent coffee consumption has been associated with a reduced risk of colorectal cancer in a number of case-control studies. Coffee is a leading source of methylxanthines, such as caffeine. The induction of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) is an essential feature of tumor angiogenesis, and the hypoxia-inducible factor-1 (HIF-1) transcription factor is known to be a key regulator of this process. In this study, we investigated the effects of caffeine on HIF-1 protein accumulation and on VEGF and IL-8 expression in the human colon cancer cell line HT29 under hypoxic conditions. Our results show that caffeine significantly inhibits adenosine-induced HIF-1α protein accumulation in cancer cells. We show that HIF-1α and VEGF are increased through A3 adenosine receptor stimulation, whereas the effects on IL-8 are mediated via the A2B subtype. Pretreatment of cells with caffeine significantly reduces adenosine-induced VEGF promoter activity and VEGF and IL-8 expression. The mechanism of caffeine seems to involve the inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and Akt, leading to a marked decrease in adenosine-induced HIF-1α accumulation, VEGF transcriptional activation, and VEGF and IL-8 protein accumulation. From a functional perspective, we observe that caffeine also significantly inhibits the A3 receptor-stimulated cell migration of colon cancer cells. Conditioned media prepared from colon cells treated with an adenosine analog increased human umbilical vein endothelial cell migration. These data provide evidence that adenosine could modulate the migration of colon cancer cells by an HIF-1α/VEGF/IL-8-dependent mechanism and that caffeine has the potential to inhibit colon cancer cell growth.

Adenosine receptors in colon carcinoma tissues and colon tumoral cell lines: Focus on the A3 adenosine subtype

Adenosine may affect several pathophysiological processes, including cellular proliferation, through interaction with A1, A2A, A2B, and A3 receptors. In this study we characterized adenosine receptors in human colon cancer tissues and in colon cancer cell lines Caco2, DLD1, HT29. mRNA of all adenosine subtypes was detected in cancer tissues and cell lines. At a protein levels low amount of A1, A2A, and A2B receptors were detected, whilst the A3 was the most abundant subtype in both cancer tissues and cells, with a pharmacological profile typical of the A3 subtype. All the receptors were coupled to stimulation/inhibition of adenylyl‐cyclase in cancer cells, with the exception of A1 subtype. Adenosine increased cell proliferation with an EC50 of 3–12 µM in cancer cells. This effect was not essentially reduced by adenosine receptor antagonists. However dypiridamol, an adenosine transport inhibitor, increased the stimulatory effect induced by adenosine, suggesting an action at the cell surface. Addition of adenosine deaminase makes the A3 agonist 2‐chloro‐N6‐(3‐iodobenzyl)‐N‐methyl‐5′‐carbamoyladenosine (Cl‐IB‐MECA) able to stimulate cell proliferation with an EC50 of 0.5–0.9 nM in cancer cells, suggesting a tonic proliferative effect induced by endogenous adenosine. This effect was antagonized by 5‐N‐(4‐methoxyphenyl‐carbamoyl)amino‐8‐propyl‐2(2furyl)‐pyrazolo‐[4,3e]‐1,2,4‐triazolo [1,5‐c] pyrimidine (MRE 3008F20) 10 nM. Cl‐IB‐MECA‐stimulated cell proliferation involved extracellular‐signal‐regulated‐kinases (ERK1/2) pathway, as demonstrated by reduction of proliferation with 1,4‐diamino‐2,3‐dicyano‐1,4‐bis‐[2‐amino‐phenylthio]‐butadiene (U0126) and by ERK1/2 phosphorylation. In conclusion this study indicates for the first time that in colon cancer cell lines endogenous adenosine, through the interaction with A3 receptors, mediates a tonic proliferative effect. J. Cell. Physiol. 211: 826–836, 2007.

Adenosine and lymphocyte regulation

Adenosine is a potent extracellular messenger that is produced in high concentrations under metabolically unfavourable conditions. Tissue hypoxia, consequent to a compromised cellular energy status, is followed by the enhanced breakdown of ATP leading to the release of adenosine. Through the interaction with A2 and A3 membrane receptors, adenosine is devoted to the restoration of tissue homeostasis, acting as a retaliatory metabolite. Several aspects of the immune response have to be taken into consideration and even though in general it is very important to dampen inflammation, in some circumstances, such as the case of cancer, it is also necessary to increase the activity of immune cells against pathogens. Therefore, adenosine receptors that are defined as ‘sensors–of metabolic changes in the local tissue environment may be very important targets for modulation of immune responses and drugs devoted to regulating the adenosinergic system are promising in different clinical situations.

Hypoxia Inhibits Paclitaxel-Induced Apoptosis through Adenosine-Mediated Phosphorylation of Bad in Glioblastoma Cells

Solid tumors contain hypoxic cells that are resistant to radiotherapy and chemotherapy. The resistance in glioblastoma has been linked to the expression of antiapoptotic Bcl-2 family members. In this study, we found that in human glioblastoma cells hypoxia induces the phosphorylation of the Bcl-2 family protein Bad, thus protecting hypoxic cells from paclitaxel-induced apoptosis. Akt activation is required for the hypoxia-induced protection. In contrast, the extracellular signal-regulated kinase 1/2 activities have only a partial effect, being able to modulate Bad phosphorylation but not paclitaxel-induced apoptosis in hypoxia. We also demonstrated that the degradation of adenosine with adenosine deaminase, the knockdown of A3 adenosine receptor expression by gene silencing, and the blockade of this receptor through A3 receptor antagonists blocked the hypoxia-induced phosphorylation of Bad and the prolonged cell survival after treatment with paclitaxel in hypoxia. Thus, the adenosinergic signaling may be an essential component in the hypoxia survival pathway. These results suggest that hypoxia-induced chemoresistance of human glioblastoma cells may occur in a novel mechanism involving activation of adenosine-A3 receptor-Akt pathway, which mediates Bad inactivation and favors cell survival.