Previously studies have demonstrated that besides its actions in the cardiovascular system, Angiotensin-(1-7) also plays a role in inhibiting tumoral growth. The role of recently described Alamandine in this field is not clear. The signaling pathways underling anti-tumoral actions of these peptides are also poorly understood. Therefore, the aim of this study was to elucidate the modulatory effect of Ang-(1-7) and Alamandine in the PI3K cascade, a well-known signaling pathway described to be involved in proliferation and cancer. To achieve this goal, we stimulate human pancreatic and lung cancer cell lineage (Miapaca and A549), as well as a control cell lineage (VERO) with Ang-(1-7) and Alamandine. Through western blotting analysis, our data suggest that both Ang-(1-7) and Alamandine activate the phosphatase pTEN (dephosphorylation of S380/T382/T383) (48±4% after 24 hours Ang-(1-7) treatment in miapaca and A549 in comparison of non-treated cells, p<0.05) (60±5 and 48±4% of phosphorylation level after 24 hours Alamandine treatment in miapaca and A549, respectively, in comparison of non-treated cells, p<0.05), which dephosphorylates PI3K, inactivating this kinase. Furthermore, AKT phosphorylation is transient, followed by a significant dephosphorylation when compared to the non-treated cells (30±5% after 24 hours Ang-(1-7) treatment in miapaca in comparison of non-treated cells, p<0.05). Ang-(1-7) also inhibits a PTEN downstream effector kinase, mTOR through dephosphorylation of T246 (70±5% after 24 hours Ang-(1-7) treatment in miapaca in comparison of non-treated cells, p<0.05). These effects were not observed in control non-tumoral cells (VERO cells). As previously demonstrated with Ang-(1-7) stimulation, Alamandine also induces the FOXO1 activation and migration to the nucleus in A549 (122 ± 8 of A.U. of fluorescence at 4 hours after alamandine treatment vs 46±4 A.U. at control, p<0.001) and Miapaca cells (67 ± 5 of A.U. of fluorescence at 4 hours after alamandine treatment vs 16±2 A.U. at control, p<0.001). These results indicate that, in contrast to normal tissues, Ang-(1-7) and Alamandine decreases, through PTEN activation, PI3K/AKT pathway in tumoral cells.
Several reports have shown the actions of ACE/ AngII/AT1 axis in the development of malignancy and also predict that RAS inhibitors could potentiate cancer therapies. On the other hand, the alternative axis of renin angiotensin system, ACE2/Ang-(1-7)/Mas, demonstrated an anti-tumoral property. This anti-tumoral effect is not clear for the recently described components of RAS, Alamandine/MrgD. The aim of this work is to characterize the expression and the enzyme activities of the RAS components in different tumoral and normal cell lines. There was a significant increase in the expression of the components (ACE and AT2) related to the formation and actions of Ang II. ACE presented 1.8 A.U. and 1.4 A.U. in A549 and MIAPACA tumoral cell lines, respectively, in comparison with 0.6 A.U. of normal cell lines (VERO) (p< 0.05). On the other hand, there was reduction in ACE2 enzyme related to the formation of Angiotensin-(1-7) and Alamandine (0.25 A.U. in MIAPACA VS 1.7 A.U. in VERO, p<0.05), which are modulatory peptides of the actions of Angiotensin II. Furthermore, there is also a significant increase in both Mas (0.18 A.U. in A549 VS 0.12 A.U. in VERO, p<0.05) and MrgD receptors (85 ±6 A.U. in A549 and 73 ±5 A.U. in MIAPACA VS 11 ±3 A.U. in VERO, p<0.001) expression in tumoral cells. Additionally, to complete the data obtained by Western Blotting, the enzymatic activity of ACE and ACE2 was evaluated in tumor cells and in Vero cells used as control. ACE enzyme activity is increased in tumor cells, while the ACE2 is reduced (950 A.U. in VERO VS 500 A.U. MIAPACA and 450 A.U. A549 p<0.01). These data suggest that RAS proliferative axis is activated in tumor cells and anti -proliferative axis is decreased compared to control cells.
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