Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

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Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53-wild type U2OS cells (and not of p53-null Saos and p53-mutant MG63 cells) by slowing-down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin-induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub-G1 population, Bcl-2 downregulation, caspase-3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination-induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine-alpha. Moreover, the doxorubicin-induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53-dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy.