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The discovery of the anti-proliferative activity of nelfinavir in HIV-free models has encouraged its investigation as anticancer drug. Although the molecular mechanism by which nelfinavir exerts antitumor activity is still unknown, its effects have been related to Akt inhibition. Here we tested the effects of nelfinavir on cell proliferation, viability and death in two human breast cancer cell lines and in human normal primary breast cells. To identify the mechanism of action of nelfinavir in breast cancer, we evaluated the involvement of the Akt pathway as well as the effects of nelfinavir on reactive oxygen species (ROS) production and ROS-related enzymes activities. Nelfinavir reduced breast cancer cell viability by inducing apoptosis and necrosis, without affecting primary normal breast cells. The antitumor activity of nelfinavir was related to alterations of the cell redox state, coupled with an increase of intracellular ROS production limited to cancer cells. Nelfinavir treated tumor cells also displayed a downregulation of the Akt pathway due to disruption of the Akt-HSP90 complex, and subsequent degradation of Akt. These effects resulted to be ROS dependent, suggesting that ROS production is the primary step of nelfinavir anticancer activity. The analysis of ROS-producers and ROS-detoxifying enzymes revealed that nelfinavir-mediated ROS production was strictly linked to flavoenzymes activation. We demonstrated that ROS enhancement represents the main molecular mechanism required to induce cell death by nelfinavir in breast cancer cells, thus supporting the development of new and more potent oxidizing molecules for breast cancer therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: The involvement of GRK2 in cancer growth and an inverse correlation with p53 levels were suggested in breast cancer. Furthermore, increased GRK2 expression and activity were detected in thyroid cancer, but its effects and mechanisms of action were not investigated yet. This study aimed to explore the role of GRK2 in thyroid cancer both in vitro and in vivo and its crosstalk with p53. We demonstrated that thyroid cancer cells bearing a mutant form of p53 but not p53 null cells rely on GRK2 as a mechanism of proliferation by regulating p53 levels. Indeed, GRK2 indirectly induces p53 degradation through means of its catalytic activity. The pharmacological inhibition of the kinase effectively inhibits cancer growth by inducing p53-dependent mitochondrial pathways of apoptosis. Our results demonstrate a p53-dependent effect of GRK2 in cancer and suggest kinase inhibition as a potential therapeutic strategy for thyroid cancer. The involvement of GRK2 in cancer cell proliferation and its counter-regulation of p53 have been suggested in breast cancer even if the underlying mechanism has not yet been elucidated. Furthermore, the possibility to pharmacologically inhibit GRK2 to delay cancer cell proliferation has never been explored. We investigated this possibility by setting up a study that combined in vitro and in vivo models to underpin the crosstalk between GRK2 and p53. To reach this aim, we took advantage of the different expression of p53 in cell lines of thyroid cancer (BHT 101 expressing p53 and FRO cells, which are p53-null) in which we overexpressed or silenced GRK2. The pharmacological inhibition of GRK2 was achieved using the specific inhibitor KRX-C7. The in vivo study was performed in Balb/c nude mice, where we treated BHT-101 or FRO-derived tumors with KRX-C7. In our in vitro model, FRO cells were unaffected by GRK2 expression levels, whereas BHT-101 cells were sensitive, thus suggesting a role for p53. The regulation of p53 by GRK2 is due to phosphorylative events in Thr-55, which induce the degradation of p53. In BHT-101 cells, the pharmacologic inhibition of GRK2 by KRX-C7 increased p53 levels and activated apoptosis through the mitochondrial release of cytochrome c. These KRX-C7-mediated events were also confirmed in cancer allograft models in nude mice. In conclusion, our data showed that GRK2 counter-regulates p53 expression in cancer cells through a kinase-dependent activity. Our results further corroborate the anti-proliferative role of GRK2 inhibitors in p53-sensitive tumors and propose GRK2 as a therapeutic target in selected cancers. [ABSTRACT FROM AUTHOR]