Anna D'Angelillo

Role of FK506-binding protein 51 in the control of apoptosis of irradiated melanoma cells

FK506-binding protein 51 (FKBP51) is an immunophilin with isomerase activity, which performs important biological functions in the cell. It has recently been involved in the apoptosis resistance of malignant melanoma. The aim of this study was to investigate the possible role of FKBP51 in the control of response to ionizing radiation (Rx) in malignant melanoma. FKBP51-silenced cells showed reduced clonogenic potential after irradiation compared with non-silenced cells. After Rx, we observed apoptosis in FKBP51-silenced cells and autophagy in non-silenced cells. The FKBP51-controlled radioresistance mechanism involves NF-κB. FKBP51 was required for the activation of Rx-induced NF-κB, which in turn inhibited apoptosis by stimulating X-linked inhibitor of apoptosis protein and promoting authophagy-mediated Bax degradation. Using a tumor–xenograft mouse model, the in vivo pretreatment of tumors with FKBP51-siRNA provoked massive apoptosis after irradiation. Immunohistochemical analysis of 10 normal skin samples and 80 malignant cutaneous melanomas showed that FKBP51 is a marker of melanocyte malignancy, correlating with vertical growth phase and lesion thickness. Finally, we provide evidence that FKBP51 targeting radiosensitizes cancer stem/initiating cells. In conclusion, our study identifies a possible molecular target for radiosensitizing therapeutic strategies against malignant melanoma.

FK506 can activate transforming growth factor-β signalling in vascular smooth muscle cells and promote proliferation

AIMS FK506-binding protein (FKBP) 12 is an inhibitor of transforming growth factor (TGF)-beta type I receptors. Several lines of evidence support the view that TGF-beta stimulates vascular smooth muscle cell (VSMC) proliferation and matrix accumulation. We investigated the effect of FK506, also known as tacrolimus, on cellular proliferation and on matrix protein production in human VSMCs. METHODS AND RESULTS We measured cell proliferation with flow cytometry using BrdU incorporation and fluorimetrically by measuring DNA concentration with Hoechst 33258. Western blot assay of whole-cell lysates was used to measure the levels of signalling proteins involved in proliferative pathways, in particular beta-catenin, pErk, pAkt, pmTOR, and cyclin D1. Collagen synthesis was also investigated by Western blotting. The TGF-beta signal was studied by both Western blotting and confocal microscopy. We used the SiRNA technique for FKBP12 gene silencing. Our results show that FK506 stimulates VSMC proliferation and collagen type I production. FK506 enhanced beta-catenin levels and activated the extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin kinase, which are important effectors of proliferation. Accordingly, cyclin D1 expression was increased. We also demonstrate that FK506 activates the TGF-beta signal in VSMCs and that, through this mechanism, it stimulates cell proliferation. CONCLUSION FK506 can act as a growth factor for VSMCs.

FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma

Melanoma is the most aggressive skin cancer; its prognosis, particularly in advanced stages, is disappointing largely due to the resistance to conventional anticancer treatments and high metastatic potential. NF-κB constitutive activation is a major factor for the apoptosis resistance of melanoma. Several studies suggest a role for the immunophilin FKBP51 in NF-κB activation, but the underlying mechanism is still unknown. In the present study, we demonstrate that FKBP51 physically interacts with IKK subunits, and facilitates IKK complex assembly. FKBP51-knockdown inhibits the binding of IKKγ to the IKK catalytic subunits, IKK-α and -β, and attenuates the IKK catalytic activity. Using FK506, an inhibitor of the FKBP51 isomerase activity, we found that the IKK-regulatory role of FKBP51 involves both its scaffold function and its isomerase activity. Moreover, FKBP51 also interacts with TRAF2, an upstream mediator of IKK activation. Interestingly, both FKBP51 TPR and PPIase domains are required for its interaction with TRAF2 and IKKγ, whereas only the TPR domain is involved in interactions with IKKα and β. Collectively, these results suggest that FKBP51 promotes NF-κB activation by serving as an IKK scaffold as well as an isomerase. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51.

The effect of FK506 on transforming growth factor β signaling and apoptosis in chronic lymphocytic leukemia B cells

Loss of response to transforming growth factor-β (TGF-β) is thought to contribute to the progression of chronic lymphocytic leukemia. This study shows that chronic lymphocytic leukemia cells do indeed escape the homeostatic control of TGF-β. Background Loss of response to transforming growth factor-beta (TGF-β ) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-β signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-β signal in chronic lymphocytic leukemia. Design and Methods We studied 62 chronic lymphocytic leukemia samples from patients with Rai/Binet stage 0 to 4 disease. The TGF-β signal was investigated by western blotting and flow cytometry. The levels of Bcl2-family members and death-associated-protein kinase were also investigated by western blotting, whereas apoptosis was studied in flow cytometry. Down-modulation of FKBP12 was obtained by gene silencing with short interfering RNA. Results Twenty-two out of 62 chronic lymphocytic leukemia samples were sensitive to TGF-β-induced apoptosis. All but two of the responsive samples underwent apoptosis also when cultured with FK506, but not with cyclosporine. Thirteen samples that were not sensitive to TGF-β were sensitive to FK506. Overall, response to FK506 occurred in 33 samples. FK506 induced Smad2 phosphorylation and nuclear translocation. Accordingly, death-associated-protein kinase, a transcriptional target of Smad, was induced. At the same time, Bcl-2 and Bcl-xL levels decreased whereas the levels of Bim and Bmf increased. A loss of mitochondrial membrane potential preceded caspase activation and cell death. FK506 removed FKBP12 from its binding to the TGF-β-receptor. FKBP12 release activated the receptor-kinase activity as suggested by the enhanced levels of phospho-Smad found in cells depleted of FKBP12. Conclusions Our study shows that most chronic lymphocytic leukemia cells escape the homeostatic control of TGF-β and that FK506 restores the TGF-β signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-β receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway.

Tirofiban induces VEGF production and stimulates migration and proliferation of endothelial cells.

Tirofiban is a fibrinogen receptor antagonist, generated using the tripeptide Arg-Gly-Asp (RGD) as a template. RGD activates integrin receptors and integrin-mediated signals are necessary for normal cells to promote survival and stimulate cell cycle progression. We investigated whether tirofiban activated growth-stimulatory signals in endothelium. For this study human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) were used. Analysis of cell proliferation, by cell counts, showed that the number of endothelial cells doubled after 72 h of culture in the absence of tirofiban, while they were tripled and even quadrupled, in the presence of increasing doses of the drug. Moreover, tirofiban-stimulated cells had a greater ability to migrate through the transwell filters of Boyden chamber, compared to unstimulated cells. The scratch assay, which mimics cell migration during wound healing, showed that tirofiban stimulated HUVECs to migrate into the leading hedge of the scratch. Western blot showed that tirofiban increased the expression levels of VEGF and the downstream effectors Erk and cyclin D. An inhibitor of VEGFR2 counteracted tirofiban-induced-proliferation, suggesting a role for VEGF in such effect. Our study shows that tirofiban stimulates the migration and proliferation of endothelial cells suggesting that it can promote endothelial repair. Ex vivo cultures of arterial rings confirmed the growth stimulatory effect of tirofiban on endothelium. Thus, the benefits of tirofiban in those with acute coronary syndromes undergoing PTCA may be due to rapid endothelization of damaged vessel, besides antiplatelet effects.

Synergy between enzastaurin doxorubicin in inducing melanoma apoptosis

Melanoma is resistant to most standard chemotherapeutics. We analysed the combined effect of doxorubicin and enzastaurin on cell death of four melanoma cell lines, namely G361, SK‐MEL3, A375 and SAN. Enzastaurin IC50 was calculated by measure of growth inhibition with MTS assay and corresponded to 2 μM; the half maximal cytotoxicity of doxorubicin was obtained at 3 μM dose. Evaluation of combination index showed synergism (CI > 1) or additive effect (CI = 1) with all melanoma cell lines, with enzastaurin doses ≥0.6 μM and doxorubicin doses ≥1 μM. Combination of the two drugs resulted in increase in caspase 3 and 8 activation, in comparison with activation by single agents. Caspase 8 activation was impaired by TNFR‐1 blocking. Our results show doxorubicin‐stimulated production of TNFα, whereas enzastaurin‐stimulated TNFR‐1 expression on plasma membrane. The effect on TNFR‐1 appeared to be mediated by PKCζ inhibition. Taken together, our findings suggest that enzastaurin increases doxorubicin‐induced apoptosis of melanoma by a mechanism involving, at least in part, activation of the TNF‐α signal.

Immunomodulatory pathways regulate expression of a spliced FKBP51 isoform in lymphocytes of melanoma patients

FKBP51 (gene FKBP5) is an immunophilin capable of immunosuppression expressed in melanoma and lymphocytes. We found increased levels of a spliced FKBP5 variant in the PBMCs of 124 patients with melanoma. This variant encodes for an unknown isoform (FKBP51s). We hypothesized that FKBP51s resulted from tumour interaction with immune cells, through PDL‐1/PD‐1. To address this issue, we performed melanoma/PBMC cocultures. Furthermore, the immunohistochemistry of 76 melanoma specimens served to investigate whether FKBP51s stained tumour infiltrating lymphocytes. Our results showed that PBMCs expressed FKBP51s when cocultured with melanoma. Tumour PDL‐1 knockdown or anti‐PD‐1 reduced FKBP51s expression in cocultured PBMCs. IHC showed a strong FKBP51s signal in tumour infiltrating lymphocytes, and lymphocytes of the invasion front of the tumour, along with melanoma PDL‐1 expression. When overexpressed in melanoma, FKBP51s facilitated PDL‐1 expression on the cell surface. In conclusion, our study shows that FKBP51s marks the PBMCs of patients with melanoma and is exploited by the tumour to immunomodulate through PDL‐1.

Effects Of Glycoprotein IIb/IIIa Antagonists: Anti Platelet Aggregation And Beyond

BACKGROUND The use of inhibitors of glycoprotein IIb/IIIa (GPIIb/IIIa) has provided dramatic results in terms of the prevention of acute stent thrombosis and a reduction in major adverse coronary events in patients subjected to percutaneous coronary intervention. GPIIb/IIIa or αIIbβ3 is a member of the β3 subfamily of integrins, which also includes αVβ3. GPIIb/IIIa functions as a receptor for fibrinogen and several adhesion proteins sharing an arginine-glycine-aspartic acid (RGD) sequence. GPIIb/IIIa antagonists, through blockade of the receptor, prevent platelet aggregation. Among the three GPIIb/IIIa antagonists used in therapy, abciximab is an anti-β3 monoclonal antibody, while tirofiban and eptifibatide mimic the binding sequence of the fibrinogen ligand. Although antiplatelet aggregation represents the central function of GPIIb/IIIa inhibitors, further actions have been documented for these compounds. OBJECTIVE The aim of the present article is to review the structures and functions of GPIIb/IIIa antagonists and to highlight the clinical outcomes and results of randomized trials with these compounds. Hypotheses on the unexplored potential of GPIIb/IIIa antagonists will be put forward. CONCLUSION GPIIb/IIIa inhibitors were developed to prevent platelet aggregation, however, these compounds can exert further biological functions, both platelet- and non-platelet-related. Large-scale studies comparing the efficacy and safety of GPIIb/IIIa antagonists are lacking. More insights into the functions of these compounds may lead to generation of novel small molecules able to antagonize platelet aggregation while promoting vascular repair.

Tirofiban counteracts endothelial cell apoptosis through the VEGF/VEGFR2/pAkt axis.

Tirofiban is used in the treatment of patients with acute coronary syndrome submitted to percutaneous coronary intervention (PCI). We have, previously, shown that tirofiban stimulates VEGF expression and promotes proliferation of endothelial cells. VEGF is a well known inhibitor of endothelial cell apoptosis. TNF-α is a pro-apoptotic cytokine released in the site of a vascular injury, including balloon angioplasty. We thought to investigate whether tirofiban was able to protect endothelial cells from cell death induced by TNF-α. For this study, we used human umbilical vein endothelial cells (HUVEC). Analysis of apoptosis was performed by propidium iodide incorporation, annexin V staining and measure of active caspase 3 levels. Western blot served for a semiquantitative measure of Akt activation, VEGF, and the pro-apoptotic Bim and Bak. Our results show that TNF-α was unable to activate caspase 3 and produce cell death in the presence of tirofiban. Activation of apoptosis was preceded by upregulation of Bim and Bak that resulted decreased after addition of tirofiban. The anti-apoptosis effect of tirofiban was reproduced by VEGF and counteracted by VEGFR2 blockade and the cation chelating agent ethylene glycol tetraacetic acid (EGTA). The use of p-Akt inhibitor, BEZ235,and Akt knockdown, suggested that pAkt mediated the prosurvival effect of tirofiban. In conclusion, tirofiban protects endothelial cells from apoptosis stimulated by TNF-α, due to its ability to stimulate VEGF production.

High expression of a spliced variant of FKBP51 in peripheral blood mononuclear cells of melanoma patients may be related to PDL-1 on tumour and predictive of response to Ipilimumab

Meeting abstracts Identifying molecular biomarkers in melanoma may provide useful diagnostic and therapeutic tools. Melanoma delivers immune suppressive stimuli through the pathway PDL-1/PD-1. Recent data suggest tumour-cell expression of PD-L1 in melanoma may be driven by constitutive oncogenic