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

Background Loss of response to transforming growth factor-beta (TGF-beta) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-beta signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-beta 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-beta 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-beta-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-beta 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-beta-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-beta and that FK506 restores the TGF-beta signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-beta receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway

The 2020 Italian Society of Arterial Hypertension (SIIA) practical guidelines for the management of primary aldosteronism

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Rapamycin controls multiple signalling pathways involved in cancer cell survival

Suppression of apoptosis by survival signals is considered a hallmark of malignant transformation and resistance to anti-cancer therapy. The phosphoinositide-3 kinase (PI3k)/Akt pathway and NF-kB transcription factors are potent mediators of tumour cell survival. The carbocyclic lactone-lactam antibiotic rapamycin, a widely used immunosuppressant, inhibits the oncogenic transformation of human cells induced by PI3k or Akt by blocking the downstream mTOR kinase. However, inhibition of the PI3k/Akt/mTOR cascade may not be the only mechanism whereby rapamycin exerts anticancer effects. We previously demonstrated that rapamycin inhibits NF-kB by acting on FKBP51, a large immunophilin whose isomerase activity is essential for the functioning of the IKK kinase complex. This suggested that rapamycin may be effective also against neoplasias that express the tumour suppressor PTEN, which, by reducing cellular levels of phosphatidyl-inositol triphosphate, antagonizes the action of PI3k. To address this issue, we over-expressed PTEN in a human melanoma cell line characterized by high phospho-Akt and phospho-mTOR levels, and examined the effect of rapamycin on the apoptotic response to the NF-kB inducer doxorubicin versus cisplatin, which does not activate NF-kB. Rapamycin increased both cisplatin- and doxorubicin-induced apoptosis. Transient transfection of PTEN remarkably decreased phospho-mTOR levels and increased sensitivity to cisplatin’s cytotoxic effect. Under these conditions, rapamycin failed to enhance cisplatin-induced apoptosis. This finding supports the notion that inhibition of a survival pathway increases the efficacy of cytotoxic drugs, and suggests that the pro-apoptotic effect of the rapamycin-cisplatin association requires activated mTOR. Rapamycin retained the capacity to enhance doxorubicin-induced apoptosis in cells over-expressing PTEN, which confirms our earlier observation that inhibition of the PI3k/Akt/mTOR pathway is not involved in the effect exerted by the rapamycin-doxorubicin association. These findings indicate that constitutive activation of mTOR is sufficient but not necessary for rapamycin’s anti-cancer effect. Finally, we show that a decrease in FKBP51 expression levels, obtained with the small interfering RNA technique in the leukemic cell line Jurkat, increased doxorubicin-induced apoptosis, suggesting that this rapamycin ligand is involved in resistance to chemotherapy-induced apoptosis. In conclusion, rapamycin affects more than one signalling survival pathway and more than one target. Our data may impact on the synthesis of rapamycin derivatives. Thus far, rapamycin derivatives used in clinical trials have been tested for their mTOR-inhibiting effect. Our study opens the door to a novel class of anti-cancer drugs that specifically target immunophilins