Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells
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To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic exocrine and endocrine β-cell fate. Human exocrine PANC-1 and rat endocrine insulin-secreting RIN-m5F cells and isolated rat islets were submitted to 1–100 nM TAC or SIR. In cultures, insulin secretion was measured as endocrine cell function marker. Apoptosis was quantified by annexin 5 and propidium iodide staining. Cleaved caspase-3, Bax apoptosis indicators, and p53, p21 cell cycle regulators were detected by Western blot. Cell cycle and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry and SA-beta-galactosidase (SA-β-gal) activity by fluorescence microscopy. Only TAC reduced insulin secretion by RIN-m5F after 24 h. TAC and SIR promoted moderate apoptosis in both PANC-1 and RIN-m5F after 24 h. Apoptosis was associated with up-regulated Bax (threefold) and cleaved caspase-3 (fivefold) but only in PANC-1, while p53 and p21 were up-regulated (twofold) in both cell lines. ΔΨm was impaired only in PANC-1 by TAC and SIR. Only SIR prompted cell cycle arrest in both cell lines. The induction of a premature senescence-like phenotype was confirmed in isolated islets by SA-β-gal activity. TAC and SIR are early inducers of pancreatic cell dysfunction and apoptosis but differentially alter endocrine and exocrine cells via mitochondrial-driven pathways. In rat islets, TAC and SIR prompt a senescence-like phenotype.
KeywordsEndocrine β-cells Exocrine cells Tacrolimus Sirolimus Mitochondria-related apoptosis Cellular premature senescence
Mitochondrial membrane potential
Dulbecco’s modified Eagle’s medium
Enzyme-linked immunosorbent assay
Fetal bovine serum
Hank’s balanced salt solution
Mammalian target of rapamycin
mTOR complex 1
Reactive oxygen species
Roswell Park Memorial Institute medium
Sodium dodecyl sulfate
We are grateful to Dr. C. Muller and Mr. D. Nabergoj from the eBIOCYT cytometry platform of Strasbourg University, and to Mr. R. Vauchelles from the PIQ quantitative imaging platform of Strasbourg University. We appreciate the kind assistance in isolation of rat islets by Prof. D. Bosco from Laboratoire d’Isolement et Transplantation Cellulaire, Hôpital Universitaire, Genève, Suisse. We thank the European Doctoral College, part of Strasbourg University, for its support during this scientific achievement.
This research was supported by the Sectorial Operational Programme Development of Human Resources 2007–2013 (POSDRU) through the Financial Agreement POSDRU/107/1.5/S/76888 of the Government of Romania, recurrent founding, by the Association Vaincre la Mucoviscidose (VLM) and by the Association d’Aide aux Insuffisants Respiratoires Alsace-Lorraine (ADIRAL).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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