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Molecular and Cellular Biochemistry

, Volume 418, Issue 1–2, pp 91–102 | Cite as

Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells

  • Andrei Alexandru ConstantinescuEmail author
  • Malak Abbas
  • Mohamad Kassem
  • Céline Gleizes
  • Guillaume Kreutter
  • Valerie Schini-Kerth
  • Ioan Liviu Mitrea
  • Florence Toti
  • Laurence KesslerEmail author
Article

Abstract

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.

Keywords

Endocrine β-cells Exocrine cells Tacrolimus Sirolimus Mitochondria-related apoptosis Cellular premature senescence 

Abbreviations

ΔΨm

Mitochondrial membrane potential

a-5

Annexin 5

a.u.

Arbitrary units

C12FDG

5-Dodecanoylaminofluorescein Di-β-D-galactopyranoside

DiOC6

3,3′-Dihexyloxacarbocyanine iodide

DMEM

Dulbecco’s modified Eagle’s medium

ELISA

Enzyme-linked immunosorbent assay

FBS

Fetal bovine serum

FDA

Fluorescein diacetate

FITC

Fluorescein isothiocyanate

HBSS

Hank’s balanced salt solution

mTOR

Mammalian target of rapamycin

mTORc1

mTOR complex 1

PBS

Phosphate-buffered saline

PI

Propidium iodide

ROS

Reactive oxygen species

RPMI

Roswell Park Memorial Institute medium

SA-β-gal

Senescence-associated beta-galactosidase

SDS

Sodium dodecyl sulfate

SIR

Sirolimus

TAC

Tacrolimus

Notes

Acknowledgments

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.

Funding

This research was supported by the Sectorial Operational Programme Development of Human Resources 20072013 (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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Andrei Alexandru Constantinescu
    • 1
    • 2
    • 6
    Email author
  • Malak Abbas
    • 1
    • 3
  • Mohamad Kassem
    • 1
  • Céline Gleizes
    • 1
  • Guillaume Kreutter
    • 1
  • Valerie Schini-Kerth
    • 4
  • Ioan Liviu Mitrea
    • 2
  • Florence Toti
    • 4
  • Laurence Kessler
    • 1
    • 5
    Email author
  1. 1.EA7293, Vascular and Tissular Stress in Transplantation, Federation of Translational Medicine of Strasbourg, Faculty of MedicineUniversity of StrasbourgIllkirch, StrasbourgFrance
  2. 2.Department of Parasitology and Parasitic Diseases and Animal Biology, Faculty of Veterinary MedicineUniversity of Agronomical Sciences and Veterinary MedicineBucharestRomania
  3. 3.Ecole Doctorale de Sciences et TechnologiesUniversité LibanaiseHadathLebanon
  4. 4.UMR7213 CNRSLaboratory of Biophotonics and PharmacologyIllkirchFrance
  5. 5.Department of DiabetologyUniversity HospitalStrasbourg CedexFrance
  6. 6.INSERM, UMR866«Equipe labellisée Ligue contre le Cancer » and Laboratoire d’Excellence LipSTICDijonFrance

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