Caspase-3 gene silencing for inhibiting apoptosis in insulinoma cells and human islet

Guofeng Cheng, Lin Zhu, Ram I. Mahato

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Although islet transplantation has great potential to treat type I diabetes, most islet grafts do not function due to the host immune rejection, nonspecific inflammatory response and poor revascularization. Since caspase-3 plays a crucial role in apoptosis of transplanted islet cells, we used chemically synthesized small interfering RNAs (siRNAs) to silence caspase-3 in insulinoma (INS-1E) cells and human islets, and then determined whether caspase-3 gene silencing can prevent these cells from cytokine-induced apoptosis. Transfection of INS-1E cells and islets with siRNAs reduced caspase-3 transcripts by 50-67% and 50%, respectively. Additionally, apoptosis in transfected insulinoma cells was markedly inhibited. Since gene silencing did not last beyond two days, we converted potent siRNA into shRNA and constructed replication deficient adenoviral (Adv) vectors encoding these shRNAs driven by a U6 or H1 promoter. Compared to chemically synthesized siRNA, Adv-caspase-3-shRNA efficiently transduced islets, showed relatively higher and prolonged levels of gene silencing beyond five days, with higher gene silencing with a U6 promoter, and protected islets from cytokine-induced apoptosis. Finally, return to normoglycemia was achieved at 1 day post-transplantation of Advcaspase-3-shRNA transduced islets under the kidney capsules of streptozotocin induced nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice and maintained beyond two weeks. Blood glucose levels returned to ≥325 mg/dL upon removal of the islet graft-bearing kidney at 32 days after transplantation, confirming that transplanted islets were functional.

Original languageEnglish (US)
Pages (from-to)1093-1102
Number of pages10
JournalMolecular Pharmaceutics
Volume5
Issue number6
DOIs
StatePublished - Dec 1 2008

Keywords

  • Apoptosis
  • Caspase-3
  • Gene silencing
  • Human islets
  • Insulinoma cells

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmaceutical Science
  • Drug Discovery

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