The HIV protease inhibitors saquinavir, ritonavir, and nelfinavir induce apoptosis and decrease barrier function in human intestinal epithelial cells.
OBJECTIVE
Diarrhoea is a frequent adverse effect of HIV protease inhibitors (PIs) which may be due to intestinal barrier disruption. We investigated whether tight junction dysregulation, apoptosis or necrosis are responsible for this epithelial damage.


METHODS
Saquinavir, nelfinavir, and ritonavir were added to the mucosal or serosal side of HT-29/B6 colon cell monolayers. Transepithelial resistance was monitored for 72 h to assess epithelial barrier function. Apoptosis and necrosis were investigated by light and electron microscopy and quantified by nucleosome ELISA and LDH measurement, respectively. Tight junction components were analysed by Western blots of occludin and zonula occludens. Apoptosis induction in normal human intestinal epithelium was examined by measurement of poly(ADP-ribose) polymerase (PARP) cleavage in Western blots of mucosal tissue explants cultured with PIs for 24 h.


RESULTS
HIV PIs decreased transepithelial resistance by more than 44% in HT-29/B6 monolayers. Histology revealed massive apoptotic body formation but no evidence for necrosis after PI treatment. Correspondingly, LDH release was lower than 0.2%/h of total LDH, independent of PI treatment, and nucleosomes were increased up to 22-fold after drug treatment versus control. Occludin and zonula occludens-1 expression in the membrane were not diminished. PARP cleavage increased in normal human intestinal tissue treated with PIs.


CONCLUSIONS
PI-induced barrier disruption in intestinal epithelial cells is not due to necrosis or tight junction alterations, but to induction of massive apoptosis which may lead to leak-flux diarrhoea in vivo. Our findings suggest that induction of apoptosis by PIs could have potential for antitumour therapy.
Bode
Hagen
H
Department of Gastroenterology/Infectious Diseases, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Berlin, Germany. hagen.bode@charite.de
Lenzner
Luzie
L
Kraemer
Oliver Holger
OH
Kroesen
Anton Josef
AJ
Bendfeldt
Kerstin
K
Schulzke
Jörg Dieter
JD
Fromm
Michael
M
Stoltenburg-Didinger
Gisela
G
Zeitz
Martin
M
Ullrich
Reiner
R
D000328:Adult; D000368:Aged; D017209:Apoptosis; D002478:Cells, Cultured; D003106:Colon; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D019888:Nelfinavir / Q000494:pharmacology; D009707:Nucleosomes / Q000378:metabolism; D019438:Ritonavir / Q000494:pharmacology; D019258:Saquinavir / Q000494:pharmacology
D017320:HIV Protease Inhibitors; D009707:Nucleosomes; D007770:L-Lactate Dehydrogenase; D019888:Nelfinavir; D019258:Saquinavir; D019438:Ritonavir