Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells.
Autophagy has emerged as another major "programmed" mechanism to control life and death much like "programmed cell death" is for apoptosis in eukaryotes. We examined the expression of autophagic proteins and formation of autophagosomes during progression of cisplatin injury to renal tubular epithelial cells (RTEC). Autophagy was detected as early as 2-4 h after cisplatin exposure as indicated by induction of LC3-I, conversion of LC3-I to LC3-II protein, and upregulation of Beclin 1 and Atg5, essential markers of autophagy. The appearance of cisplatin-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in RTEC provided further evidence for autophagy. The autophagy inhibitor 3-methyladenine blocked punctated staining of autophagosomes. The staining of normal cells with acridine orange displayed green fluorescence with cytoplasmic and nuclear components in normal cells but displayed considerable red fluorescence in cisplatin-treated cells, suggesting formation of numerous acidic autophagolysosomal vacuoles. Autophagy inhibitors LY294002 or 3-methyladenine or wortmannin inhibited the formation of autophagosomes but induced apoptosis after 2-4 h of cisplatin treatment as indicated by caspase-3/7 and -6 activation, nuclear fragmentation, and cell death. This switch from autophagy to apoptosis by autophagic inhibitors further suggests that the preapoptotic lag phase after treatment with cisplatin is mediated by autophagy. At later stages of cisplatin injury, apoptosis was also found to be associated with autophagy, as autophagic inhibitors and inactivation of autophagy proteins Beclin 1 and Atg5 enhanced activation of caspases and apoptosis. Our results demonstrate that induction of autophagy mounts an adaptive response, suppresses cisplatin-induced apoptosis, and prolongs survival of RTEC.
Yang
Cheng
C
Department of Medicine, Central Arkansas Veterans Healthcare System and University of Arkansasfor Medical Sciences, Little Rock, Arkansas 72205, USA.
Kaushal
Varsha
V
Shah
Sudhir V
SV
Kaushal
Gur P
GP
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D001343:Autophagy / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002945:Cisplatin / Q000633:toxicity*; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology*; D007684:Kidney Tubules / Q000187:drug effects / Q000473:pathology*; D018374:LLC-PK1 Cells; D013552:Swine
D000970:Antineoplastic Agents; C025946:3-methyladenine; D020169:Caspases; D000225:Adenine; D002945:Cisplatin