Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy☆

Background/Aims

Liver pathology induced by chemotherapy (steatosis or vascular injury) is known to increase the liver’s sensitivity to ischemia/ reperfusion (I/R) injury, thereby increasing morbidity and mortality after liver resection. Our aim was to assess whether ischemic preconditioning (IP) reduces I/R injury to livers with chemotherapy-induced pathology.

Methods

We analyzed a series of livers from patients treated with chemotherapy for colorectal cancer who underwent IP (n=30) or not (n=31) before hepatectomy. All but one of the livers exhibited chemotherapy-induced steatosis and/ or peliosis before the I/R insult.

Results

Necrosis was less frequent (p=0.038) in livers with IP than in the others. IP had no influence on apoptosis as assessed by terminal transferase uridyl nick-end labeling (TUNEL) assay or caspase-3, -8 and -9 expression. IP induced a twofold increase in B-cell leukemia/ lymphoma 2 (Bcl-2; p<0.05), which was localized to hepatocytes of centrolobular and peliotic areas and colocalized with the autophagy protein beclin-1 in livers with IP, suggesting their coordinated role in autophagy. Increased expression of the phosphorylated Bcl-2 was observed in preconditioned livers and was associated with a decreased immunoprecipitation of beclin-1 and the increased expression of light chain 3 type II (LC3-II). The increased number of autophagic vacuoles seen by electron microscopy confirmed an association of autophagy in chemotherapy-injured livers following IP. However, the differences in protein expression were not reflected in postresection liver-injury tests or measure of patient morbidity.

Conclusions

IP is associated with a reduction in necrosis of hepatocytes already damaged by chemotherapy and an activation of autophagy. Bcl-2 and beclin-1 could be major targets in the regulation of cell death during I/R injury.

Keywords: Cell death, Ischemia/reperfusion, Ischemic preconditioning, Chemotherapy, Bcl-2, Beclin-1, Autophagy, Liver

Abbreviations: ATP, adenosine triphosphate, BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, Bcl-2, B-cell leukemia/lymphoma 2, IP, ischemic preconditioning, I/R, ischemia/reperfusion, LC3-II, light chain 3 type II, SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, TUNEL, terminal transferase uridyl nick-end labeling