[Effect of aminopeptidase inhibitor on differentiation induction activity of all-trans retinoic acid in human acute promyelocytic leukemia NB4 cells and its mechanism].
This study was purposed to investigate whether aminopeptidase inhibitor, bestatin, can potentiate all-trans retinoic acid (ATRA)-inducing differentiation in NB4 cells, and to explore its mechanism. The NB4 cells were exposed to either bestatin and ATRA alone or in combination, the morphological changes of NB4 cells were observed by optical microscopy, the CD11b expression was measured by flow cytometry, the function of defferentiation cells was analyzed by nitroblue-tetrazolium (NBT) reduction assay, the mRNA expressions of c-myc and c-EBPepsilon in NB4 cells were detected by RT-PCR, the c-Myc protein expression was determined by Western blot. The results showed that treatment with bestatin alone induced no significant changes in morphology, NBT reduction activity and CD11b expression in NB4 cells. NB4 cells incubated with 10 nmol/L ATRA plus 100 microg/ml bestatin showed more morphologic feature of metamyelocyte and band neutrophil than ATRA alone treated cells. 100 microg/ml bestatin enhanced the NBT reduction activity in NB4 cells induced by various concentrations of ATRA (10, 20, 40 nmol/L). The effects of various concentrations of ATRA in combination with 100 microg/ml bestatin were statistically different from the effect of ATRA alone (P < 0.01). From 48 to 96 hours, 100 microg/ml bestatin time-dependently increased NBT reduction in NB4 cells induced by 10 nmol/L ATRA (P < 0.01). 10 nmol/L ATRA plus 100 microg/ml bestatin for 72 hours prominently elevated CD11b expression in NB4 cells as compared with ATRA alone treated NB4 cells (P < 0.01). There was a substantial decrease in c-myc mRNA levels when 100 microg/ml bestatin was added to 10 nmol/L ATRA (P < 0.05). Various concentrations (50, 75, 100 microg/ml) of bestatin combined with 10 nmol/L ATRA down-regulated the expression of c-Myc protein, which was negatively correlated with the NBT reduction activity of NB4 cells induced by 10 nmol/L ATRA alone or plus bestatin at various concentrations (r = -0.940, P = 0.017). However, 100 microg/ml bestatin plus 10 nmol/L ATRA could not induce any significant changes in the levels of c-EBPepsilon mRNA as compared with ATRA alone treated NB4 cells. It is concluded that an aminopeptidase inhibitor bestatin can potentiate ATRA-inducing differentiation of NB4 cells, possibly by down-regulating c-myc expression in synergy with ATRA.
Lin
Mao-Fang
MF
Department of Hematology, The First Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310003, China.
Qian
Xi-Jun
XJ
D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D015536:Down-Regulation; D004357:Drug Synergism; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015473:Leukemia, Promyelocytic, Acute / Q000473:pathology*; D016271:Proto-Oncogene Proteins c-myc / Q000187:drug effects / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D014212:Tretinoin; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex