tretinoin and ubenimex

This study was purposed to investigate the effect of aminopeptidase N/CD13 on bestatin enhancing all-trans-retinoic acid (ATRA)-inducing differentiation in NB4 cells. The nitroblue-tetrazolium (NBT) reduction assay was performed to determine the differentiation of NB4 cells, MR2 cells and primary APL blasts. The expression of P38 MAPK protein and the phosphorylation of P38 MAPK protein in NB4, MR2 and K562 cells were detected by Western blot. The results showed that pre-incubation with 5 µg/ml WM-15 blocked the enhancement effect of bestatin on differentiation of NB4 cells induced by ATRA. 5 µg/ml CD13 antibody WM-15 partly blocked the inhibition of bestatin on the phosphorylation of P38 MAPK in NB4 cells. 100 µg/ml bestatin inhibited the phosphorylation of P38 MAPK in NB4 cells and MR2 cells in a time-dependent manner. 100 µg/ml bestatin had no effect on the phosphorylation of P38 MAPK in K562 cells with low level of CD13. Bestatin could not restore the sensitivity to ATRA in ATRA-resistant primary APL blasts and MR2 cells. It is concluded that aminopeptidase N/CD13 inhibitor bestatin may enhance the differentiation-inducing activity of ATRA through inhibiting the phosphorylation of P38 MAPK in NB4 cells mediated by the cell surface APN/CD13.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; CD13 Antigens; Cell Differentiation; Cell Division; Cell Line, Tumor; Humans; Leucine; Leukemia, Promyelocytic, Acute; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Tretinoin

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.

Topics: Aminopeptidases; Antibiotics, Antineoplastic; Cell Transformation, Neoplastic; Down-Regulation; Drug Synergism; Humans; Leucine; Leukemia, Promyelocytic, Acute; Proto-Oncogene Proteins c-myc; Tretinoin; Tumor Cells, Cultured

To investigate the effect of aminopeptidase N inhibitor ubenimex on differentiation induction of all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia (APL) cells and its mechanism.. The expression of CD11b was analyzed by flow cytometry and nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cell differentiation of APL cells. The expressions of c-Myc, ERK1/2, p38MAPK protein and the phosphorylation of ERK1/2, p38MAPK protein in NB4 cells were detected by Western blot assay.. Ubenimex alone induced no significant changes in NBT reduction activity and CD11b expression but potentiated the differentiation induction activity of ATRA in APL cells. 100 microg/ml of ubenimex could enhance the NBT reduction activity induced by 10 nmol/L of ATRA, intensify the down-regulation of c-Myc protein expression and inhibit the phosphorylation of p38MAPK protein induced by 10 nmol/L of ATRA in NB4 cells.. Ubenimex could potentiate ATRA induced differentiation in APL cells, which may be correlated with the inhibition of p38 MAPK protein phosphorylation and regulation of c-Myc protein expression.

Topics: Aminopeptidases; Cell Differentiation; Drug Synergism; Flow Cytometry; Humans; In Vitro Techniques; Leucine; Leukemia, Promyelocytic, Acute; Tretinoin

All-trans retinoic acid (ATRA) induces the differentiation of acute promyelocytic leukemia (APL) cells into neutrophils. We found that bestatin, an inhibitor of CD13/aminopeptidase N, enhanced the sensitivity of APL NB4 cells to ATRA at concentrations of 0.1-1000ng/ml. A structurally different aminopeptidase N inhibitor, actinonin, also increased the effect of ATRA on differentiation, but an inactive stereoisomer of bestatin, (2R,3S)-AHPA-(R)-Leu, did not. Bestatin synergistically enhanced the cytostatic effect of ATRA on NB4 cells. Masking of the cell-surface CD13 by anti-CD13 antibody WM15 blocked the synergistic effect of bestatin and ATRA on differentiation. Thus bestatin, an immunomodulator clinically used for nonlymphocytic leukemia, synergistically increased the ATRA-induced differentiation of NB4 cells by inhibiting CD13/aminopeptidase N on the cell-surface.

Topics: Aminopeptidases; Antineoplastic Combined Chemotherapy Protocols; CD13 Antigens; Cell Differentiation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Humans; Leucine; Leukemia, Promyelocytic, Acute; Protease Inhibitors; Stereoisomerism; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured