Inhibition of p38 MAPK Phosphorylation Is Critical for Bestatin to Enhance ATRA-Induced Cell Differentiation in Acute Promyelocytic Leukemia NB4 Cells.

Bestatin has been known as an immunomodulating agent in anti-leukemia treatment. The mechanism by which Bestatin enhances all-trans retinoic acid (ATRA)-induced cell differentiation of acute promyelocytic leukemia (APL) cells is generally attributed to inhibition of cell surface CD13/aminopeptidase N activity. Bestatin also exerts its biological activities besides its ability to inhibit aminopeptidase N enzymatic activity. This article provides data to support an alternative mechanism regarding an important role of inhibition of p38 mitogen-activated protein kinase (MAPK) signal pathway in Bestatin's anti-leukemia effect. Bestatin enhanced ATRA-induced differentiation and inhibited ATRA-driven phosphorylation of p38 MAPK in ATRA-sensitive APL NB4 cells. In contrast, Bestatin could not reverse the differentiation block in ATRA-resistant APL MR2 cells, in which ATRA was unable to induce phosphorylation of p38 MAPK. Moreover, CD13 ligation with anti-CD13 antibody WM-15 resulted in phosphorylation of p38 MAPK, reduced the inhibition of Bestatin on the phosphorylation of p38 MAPK, and completely abolished the enhancement of Bestatin on ATRA-inducing differentiation in NB4 cells. This study shows that inhibition of p38 MAPK phosphorylation is critical for Bestatin to enhance ATRA-induced cell differentiation in ATRA-sensitive APL NB4 cells. Results suggested that pharmacological inhibition of the p38 MAPK pathway might enhance ATRA-dependent differentiation.

[Effect of APN/CD13 on bestatin enhancing all-trans-retinoic acid-inducing differentiation in NB4 cells].

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.

[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.

[Experimental study of the enhancement effect of aminopeptidase N inhibitor ubenimex on the differentiation induction activity of all-trans-retinoic acid in acute promyelocytic leukemia cells and its mechanism].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Effect of tetrandrine combined with Droloxifen on the expression of bcr/abl of K562 at both mRNA and protein levels].

To observe the effect of Tetrandrine (tet) combined with Droloxifen (DRL) on the expression of bcr/abl mRNA and P(210) BCR/ABL protein of K562 cell line, after K562 cells were cultured in the medium containing Tet (1 micromol/L), DRL (5 micromol/L) separately or in their combination for some time, the changes of bcr/abl mRNA and protein expression were detected by RT-PCR and Western blot respectively. The results showed that the application of single drug of Tet or DRL had no effect on bcr/abl mRNA and BCR/ABL protein expression in K562 cell line. However, Tet in combination with DRL began to downregulate bcr/abl mRNA and P(210) BCR/ABL expression of K562 cells at 48 h and 72 h, respectively. It is concluded that tetrandrine in combination with Droloxifen can downregulate the expression of bcr/abl mRNA and P(210) BCR/ABL protein and the combination may be involved in the mechanism underlying the reverse effects on multidrug resistance in leukemia.

[Study on the relationship between [Ca2+]i and the MDR formation in K562/A02 cells].

To explore the relationship of multidrug resistance formation in K562/A02 cells with the intracellular concentration of [Ca(2+)]i, the cytotoxicities of daunorubicin (DNR) were assayed by MTT method, the variations of [Ca(2+)]i of K562 cells and K562/A02 cells after treatment of Tet, DRL and DNR alone or in combination were detected by using Fura-2/AM. The results showed as follows: (1) The cytotoxicities of DNR to cell line K562/A02 were enhanced by 1 micro mol/L Tet or 5 micro mol/L DRL. Their IC(50) was (7.28 +/- 2.06) micro g/ml and (7.58 +/- 3.44) micro g/ml; multiple of their reversal effect was 2.94 and 2.82, but IC(50) of combined Tet and DRL was (1.66 +/- 0.41) micro g/ml. Its reverse effect distinctly increased by 12.9 times. (2) The [Ca(2+)]i in K562/A02 cells were higher than that in K562 cells. (3) One micro mol/L Tet and 5 micro mol/L DRL alone increased the [Ca(2+)]i in K562/A02 cells time-dependently and there was antagonism when both were used. It is concluded that high [Ca(2+)]i is supposed to be a reason of MDR in K562/A02 cells, the action of resistance modifying agents (RMA) in MDR reverse course, however, needs further research.