No abstract

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.

The bcr/abl fusion gene in 20 patients with chronic myeloid leukemia (CML) was detected by RNA polymerase chain reaction, which used mRNA as the starting material to generate cDNA with reverse transcriptase followed by PCR amplification (RNA/PCR). Amplification of a sequence spanning the bcr/abl junction region was achieved by using peripheral blood cells as the source of mRNA from all 20 patients with CML, including 3 cases of Ph (-) CML, and cell line K562 was derived from patients with CML. No amplification was seen when mononuclear cells from 3 normal individuals, 2 patients with lymphoma and cell line HL-60 were used. The presence or absence of bcr exon 3 in the fusion mRNA was determined by the size of the amplified fragments. Of the 20 CML patients, 15 showed only the 165-bp amplified band (indicating retention of bcr exon 3), one showed only the 90-bp amplified band, and 4 showed both 165-bp and 90-bp bands. Both bands were seen more frequently in blast crisis than in remission and chronic phase.

All-trans-retinoic acid (ATRA) represents the sole example of clinically useful cyto-differentiating agent. ATRA treatment alone results in complete remission of nearly 80% patients with acute promyelocytic leukemia (APL). However, the therapeutic use of this compound is limited by a number of problems, including the systemic toxicity and ATRA resistant leukemia. One way to circumvent these problems is to identify the agents capable of enhancing the pharmacologic activity of ATRA. As we know, an aminopeptidase inhibitor, bestatin, had been used as an immunomodulator in anti-tumor therapy. Recently, we have reported bestatin can induce apoptosis in HL-60 and K562 cells. In the present study, we investigated whether bestatin can potentiate the ATRA induced-differentiation of APL cell line NB4 cells and whether changes of transcription factors expression are involved in this course. The cellular morphology observed by optical microscopy, the expression level of CD11b measured by flow cytometry and the nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cyto-differentiation in NB4 cells. The mRNA expression levels of c-myc and c-EBPε in NB4 cells were detected by RT-PCR. NB4 cells incubated with 10nM ATRA plus 100μg/ml bestatin showed more morphologic character of metamyelocyte and band neutrophil than that of the cells treated by ATRA alone. Compared with 10nM ATRA used alone, after treating NB4 cells for 72 hours, the addition of various concentration of bestatin (50μg/ml, 75μg/ml, 100μg/ml) dose-dependently enhancesd NBT reduction of NB4 cells (17.6±2.5 vs. 12.0±2.2, p<0.05; 23.5±3.2 vs. 12.0±2.2, p<0.01; 36.0±8.3 vs. 12.0±2.2, p<0.01, respectively). 100μg/ml bestatin time-dependently increased 10nM ATRA induced NBT reduction of NB4 cells from 24 to 72 hours (p<0.01). The effect of various concentration of ATRA in combination with 100μg/ml bestatin was statistically different with the sum of the effects of individual drugs after subtracting the value of background (31.2±9.1 vs. 12.7±4.3, p<0.01; 39.5±5.0 vs.16.0±1.8, p<0.001; 49.6±5.3 vs. 22.1±1.6, p<0.001, respectively). Moreover, 10nM ATRA plus 100μg/ml bestatin could prominently elevate CD11b expression in NB4 cells compared with ATRA alone treated NB4 cells group(60.58±9.18% vs. 31.95±5.52%, p<0.01), while 100μg/ml bestatin could not induced significant changes in the expression level of CD11b in NB4 cells after 72 hours incubation. The various concentration (50μg/ml, 75μg/ml, 100μg/ml) of bestatin synergizes with 10nM ATRA to down-regulate the expression level of c-myc mRNA (p<0.01), which was inversely correlated with the NBT reduction activity of NB4 cells induced by 10nM ATRA plus various concentration bestatin (r=−0.917, p=0.028). However, 100μg/ml bestatin plus 10nM ATRA could not induce any significant changes in the expression level of c-EBPε mRNA compared with ATRA treated alone group. In conclusion, an aminopeptidase inhibitor bestatin can potentiate ATRA-induced differentiation of NB4 cells, which may be through down-regulating the expression of c-myc in concert with ATRA. Bestatin would be useful in anti-APL therapy by enhancing the pharmacologic activity of ATRA.