| Acute promyelocytic leukemia (APL) is a subtype of acute myelogenous leukemia (AML). This disease has distinct clinical, cyto-genetic and molecular characteristics. Without treatment, APL is rapidly fatal due to life-threatening coagulopathy. Since the introduction of all-trans retinoic acid (ATRA) to the treatment of APL by Chinese hematologists in 1988, this disease has become a clinical curable subtype of AML. However, the therapeutic effect of ATRA as a signal agent is frequently transient, and relapse of APL is often associated with the acquisition of resistance to ATRA. The clinical use of ATRA is also limited by serious systemic toxicity. In addition, ATRA alone has not been effective in non-APL AML treatment, as all the other French-American-British subtypes are generally refractory to its cyto-differentiating action. One way to circumvent these problems is to identify agents capable of enhancing the pharmacologic activity of ATRA and to reveal the mechanism of such effect. These issues are always the spotlight in the differentiation therapy of acute leukemia.Recent advances in the understanding of the mechanisms of ATRA have shownthat this agent exerts its biological activities mainly through ligand dependent activation of retinoid receptors to modulate gene expression and the degradation of the PML-RARa fusion protein. In addition, several signal pathways are also involved in the course of ATRA-induced differentiation. In vitro study has demonstrated that many cytokines such as granulocyte colony-stimulating factor, interferon y and some medicines such as tyrosine kinase inhibitor STI571, lithium chloride could potentiate the pharmacologic activity of ATRA in acute promyelocytic leukemia cells. However, the clinical application is limited by their severe adverse effects.Aminopeptidase N/CD13 (APN/CD13) is a ubiquitously occurring Zn~2+-dependent metalloprotease. Enhanced APN levels are observed in tumor cells such as AML. APN/CD13 not only exerts its biological activity as a protease, but also is involved in signal transduction, tumor cell invasion and regulating cell proliferation and immune response. Ubenimex inhibits aminopeptidase N, aminopeptidase B, and leucine aminopeptidase in mammalian cells and has antitumor activity through its immunomodulatory functions and its activity of inducing tumor cells apoptosis. Ubenimex has been applied to treat acute non-lymphocytic leukemia and its adverse effects are mild and infrequent. Recently, Hirano et al suggested that ubenimex could enhance the sensitivity of acute promyelocytic leukemia NB4 cells to all-trans retinoic acid. For further study about the enhancement of ubenimex on the inducing-differentiation activity of ATRA, we therefore conducted this study to address the following questions: a) Whether ubenimex has the same effect on the primary APL cells and other AML subtype cell line? b) Could ubenimex restore the sensitivity of ATRA-resistant APL cell line to the ATRA? c) Are there some common pathways in the effects of ATRA and ubenimex targeting APN/CD13? d) How APN/CD13 is involved in this course?In part I, the effects of ubenimex on the pharmacologic activity of ATRA on the several cell lines were investigated. Firstly, the expression of CD 13 was measured byflow cytometry and the level of PML-RARa mRNA was detected by PT-PCR to demonstrate the situation of ATRA and ubenimex targets in these cell lines. The levels of CD 13 inNB4 cells, MR2 cells and HL-60 cells were 99.8%, 99.5%, 99.7%, respectively. The expression of CD 13 in the K562 cells as the negative control was very low, only 3.36%. PML-RARa mRNA was amplified in NB4 cells and MR2 cells. Meanwhile, PML-RARa mRNA could not be detected in HL-60 cells and K562 cells. lOOug/ml ubenimex alone had no effect on the differentiation of NB4 cells, primary APL cells, MR2 cells, HL-60 cells and K562 cells. NB4 cells incubated with lOnmol/L ATRA plus lOOug/ml ubenimex showed more morphologic character of metamyelocyte and band neutrophil, while NB4 cells exposed to lOnmol/L ATRA alone showed the morphology of partial differentiation. After treatment for 72 hours, various concentration of ubenimex (50^g/ml, 75ng/ml and 100ug/ml) enhanced lOnmol/L ATRA induced NBT reduction activity of NB4 cells in a dose dependent manner (17.6±2.5, 23.5±3.2, 36.0±8.3 vs. 12.0±2.2, respectively). Meanwhile, lOOug/ml ubenimex augmented the inducing NBT reduction activity of various concentration of ATRA (lOnmol/L, 20nmol/L and 40nmol/L) in NB4 cells. The effect of various concentration of ATRA in combination with lOOug/ml ubenimex was statistically different with the effect of various concentration ATRA alone (32.3±9.4 vs. 12.4±2.5;40.7±5.3 vs. 15.7±2.6;50.8±5.5 vs. 21.8±2.4;p<0.0\, respectively). From 48 to 96 hours, lOOug/ml ubenimex potentiated lOnmol/L ATRA induced NBT reduction activity of NB4 cells in a time dependent manner (23.0±5.0 vs. 8.9±1.8;32.0±7.1 vs. 14.4±0.8;61.4±4.0 vs. 17.2±1.2;p<0.0\, respectively). lOnmol/L ATRA plus 100ug/ml ubenimex for 72 hours could prominently elevate CD lib expression in NB4 cells compared with ATRA alone treated NB4 cells (60.58±9.18% vs. 31.95±5.52%,/?<0.01). In addition, 100ng/ml ubenimex significantly enhanced lOnmol/L ATRA induced NBT reduction activity of primary APL blasts (55.3±10.2 vs. 37.5±8.3,^<0.05). The NBT reductionactivity of primary APL blasts induced by lOOug/ml ubenimex in combination with lOnmol/L ATRA was similar to that induced by lOOnmol/L ATRA. In ATRA-resistant MR2 cells and primary APL blasts obtained from a refractory relapsed APL patient, lOOnmol/L ATRA alone or in combination with lOOug/ml ubenimex could not significantly induce the NBT reduction activity. Under the conditions in our study, ubenimex could not restore the sensitivity of ATRA in ATRA-resistant primary APL blasts and MR2 cells. Compared with the control group, HL-60 cells incubated with lOOnmol/L ATRA for 96h possessed the elevated NBT reduction activity (16.8±2.5 vs. 0.3±0.1, p<0.0\). Various concentration (50ug/ml, 75ug/ml and 100ng/ml) ubenimex had no effect on the NBT reduction activity of HL-60 cells induced by lOOnmol/L ATRA. In K562 cells as the negative control, neither ubenimex nor ATRA could increase the NBT reduction activity.Previous research reported that p38 mitogen activated protein kinase (MAPK) pathway was activated in an ATRA-dependent manner in NB4 cells and exhibited negative regulatory effects on inducing-differentiation activity of ATRA. To explore the role of p38 MAPK pathway in the enhancement of ubenimex on ATRA pharmacologic activity, the phosphorylation levels of p38 MAPK in NB4 cells and MR2 cells were detected by Western blot assay. From 24 to 96 hours, lOOug/ml ubenimex inhibited the phosphorylation of p38 MAPK in NB4 cells in a time dependent manner. After treatment for 72 hours, lOOug/ml ubenimex inhibited the phosphorylation of p38 MAPK induced by lOnmol/L ATRA alone in NB4 cells. Ubenimex might enhance the inducing-differentiation activity of ATRA through inhibiting the phosphorylation of p38 MAPK induced by ATRA and weakening its negative regulatory effects. From 24 to 96 hours, lOOug/ml ubenimex also inhibited the phosphorylation of p38 MAPK in MR2 cells in a time dependent manner. However, the phosphorylation of p38 MAPK in MR2 cells appeared no changes after treatment by lOOnmol/L ATRA for 72 hours. So ubenimex could not potentiatethe effect of ATRA in MR2 cells through inhibiting the phosphorylation of p38 MAPK. 5ug/ml CD13 antibody WM-15 induced the phosphorylation of p38 MAPK in NB4 cells and partly blocked the inhibition of ubenimex on the phosphorylation of p38 MAPK in NB4 cells. 5ug/ml WM-15 had no effect on the NBT reduction activity in the NB4 cells and ATRA-treated NB4 cells. However, pre-incubation of 5ug/ml WM-15 blocked the enhancement of ubenimex on NBT reduction activity of NB4 cells induced by ATRA (12.8±5.2 vs. 40.3±6.3, p<0.0\). These results suggested that ubenimex might enhance the inducing-differentiation activity of ATRA through inhibiting the phosphorylation of p38 MAPK induced by ATRA via the cell surface CD 13 in NB4 cells. Repression of the proto-oncogene c-myc was required for terminal differentiation in many myelogenous leukemia cell lines induced by ATRA. In our study, lOOug/ml ubenimex cooperated with ATRA to significantly down-regulate c-myc mRNA expression in NB4 cells after 4 hours treatment (0.36±0.11 vs. 0.68±0.05, p<0.05). Various concentration (50ug/ml, 75ug/ml and lOOug/ml) of ubenimex synergized with lOnmol/L ATRA to decrease the expression of c-Myc protein, which was inversely correlated with NBT reduction activity of NB4 cells induced by lOnmol/L ATRA alone or with various concentration ubenimex (r=-0.940, p=0.0l7). Compared with the control group, the expressions of C/EBPe mRNA in the NB4 cells exposed to lOnmol/L ATRA alone or in combination with lOOug/ml ubenimex for 12 hours increased significantly (p<0.0\). But there was no difference between the two groups. After treatment of lOnmol/L ATRA for 72 hours, the expression of PML-RARa fusion protein was down-regulated and that of RARa protein had no changes in NB4 cells. lOnmol/L ATRA for 72 hours activated the extracellular signal-regulated kinase (ERK)i/2 MAPK and did not alter the total levels of ERK1/2. The expression of statl mRNA had no changes due to lOnmol/L ATRA for 72 hours. lOOug/ml ubenimex had no effect on the expression of PML-RARa fusion protein, RARa protein, statl mRNA,ERKi/2 protein and the phosphorylation of ERK1/2 protein in NB4 cells and ATRA-treated NB4 cells.In summary, some conclusion could be drawn from our results. 1. Ubenimex enhanced the inducing-differentiation of ATRA in NB4 cells in a dose and time dependent manner and significantly augmented ATRA induced NBT reduction activity of primary APL blasts. 2. Ubenimex inhibited the phosphorylation of p38 MAPK in NB4 cells and MR2 cells. 3. Ubenimex inhibited the phosphorylation of p38 MAPK induced by ATRA in NB4 cells. 4. CD 13 antibody WM-15 partly blocked the inhibition of ubenimex on the phosphorylation of p38 MAPK in NB4 cells. 5. CD 13 antibody WM-15 blocked the enhancement of ubenimex on NBT reduction activity of NB4 cells induced by ATRA. 6. Ubenimex synergized with ATRA to down-regulate the c-myc expression, and the levels of c-Myc protein correlated inversely with the NBT reduction activities of NB4 cells in ATRA alone or in combination with various concentrations ubenimex groups. 7. Ubenimex had no impact on the effect of ATRA on the expression of C/EBPe mRNA, statl mRNA, RARa protein, PML-RARa protein, ERK1/2 protein and the phosphorylation of ERK1/2 protein.
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