Study On The Effect And Mechanism Of Bortezomib Induced Differentiation Of Acult Myeloid Leukemia Cells

Objective:The proteasome inhibitor Bortezomib (Velcade, formerly PS-341) is known to be effective in therapy of various neoplasms and is approved by the FDA for use in multiple myeloma. Clinical trials in acute myeloid leukemia (AML) patients given bortezomib also have revealed a definite clinical activity. Although all of the mechanisms by which this novel drug acts as an antineoplastic agent are not fully understood. All-trans-retinoic acid (ATRA) represents the sole example of clinically useful cytodifferentiating agent. However the therapeutic efficacy of this compound is still burdened by problems such as toxicity and resistance. One possible strategy to increase the therapeutic index of ATRA is the development of ATRA-based pharmacologic combinations that are more powerful and easily tolerated than the individual components. The aim of this study was to investigate the function and mechanisms of Bortezomib-induced differentiation as well as the synergistic activity of Bortezomib on ATRA-induced differentiation of AML cells.Methods:1) Primary patient AML blasts isolated from bone marrow by LSM. For proliferation assay, human myeloid leukemia cells HL60 and U937 were treated with Bortezomib, cell growth curve was performed by counting cells each day. Giemsa staining was performed to evaluate morphologic changes. NBT-reducing activity as a potent differentiation marker of leukemia cells was used and the blue cells were counted by light microscopy for at least 200 cells. Cell-surface markers (CD11b and CD14) were detected by flow cytometry. The expressions of MAPK related proteins (p-MEK, p-ERK, MEK, p-JNK, JNK, p-p38, p38) and transcription factors (STAT1, p-STAT1, C/EBPa) were shown by Western blotting. The transcription level of STAT1 was determined by realtime PCR. Lentivirus-mediated shRNA-STAT1 transfected into HL60 cells to support the concept that the induced differentiation effect of bortezomib is related to the up-regulation of STAT1. Inhibitors (PD98059, SP600125, SB203580 and CHX) were used to further explore the involvement of MAPK pathway and STAT1 in Bortezomib-induced differentiation. The ratio of apoptosis was assessed by annexinⅤ-fluorescein and propidium iodide staining, analyzed by Flow Cytometry. cos7 cells were transiently co-tranfected, with RARE-pGL4-LUC reporter plasmids and with the Renilla-encoding pRL-TK plastmid. Luciferase antivity quantified using the Dual Luciferase Reporter kit.2) MTT method and CCK8 assay were used in proliferation assay to evaluate the inhibition effect of ATRA and Bortezomib on HL60 and NB4 cells or primary patient AML blasts. Cell growth curve was performed by counting cells each day after staining with trypan blue. Giemsa staining was performed to evaluate morphologic changes. NBT-reducing activity was used and the blue cells were counted by light microscopy for at least 200 cells. Cell-surface markers CD11b was detected by flow cytometry.20S proteasome activity was determined by using fluorogenic peptide substrate Suc-LLVY-AMC. The mRNA level of RARαand STAT1 were shown by realtime PCR. Immunofluorescence analyse was used in the detection of STAT1 and p65 distribution. CD11b expression was examined in HL60 cells transfected with lentivirus-mediated shRNA-STAT1. All the proteins expression and activation were measured by western blotting. In vivo antiproliferation of ATRA and Bortezomib was evaluated using human tumor models xenografted HL60 in nude mice, the weight of tumor were measured and the inhibitory rate were calculated. C/EBPεprotein expression were confirmed by immunohistochemistry in HL60 tumors. The radio of apoptosis was assessed by TUNEL staining in HL60 tumors. Results:Part 1 Involvement of MAP kinase in STAT1-mediated differentiation induced by Bortezomib in AML cells.1) Bortezomib inhibits growth and induces cytodifferentiation in AML cell lines and primary patient AML blasts.It was observed that Bortezomib induces morphologic, biochemical and functional changes indicative of macrophage differentiation in HL60 and U937 cells. By the third day, a significant increase in cell growth inhibiton induced by Bortezomib was evident at 5 nM (44.6% of vehicle-treated cells in HL60 cells and 60.6% in U937 cells. HL60, U937 cells and primary patient cells treated with Bortezomib for 3 days underwent striking changes consistent with myeloid maturation with condensation and lobulation of the nucleus, which was assessed by Giemsa staining. Similar results were observed on primary patient cells. Besides, time and dose-dependent effect of Bortezomib were showed on monocytic maturation associated myeloid cell surface markers and NBT reduction assay.2) Bortezomib activates the MEK/ERK cascade, which further up-regulates the expression and activity of the key myeloid transcription factor STAT1, thus promoting other events in myeloid differentiation.MAPK pathways, including MEK/ERK, JNKp46, and p38, were activated during Bortezomib-induced myeloid differentiation of both HL60 cells and U937 cells in a time-dependent manner. PD98059, a specific MEK inhibitor, or SP600125, a specific INK inhibitor was employed to examine the role of MEK signaling in Bortezomib-induced differentiation. Both inhibitors completely suppressed both CD 14 and CDllb expression induced by Bortezomib, in condition of suppression of MEK, ERK or JNKp46 activation. Western blotting results indicated that MEK/ERK cascade activation may be crucial for the induction of the cytodifferentiation effect by the Bortezomib, however it was conjectured that JNK may participate in the process as a downstream kinase. However, activation of p38 might play a negative role in the process of monocytic differentiation of AML cells induced by Bortezomib.Treatment of HL60 cells with Bortezomib led to a significant increase in the amounts of STAT1. as well as the level of phosphorylation of Tyr701. Realtime PCR and the employment of CHX proved that increase of STAT1 accumulation in HL60 cells treated with Bortezomib was caused by the combined effects of its synthesis and degradation inhibition. In addition, an increased level of C/EBPa, a downstream factor of STAT1, was also observed with treatment of Bortezomib. HL60 cells were transduced with lenti viral-vector, expressing shRNA against STAT1 or control vector using established method. The level of STAT1 protein was decreased in cells transfected with STAT1-shRNA, resulting in inhibition of bortezomib-dependent differentiation.We further found that the activation of STAT1 might be mediated by MEK/ERK-JNK, as pre-treatment with PD98059 or SP600125 attenuated the increased level of STAT1 and its phosphorylation form in Bortezomib-treated HL60 cells, which was in consistence with block of differentiation.3) Bortezomib induced differentiation is independent on RARa.An increased level of RARa was observed without the transcription activity. And HL60R went into myeloid maturation with the treatment of Bortezomib.4) Bortezomib-induced differentiation and apoptosis are two independent processes.A time-dependent study showed that Bortezomib at 5 nM induced apoptosis in HL60 or U937 cells. And differentiation and apoptosis induced by Bortezomib seemed to be independent responses, as the rate of apoptosis cells was not declined when Bortezomib-induced differentiation was suppressed.Part 2 Bortezomib enhances the cytodifferentiating properties of ATRA via stabilization of RARa1) Bortezomib enhances terminal differentiation of ATRA-treated AML cells.Bortezomib enhanced the growth-inhibitory and cyto-differentiation effects of ATRA on HL60 and NB4 cells, which were based on inhibition of proliferation, morphologic assessment, differentiation marker CDllb expression, and nitro-blue tetrazolium reduction.Bortezomib enhanced the apoptogenic and cyto-differentiation effect of ATRA on primary patient cells. As for the three leukemic samples tested, one patient sample was derived from a patient with AML-M3 with relapsed leukemia, another one was from a patient with AML/APL, and the third one was from a patient with AML-resistant. Bortezomib and ATRA combination caused a significant decrease in cell viability relative to what is bserved in cultures treated with on drug alone. This was associated with a stronger induction of NBT-reducing activity in combination than in ATRA-differentiation cells.2) The combination of Bortezomib and ATRA inhibits tumor growth via differentiation induction in nude mice implanted with human HL60 xenograftsThe combination therapy of ATRA (5 mg/kg) and Bortezomib (0.1 mg/kg) notably inhibited the tumor growth of the HL60 xenografts, with T/C value 45% and inhibition rate 58.9%, which was prominently stronger than ATRA-administrated group (T/C value:58%, inhibition rate:40.0%), and Bortezomib-treated group (T/C value:62%, inhibition rate:20.0%). The tumor volume of combination group was remarkably decreased from that of vehicle group, and the mono-treated groups. Combination group exerted significantly more potent activities on induction of CD11b expression comparing with the mono-treated groups. And similar observation was obtained on C/EBPεby immunohistochemistry, while no apoptosis was detected after TUNEL staining.3) Mechanism of the enhanced differentiation effect of ATRA in presence of Bortezomib in AML cells.Upregulation of proteasome activity by ATRA was inhibited in presence of Bortezomib. Bortezomib suppressed ATRA-induced UPP-dependent degradation of RARαin HL60 and NB4 cells, but enhanced the transcription of ATRA-responsive genes, represented by STAT1. Western blotting showed the synergistic effect of this combination on the HL60 xenografts might be related to RARαsatabilization.Bortezomib enhanced the ATRA-dependent induction of STAT1, and also in the transcriptional active state, as indicated by the level of phosphorylation of Tyr701 and nuclear translocation. Downregulation of STAT1 expression partially suppressed the potentiates differentiation effect of Bortezomib in HL60 cells transfected with lentivirus-mediated shRNA-STAT1.The progress that Bortezomib synergized ATRA-induced differentiation was NFκB independent, as well as MAPK pathways.Conclusion:In this study, we first report the unexpected pharmacologic effects of Bortezomib in acute myelogenous leukemia cells, which is that Bortezomib induces myeloid differentiation and apoptosis, and also enhaces the cyto-differentiation, and antiproliferative activity of ATRA. These results indicate a potential mechanism of action whereby Bortezomib warrants clinical efficacy for patients with AML and may provide some hints toward developing new treatment approaches for human myeloid leukemia.