Study On The Effects And Mechanisms Of ATRA And Proteasome Inhibitors In Combination To Induce Differentiation And Apoptosis Of Acute Myeloid Leukemia Cells

IntroductionA characteristic abnormality of tumor cells is that they have been arrested at an immature or less differentiated state, lack the ability to control their own growth, which results in an abnormally fast proliferation rate. Differentiation therapy aims to force the malignant cells to resume the process of maturation, thus restraining their growth, invasion and metastasis. As the first example of clinically cyto-differentiating agent, all-trans retinoic acid (ATRA) triggers growth arrest and terminal differentiation of several human myeloid leukemia cell lines in vitro, and elicits complete remission of acute promyelocytic leukemia (APL) in 90% of patients. ATRA tend to have less toxicity than conventional cancer treatments. The striking clinical benefits of ATRA in treating APL gave rise to enthusiasm in clarifying the mechanisms of its action and a variety of ATRA targets have been reported, the mechanisms responsible for its beneficial effects remain unclear. Knowledge pertaining to this process may lead to more efficacious therapies for leukemia and other types of cancer.The ubiquitin-proteasome pathway (UPP) is an intracellular non-lysosomal protein-degradation system in most eukaryotic cells, thus the UPP plays an important role in various cellular processes including cell cycle progression, differentiation, signaling transduction, apoptosis and gene transcription. Previously, the proteasome has mainly been considered to be a promising target for apoptosis induction; however, the roles of ubiquitin-proteasome pathway in regulation of cell differentiation have received increased attention during recent years. The ubiquitin-proteasome pathway has been demonstrated to play important roles in regulating the differentiation of rat lens explants, neuronal cells and oligodendroglial cells. Whereas, the effects of proteasome inhibition on cell differentiation are different and mainly depend on cell types. Previous study has reported that genes related with UPP were significantly up-regulated in ATRA-treated NB4 cells. Numerous studies have demonstrated that RARαand its fusion protein PML-RARαare degraded by the ubiquitin-proteasome pathway, which might limit the full differentiation of myeloid cells in response to ATRA. Thus the ubiquitin-proteasome pathway might play important roles in ATRA-induced differentiation of leukemia cells, however, the mechanisms has not been elucidated yet.The aim of this study was to investigate the involvement of ubiquitin-proteasome pathway in ATRA-induced differentiation of leukemia cells as well as the effects of proteasome inhibitors on ATRA-induced cell differentiation. The current study include three sections:1) To demonstrate the changes of levels of UPP components in ATRA-induced differentiation of acute myeloid leukemia cells (AML), and then investigate the effect of proteasome inhibition on ATRA's biologic effects.2) To investigate the synergistic effect of proteasome inhibitors and ATRA on inducing the differentiation of acute myeloid leukemia cells in vitro and in vivo as well as its mechanisms.3) To investigate the effect of proteasome inhibitors and ATRA in combination to induce apoptosis of leukemia cells in vitro. Section 1 Evaluation of the involvement of ubiquitin-proteasome pathway in ATRA-induced differentiation of acute myeloid leukemia cellsObjective:The striking clinical benefits of ATRA in treating APL have attracted much attention in clarifying the mechanisms of its action; however, the mechanisms responsible for its beneficial effects remain unclear. This study was to investigate the activity of ubiquitin-proteasome pathway in ATRA-induced differentiation of AML and evaluate the influence of proteasome inhibition on ATRA's biological effect.Methods and results:ATRA-induced differentiation of HL60 and NB4 cells was coincident with a general elevation of ubiqui tin-protein conjugates, which were not the results of the malfunction of proteasome, because the activities of 20S proteasome were also enhanced by ATRA during this period. The proteasome activity might be elevated to satisfy the degradation of the increased ubiquitin-protein conjugates. In addition, the transcriptional level of ubiquitin was also augmented by ATRA, which might to meet the increased requirement for ubiquitylation of target proteins. These observations that the UPP components were up-regulated in parallel with ATRA-induced HL60 differentiation progression suggested the UPP might play an essential role in ATRA-induced differentiation, thus proteasome inhibitor MG132 was employed to determine this presumption. We found that inhibition of ATRA-elevated proteasome activity significantly enhanced the myeloid maturation program activated by ATRA, suggesting that ATRA induced proteasome activation was not beneficial for leukemia cell differentiation. We further determined that inhibition of ATRA-elevated proteasome activation was sufficient to suppress the degradation of RARa and PML-RARa, thus increase the differentiation of myeloid leukemia cells in response to ATRA. Moreover, we found that RARa overexpression mimics the enhancement of MG132 on ATRA-induced differentiation of HL60 cells. It not only decreased the concentration of ATRA required for differentiation induction, but also accelerated the differentiation progression, suggesting the cellular level of retinoic acid receptors contributed to the sensitivity of leukemia cells to ATRA.Conclusion:ATRA-induced differentiation of AML cells is accompanied with the activation of ubiquitin-proteasome pathway, however, the overactivation of the proteasome is not the cellular target of ATRA to induce cell differentiation; in contrast, it limits the sensitivity of myeloid leukemia to ATRA. Inhibition of the elevated proteasome activity, attenuating proteasome-dependent loss of RARa, synergistically enhances the differentiating effect of ATRA. Together, by linking the UPP to ATRA-dependent signaling, this study provide a novel sight in studying the mechanisms of ATRA-elicited cellular effects, and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy.Section 2 Study on the synergistic effect of proteasome inhibitors and ATRA in combination to induce the differentiation of acute myeloid leukemia cells in vitro and in vivo as well as its mechanismsObjective:Given that inhibition of the elevated proteasome activity could enhance the differentiating effect of ATRA, here we aim to evaluate the synergistic effect of proteasome inhibitors and ATRA in combination on inducing the differentiation of acute myeloid leukemia cells in vitro and in vivo as well as its mechanisms, which imply a possibility of the combination of ATRA and proteasome inhibitors in leukemia therapy.Methods and results:Proeasome inhibitors PS341, Lactacystin and LLnL significantly enhanced the growth-inhibitory and cyto-differentiation effects of ATRA on HL60 cells, as evidenced by the enhanced proliferation inhibition, nuclear segmentation, the ability of NBT reduction and expression of differentiation marker CDllb. The synergistic effect of PS341 and ATRA in combination to inhibit tumor growth via differentiation induction was also observed in nude mice implanted with human HL60 xenografts. Here we observed that the combination of PS341 and ATRA could attenuate proteasome-dependent loss of RARa proteins in HL60 cells and in tumor tissues. Then the accumulated RARa proteins enhanced the transcription of its target genes STAT1 and PU.1, thus leading to the enhanced differentiation of leukemia cells. PS341 also enhanced the expression of transcription factors C/EBPa and C/EBPs, which was induced by ATRA during granulocytic differentiation of myeloid leukemia cells.In addition, the down-regulation of c-Myc triggered by ATRA was further enhanced by PS341, subsequently followed with down-regulation of Cyclin E and up-regulation of p27, thus leading to the enhanced G0/G1 phase arrest.Conclusion:This study indicated the synergistic effect of proteasome inhibitors and ATRA on inducing the differentiation of acute myeloid leukemia cells in vitro and in vivo. We also elucidated the possible mechanisms of the combination of proteasome inhibitors and ATRA to enhance the differentiation of leukemia cells. Together, these findings build the rationale for further (pre)clinical development of proteasome inhibitors and ATRA in combination for differentiation therapy of AML.Section 3 Study on the effect of proteasome inhibitors and ATRA in combination to induce apoptosis of acute myeloid leukemia cellsObjective:ATRA-base chemotherapy has been considered to be efficient in AML, especially for APL. As the first proteasome inhibitor used in clinic, PS341 has exhibited a promising anti-cancer effect. We have demonstrated the effect of PS341 to enhance ATRA-induced differentiation of AML cells in quite a low concentration in Section 2. Here we aim to investigate the combination effect of PS341 and ATRA to induce apoptosis of leukemia cells and compare the anti-tumor effect of sequential versus the simultaneous ATRA plus PS341 schedule.Methods and results:We found that simultaneous treatment with ATRA and PS341 exerted synergistic anti-proliferative and apoptotic effect against HL60 cells in vitro. However, sequential treatment of ATRA followed by PS341 resulted in a decreased apoptosis of HL60 (vs. PS341 treatment alone). Comparing the proteasome activities of PS341 treated AML cells with and without ATRA pretreatment, we found that PS341 could effectively inhibited the proteasome activities of both cells; suggesting ATRA activated proteasome did not cause the resistance of AML cells to PS341. Bcl-2 family proteins Bcl-2, Bax and Mcl-1 were not involved in regulating the decreased anti-tumor effect of sequential ATRA plus PS341 schedule. Interestingly, we found that the protein levels of p-MEK, c-FLIP and Procaspase-8 were significantly up-regulated by ATRA treatment. Thus MEK inhibitor PD98059 was employed for further examination. Of note, we observed that inhibition of MEK phosphorylation could partially increase the sensitivity of ATRA-pretreated HL60 cells to PS341, which was accompanied with an inhibition of ATRA-induced up-regulation of c-FLIP and Procaspase-8. Taken together, the decreased anti-tumor effect of sequential ATRA plus PS341 schedule might be attributed to ATRA-induced MEK phosporylation followed with the up-regulation of c-FLIP and Procaspase-8 as well as the inhibition of Procaspase-3 activation.Conclusion:This study demonstrated the synergistic apoptotic effect of ATRA and PS341 against HL60 cells in vitro, which requires a simultaneous ATRA plus PS341 treatment. However, sequential treatment of ATRA followed by PS341 significantly decreased the apoptotic effect of PS341, which could be attributed to ATRA-induced MEK phosporylation followed with the up-regulation of c-FLIP and Procaspase-8 as well as the inhibition of Procaspase-3 activation. In all, our research suggests that ATRA and proteasome inhibitors should be given simultaneously when they are employed to treat leukemia through the apoptotic effect of PS341.