| Background:Acute promyelocytic leukemia(APL) is a relatively rare disease, which is a special type of acute myeloid leukemia(AML). APL is one of the most clearly known malignant tumors at present, and at the same time it is also the only malignancy that can be cured by targeted therapy. There have been some patients with APL cured by Anthracyclines in the 1970 s, but treatment with Anthracyclines is easy to cause DNA damage. The introduction of all-trans retinoic acid(ATRA), since 1985, has improved therapeutic effects of these patients with APL, and the prognosis of patients with APL has come to a revolutionary progress. Furthermore, in Clinical trials, under treatment with ATRA and other drugs, more than 90 percent of patients out of treatment have a disease-free survival date for more than five years. ATRA can induce APL cells to rapidly differentiate into granular cells in vitro and in vivo, and patients can gain remission with the treatment of ATRA within only three to five weeks. The therapeutic mechanism of ATRA has been a hot research topic, and has not been completely illuminated. In recent years, NEDD8, one of the ubiquitin-like proteins, has been attracting increasing attention, and NEDD8 is a kind of small molecule protein which contains only 81 amino acids. The process of covalent bonding between NEDD8 and its corresponding substrates is called Neddylation. NEDD8 activating enzyme E1 is a heterodimer formed by UBA3 and APPBP1, and UBA3 is the catalytic subunit.Thus UBA3 is crucially important for the normal Neddylation process. And cullins are the classic substrates to NEDD8, and cullins mainly regulate the cell cycle and cell apoptosis. E3 ligase-catalyzed Neddylation of cullins can enhance the activity of the corresponding enzyme complex, and Rbx1(RING box protein 1) is the E3 ligase. Previous studies have shown that the occurrence and development of various tumors may be attributed to dysfunctional Neddylation system. MLN4924, the specific inhibitor of the NEDD8 activating enzyme E1, has been shown to have a good threpeutic effect in many solid tumors and leukemia, and has been under phase II clinical trials. Therefore, treatment targeting the Neddylation system is expected to bring a new dawn for cancer treatment. There have been studies indicating that Neddylation promotes the survival and proliferation of AML cells, but it is not clear whether Neddylation has an effect on the differentiation of APL cells. And what is the role of Neddylation in the therapeutic effects of ATRA on the APL patients? In order to solve the above scientific questions, we have carried out this study.Objective:To explore the mechanism of Neddylation involved in the treatment of ATRA in APL cells; To investigate the regulatory effect of ATRA on the expression of UBA3 in APL cells and the underlying molecular mechanism; To explore the role of UBA3 in the malignant growth of APL cells and the underlying molecular mechanism; To exploe new therapeutic targets for APL patients.Method:To study the effect of ATRA on the expression of UBA3 in APL cells, two APL cell lines, HL60 and NB4, were used and were treated with ATRA. Then Neddylation-related protein levels(Neddylated cullins, UBA3 and APPBP1) were assessed by Western Blot(WB) using antibodies against NEDD8, UBA3, and APPBP1, respectively. And UBA3 gene expression was detected by QPCR. In order to study the molecular mechanism by which ATRA regulates the expression of UBA3 in APL cells, NB4 and HL60 cells were treated with proteasome inhibitor MG132 and lysosomal inhibitor E64d/pepstatinA. Then the expression of UBA3 and Neddylated cullins were detected by WB. In order to verify that ATRA can induce autophagy, HL60 and NB4 cells were treated with ATRA. Then autophagy index LC3 BII was assessed by WB with anti-LC3 B antibody. And HL60 and NB4 cells were treated with autophagy inducer rapamycin, then the expression of LC3 BII,UBA3 and Neddylated cullins was assessed by WB. In order to study the role of Neddylation system in the malignant growth of APL cells and the underlying mechanisms, HL60 and NB4 cells were treated with MLN4924 or shRNA against UBA3 to inhibit the global Neddylation. Then the effects of Neddylation on the malignant growth of APL cells were detected by growth curve analysis and soft agar assays. To explore the mechanisms of Neddylation on the malignant growth of APL cells, apoptosis detection and cell cycle analysis were perforemed. To explore the effect of Neddylation on the undifferentiation status of APL cells, Giemsa staining and flow cytometry testing surface Marker CD11 b were performed. In order to analyze the role of the classical Neddylation substrates-cullins, HL60 cells were transduced with lentivirus carrying Rbx1 shRNA for silencing of endogenous Rbx1 to specifically inhibit cullins Neddylation. Then the changes about HL60 cell growth curve, apoptosis, cell cycle progression, cellular morphology and CD11 b expression were measured as described previously.Results:1) ATRA inhibits the expression of UBA3After treating HL60 and NB4 cells respectively with ATRA for 0d?1d?2d?3d?4d?5d, WB showed that the expression of UBA3 decreased gradually. However, QPCR assay showed that the mRNA level of UBA3 was gradually increased under the same conditions. Thus the protein level of UBA3 is opposite to that of the mRNA level after ATRA treatment, suggesting that the decrease of UBA3 expression may be due to the decrease of protein stability.2) ATRA inhibits the expression of UBA3 by inducing autophagy, and thereby inhibits Neddylation.Proteasome inhibitor MG132 could not reverse the decrease of UBA3 expression induced by ATRA, suggesting that the decline in the amount of UBA3 induced by ATRA is not through the proteasome pathway.The lysosomal inhibitor E64 d and pepstatinA could well reverse the decline in the amount of UBA3 induced by ATRA in HL60 cells and there was also partial reversal in NB4 cells, indicating that the decline in the amount of UBA3 induced by ATRA is through the lysosomal pathway. After treating HL60 and NB4 cells with ATRA, WB showed that autophagy index LC3 BII increased, which verifies that ATRA could induce autophagy. After treating HL60 and NB4 cells with rapamycin, WB showed that UBA3 and Neddylated cullins both decreased, suggesting that ATRA can enhance the activity of lysomal enzymes by inducing autophagy, which leads to the reduction of UBA3 expression in APL cells.3) UBA3 and Neddylation mediated by UBA3 promote cell growth, survival and cell cycle progression of APL cells, and can also maintain the undifferentiation status of APL cells.After treating APL cells with MLN4924 or knocking down UBA3 to inhibit Neddylation, we carried out the following analysis: ? Growth curve analysis showed that MLN4924 treatment or knockdown of UBA3 could both significantly inhibit the growth of HL60 and NB4 cells. Soft agar assays showed that MLN4924 significantly inhibited the colony forming ability of HL60 and NB4 cells; ? Apoptosis assay and cell cycle analysis showed that MLN4924 treatment or knockdown of UBA3 both significantly promoted the apoptosis of HL60 and NB4 cells, and also induced cell cycle arrest; ? Giemsa staining showed that MLN4924 treatment or knockdown of UBA3 increased the ration of cytoplasmic/nuclear area, increased the number of cytoplasmic vacuoles in HL60 and NB4 cells; Analysis of surface Marker CD11 b showed that MLN4924 treatment or knockdown of UBA3 both upregulated CD11 b expression in HL60 and NB4 cells. Therefore, UBA3 and Neddylation mediated by UBA3 promote cell growth, survival and cell cycle progression of APL cells, and can also maintain the undifferentiation status of APL cells.4) UBA3 and Neddylation mediated by UBA3 promote the malignant growth of APL cells via classical substrates-cullins.After knocking down Rbx1 in HL60 cells, growth curve analysis showed that the growth of HL60 cells was significantly inhibited; Apoptosis detection showed that the apoptosis of HL60 cells increased; Cell cycle progression analysis showed that the cell cycle arrest at the G0/G1 phase; Giemsa staining showed that morphological changes(the ration of cytoplasmic/nuclear area increased, and the number of cytoplasmic vacuoles increased) were similar to global Neddylation inhibition; Analysis of surface Marker CD11 b showed that the expression of CD11 b significantly increased in HL60 cells. Therefore, knocking down Rbx1 to weaken the cullins Neddylation has a similar effect to the suppression of the overall Neddylation within APL cells, suggesting that Neddylation promotes the malignant growth of APL cells by classical substrates-cullins.Conclusion:ATRA inhibits the expression of UBA3 by inducing autophagy and thereby inhibits Neddylation. UBA3 and Neddylation mediated by UBA3 promote cell growth, survival and cell cycle progression of APL cells, and can also maintain the undifferentiation status of APL cells. The inhibitory effect of ATRA on Neddylation is an important mechanism for the significant therapeutic effect of ATRA in APL patients. Knocking down Rbx1 to weaken the cullins Neddylation has a similar effect to the inhibition of UBA3 in APL cells, suggesting that Neddylation promotes the malignant growth of APL cells by the classical substrates-cullins.The creative points of this paper:1. We discover that ATRA inhibits the expression of UBA32. We discover that the inhibitory effect of ATRA on Neddylation is an important mechanism for the significant therapeutic effect of ATRA in APL patients3. We discover that Rbx1 may be a new target for the treatment of APL... |
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