ITRAQ-based proteomics analysis of human ectopic endometrial stromal cells treated by Maqian essential oil.

BACKGROUND: Endometriosis is a common and complex syndrome characterized by the presence of endometrial-like tissue outside the uterus. Chinese medicine has been recently found to show good efficacy in treating endometriosis. Our previous results revealed that Maqian fruit essential oil (MQEO) could inhibit the proliferation and induce apoptosis of ectopic endometrial stromal cells (EESCs), but the mechanisms remain unclear. In this study, we aim to explore the molecular mechanism of MQEO's specific effects in EESCs. METHODS: We conducted a quantitative proteomics analysis by iTRAQ on EESCs treated with MQEO or DMSO. Then deep analysis was performed based on differentially expressed proteins, including Gene Ontology enrichment analysis, pathway enrichment analysis and protein interaction analysis. Candidate protein targets were subsequently verified by western blotting. RESULTS: Among 6575 identified proteins, 435 proteins exhibited altered expression levels in MQEO-treated EESCs. Of these proteins, most were distributed in signal transduction as well as immune system and the most significantly altered pathway was complement and coagulation cascades. Moreover, two differentially expressed proteins (Heme oxygenase 1 and Acyl-CoA 6-desaturase) were verified and they can be potential biomarkers for endometriosis treatment. CONCLUSIONS: Our proteomic analysis revealed distinct protein expression patterns induced by MQEO treatment in EESCs, highlighting the potential of MQEO for endometriosis treatment and biomarker discovery.

Expression of the promyelocytic leukemia protein without the nuclear localization signal as a novel diagnostic marker for acute promyelocytic leukemia.

Promyelocytic leukemia-retinoic acid receptor α (PML-RARα) is a fusion protein generated by the t(15;17)(q22;q12) translocation associated with acute promyelocytic leukemia (APL). PML-RARα is cleaved by neutrophil elastase, an early myeloid-specific serine protease, leading to translocation of the nuclear localization signal (NLS) of the PML protein to the N-terminal of RARα, and the mutational product PML(NLS-). The present study was designed to analyze the role of the NLS in mediating PML transport into the nucleus and to evaluate the value of measuring NLS translocation in the early diagnosis of APL. PML and PML(NLS-) localization was examined by immunofluorescence (IF). The interaction between PML/PML(NLS-) and importin α was detected by an in vivo binding assay using co-immunoprecipitation and double IF labeling. Twenty-seven untreated APL patients with PML-RARα and 22 non-APL controls were evaluated. PML(NLS-) was detected in primary APL, but not non-APL cells. IF showed that PML was localized to the nucleus, interacted with importin α in vivo, and co-localized in the PML nuclear bodies. PML(NLS-) was primarily localized in the cytoplasm and the interaction with importin α was lost. IF had a sensitivity and specificity of 92.6 and 77.3%, respectively, for diagnosing APL. These data suggest that PML(NLS-) may be a novel diagnostic biomarker for APL.

PML(NLS¯) protein: A novel marker for the early diagnosis of acute promyelocytic leukemia.

Promyelocyte leukemia­retinoic acid receptor α (PML­RARα) is known as a fusion gene of acute promyelocytic leukemia (APL). Previous studies have reported that neutrophil elastase (NE) cleaves PML­RARα in early myeloid cells, which leads to the removal of the nuclear localization signal (NLS) in PML and increases the incidence of APL. The resultant PML without the NLS is termed PML(NLS­). The aim of the present study was to verify the existence and location of the PML(NLS­) protein in NB4 cells. NB4 cells underwent electroporation with the pCMV­HA­NE plasmid to form NB4­HA­NE cells, which were then transplanted to produce tumors in nude mice and samples were collected from patients with APL. Western blot analysis, an immunofluorescence assay, confocal laser microscopy and immunohistochemistry were performed to detect the expression and localization of the PML(NLS­) protein. The findings demonstrated that PML(NLS­) was detectable in the cytoplasm of NB4­HA­NE cells, the tumors in nude mice and in neutrophils from patients with APL. This indicated that PML(NLS­) may be an effective and novel target for the diagnosis of APL.

Location of NLS-RARα protein in NB4 cell and nude mice.

In the majority of acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RAα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. NLS-RARα promotes cell growth and inhibits differentiation in response to ATRA. However, the mechanisms by which NLS-RARα affects cell biological characteristics are yet to be fully elucidated. The present study found that the location of RARαwas altered after it was cleaved by NE. Firstly, NE was overexpressed during the preparation of recombinant plasmid NB-4/pCMV6-NE-Myc to cleave PML-RARα. The total protein expression levels of myc and NE and expression levels of NLS-RARα in nucleoprotein were detected by western blotting. Location of NLS-RARα protein was detected by immunofluorescence and confocal laser scanning. Secondly, a nude mice model was constructed and NE protein, NLS-RARα and RARα protein assays, and the location of NLS-RARα and RARα proteins were assessed as described. The present results showed that, compared with the control groups, the location of NLS-RARα protein was predominantly detected in the nucleus, whereas RARα was mainly distributed in the cytoplasm. These findings were consistent with those of the nude mice model, and these may be used as a foundation to explain the occurrence mechanism of APL.

NLS-RARα is a novel transcriptional factor.

Acute promyelocytic leukemia (APL) is characterized by the presence of the promyelocytic leukemia (PML)-retinoic acid receptor-α (RAR-α) fusion protein. PML-RARα can be cleaved by neutrophil elastase (NE) in several positions in cells in the promyelocytic stage, nuclear location signal (NLS)-negative PML and NLS-RARα may be the products of PML-RARα by NE. The function of NLS-RARα may be affected by the addition of NLS, which would alter its localization in cells, as the role of NLS is to identify proteins for transport to the nucleus. Preliminary experiments demonstrated that the overexpression of NLS-RARα in HL-60 cells could promote cellular proliferation and inhibit cellular differentiation. Following treatment with all-trans retinoic acid (ATRA), the degree of cellular differentiation was enhanced. In the present study, the localization of NLS-RARα was identified and its activity as a novel transcriptional factor was assessed, which may be critical in the development of APL. The location of NLS-RARα was detected in the nucleus and cytoplasm by indirect immunofluorescence and western blot analysis, with expression in the nucleus revealed to be increased compared with that in the cytoplasm. Next, native-PAGE was performed and NLS-RARα and RXRα were revealed to form heterodimers in the nucleus. In addition, co-immunoprecipitation revealed an interaction between NLS-RARα and retinoid X receptor-α (RXRα). An electrophoresis mobility shift assay (EMSA) indicated that NLS-RARα could bind retinoic acid response elements (RAREs) in the presence of ATRA. Indeed, NLS-RARα could bind RAREs just as WTRARα could, including the RAREs direct repeat-2 (DR-2) and DR-5. In addition, results from a luciferase reporter gene assay demonstrated that NLS-RARα could mediate the activity of RAREs that it bound. Together, these results indicated that NLS-RARα may be a novel transcription factor that contributes to leukemogenesis by competitively binding RAREs as heterodimers with RXRα, just as PML-RARα does, thus repressing the gene transcription essential for myeloid differentiation. These findings indicate the potential role of NLS-RARα targeted therapy in APL.

Neutrophil elastase and its therapeutic effect on leukemia cells.

Neutrophil elastase (NE) is an early myeloid-specific serine protease, which is predominantly produced by promyelocytes. A previous study demonstrated that NE has an important role in the development of acute promyelocytic leukemia (APL). The process of APL was shown to be accelerated in animals that expressed abundant NE, whereas NE­deficient mice were protected from APL development; thus suggesting an important role for NE in the development of APL. The present study aimed to investigate the effects and possible mechanisms of NE. Up- and downregulation of NE in various leukemia cell lines was conducted in order to explore its significance in the occurrence and procession of leukemia, with the aim of identifying novel targeted therapeutic drugs for the treatment of leukemia. NE was overexpressed in cells following infection with an adenovirus, and Cell Counting kit­8 and flow cytometry results demonstrated that cell proliferation was promoted, and cell apoptosis was inhibited, as compared with the untreated cells. NE was downregulated in the cells by both RNA interference and treatment with GW311616A, a specific inhibitor of NE, following which cell growth was shown to be inhibited and apoptosis was induced. These results suggested that NE may promote the development of APL, therefore, NE may be a therapeutic target and its inhibitor GW311616A may be a potential therapeutic drug for leukemia. Furthermore, the apoptosis­associated protein B­cell lymphoma 2 (Bcl­2)­associated X protein was significantly increased, whereas Bcl­2 was markedly decreased in the cells with downregulated NE. Further experiments revealed that the probable apoptosis­associated signaling pathway was the phosphoinositide 3­kinase/AKT pathway. The present study is the first, to the best of our knowledge, to demonstrate that GW311616A, a specific NE inhibitor, may act as a potential targeted drug for leukemia, which may have a profound impact on the future of leukemia-targeted therapy.

[Neutrophil elastase inhibitor on proliferation and apoptosis of U937 cells].

OBJECTIVE: To study and compare the effect of neutrophil elastase inhibitors (GW311616A and sivelestat) on the proliferation and apoptosis of U937 cells. METHODS: Inhibitory effects of GW311616A and sivelestat on the proliferation of U937 cells were assayed by MTT assay. The morphologic changes of U937 cells were detected by transmission electron microscope, and apoptosis was observed by AnnexinV-FITC/PI staining. The changes of cell cycle and apoptosis were detected by flow cytometry. The expression of NE in U937 cells was observed by indirect immunofluorescence, the variations of content and activity of NE in U937 cells were measured through ELISA assay and colorimetric method. RESULTS: MTT showed that both NE inhibitors could inhibit the proliferation of U937 cells in a dose dependent manner. The IC50 of GW311616A and sivelestat were 150 and 214 µmol/L respectively. The inhibition effect of GW311616A was significantly higher than of sivelestat (P<0.01). Typical apoptosis morphological changes of U937 cells was observed through electron microscope. AnnexinV-FITC/PI staining showed that U937 cells could be induced to undergo apoptosis by the two inhibitors, the apoptosis ratio of 150µmol/L GW311616A group (13.60%) was significantly higher than that of 150µmol/L sivelestat group (3.69%)(P<0.01). The result of flow cytometry indicated that the apoptosis ratio of 150 µmol/L GW311616A group was 14.61%, U937 cell cycle was mainly blocked in G2/M phase; meanwhile 150 µmol/L sivelestat group as 4.25% with cell cycle in S phase. The fluorescence intensity of GW311616A group obviously decreased than of sivelestat group. And the two inhibitors could reduce the content and activity of NE in U937 cells, but the effect of GW311616A was significantly higher than of sivelestat (P<0.01). CONCLUSION: GW311616A and sivelestat could inhibit the proliferation and cause apoptosis of U937 cells. Furthermore, GW311616A was more effective and harmful to cells than sivelestat.

Cloning and characterization of a novel gene SRG-L expressed in late stages of mouse spermatogenic cells.

A cDNA expressed specifically in late stages of mouse spermatogenic cells during spermatogenesis was cloned by using overlapping RT-PCR and RACE. The cDNA contained an open reading frame (ORF) of 2625 base pairs that encoded an 874 amino acids protein. Comparison analysis of amino acid sequence showed 91% and 80% identity to a rat homologue XP-226242 and a monkey homologue BAB63115 respectively. The expression of the mRNA was only observed in pachytene spermatocytes, round, and elongating spermatids. We named this gene as SRG-L (spermatogenesis related gene expressed in the late stages of spermatogenic cells, GenBank accession No. AY352586). The tissue-specific analysis showed that the SRG-L was highly expressed in spleen and testis. The results suggested that SRG-L might play special roles during spermatogenesis, particularly related to meiosis and spermiogenesis. Analysis of the amino acid sequence showed there was a coiled-coil region near the N-terminal region and rich phosphorylation sites, suggesting SRG-L might function as a transmembrane protein mediating signal transduction. This study also demonstrated that gene cloning by RT-PCR was applicable and convenient when its homologous gene was known.