Liproxstatin­1 induces cell cycle arrest, apoptosis, and caspase­3/GSDME­dependent secondary pyroptosis in K562 cells.

Leukemia is a fatal hematopoietic disorder with a poor prognosis. Drug resistance is inevitable after the long­term use of chemotherapeutic agents. Liproxstatin­1, commonly known as a ferroptosis inhibitor, has never been reported to have anticancer effects. In the present study, the antileukemic role of liproxstatin­1 in K562 leukemia cells was investigated. Liproxstatin­1 inhibited K562 cell proliferation in a dose­ and time­dependent manner. RNA sequencing revealed several pathways that were affected by liproxstatin­1, such as the G1/S transition of the mitotic cell cycle and extrinsic or intrinsic apoptotic signaling pathways. The results of flow cytometry indicated that liproxstatin­1 arrests the cell cycle at the G1 phase, and even at the G2/M phase. p21WAF1/CIP1, a cyclin­dependent kinase inhibitor, was upregulated. It was also determined that liproxstatin­1 induced BAX and TNF­α expression, which was accompanied by cleavage of caspase­3 and PARP. The caspase­3­specific inhibitor z­DEVD­FMK rescued some of the apoptotic cells. Interestingly, K562 cells were characterized by swelling and plasma membrane rupture when treated with a high concentration of liproxstatin­1, which was inconsistent with the typical apoptotic appearance. Thus, it was hypothesized that apoptosis­mediated pyroptosis occurs during liproxstatin­1­induced cell death. The expression of the hallmark of pyroptosis, the cleaved N­terminal GSDME, increased. Additionally, it was observed that endoplasmic reticulum stress and autophagy were involved in liproxstatin­1­induced cell death. Collectively, liproxstatin­1 induced cell cycle arrest, apoptosis, and caspase­3/GSDME­dependent secondary pyroptosis in K562 leukemia cells, which provides new hope for the treatment of leukemia.

Dipetidyl peptidase-4 and transferrin receptor serve as prognostic biomarkers for acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the most common hematological malignancy in adult patients. Ferroptosis-related signatures have been shown to act as regulators of the progression of multiple cancer types, but the role of ferroptosis in AML remains to be elucidated. We performed the present study to preliminarily investigate the roles of ferroptosis-related genes (FRGs) in AML. METHODS: The transcriptome data of AML patients was downloaded from The Cancer Genome Atlas (TCGA) and the transcriptome data of normal samples was obtained from the Genotype-Tissue Expression (GTEx) database. FRGs were selected via public articles. Expression levels of FRGs between AML and normal samples were analyzed. The prognostic model based on FRGs was constructed via lasso regression. The expression levels and prognostic role of FRGs were identified from the risk model. We also performed validation experiments to verify the expression levels of the final selected genes via immunohistochemistry, polymerase chain reaction (PCR), and RNA-seq. Finally, we explored the associations between immune infiltration, drug sensitivity, and the selected FRGs. RESULTS: The transcriptome data of 151 AML samples were retrieved from TCGA and 70 bone marrow normal samples were retrieved from the GTEx database. Additionally, 23 FRGs were collected from the published articles. There were 22 differentially expressed FRGs, and among them, dipetidyl peptidase-4 (DPP4) (P= 0.011, HR =1.504), GPX4 (P=0.055, HR =1.569), LPCAT3 (P<0.001, HR =2.243), SLC7A11 (P=0.012, HR =2.243), and transferrin receptor (TFRC) (P=0.029, 0.774) had a significant influence on the prognosis of AML patients via lasso regression. The area under the curve (AUC) values of the 1-, 3-, and 5-year receiver operating characteristic (ROC) curves of the FRG signatures indicated that this model is novel and effective method for predicting the prognosis of AML patients. DPP4 (P<0.001) was overexpressed while LPCAT3 (P<0.001), TFRC (P<0.001), GPX4 (P<0.001), and SLC7A11 (P<0.001) were downregulated, further validation experiment results indicated that DPP4 was significantly downregulated but TFRC was upregulated in AML samples. Dysregulation of DPP4 and TFRC influence numbers of chemotherapy regimens sensitivity. CONCLUSIONS: DPP4 and TFRC act as biomarkers for predicting and diagnosing AML, and their expression levels also have significant correlations with drug resistance in AML.

Key immune-related gene ITGB2 as a prognostic signature for acute myeloid leukemia.

BACKGROUND: The tumor microenvironment (TME) has an essential role in tumorigenesis, progression, and therapeutic response in many cancers. Currently, the role of TME in acute myeloid leukemia (AML) is unclear. This study investigated the correlation between immune-related genes and prognosis in AML patients. METHODS: Transcriptome RNA-Seq data for 151 AML samples were downloaded from TCGA database (https://portal.gdc.cancer.gov/), and the immune related genes (irgs) were selected from Immport database. Bioinformatics screening was used to identify irgs for AML, and genes with a critical role in the prognosis of AML were selected for further analysis. To confirm the prognostic role of irgs in AML, we undertook protein-protein interaction (PPI) network analysis of the top 30 interacting genes. We then investigated associations between immune cell infiltration and prognosis in AML patients. Immunohistochemistry was used to validate protein expression levels between AML and normal bone marrow samples. Analysis of the drug sensitivity of the selected gene was then performed. RESULTS: The integrin lymphocyte function-associated antigen 1 (CD11A/CD18; ITGAL/ITGB2) was identified as the key immune-related gene that significantly influenced prognosis in AML patients. Overexpression of ITGB2 indicated poor prognosis in AML patients (P=0.007). Risk modeling indicated that a high-risk score led to poor outcomes (P=3.076e-08) in AML patients. The risk model showed accuracy for predicting prognosis in AML patients, with area under curve (AUC) at 1 year, 0.816; AUC at 3 years, 0.82; and AUC at 5 years, 0.875. In addition, we found that ITGB2 had a powerful influence on immune cell infiltration into AML TME. The results of immunohistochemistry showed that AML patients had significantly higher ITGB2 protein expression than normal samples. The AML patients were divided into 2 groups based on ITGB2 risk scores. Drug sensitivity test results indicated that the high-risk group was sensitive to cytarabine, axitinib, bosutinib, and docetaxel, but resistant to cisplatin and bortezomib. CONCLUSIONS: In the present study, we found that ITGB2 may be able to serve as a biomarker for assessing prognosis and drug sensitivity in AML patients.

Comprehensively analyze the expression and prognostic role for ten-eleven translocations (TETs) in acute myeloid leukemia.

BACKGROUND: The ten-eleven translocation (TET) family oxidize 5-methylcytosines (5mCs) and promote the locus-specific reversal of DNA. The role of TETs in acute myeloid leukemia (AML) is mostly unknown. METHODS: TETs mRNA expression levels were analyzed via Gene Expression Profiling Interactive Analysis (GEPIA). The association TETs expression levels and methylation with prognosis by UALCAN GenomicScape, and METHsurv. We analyzed TETs' aberration types, located mutations, and structures via cBioPortal. GeneMANIA performed the functional network. Gene ontology (GO) enrichment was analyzed via LinkedOmics. MiWalK identified miRNAs, miTarbase, and TargetScan. Transcription factor (TF) targets were analyzed via ChEA3. GSCAlite analyzed the role of these defined genes in cancer pathways and potential drug targets. Finally, we selected AML patients in our department to investigate the mutated types of TETs. RESULTS: TETs expression level results showed TET1 (P=0.003) and TET2 (P=0.004) overexpressed in Haferlach leukemia samples, TET3 (P=4.04e-8) downregulation in Andersson leukemia samples. TET2 and TET3 overexpression but TET1 downregulation in the GEPIA database. Overexpression of TET2 leads to positive outcomes (P=0.0091). The upregulation of TET2 led to poor survival for CN-AML patients, but downregulation of TET3 indicated a satisfactory prognosis. The hypermethylation of TETs like cg24705708 (P=0.036), cg05976228 (P=0.022), cg19127638 (P=0.022), cg15254238 (P=0.025), cg07669489 (P=0.037) indicate poor outcomes. Overexpression of GALNS (P=0.024) as an adverse biomarker, downregulation of E2F5 (P=0.037), MAP7 (P=0.019), and NRIP1 (P=0.0013) indicated good prognosis. Regulatory network analysis indicated TETs' functions, including covalent chromatin modification, histone modification, DNA methylation, or demethylation. Enrichment functions involving. TETs participate in several cancer pathways, including DNA repair response and receptor tyrosine kinase (RTK) signaling pathway. TETs are sensitive to belinostat, ceranib-2, docetaxel, tivantinib, and vincristine. CONCLUSION: Present study showed that TETs have different expressions in AML, and the expression levels of TETs lead to different outcomes of AML. The TETs cancer pathway analysis will also provide potential therapy methods for AML patients with TETs aberrations.

[Significance of Tissue Factor-Bearing Microparticle Procoagulation Activity and Antithrombin â ¢ Detection in Thalassemia Patients].

OBJECTIVE: To explore whether the high risk factors possibly leading to hypercoagulative status and thrombosis exist in Thalassemia patients of Guangxi region through detecting plasma tissne factor-bearing microparticles (TF+MP), procoagulatima activity, coagulation and anticoagulation function, fibrinolytic function, endothelial function and platelet count. METHODS: The TF+MP procoagulation activity was detected by chromogenic saubstract method, the levels of tissue factors (TF), tissue factor pathway inhibitor(TFPI), protein C (PC), protein S (PS), antithrombin Ⅲ(AT-Ⅲ), tissue plasminogen activator (tPA), thrombin-activated fibrinolysis inhibitor (TAFI), soluble E-selectin (sE-sel), intercellular adhesion molecule-1 (ICAM-1) and thrombomodulin (TM) were detected by ELISA in thalassemia group (n=71) and control group (n=20 heathy persons). RESULTS: Compared with control group, the AT-Ⅲ level decreased in ß-thalastemia major group (TM) (P<0.05), the AT-Ⅲ level in TM group independeutly posstiody correlated with plt count (r=0.37, P<0.05); the levels of TF and sICAM in α-thalassenia intermediate group (TA) significantly decteased (P<0.05), the procoagulatim activity of TF+MP in ß-thalassemia intermediate group (TI) increased sngnificantly (P<0.05)， moreover positively corretated with AT-Ⅲ level (r=0.77, P<0.05). The TF and sICAM-1 levels in normal liver functim group of Thalassemia patients were lower tham those in control group (P<0.01 and P<0.05, respectively), the TF+MP activity between normal and abnormal liver function was significantly different (P<0.05), while there were no significant difference in other correspoding indexes beween thalassemia group and control group as well as between each thalassemia groups. CONCLUSION: The damage of liver function and reduction of anticoagylation substances exist in patients with ß-thalassenia major in Guangxi region, the procoagulation activity of plasma TF+MP in patients with ß-thalassemia intermedia abnormally increases. All the above-mentioned factors may increase the risk of high coagulation status or thrombosis is thalassemia patients, the decrease of TF and SICAM-1 levels in patients with α-thalassemia intermedia may be factor against thrombosis.

[Clinical Significance of Detecting Tissue Factor and Tissue Factor Pathway Inhibitor in Thalassemia Patients].

OBJECTIVE: To investigate the changes of E-selectin, thrombin-antithrombin complex（TAT）, prothrombin fragment 1+2（F1+2）, tissue factor（TF）and tissue factor pathway inhibitor（TFPI）before and one year after splenectomy in thalassemia patients. METHODS: A total of 30 thalassemia patients undergoing electric laparoscopic splenectomy and 30 normal controls（NC） were enrolled in the study．Plasma levels of E-selectin, TAT, F1+2, TF and TFPI were detected by enzyme-linked immuno sorbent assay（ELISA）. RESULTS: One year after splenectomy，the plasma concentrations of E-selectin, TAT, F1+2, TF, TFPI were significantly higher than those in both preoperative and NC groups．Levels of E-selectin, TAT, F1+2 before splenectomy were significantly higher than those in NC groups. In addition, there was a positive correlation between plasma TF and TFPI level before and after splenectomy, and the levels of TF and TFPI positively correlated with TAT and F1+2, respectively. CONCLUSION: After splenectomy, the platelet count increases, the activity of endothelial cells is injured, the procoagulant factor increases, the blood is in procoagulant state, the TF/TFPI shows an importent role in the thrombosis of thalassemia patieints after splenectomy and may be used to evaluate the prothrombotic state of this diasease.

[Research Advance of Microparticles in Hypercoagulation of Hemolytic Anemia-Review].

Microparticles (MP) are small membrane vesicles released from many different cell types in response to cellular activation or apoptosis, which have the procoagulant effect. Hemolytic anemia(HA) is a type of anemia that have a short life expectancy of red blood cells due to the destruction which exceed the hematopoietic compensatory capacity of bone marrow. Sickle cell anemia(SCD), thalassemia and paroxysmal nocturnal hemoglobinuria(PNH) are all characterized by hypercoagulation and thromboembolism (TE). Research shows that MP can promote the formation of hypercoagulative state which in turn increases the risk of thromboembolism in HA. This review mainly summarized the advance research of MP in HA in the past 5 years. Moreover the relationship between the abnormal MP and hypercoagulation in HA, the impact of the related treatment to the MP, the research of MP in animal model of HA and the application of the MP-proteomics in HA are also disscussed.

[Expression of miR-550a-5p in Myelodysplastic Syndrome and Its Prediction of Target Genes].

OBJECTIVE: To investigate the expression of miR-550a-5p in bone marrow of patients with myelodysplastic syndrome (MDS), and to predict its target genes and function by bioinformatics analyses, so as to provide the evidence to furthre explore the role of miR-550a-5p and its target genes in pathogenesis of MDS. METHODS: Real-time PCR was used to detect the expression of miR-550a-5p in 54 MDS patients, 16 acute myeloid leukemia transformed from MDS (sfAML) and 19 healthy controls, and the correlation between the expression of miR-550a-5p and clinical pathologic characteristics of MDS, including chromosome, percentage of marrow blasts, absolute neutrophil count, platelet count and hemoglobin levels were analyzed. The sequence of miR-550 was searched in miRBase database. Target genes of miR-550a-5p were predicted by Microcosm,Miranda and Targetscan, and the predective results were collected, then the enrichment analyses of target gene function(GO) and signalling pathway(pathway of miR-550a-5p) were carried out by using gene ontology darabase and KEGG database. RESULTS: The expression of miR-550a-5p in bone marrow of all MDS patients was higher than that in controls: the expression level of miR-550a-5p in low risk MDS and middl risk 1 MDS was 1.7 times of controls (P=1.23×10-10); the expression of miR-550a-5p in midde risk 2 MDS and high risk MDS was 1.9 times of controls (P=1.20×10-10); the expression of miR-550a-5p in tAML was 2.0 times of controls (P=5.61×10-10). The miR-550a-5p expression level was up-regulated gradually with the enhancement of disease risk of MDS, but there was no correlation between the expression level of miR-550a-5p and clinical pathologic characteristics of MDS(chromosome: Normal: 1.11±0.19, Abnormal:1.26±0.15, P>0.05; Percentage of Marrow Blasts: r=0.29,P=0.07; absolute neutrophil count: r=-0.02,P=0.89; hemoglobin level: r=0.09,P=0.57; platelet count: r=0.25,P=0.08). The sequence of miR-550 was conservative among different species, and the prediced results indicated that there were 19 target genes in intersection. The functions of target genes were enriched in regulation of stress-activated cascade, MAPK pathway, regulation of muscle organ development, regulation of protein homodimerization activity and other biological processes; they participated in some molecular functions including enzyme activity, combination processes of some molecules as protein, cAMP and domain existed in cell junction, synapse, coated vesicle, dendrite and other cellular components. Two of them-PDLIM2 and PSME1 were selected which might play a role in pathologic mechanism of MDS regulated by miR-550a-5p. CONCLUSION: The expression of miR-550a-5p in bone marrow of MDS patients increases specifically, and miR-550a-5p may play a role in the pathogenesis of MDS through regulation of target genes, PDLIM2 and PSME1.

Analysis of the genetic variants associated with recurrent thromboembolism in a patient with hemoglobin H disease following splenectomy: A case report.

Reports of recurrent thromboembolism in thalassemia, particularly in hemoglobin H (HbH) disease associated with congenital thrombophilic mutations, are scarce. However, several mutations were detected in a 22-year-old woman with HbH disease. The patient experienced the first thrombotic event at the age of 20 years and had four recurrent thromboses in a short time interval, despite receiving anticoagulant treatment. The present study reports a case with six nucleotide substitutions, including a missense 565C>T (Arg189Trp) mutation and two synonymous mutations, 66T>C (Pro22Pro) and 423G>T (Ser141Ser), identified in the protein C gene. The other three mutations, 947G>A (Arg316His), 981A>G (Val327Val), and 775C>A (rs13146272), were identified in the protein S, antithrombin and cytochrome P450, family 4, subfamily V, polypeptide 2 genes, respectively. These findings suggest that if thrombotic events repeatedly occur in a patient with thalassemia, not only the risk factors associated with a hypercoagulable state, but the acquired and congenital thrombophilia should be screened for.

[Advances in Pathogenesis and Related Clinical Research of Thromboembolism in Patients with Thalassemia after Splenectomy].

Thalassemia is the most common human hereditary hemolytic anemia. Due to splenomegaly and hypersp-lenism, splenectomy can be used as a means of treatment for thalassemia. Various complications following splenectomy, however, especially thromboembolic complications are remarkable. This review summarizes the incidence, clinical manifestations and development time of thromboembolism. The pathogenesis of thromboembolism after splenectomy in thalassemia, such as abnormal platelet number and function, changes in red cell membrane, endothelial cell damage, dysfunction of other procoagulant and anticoagulant factors, and local factors associated with splenectomy are elaborated and the trategies to prevent and treat the thromboembolic events in thalassemia after splenectomy, including the attention to risk factors associated with splenectomy, a reassessment of splenectomy, regular blood transfusion to reduce the ratio of abnormal red blood cells, treatment with anticoagulant and antiplatelet drugs, application of hydroxyurea and stem cell transplantation are discussed.

Favorable prognosis of biallelic CEBPA gene mutations in acute myeloid leukemia patients: a meta-analysis.

OBJECTIVES: Increasing number of studies suggested that biallelic CEBPA (bi CEBPA) mutations were associated with favorable prognosis in patients with acute myeloid leukemia (AML), but the results remain inconclusive. We therefore present a meta-analysis to evaluate the prognostic value of bi CEBPA mutations in patients with AML. METHODS: A comprehensive literature search was undertaken through August 2014 looking for eligible studies. Pooled hazard ratios (HRs) estimates and 95% confidence intervals (95% CIs) in overall survival (OS) and event-free survival (EFS) were used to calculate estimated effect. RESULTS: Ten studies covering a total of 6219 subjects were included in this analysis. Overall, bi CEBPA mutations were associated with favorable clinical outcome in patients with AML (HR for EFS: 0.41, 95% CI: 0.32-0.52; for OS: 0.37, 95% CI: 0.27-0.50), in cytogenetically normal (CN)-AML (HR for EFS: 0.38, 95% CI: 0.29-0.49; for OS: 0.32, 95% CI: 0.23-0.43). When took the cohort of monoallelic CEBPA (mo CEBPA) mutated and wild-type CEBPA (wt CEBPA) AML as a reference group, bi CEBPA mutated AML also shown beneficial outcomes (HR for OS: 0.52, 95% CI: 0.37-0.72). No significant difference was found between mo CEBPA mutation and wt CEBPA in patients with AML or CN-AML (P > 0.05). CONCLUSION: Bi CEBPA mutations in patients with AML are strongly associated with a favorable prognosis, which suggested that bi CEBPA mutations would potentially serve as a novel prognostic marker in AML.