UNC13B Promote Arsenic Trioxide Resistance in Chronic Lymphoid Leukemia Through Mitochondria Quality Control.

In clinical practice, arsenic trioxide can be used to treat a subset of R/R CML patients, but resistance tends to reappear quickly. We designed an experiment to study arsenic trioxide resistance in K-562 cells. Previously, we identified the UNC13B gene as potentially responsible for arsenic trioxide resistance in K-562 cells via gene chip screening followed by high-content screening. We aimed to investigate the role and mechanism of the UNC13B gene in K-562 cells, an arsenic trioxide-resistant chronic myeloid leukemia cell line. In vitro lentiviral vector-mediated UNC13B siRNA transfection was performed on K-562 cells. The roles of UNC13B in cell proliferation, apoptosis and cell cycle pathways, and colony formation were analyzed by CCK-8 assay, fluorescence-activated cell sorting, and soft agar culture, respectively. Gene chip screening was used to define the possible downstream pathways of UNC13B. Western blot was performed to further validate the possible genes mediated by UNC13B for arsenic trioxide resistance in patients with chronic myeloid leukemia. UNC13B downregulation significantly inhibited growth, promoted apoptosis, decreased colony formation, reduced the duration of the G1 phase, and increased the duration of the S phase of K-562 cells. Western blot results confirmed that UNC13B may modulate the apoptosis and proliferation of arsenic trioxide-resistant chronic myeloid leukemia cells through the mediation of MAP3K7, CDK4, and PINK1. UNC13B is a potential therapeutic target for patients with arsenic trioxide-resistant chronic myeloid leukemia.

Prevention of acute graft-vs.-host disease by targeting glycolysis and mTOR pathways in activated T cells.

Graft-versus-host disease (GvHD) is a common life-threatening complication that can occur following allogeneic hematopoietic stem cell transplantation. This occurs if donor T cells recognize the host as foreign. During acute GvHD (aGVHD), activated T cells utilize glycolysis as the main source of energy generation. Therefore, inhibition of T cell glycolysis is a potential treatment strategy for aGVHD. In the present study, the effects of the combination of the glycolysis inhibitor 3-bromopyruvate (3-BrPA) and the mTOR inhibitor rapamycin (RAPA) on a mode of aGVHD were explored. In vitro mixed lymphocyte culture model was established by using splenocytes from C57BL/6 (H-2b) mice as responder and inactivated splenocytes from BALB/c (H-2d) mice as stimulator. In this model, 3-BrPA treatment (0-100 µmol/l) was found to suppress cell viability, increase cell apoptosis and reduce IFN-γ secretion, in a concentration-dependent manner. 3-BrPA treatment (0-100 µmol/l) was found to suppress cell viability, increase cell apoptosis and reduce IFN-γ secretion, in a concentration-dependent manner. In addition, combined treatment with 3-BrPA (0-100 µmol/l) alongside RAPA (20 µmol/l) exhibited synergistic effects on inhibiting cell viability and IFN-γ production, compared with those following either treatment alone. An aGVHD model was established by injection of bone marrow cells and spleen cells from the donor-C57BL/6(H-2b) mice to the receptor-BALB/c(H-2d) mice which were underwent total body irradiation first. In the aGVHD model, 3-BrPA (10 mg/kg/day), RAPA (2.5 and 5 mg/kg/day) and both in combination (5 and 2.5 mg/kg/day for 3-BrPA and RAPA, respectively) were all found to alleviate the damage caused by aGVHD, in addition to prolonging the survival time of mice with acute GvHD. In particular, the combined 3-BrPA and RAPA treatment resulted in the highest median survival time among all groups tested. In addition, the effects induced by combined 3-BrPA and RAPA treatment were found to be comparable to those in the 5 mg/kg/day RAPA group but superior to the 3-BrPA group with regards to the cumulative survival profile, GvHD score and lung histological score. The 3-BrPA and RAPA combination group also exhibited the lowest IFN-γ levels among all groups. Therefore, the combination of inhibiting both glycolysis and mTOR activity is a promising strategy for acute GvHD prevention.

Features and impacts on the prognosis of gene mutations in patients with acute myeloid leukemia.

To explore features and impacts on the prognosis of common gene mutations in acute myeloid leukemia (AML), we assessed mutation status as well as variant allele frequency (VAF) of 24 genes in 81 AML patients by next-generation sequencing (NGS) technology. Eighty-six percentages of patients showed at least one mutation. Mutation in BCOR was associated with lower complete remission (CR) rate, whereas double mutation in CEBPA was associated with a favorable odds ratio for CR achievement. TP53 mutation was associated with inferior overall survival (OS) in univariate analysis. Multivariate analysis confirmed the negative effect of adverse cytogenetic abnormalities on survival. Mutation in RUNX1 and ZRSR2 had negative impacts on OS in patients with wild-type TP53. VAF of SRSF2 mutation was observed negatively correlated with OS. In conclusion, our study suggested that mutations in BCOR and spliceosomes might predict worse outcomes, and VAF of gene mutations may play a crucial role in outcomes of AML patients.

Compound heterozygosity for two novel mutations of the SEC23B gene in congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDA II), a rare genetic disorder, results from SEC23B gene mutations according to previous studies. Here, we present a case of CDA II involving two novel pathogenic mutations of SEC23B that have not previously been reported. The patient suffered from jaundice, tea-colored urine, and weakness. Laboratory data indicated moderately decreased hemoglobin, iron overload, and abnormal erythroblast morphology. Therefore, a diagnosis of CDA II was considered. Peripheral blood samples were used to perform whole exome sequencing, and the results showed compound heterozygosity of the SEC23B gene with the following mutations: c.1162T>A (p.F388I) and c.1603delC (p.R535del). The mutant proteins were predicted to be deleterious and resulted in decreased structural stability. PyMOL software was used to analyze the structural change caused by the p.F388I missense mutation, and the results indicated a deficiency in π-π interactions. In conclusion, our report extends the mutation spectrum of SEC23B in the diagnosis of CDA II.

JMJD3-regulated expression of IL-6 is involved in the proliferation and chemosensitivity of acute myeloid leukemia cells.

Emerging evidence shows that histone modification and its related regulators are involved in the progression and chemoresistance of multiple tumors including acute myeloid leukemia cells (AML). Our present study found that the expression of histone lysine demethylase Jumonji domain containing-3 (JMJD3) was increased in AML cells as compared with that in human primary bone marrow (HPBM) cells. Knockdown of JMJD3 can decrease the proliferation of AML cells and increase the chemosensitivity of daunorubicin (DNR) and cytarabine (Ara-C). By screening the expression of cytokines involved in AML progression, we found that knockdown of JMJD3 can inhibit the expression of interleukin-6 (IL-6). Recombinant IL-6 (rIL-6) can attenuate si-JMJD3-suppressed proliferation of AML cells. Mechanistically, JMJD3 can positively regulate the promoter activity and transcription of IL-6 mRNA, while had no effect on its mRNA stability. Further, JMJD3 can regulate the expression of p65, which can directly bind with promoter of IL-6 to increase its transcription. Over expression of p65 significantly attenuated si-JMJD3-suppressed expression of IL-6. Collectively, we revealed that JMJD3 can regulate the proliferation and chemosensitivity of AML cells via upregulation of IL-6. It suggested that JMJD3 might be a potential therapy target for AML treatment.

[Synergistic Killing Effects of Arsenic Trioxide Combined with Itraconazole on KG1a Cells].

OBJECTIVE: To explore the effect of arsenic trioxide combined with itraconazole on proliferation and apoptosis of KG1a cells and its potential mechanism. METHODS: The cell morphology was observed with Wrighe-Giemsa staining; cell survival rate was examined by CCK-8; and colony formation capacity was measured by methylcellulose colony formation test; the flow cytometry was used to analyse the cell apoptosis rate and cell cycle; the protein expressions of BCL-2,caspase-3,BAX,SMO,Gli1 and Gli2 were detected by Western-blot. RESULTS: The arsenic trioxide and itraconazole alone both could inhibit the KG1a cell proliferation in dose-and time-dependent manner. In comparison between single and combined drug-treatment group, both the cell survival rate and the colony number of the single drug-treatment group were significantly lower(P<0.05), and the apoptosis rate was higher in the combined drug-treatment group. In the combined-treatment group, the protein expression of Caspase-3 and BAX was upregulated, while the protein expression of BCL-2,SMO,Gli1 and Gli2 was downregulated. CONCLUSION: Arsenic trioxide combined with itraconazole can inhibit the KG1a cell proliferation and induce apoptosis, which may be related with the inhibition of Hh signaling pathway and upregulation of both Caspase-3 and BAX protein expression, and provided experimental data of arsenic trioxide combined with itraconazole for the treatment of refractory AML.

[Establishment of a Xenografted Acute Myeloid Leukemia Model by using Zebrafish].

Objectve: To investigate the feasibility of establishing xenografted leukemia model by zebrafish, so as to provide the more direct model in vitro and experimental evidence for study of acute myeloid leukemia and screening of the drugs for targeting therapy. METHODS: Acute myeloid leukemia cell line KG-1a was labeled with red fluorescent dye-MitoRed, then the labeled cells were injected into the yolk sac of zebrafish embryos. Morphological observation, cell count and histopathological detection were used to analyse the infiltration and metastasis of KG-1a cells in zebrafish. RESULTS: KG1a cells could proliferate and gradually spread to the entire abdominal cavity of the zebrafish after KG-1a cells were injected into the yolk sac during 1-7, the results of cell counting in vitro also proved a significant proliferation of KG-1a cells in zebrafish, suggesting that the implanted leukemia stem cells could survive, proliferate and spread in zebrafish. Further study showed that the implanted cells could be transfered to the liver of zebrafish, these cells displayed the signature of KG-1a cells by hematoxylin-eosin(HE) staining. CONCLUSIONS: Human acute myeloid leukemia cells KG1a can survive, proliferate and migrate in zebrafish, suggesting xenografted leukemia model of zebrafish has been successfully established. This model may be benefitcial for the study of acute myeloid leukemia and the screening of the drugs for targeting therapy of acute myeloid leukemia.

Conditioning with Fludarabine-Busulfan versus Busulfan-Cyclophosphamide Is Associated with Lower aGVHD and Higher Survival but More Extensive and Long Standing Bone Marrow Damage.

Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a major cause of nonrelapse mortality after allo-HSCT. A conditioning regimen plays a pivotal role in the development of aGVHD. To provide a platform for studying aGVHD and evaluating the impact of different conditioning regimens, we established a murine aGVHD model that simulates the clinical situation and can be conditioned with Busulfan-Cyclophosphamide (Bu-Cy) and Fludarabine-Busulfan (Flu-Bu). In our study, BALB/c mice were conditioned with Bu-Cy or Flu-Bu and transplanted with 2 × 107 bone marrow cells and 2 × 107 splenocytes from either allogeneic (C57BL/6) or syngeneic (BALB/c) donors. The allogeneic recipients conditioned with Bu-Cy had shorter survivals (P < 0.05), more severe clinical manifestations, and higher hepatic and intestinal pathology scores, associated with increased INF-γ expression and diminished IL-4 expression in serum, compared to allogeneic recipients conditioned with Flu-Bu. Moreover, higher donor-derived T-cell infiltration and severely impaired B-cell development were seen in the bone marrow of mice, exhibiting aGVHD and conditioned with Flu-Bu. Our study showed that the conditioning regimen with Bu-Cy resulted in more severe aGVHD while the Flu-Bu regimen was associated with more extensive and long standing bone marrow damage.

[Establishment of a mouse model of acute graft-versus-host disease by busulfan combined with cyclophosphamide].

OBJECTIVE: To develop a stable mouse model of acute graft-versus-host disease (aGVHD) by preconditioning with busulfan (BS)-cyclophospha mide (CP). METHODS: Male BALB/c (H-2kd) as recipients were conditioned with 100 mg/kg BS and 200 mg/kg CP followed by being transplanted with bone marrow cells (2×10(7)) or bone marrow cells (2×10(7))-spleen cells (2×10(7)) from female C57BL/6(H-2Kb) as donors. The general data of the transplanted mice were recorded and the manifestations of aGVHD were evaluated. Histopathological changes were observed using HE staining and chimerism in spleen and bone marrow cells was studied using flow cytometry. RESULTS: Mice transplanted with bone marrow cells-spleen cells developed classic aGVHD manifestations starting from the 7th day after transplantation with full donor chimerism. GVHD histopathological changes were seen in liver, spleen, intestine, skin and lung of these mice. The median survival time of these aGVHD mice was 10 days, and they all died within 30 days. Mice transplanted with only bone marrow cells survived over 45 days without any aGVHD signs. CONCLUSION: We developed a stable and reliable aGVHD murine model successfully, which can be used to study the pathogenesis, prevention and therapeutic effect of aGVHD.

Low-dose arsenic trioxide combined with aclacinomycin A synergistically enhances the cytotoxic effect on human acute myelogenous leukemia cell lines by induction of apoptosis.

Acute myeloid leukemia (AML) is a common disorder in the elderly. Although remarkable progress has been made over recent decades, the outcome remains poor. Thus, the development of a more effective method to overcome this problem is necessary. In this study, we aimed to investigate the synergistic cytotoxic effect of low-dose arsenic trioxide (As2O3) combined with aclacinomycin A (ACM) on the human AML cell lines KG-1a and HL-60, and to clarify the underlying mechanism. Results showed that As2O3 combined with ACM exerted a synergistic cytotoxic effect by activation of the apoptosis pathway. Additionally, we found that the combination treatment decreased Bcl-2, c-IAP and XIAP expression but increased SMAC and caspase-3 expression more significantly than the single drug treatments. Furthermore, combination index (CI) values were < 1 in all matched combination groups. Additional evaluation of As2O3 combined with ACM as a potential therapeutic benefit for AML seems warranted.

[Honokiol combined with Gemcitabine synergistically inhibits the proliferation of human Burkitt lymphoma cells and induces their apoptosis].

This study was aimed to investigate the effect of Honokiol (HNK) combined with Gemcitabine (GEM) on the proliferation and apoptosis of human Burkitt lymphoma Raji cells. Cell proliferation was detected by CCK-8 method to study the role of Honokiol and Gemcitabine in Raji cells. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The level of apoptosis-related protein BCL-2 was measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (P < 0.01) and the inhibition rate was related to the concentration and action time of HNK; and apoptosis rate markedly increased (P < 0.01), while most Raji cells were arrested at G0/G1 phase and decreased in S phase after treatment with combination of two drugs; the expression of BCL-2 protein decreased (P < 0.01). It is concluded that Honokiol combined Gemcitabine can synergistically inhibit the proliferation, induce cell apoptosis, and down-regulate the expression of BCL-2 in Raji cells. The possible mechanism of synergistic effect may be related with arrest of cell cycle at G0/G1 phase and downregulation of the expression of BCL-2.

γ-secretase inhibitor-I enhances radiosensitivity of glioblastoma cell lines by depleting CD133+ tumor cells.

BACKGROUND AND AIMS: Glioblastoma is a deadly primary brain tumor with great resistance to radiotherapy. To reverse its radioresistance is important for improving prognosis. Gamma-secretase inhibitors (GSI) have been proven to have anti-tumor effects, yet the knowledge of their influences on glioblastomas is still limited. METHODS: The cytotoxic effects of GSI-I (a tripeptide GSI) on glioblastoma cell lines U87 and U251 were assessed by MTT assay, and the low concentration that did not induce significant cell death was determined. The in vitro radiosensitization effects of this low concentration of GSI-I were evaluated by cell colony forming assays. The CD133+ cell fractions before and after radiation with or without treatment of GSI-I were analyzed by flow cytometry. Then CD133+ and CD133- glioblastoma cells were sorted by magnetic activated cell sorting (MACS), and the radiosensitization effects of GSI-I on the two cell subtypes were investigated separately. Finally, the cytotoxic effects of GSI-I on CD133+and CD133- glioblastoma cells were examined, respectively, and the expression of the Notch pathway components between the two cell subtypes were compared. In addition, the anti-tumor effects of GSI-I were confirmed by in vivo experiments. RESULTS: GSI-I at a low concentration sensitized U87 and U251 cells to radiation by depletion of radioresistant CD133+ cells. CD133+ U87/U251 cells displayed preferential sensitivity to low concentrations of GSI-I, which may be related to the higher expression of the Notch signaling pathway in these cells. CONCLUSIONS: Combining GSI-I with radiotherapy may represent a promising strategy for treating radioresistant gliomas.