Transcriptome- and metabolome-based candidate mechanism of BCR-ABL-independent resistance to olverembatinib in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Olverembatinib represents the third-generation breakpoint cluster region protein-Abelson-murine leukemia 1 (BCR-ABL1) tyrosine kinase inhibitor with oral bioavailability, which can be used to overcome the T315I mutation in Philadelphia chromosome-positive (Ph +) leukemia. BCR-ABL-independent resistance to olverembatinib has been reported among patients in various clinical cases. However, the mechanism of olverembatinib resistance has rarely been reported. This study has illustrated bone marrow cell transcriptome and metabolome profiles among Ph + acute lymphoblastic leukemias (ALL) cases pre- and post-olverembatinib resistance. The transcriptome studies demonstrated that PI3K/AKT, purine metabolism, and other signaling pathways could play a vital role in olverembatinib resistance. As suggested by metabolomics, olverembatinib resistance in Ph + ALL was associated with purine metabolism alterations. Subsequently, high-performance liquid chromatography along with real-time quantitative PCR was utilized to measure purine metabolism-related mRNA levels and metabolism expression levels between olverembatinib resistance and sensitive cell lines. Our results elucidate the mechanism of olverembatinib resistance in Ph + ALL at transcriptome and metabolome levels, which facilitate a better understanding of olverembatinib resistance and hence may prove crucial in identifying novel drugs to tackle this conundrum.

Busulfan Plus Cyclophosphamide Versus Total Body Irradiation Plus Cyclophosphamide for Adults Acute B Lymphoblastic Leukemia: An Open-Label, Multicenter, Phase III Trial.

PURPOSE: It remains controversial whether busulfan-based versus total body irradiation (TBI)-based regimens have comparable outcomes in patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic hematopoietic stem-cell transplantation (allo-HSCT). We investigated the efficacy and toxicity of busulfan plus cyclophosphamide (BuCy) and TBI plus cyclophosphamide (TBI-Cy) conditioning in allo-HSCT for adult standard-risk B-cell-ALL in first complete remission (CR1). PATIENTS AND METHODS: We performed an open-label, randomized phase III trial at 13 hospitals in China. Eligible patients (age 14-65 years) had standard-risk ALL in CR1. Patients were randomly assigned (1:1) to BuCy (0.8 mg/kg four times per day on days -7 to -4 and cyclophosphamide 60 mg/kg once daily on days -3 to -2) or TBI-Cy (4.5 Gy TBI on days -5 to -4 and cyclophosphamide 60 mg/kg once daily on days -3 to -2). The primary end point was 2-year overall survival. Analysis was per protocol. This trial is registered with ClinicalTrials.gov (identifier: NCT02670252) and is complete. RESULTS: Between January 2016 and February 2020, 275 patients were assigned to receive BuCy (273 assessed) and 275 to TBI-Cy (272 assessed). The 2-year overall survival was 76.6% (95% CI, 71.7 to 81.8) and 79.4% (74.7 to 84.4; P = .457; difference 2.9%; 95% CI, -4.1 to 9.8; P = .022), indicating noninferiority of BuCy. The 2-year relapse was 20.2% (95% CI, 15.6 to 25.1) and 18.4% (14.0 to 23.2; P = .616), and the nonrelapse mortality was 11.0% (95% CI, 7.6 to 15.0) and 11.0% (7.7 to 15.1; P = .988) in the BuCy and TBI-Cy groups, respectively. There were no differences in regimen-related toxicity, graft-versus-host disease, or late effects between the two groups. CONCLUSION: The BuCy regimen has noninferior efficiency and safety as TBI-Cy (4.5 Gy × 2) for patients with adult standard-risk B cell-ALL in CR1 undergoing HLA-matched allo-HSCT.

Quercetin induces autophagy-associated death in HL-60 cells through CaMKKß/AMPK/mTOR signal pathway.

Acute myeloid leukemia (AML) is one of the most common malignancies of the hematopoietic progenitor cell in adults. Quercetin has gained recognition over the years because of its anti-cancer effect with minimal toxicity. Herein, we aim to investigate the anti-leukemia mechanism of quercetin and to decipher the signaling pathway of quercetin in HL-60 leukemic cells. We observed that quercetin induces apoptosis and autophagic cell death, in which both pathways play an important role in suppressing the viability of leukemia cells. Phosphorylated AMPK (p-AMPK) protein expressions are lower in primary AML cells, HL-60 cells, KG-1 and THP-1 cells than in peripheral blood monocular cells. After quercetin treatment, the expression of p-AMPK is increased while the expression of p-mTOR is decreased in a dose-dependent manner. Mechanistically, compound C, an AMPK phosphorylation inhibitor, upregulates the phosphorylation of mTOR and inhibits autophagy and apoptosis in quercetin-induced HL-60 cells, while silencing of CaMKKß inhibits the quercetin-induced phosphorylation of AMPK, resulting in increased mTOR phosphorylation. Furthermore, silencing of CaMKKß inhibits the autophagy in HL-60 cells. Taken together, our data delineate that quercetin plays its anti-leukemia role by inhibiting cell viability and inducing apoptosis and autophagy in leukemia cells. Quercetin inhibits the phosphorylation of mTOR by regulating the activity of AMPK, thus playing a role in the regulation of autophagy and apoptosis. CaMKKß is a potential upstream molecule for AMPK/mTOR signaling pathway, through which quercetin induces autophagy in HL-60 cells.

Lineage switch from lymphoma to myeloid neoplasms: First case series from a single institution.

Lymphoma relapse is very common in clinical work, but lineage switch at relapse is rare. Although some cases have reported acute lymphocytic leukemia (ALL) switch to acute myeloid leukemia (AML) or myeloid sarcoma upon relapse, phenotype switch seldom occurs in other types of lymphoma. Here we report six cases with lineage switch from lymphoma to myeloid neoplasms. In our cohort, three cases were mantle cell lymphoma (MCL), and the other three cases were T-cell lymphoblastic lymphoma (T-LBL), B-cell lymphoblastic lymphoma (B-LBL), and diffuse large B-cell lymphoma (DLBCL) at the initial diagnosis. When linage switch occurred, most cases were AML M5 phenotypes, and only one case was myelodysplastic syndrome (MDS) phenotype. 11q23/mixed-lineage leukemia (MLL) rearrangement was negative in all cases. Although intensive therapy and stem cell transplantation have been applied in most cases, the poor outcome cannot be reversed. Therefore, we found that lineage switch could occur not only from ALL to AML or vice versa, but also from MCL or DLBCL to AML. Moreover, the incidence of MLL rearrangement in lineage switch is lower in adult hematologic malignancies as compared with pediatric patients.

The short-term predictive value of CD4+ cells for combination therapy with high-dose dexamethasone and immunoglobulin in newly diagnosed primary immune thrombocytopenia patients.

INTRODUCTION: Dexamethasone (DXM) or immunoglobulin (IVIg) are first-line therapies for primary immune thrombocytopenia (ITP), with an effective rate of 80 %. Some patients with both severe bleeding symptoms and platelet counts of <30 × 109/L received a combination of DXM and IVIg. Autoimmune disorders, especially involving CD4+ T-cells, play a key role in the pathogenesis of ITP. We assumed that variations in the immune status of CD4+ T-cells will lead to different treatment responses. Until now, there have been few relevant clinical studies on CD4+ T-cells and the outcome of first-line therapies. METHODS: A prospective study enrolling 42 newly diagnosed ITP patients and 30 normal control volunteers was performed. The profiles of major CD4+ T-cells, including T helper (Th)1, Th2, Th17, and regulatory T (Treg) cells, and the related levels of interleukin (IL)-2, IL-17, and IL-23 were examined. The platelet number was recorded at the time point of day 0, day 14, and day 30. RESULTS: Greater concentrations of Th1 and Th17 cells and lower relative numbers of Treg cells were found in the ITP group. As for the treatment outcome on day 14, the profiles of Th2 and IL-2 were significantly greater in the NR group, while the expression of IL-17 was elevated in the CR group. As for the treatment outcome on day 30, higher levels of Th2 cells were observed in those patients who needed 2× pulses of HD DXM compared to those who needed only 1× pulse of HD DXM and IVIg, and receiver operating characteristic curve analysis showed that lower Treg cell may predict favorable values. Meanwhile, the higher IL-23 value may predict a poor early response. CONCLUSIONS: Our results indicate that Th1, Th17, and Treg cells and IL-2 and IL-23 participate in the onset of ITP. Higher profiles of Th2, IL-2 and IL-23 may predict poor treatment outcomes. Higher levels of IL-17 and lower profile of Treg may predict sensitivity to HD DXM and IVIg combination therapy.

Growth of T-cell lymphoma cells is inhibited by mPGES-1/PGE2 suppression via JAK/STAT, TGF-ß/Smad3 and PI3K/AKT signal pathways.

Background: T-cell lymphoma (TCL) has a very poor prognosis with limited treatment options and novel therapeutic target is urgently needed. Our previous studies have found that suppression of membrane-bound prostaglandin E2 synthase l/prostaglandin E2 (mPGES-1/PGE2) exerted anti-neoplastic effects in leukemia cells by suppressing AKT signal pathway. Here, we aim at evaluating the role and mechanism of mPGES-1/PGE2 signaling in TCL. Methods: Expression of mPGES-1 in TCL cell line Hut78 was analyzed by Western blot and immunofluorescence. CAY10526, a selective mPGES-1 inhibitor, was used to treat Hut 78 cells. Cell viability assays was performed by using cell counting kit-8 (CCK-8). Cell apoptosis rate was examined by flow cytometer. PGE2 synthesis was detected by enzyme immunoassay (EIA). The expression of mPGES-1, cleaved caspase-3, Janus kinase/signal transduction and transcription (JAK/STAT), transforming growth factor-ß (TGF-ß)/Smad3 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway of Hut 78 cells after exposed to CAY10526 was analyzed by Western blot. Results: mPGES-1 was highly expressed in Hut78 cell compared to normal peripheral blood mono-nuclear cells. CAY10526 inhibited cell proliferation and induced apoptosis in Hut78 cells. These effects may be partially attributed to the activation of the Caspase family and the inhibition of JAK/STAT, TGF-ß/Smad3 and PI3K/AKT signal pathways. Conclusions: Our results suggested that mPGES-1/PGE2 could be a potential therapeutic target for TCL.

Prognostic and therapeutic significance of XPO1 in T-cell lymphoma.

T-cell lymphoma (TCL) is a highly heterogeneous group of invasive non-Hodgkin lymphoma with adverse prognosis and limited treatment options. The relationship between TCL and Exportin-1 (XPO1), a major nuclear export receptor, has not been established yet. We here investigated the prognostic role and therapeutic implication of XPO1 in TCL. We analyzed XPO1 expression in a cohort of 69 TCL tumors and found that XPO1 was over-expressed in 76.8% of TCL and correlated with decreased progression-free survival (PFS) and overall survival (OS). In vitro treatment of TCL cell lines with KPT-8602, the second-generation selective inhibitor of nuclear export (SINE), inhibited XPO1 expression and showed significant anti-proliferative, cell-cycle arrest and pro-apoptotic efficacy. In mechanism, KPT-8602 restored the localization of cytoplasmic FOXO3A, p27, p21, IκBα and PP2A into the nucleus, leading to AKT and NF-κB deactivation. Our data demonstrate for the first time that XPO1 could be an unfavorable prognostic factor for TCL, and provide a rationale for further investigation of the efficacy of KPT-8602 in TCL patients.

Comparative efficacy and safety of eleven induction chemotherapy regimens for young adult patients with newly diagnosed acute myeloid leukemia: a network meta-analysis.

The optimal induction chemotherapy regimens for young adult patients with newly diagnosed acute myeloid leukemia (AML) are not well-defined since the lack of direct comparisons between emerging treatments. Network meta-analysis (NMA) is a statistical tool to integrate direct and indirect evidence to evaluate the effect of multiple interventions. Thus, we conducted an NMA to systematically assess the efficacy and safety of different inductions for these patients. PubMed, Embase, Cochrane Library, and Web of Science were searched from establishment to 2020-03-11. Randomized controlled trials (RCTs) using different inductions were included. We deemed 11 trials eligible, including 11 inductions with 5052 participants. Relative risk (RR) and 95% confidence intervals (CIs) were calculated. In terms of complete remission (CR) rate, DAC ranked highest and was significantly higher than IA (RR = 1.27, 95% CI (1.09-1.48)) and DA (RR = 1.28, 95% CI (1.13-1.46)) (p < 0.05). The ranking of DA + Pioglitazone was second only to that of DAC, followed by HAA. For early mortality, HAD, HAA, and DA + GO were significantly higher than DA/IA (p < 0.05). DAC and DA + Pioglitazone showed similar early mortality compared to DA/IA (p > 0.05). Regarding incidence of early grade 3-4 infection, no significant differences between interventions were observed. To conclude, among the included 11 induction regimens, DAC was potentially the top choice for young adult patients with newly diagnosed AML, with highest CR rate, low early mortality, and incidence of early infection. DA + Pioglitazone and HAA also showed a superiority over the others to achieve higher CR rate, while caution should be kept in mind due to the higher early mortality of HAA.

Clinical analysis of 11 cases of nocardiosis.

Nocardiosis is a rare, life-threatening, opportunistic, and suppurative infection. Its clinical manifestation lacks specificity, which makes early diagnosis difficult. A retrospective analysis of the clinical records of 11 patients with nocardiosis admitted to our hospital from January 2013 to November 2018 was conducted. All patients had at least one underlying disorder, such as an autoimmune disease (6/11), a blood malignancy (2/11), avascular necrosis of the femoral head (1/11), bronchiectasis (1/11), or pneumonia (1/11). The first-line treatment was trimethoprim-sulfamethoxazole (TMP-SMX); one or two additional antibiotics were given according to the drug-sensitive test. The median time from onset to treatment was 3 weeks (ranging from 1 to 9 weeks). The median duration of treatment after diagnosis was 20.5 weeks (ranging from 7 to 47 weeks). Eight patients were discharged and survived, and three patients died. This indicates that early use of TMP-SMX combined with sensitive antibiotics could improve the condition of patients and improve the cure rate (8/11). Clinically, it is necessary to consider the possibility of nocardiosis in patients with long-term use of immunosuppressants and poor response to treatment of common bacterial infections. Early diagnosis, timely treatment, and combination drug therapy are keys to improving the outcomes of patients with nocardiosis.

mPGES-1/PGE2 promotes the growth of T-ALL cells in vitro and in vivo by regulating the expression of MTDH via the EP3/cAMP/PKA/CREB pathway.

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy that is characterized by a high frequency of induction failure and by early relapse. Many studies have revealed that metadherin (MTDH) is highly expressed in a variety of malignant solid tumours and plays an important role in the occurrence and development of tumours. However, the relationship between the expression of MTDH and T-ALL has not yet been reported, and the regulatory factors of MTDH are still unknown. Our previous studies found that mPGES-1/PGE2 was important for promoting the growth of leukaemia cells. In the present study, we found that MTDH was highly expressed in primary T-ALL cells and in the Jurkat cell line. Our results showed that mPGES-1/PGE2 regulates the expression of MTDH through the EP3/cAMP/PKA-CREB pathway in T-ALL cells. Downregulation of MTDH inhibits the growth of Jurkat cells in vitro and in vivo. Our results suggest that MTDH could be a potential target for the treatment of T-ALL.

Regulation of mPGES-1 composition and cell growth via the MAPK signaling pathway in jurkat cells.

Previous studies have suggested that microsomal prostaglandin E synthase-1 (mPGES-1) is highly expressed and closely associated with mitogen-activated protein kinase (MAPK) signaling pathways in various types of malignant cells. However, their expression patterns and function with respect to T-cell acute lymphoblastic leukemia (T-ALL) remain largely unknown. The present study investigated whether mPGES-1 served a crucial role in T-ALL and aimed to identify interactions between mPGES-1 and the MAPK signaling pathway in T-ALL. The results indicated that mPGES-1 overexpression in T-ALL jurkat cells was significantly decreased by RNA silencing. Decreasing mPGES-1 on a consistent basis may inhibit cell proliferation, induce apoptosis and arrest the cell cycle in T-ALL jurkat cells. Microarray and western blot analyses revealed that c-Jun N-terminal kinase served a role in the mPGES-1/prostaglandin E2/EP4/MAPK positive feedback loops. In addition, P38 and extracellular signal-regulated kinase 1/2 exhibited negative feedback effects on mPGES-1. In conclusion, the results suggested that cross-talk between mPGES-1 and the MAPK signaling pathway was very complex. Therefore, the combined regulation of mPGES-1 and the MAPK signaling pathway may be developed into a new candidate therapy for T-ALL in the future.

Cyanidin-3-o-ß-Glucoside Induces Megakaryocyte Apoptosis via PI3K/Akt- and MAPKs-Mediated Inhibition of NF-κB Signalling.

Apoptotic-like phase is an essential step in thrombopoiesis from megakaryocytes. Anthocyanins are natural flavonoid pigments that possess a wide range of biological activities, including protection against cardiovascular diseases and induction of tumour cell apoptosis. We investigated the effects and underlying mechanisms of cyanidin-3-o-ß-glucoside (Cy-3-g, the major bioactive compound in anthocyanins) on the apoptosis of human primary megakaryocytes and Meg-01 cell line in vitro. We found that Cy-3-g dose-dependently increased the dissipation of the mitochondrial membrane potential, caspase-9 and caspase-3 activity in megakaryocytes from patients with newly diagnosed acute myeloid leukaemia but not in those from healthy volunteers. In Meg-01 cells, Cy-3-g regulated the distribution of Bak, Bax and Bcl-xL proteins in the mitochondria and cytosol, subsequently increasing cytochrome c release and stimulating caspase-9 and caspase-3 activation and phosphatidylserine exposure. However, Cy-3-g did not exert significant effects on factor-associated suicide (Fas), Fas ligand, caspase-8 or Bid expression. Cy-3-g inhibited nuclear factor kappa B (NF-κB) p65 activation by down-regulating inhibitor of NF-κB kinase (IKK)α and IKKß expression, followed by the inhibition of inhibitor of NF-κB (IκB)α phosphorylation and degradation and subsequent inhibition of the translocation of the p65 sub-unit into the nucleus, and finally stimulating caspase-3 activation and phosphatidylserine exposure. The inhibitory effect of Cy-3-g on NF-κB activation was mediated by the activation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinase (MAPK) and the inhibition of phosphoinositide 3-kinase (PI3K)/Akt signalling. U0126 (Erk1/2 inhibitor), SB203580 (p38 MAPK inhibitor) and 740 Y-P (PI3K agonist) significantly reversed Cy-3-g-reduced phosphorylation of p65. Taken together, our data indicate that Cy-3-g induces megakaryocyte apoptosis via the inhibition of NF-κB signalling, which may play important roles in regulating thrombopoiesis.

KPT-330 inhibition of chromosome region maintenance 1 is cytotoxic and sensitizes chronic myeloid leukemia to Imatinib.

As tyrosine kinase inhibitors (e.g., Imatinib, IM) fail to induce long-term response in some chronic myeloid leukemia (CML), novel therapies targeting leukemia-dysregulated pathways are necessary. Nuclear-cytoplasmic trafficking of proteins play a key role in the development of leukemia and drug resistance. KPT-330 (Selinexor), an inhibitor of chromosome region maintenance 1 (CRM1, nuclear receptor exportin 1, XPO1), demonstrated activities against a few hematological malignancies. We examined the anti-leukemic efficacy of KPT-330 in IM-resistant CML. Cell viability was examined by MTS assay. Apoptosis and cell cycle were assessed by flow cytometry. CRM1 mRNA was detected by PCR. Expression of CRM1 protein and its cargo proteins were determined by western blot or immunofluorescent staining. Furthermore, we engrafted nude mice subcutaneously with IM-resistant CML K562G. Mice were treated with IM, KPT-330 alone or in combination. Expression of CRM1 in CML were markedly higher than control. KPT-330 inhibited proliferation, induced cell cycle arrest and apoptosis of K562 and K562G. IC50 of IM on K562G was reduced by KPT-330. Mechanistically, KPT-330 inhibited CRM1 and increased the nuclear/cytoplasm ratio of BCR-ABL and P27. p-AKT was downregulated while p-STAT1 and caspase-3 were upregulated. Furthermore, KPT-330 showed anti-leukemic effect in primary IM-resistant CML with T315I mutation in CRM1-dependent manner. In K562G xenograft mice model, KPT-330 inhibited tumor growth and sensitized K562G to IM in vivo. To conclude, KPT-330 showed anti-leukemic activity and sensitized CML to IM in CRM1-dependent manner in vitro and in vivo. KPT-330 represents an alternative therapy for IM-refractory CML, warranting further investigation of CRM1 as therapeutic target.

[Expression of HMGB1 in Spleen of Adult Patients with Chronic and Refractory Immune Thrombocytopenia and Its Significance].

OBJECTIVE: To investigate the expression and clinical significance of high mobility group box 1(HMGB1) in spleen of adult patients with chronic and refractory immune thrombocytopenia(ITP). METHODS: Twenty chronic and refactory ITP patients received splenectomy were enrolled in ITP group and 20 cases of traumatic spleen rupture were enrolled in control group. The splenectomy efficacy in ITP patients was analyzed retrospectively. The HMGB1 expression in spleen tissue was detected by immunohistochemistry, and the correlation between different expression levels of HMGB1 and splenectomy efficacy were analysed. Meanwhile, the protein expression levels of HMGB1 in peripheral blood serum and mononuclear cells(PBMNC) of 25 patients with chronic and refractory ITP were detected by ELISA and Western blot. RESULTS: The median platelet count before splenectomy was 7.5 (0-20) ×109/L; all the patients showed that the initial response to splenectomy within the first month after operation was 100%, the median time of response was 1 day (1-6 days). The median peak platelet count post splenectomy was 448.5 (161-1272)×109/L. In the median time of 10(3-30) months, the platelets count in 8 patients was reduced to varying degrees. After a median follow-up of 69.5 months (22-195), complete response was found in 12 patients, 4 cases showed response and 4 did not. The HMGB1 expression positive rate in spleen of ITP patients was significantly higher than that in control group (85.0% vs 15.0%)(P<0.001). There were a negative correlation between the HMGB1 expression in ITP and therapeutic outcome after splenectomy (r=-0.791, P<0.01). In addition, HMGB1 expression levels in serum and PBMNC of the patients with chronic refractory ITP were also significantly higher than that in healthy controls (P<0.01). CONCLUSION: The splenectomy has been found to be effective therapeutic method for patients with ITP, the HMGB1 highly express in the spleen of the patients with chronic refractory ITP, but negatively correlats with the therapeutic outcome after splenectomy.

Assessment of Th17/Treg cells and Th cytokines in an improved immune thrombocytopenia mouse model.

OBJECTIVES: The improved passive immune thrombocytopenia (ITP) mouse model has been extensively utilized for the study of ITP. However, how closely this model matches the human inflammation state and immune background is unclear. Our study aimed to explore the profile of Th cytokines and Th17/Treg cells in the model. METHODS: We induced the ITP mouse model by dose-escalation injection of MWReg30. The serum levels of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17A, and TGF-ß1) were measured by enzyme-linked immunosorbent assay and the frequency of Th17 and Treg cells was measured by flow cytometry. The mRNA expression of Foxp3 and RORrt was measured by real-time PCR. RESULTS: The serum levels of cytokines IFN-γ, TGF-ß1, IL-4, and IL-10 were significantly lower in ITP mice. The secretion of serum proinflammatory cytokines IL-2 and IL-17A and the percentage of Th17 cells showed no statistically significant increase. In ITP mice the frequency of Treg cells and mRNA expression of Foxp3 was significantly lower in splenocytes. CONCLUSION: Our data suggest that the improved passive ITP mouse model does not mimic the autoimmune inflammatory process of human ITP. Compared with human ITP, this model has a similar change in frequency of Treg cells, which may directly or indirectly result from antibody-mediated platelet destruction due to attenuated release of TGF-ß.

[Effects of shRNA Targeting mPGES-1 on Tumorigenicity of K562 Cells in Nude Mice In Vivo].

OBJECTIVE: To investigate the effects of shRNA targeting mPGES-1 on tumorigenicity of human acute leukemia K562 cells in nude mice in vivo and its mechanisms. METHODS: For experiment 3 groups including KD group(expression of mPGES-1 in K562 cells was down-regulated by shRNA), CON (cells without any treatment) and NC group (cells treated with nonspecific-sequence shRNA) were set-up. Western blot was used to test the expression of ß-catenin and cyclinD1 in cells. Then the cells of 3 groups were implanted into BALB/c nude mice subcutaneously to establish murine xenograft model. The growth state of the mice and the size of the xenograft tumor were recorded. HE staining was used to observe the morphology of xenograft tumor. Expressions of ß-catenin and cyclinD1 in xenograft tumor were detected by immunohistochemical staining. RESULTS: In vitro the expression of ß-catenin and cyclinD1 in KD group were lower than the CON group and NC group (P<0.05). In vivo the tumor volume and weight of KD group were significant smaller than the other two groups (P<0.01). HE staining showed that tissues in the KD group were relatively looser in arrangement with smaller cell nucleus and less cytoplasm. The expression of ß-catenin and cyclinD1 in the KD group were remarkable weak as compared with that in CON group and NC group (P<0.05). CONCLUSION: Down-regulating the expression of mPGES-1 by shRNA may significantly inhibit the tumorigenicity of K562 cells in nude mice in vivo and its mechanism may be related with the inhibition of expression of ß-catenin and cyclinD1.

Strains of Group B streptococci from septic patients induce platelet activation via Toll-like Receptor 2.

Group B Streptococcus (GBS) causes life-threatening bacterial sepsis, especially in newborns and pregnant women. Patients suffering from sepsis often display low platelet counts, characterized by thrombocytopenia, because of platelet activation. In the present study, the roles of six GBS strains from septic patients in platelet aggregation, as well as the underlying mechanisms, were investigated. Incubation of platelets with three of the strains induced platelet aggregation, increased the secretion of cellular adhesin molecule CD62P and activation of GPIIb/IIIa. Furthermore, the GBS strains that induced platelet activation also caused an increase in the expression of Toll-like receptor (TLR) 2 in platelets. Pre-incubation of platelets with anti-TLR2 monoclonal antibody, but not anti-TLR4 monoclonal antibody, inhibited these functional responses induced by GBS. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signalling pathway in platelets, and inhibition of PI3-K significantly reduced GBS-induced platelet responses. Our results indicate that three of the GBS strains from the septic patients can trigger platelet activation by interacting with platelets, which involves the elevation of platelet TLR2 expression.

[Role of Treg Cells in Pathogensis of Mouse ITP].

OBJECTIVE: To explore the role of Treg cells in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). METHODS: The ITP mouse model was established, the Treg cell ratio in peripheral blood and spleen was detected by flow cytometry, the CD4+ CD25+ T cells were sorted by immunomagnetic beads, the Treg cell associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-ß) in CD4+ CD25+ T cells were enriched from spleen mononuclear cells, and the mRNA expression of Treg cell was measured by real-time PCR. RESULTS: The ratio of Tregs in peripheral blood and spleen decreased significantly in ITP mouse, as compared with the controls (P<0.01). In addition, the mRNA expression of IL-10, TGF-ß and Foxp3 decreased significantly in spleen CD4+ CD25+ T cells (P<0.05). Expression of Smad 7 mRNA was higher than that of controls. CONCLUSION: The alteration in Treg frequency and function may be responsible for the immune dysfunction in ITP disease. It is also speculated that the lower mRNA expression of Foxp3 and higher mRNA expression of Smad 7 may inhibit the proliferation and differentiation of Treg cells.

The role of AMP-activated protein kinase in quercetin-induced apoptosis of HL-60 cells.

Our previous studies have shown that quercetin inhibits Cox-2 and Bcl-2 expressions, and induces human leukemia HL-60 cell apoptosis. In order to investigate the role of AMP-activated protein kinase (AMPK) on quercetin-induced apoptosis of HL-60 cells, we used flow cytometry to detect cell apoptosis. The expressions of LKB1, phosphorylated AMPK (p-AMPK), and Cox-2 protein were detected in HL-60 cells and normal peripheral blood mononuclear cells (PBMCs) by western blot. The expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin. The expressions of p-AMPK were detected in HL-60 cells after culture with AMPK inhibitor Compound C. Then, the expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin alone or quercetin + Compound C. It was found that there was no significant difference in LKB1 between PBMCs and HL-60. p-AMPK in PBMCs was higher than that in HL-60, while Cox-2 was lower. After culture of HL-60 with quercetin, p-AMPK was increased, Cox-2 was decreased, but LKB1 remained unchanged. After culture of HL-60 with Compound C, p-AMPK was decreased. There was no significant difference in LKB1 between the quercetin-alone and the quercetin + Compound C groups. p-AMPK decreased more significantly, while Cox-2 increased more significantly in the quercetin + Compound C groups than those in the quercetin-alone groups. Taken together, these findings suggested that quercetin activates AMPK expression in HL-60 cells independent of LKB1 activation, inhibits Cox-2 expression by activating AMPK, and further regulates the Bcl-2-dependent pathways of apoptosis to exert its anti-leukemia effect.