Shenghui Zhang

Matrine Induces Apoptosis in Human Acute Myeloid Leukemia Cells via the Mitochondrial Pathway and Akt Inactivation

Acute myeloid leukemia (AML) is a hematological malignancy characterized by a rapid increase in the number of immature myeloid cells in bone marrow. Despite recent advances in the treatment, AML remains an incurable disease. Matrine, a major component extracted from Sophora flavescens Ait, has been demonstrated to exert anticancer effects on various cancer cell lines. However, the effects of matrine on AML remain largely unknown. Here we investigated its anticancer effects and underlying mechanisms on human AML cells in vitro and in vivo. The results showed that matrine inhibited cell viability and induced cell apoptosis in AML cell lines as well as primary AML cells from patients with AML in a dose- and time-dependent manner. Matrine induced apoptosis by collapsing the mitochondrial membrane potential, inducing cytochrome c release from mitochondria, reducing the ratio of Bcl-2/Bax, increasing activation of caspase-3, and decreasing the levels of p-Akt and p-ERK1/2. The apoptotic effects of matrine on AML cells were partially blocked by a caspase-3 inhibitor Z-DEVD-FMK and a PI3K/Akt activator IGF-1, respectively. Matrine potently inhibited in vivo tumor growth following subcutaneous inoculation of HL-60 cells in SCID mice. These findings indicate that matrine can inhibit cell proliferation and induce apoptosis of AML cells and may be a novel effective candidate as chemotherapeutic agent against AML.

Nucleotide-Binding Oligomerization Domain 2 Receptor Is Expressed in Platelets and Enhances Platelet Activation and Thrombosis

Background— Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results— Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1&bgr; maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions— NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored.

Th17 cells and interleukin-17 increase with poor prognosis in patients with acute myeloid leukemia.

Although Th17 cells play crucial roles in the pathogenesis of many autoimmune and inflammatory disorders, their roles in malignancies are currently under debate. The role and mechanism of Th17 cells in patients with acute myeloid leukemia (AML) remain poorly understood. Here we demonstrated that the frequency of Th17 cells was significantly increased in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells from AML patients compared with healthy donors. Plasma levels of interleukin (IL)‐17, IL‐22, IL‐23, IL‐1β, IL‐6, and transforming growth factor (TGF)‐β1 were significantly increased in blood and bone marrow in AML patients compared with healthy donors. The in vitro experiments demonstrated that IL‐1β, IL‐6, IL‐23, but not TGF‐β1 promoted the generation and differentiation of Th17 cells from naive CD4+ T cells in humans. IL‐17A, a signature cytokine secreted by Th17 cells, induced the proliferation of IL‐17 receptor (IL‐17R)‐positive AML cells via IL‐17R, in which activation of PI3K/Akt and Jak/Stat3 signaling pathway may play important roles. In addition, combination of IL‐17A and IL‐22 significantly reduced the generation of Th1 cells and the production of interferon (IFN)‐γ from healthy donor or AML patient peripheral blood mononuclear cells. Patients with high Th17 cell frequency had poor prognosis, whereas patients with high Th1 cell frequency had prolonged survival. Combined analysis of Th1 and Th17 cell frequencies improved the ability to predict patient outcomes. In conclusion, Th17 cells play a crucial role in the pathogenesis of AML and may be an important therapeutic target and prognostic predictor.

Salvianolic acid B inhibits platelets as a P2Y12 antagonist and PDE inhibitor: Evidence from clinic to laboratory

Salviae miltiorrhiza (Danshen) has been used for thousands of years in China and some other Asian countries to treat atherothrombotic diseases. Salvianolate which consists of three water-soluble ingredients purified from Salviae miltiorrhiza, has been approved by Chinese SFDA to treat coronary artery disease. So far, there is no evidence clearly showing the clinical efficiency of salvianolate and the underlying mechanism. This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome and explore the underlying mechanism. We evaluated the effects of salvianolate on platelets in patients with acute coronary syndrome by measuring ADP-induced PAC-1 binding and P-selectin expression on platelets. Salvianolate significantly potentiated the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of salvianolatic acid B (Sal-B), the major component which composes 85% of salvianolate. Sal-B inhibits human platelet activation induced by multiple agonists in vitro by inhibiting phosphodiesterase (PDE) and antagonizing P2Y12 receptor. For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12 antagonism which may account for the clinical antiplatelet effects of salvianolate. Our results suggest that Sal-B may substitute salvianolate for clinical use.

Tumor vascular disrupting agent 5,6‐dimethylxanthenone‐4‐acetic acid inhibits platelet activation and thrombosis via inhibition of thromboxane A2 signaling and phosphodiesterase

5,6‐Dimethylxanthenone‐4‐acetic acid (DMXAA) is a tumor vascular disrupting agent under clinical trials as an adjacent antitumor agent. DMXAA is structurally similar to flavone‐8‐acetic acid (FAA), an old tumor vascular disrupting agent with antiplatelet and antithrombotic effects. In contrast to FAA, which causes bleeding in tumor patients, no bleeding has been reported in patients receiving DMXAA. Whether DMXAA also affects platelet function is not clear.

Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12

Summary.  Background:  In our previous in vitro study, we reported a constitutively active chimeric P2Y12 (cP2Y12) and found that AR‐C78511 is a potent inverse agonist at this receptor. The role of cP2Y12 in platelet activation and thrombosis is not clear.

Musashi-2 Silencing Exerts Potent Activity against Acute Myeloid Leukemia and Enhances Chemosensitivity to Daunorubicin.

RNA-binding protein Musashi-2 (Msi2) is known to play a critical role in leukemogenesis and contributes to poor clinical prognosis in acute myeloid leukemia (AML). However, the effect of Msi2 silencing on treatment for AML still remains poorly understood. In this study, we used lentivirus-mediated RNA interference targeting Msi2 to investigate the resulting changes in cellular processes and the underlying mechanisms in AML cell lines as well as primary AML cells isolated from AML patients. We found that Msi2 was highly expressed in AML cells, and its depletion inhibited Ki-67 expression and resulted in decreased in vitro and in vivo proliferation. Msi2 silencing induced cell cycle arrest in G0/G1 phase, with decreased Cyclin D1 and increased p21 expression. Msi2 silencing induced apoptosis through down-regulation of Bcl-2 expression and up-regulation of Bax expression. Suppression of Akt, Erk1/2 and p38 phosphorylation also contributed to apoptosis mediated by Msi2 silencing. Finally, Msi2 silencing in AML cells also enhanced their chemosensitivity to daunorubicin. Conclusively, our data suggest that Msi2 is a promising target for gene therapy to optimize conventional chemotherapeutics in AML treatment.

P2Y12 protects platelets from apoptosis via PI3k-dependent Bak/Bax inactivation.

Summary.  Background: Platelet ADP receptor P2Y12 is well studied and recognized as a key player in platelet activation, hemostasis and thrombosis. However, the role of P2Y12 in platelet apoptosis remains unknown. Objectives: To evaluate the role of the P2Y12 receptor in platelet apoptosis. Methods: We used flow cytometry and Western blotting to assess apoptotic events in platelets treated with ABT‐737 or ABT‐263, and stored at 37 °C, combined with P2Y12 receptor antagonists or P2Y12‐deficient mice. Results: P2Y12 activation attenuated apoptosis induced by ABT‐737 in human and mouse platelets in vitro, evidenced by reduced phosphatidylserine (PS) exposure, diminished depolarization of mitochondrial inner transmembrane potential (ΔΨm) and decreased caspase‐3 activation. Through increasing the phosphorylation level of Akt and Bad, and changing the interaction between different Bcl‐2 family proteins, P2Y12 activation inactivated Bak/Bax. This antiapoptotic effect could be abolished by P2Y12 antagonism or PI3K inhibition. We also observed the antiapoptotic effect of P2Y12 activation in platelets stored at 37 °C. P2Y12 activation improved the impaired activation responses of apoptotic platelets stressed by ABT‐737. In platelets from mice dosed with ABT‐263 in vivo, clopidogrel or deficiency of P2Y12 receptor enhanced apoptosis along with increased Bak/Bax activation. Conclusions: This study demonstrates that P2Y12 activation protects platelets from apoptosis via PI3k‐dependent Bak/Bax inactivation, which may be physiologically important to counter the proapoptotic challenge. Our findings that P2Y12 blockade exaggerates platelet apoptosis induced by ABT‐263 (Navitoclax) also imply a novel drug interaction of ABT‐263 and P2Y12 antagonists.

NOD2 Receptor is Expressed in Platelets and Enhances Platelet Activation and Thrombosis

Background— Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results— Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1&bgr; maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions— NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored.

Nucleotide-Binding Oligomerization Domain 2 Receptor Is Expressed in Platelets and Enhances Platelet Activation and ThrombosisCLINICAL PERSPECTIVE

Background— Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results— Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1&bgr; maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions— NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored.