A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

APDL1-CART cells exhibit strong PD-L1-specific activity against leukemia cells.

Chimeric antigen receptor (CAR) T cells target specific tumor antigens and lyse tumor cells in an MHC-independent manner. However, the efficacy of CAR-T cell and other cancer immunotherapies is limited by the expression of immune-checkpoint molecules such as programmed death-ligand 1 (PD-L1) on tumor cells, which binds to PD-1 receptors on T cells leading to T cell inactivation and immune escape. Here, we incorporated a PD-L1-targeted single-chain variable fragment (scFv) fusion protein sequence into a CAR vector to generate human anti-PD-L1-CAR-T cells (aPDL1-CART cells) targeting the PD-L1 antigen. Unlike control T cells, aPDL1-CART cells significantly halted the expansion and reduced the viability of co-cultured leukemia cells (Raji, CD46, and K562) overexpressing PD-L1, and this effect was paralleled by increased secretion of IL-2 and IFN-γ. The antitumor efficacy of aPDL1-CART cells was also evaluated in vivo by co-injecting control T cells or aPDL1-CART cells along with PDL1-CA46 cells to generate subcutaneous xenografts in NCG mice. Whereas large tumors developed in mice inoculated with PDL1-CA46 cells alone or together with control T cells, no tumor formation was detected in xenografts containing aPDL1-CART cells. Our data suggest that immune checkpoint-targeted CAR-T cells may be useful for controlling and eradicating immune-refractory hematological malignancies.

Calreticulin increases growth and progression of natural killer/T-cell lymphoma.

In this study, we investigated the role of calreticulin (CALR) in the pathogenesis of natural killer/T-cell lymphoma (NKTCL). CALR expression was significantly higher in the NKTCL tissues than normal control tissues in the GSE80632 dataset. High CALR expression correlated with poorer overall survival of NKTCL patients (P = 0.0248). CALR mRNA and protein levels were significantly higher in NKTCL cell lines (NK92, SNK6, and SNT8) than normal NK cells. CALR-silenced SNK6 cells generated significantly smaller xenograft tumors in immunodeficient NCG mice than control SNK6 cells. CALR-knockdown NKTCL cells showed significantly less in vitro proliferation and Transwell migration than the controls. CALR knockdown inhibited G1-to-S phase cell cycle progression by increasing the levels of p27 cell cycle inhibitor and reducing the levels of cyclin E2 and cyclin-dependent kinase 2 (CDK2). CALR knockdown inhibited epithelial-to-mesenchymal transition (EMT) by decreasing the levels of ß-catenin and TCF/ZEB1 and upregulating E-cadherin. These data demonstrate that CALR regulates the growth and progression of NKTCL cells by modulating G1-to-S cell cycle progression and EMT.

Role and mechanism of PTEN in Burkitt's lymphoma.

The aim of the present study was to explore the possible mechanisms of phosphatase and tensin homolog (PTEN) in the pathogenesis of Burkitt's lymphoma, and provide novel information that can be used in the targeted treatment of this disease. PTEN lentiviral overexpression vector and short­hairpin PTEN silencing vectors were constructed. The effect of PTEN on the growth and proliferation of CA46 and RAJI cells was analyzed using a Cell Counting Kit­8 assay. Apoptosis was detected by Hoechst 33342 and propidium iodide double staining. Flow cytometry was used to analyze the cell cycle. A Transwell chamber was used to detect cell migration and invasion abilities. Western blot analysis was used to detect related protein changes. The mechanism of the effect of PTEN on the biological characteristics of Burkitt's lymphoma cells was subsequently analyzed. The results revealed that PTEN inhibited the proliferation of CA46 and RAJI cells by downregulating the expression of p­AKT, It was indicated that the upregulation of proapoptotic proteins (including Bad and Bax) induced apoptosis, regulated cyclin (including P53, P21, CDK4, CDK6, cyclin D3 and cyclin H) to inhibit cell cycle progression, and mediated epithelial­mesenchymal transition­like cell markers (including E­cadherin, N­cadherin, ß­catenin, TCF­8, vimentin, Slug and Snail) to inhibit cell migration and invasion. In conclusion, the tumor­suppressor gene PTEN inhibited the phosphoinositide 3­kinase/protein kinase B (PI3K/AKT) signaling pathway and inhibited the proliferation and migration of Burkitt's lymphoma cells, induced apoptosis and cell cycle arrest, thus playing a crucial role in the pathogenesis of Burkitt's lymphoma.

Protective autophagy or autophagic death: effects of BEZ235 on chronic myelogenous leukemia.

PURPOSE: To investigate the effects of BEZ235 on chronic myeloid leukemia (CML) cells. METHODS: MTS assay was used to detect the proliferation of CML cells. The proteins expression were detected by Western blot assay. The effects of BEZ235 on autophagy in CML cells were verified through transmission electron microscopy and evaluated by laser confocal microscopy. Annexin V-FITC/PI double staining flow cytometry was used to detect apoptosis. A xenograft model was established to observe the therapeutic effect of BEZ235 in vivo. RESULTS: BEZ235 could inhibit the proliferation of CML cells; CQ and 3-MA could increase the proliferation inhibition and Z-VAD-FMK can reduce the proliferation inhibition of BEZ235 on CML cells (P<0.05). Results of TEM showed that the autophagosomes of CML cells treated with BEZ235 increased (P<0.05). The results by confocal microscopy showed that the autophagic activity of K562 cells increased with BEZ235 treatment. When BEZ235 combined with CQ, BEZ235-induced autophagic flow was blocked. FCM results showed that BEZ235 could induces apoptosis in CML cells. Z-VAD-FMK could decrease the apoptosis of CML cells induced by BEZ235. CQ increased the apoptosis of CML cells induced by BEZ235 (P<0.05). Western blot showed that BEZ235 inhibited the phosphorylation of AKT and S6K. BEZ235 alone could upregulate the expression of cleaved caspase-3 and LC3II. When combined with Z-VAD-FMK, the expression of cleaved caspase-3 was lower than that of BEZ235 alone. When combined with CQ, the expression of cleaved caspase-3 and LC3II were higher than those of BEZ235 alone (P<0.05). BEZ235 could inhibit the growth of xenografts of CML cell line. CONCLUSION: BEZ235 can inhibit the proliferation of CML cells, induce apoptosis, and enhance autophagy activity. It induces protective autophagy. The combination of CQ can enhance the apoptosis and proliferation inhibition of CML cells induced by BEZ235.

Sonidegib, a Smoothened Inhibitor, Promotes Apoptosis and Suppresses Proliferation of Natural Killer/T-Cell Lymphoma.

BACKGROUND Dysregulation of the Hedgehog (Hh) pathway modulates various aspects of hematologic and solid tumors, but its effects in human Natural killer/T-cell lymphoma (NKTCL) are unclear. Moreover, no study has examined the consequences of pharmacologically inhibiting Hh signaling in NKTCL cell lines. MATERIAL AND METHODS In this study, the expression of Smoothened (Smo) and Glioma-associated oncogene 1 (Gli1) in NKTCL tissue were scrutinized. Two human NKTCL cell lines, SNK6 and SNT8, were subjected to various doses of sonidegib (a Smo inhibitor) and incubated for distinct durations. The cell apoptosis was examined by flow cytometry, CCK-8 assay was run to assess proliferation, and protein levels were quantified by Western blotting. RESULTS Both Smo and Gli1 expression were higher in NKTCL tissue than in Lymphoid Reactive Hyperplasia (LRH). Sonidegib significantly suppressed proliferation in NKTCL cells and the effect was dose-dependent. Further analysis revealed that sonidegib treatment elevated the number of apoptotic cells in a dose- and time-dependent manner. In addition, sonidegib downregulated Smo and Gli1expression in NKTCL cells. CONCLUSIONS The Hh pathway is crucial to the development of NKTCL and thus holds huge promise as a treatment for this disease.

[Evaluation and Comparison of Thromboelastography and Conventional Coagulation Tests for Blood Coagulation Function in Children with Henoch-Schönlein Purpura].

OBJECTIVE: To investigate the efficacy of disease control, survival time and safely in treatment of newly diagnosed multiple mycloma patients with different dose of tenalidomide regimens. METHODS: The clinical data of 116 patients with multiple myeloma from June 2011 to June 2015 were collected and analyzed retrospectively. According to doses of used lenalidomide based on dexamethasone plus lenalidomide regimen 116 patients were divided into 2 groups: conventional dose group (58 cases) and low dose group (58 cases). The ORR, PFS rate and OS rate during followed-up for 3 years, KPS score, RNS score and immunophenotypic index before and after treatment and drug toxicity incidence were compared between 2 groups. RESULTS: The ORR for 2 treatment courses of low dose group was significantly lower than that in conventienal dose group (P＜0.05). The ORR for 4 and 6 treatment courses was not significantly different between 2 groups (P＞0.05). The PFS rate and OS rate during followed-up for 3 years was no significantly different between 2 groups (P＞0.05). The KPS score and RNS score after treatment of low dose group were significantly better than those in conventional dose group and before treatment (P＜0.05). The levels of immunophenotypic index after treatment of both groups were significantly better than those before treatment (P＜0.05). The incidence of III-IV grade hematological toxicity, pulmonary infection and herpes were not significantly different between 2 groups (P＞0.05). The incidence of peripheral neuropathy and gastrointestinal reactions in the low dose group were significantly lower than that in conventional dose group (P＜0.05). CONCLUSION: Conventional and low doses of lenalidomide possess the same control effects and survival time for treatment of newly dingnosed patients with multiple myeloma; Despite, the initiation of effects from the low dose lenalidomide is relatively slower, it contributes to raise the overall quality of life and reduce the risk of drug toxicity.

[Effects of mTOR Inhibitor Rapamycin on Burkitt's Lymphoma Cells].

OBJECTIVE: To explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin. RESULTS: Rapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G1/G0 phase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G2/M phase, the decreased population was accompanied by the increase of G1/G0 phase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6. CONCLUSION: The rapamycin can effectively inhibit cell proliferation, arrest Raji and CA46 cells in G1/G0 phase, and this effect associates with inhibiting the activation of mTOR/RPS6 signal pathway through down-regulating the expression of phosphorylated RPS6, i.e. mTOR downstream signal pathway. It also can induce apoptosis by down-regulating the expression of anti-apoptotic protein survivin and activating the intrinsic pro-apoptotic protein caspase-3.

Efficacy of the dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with imatinib mesylate against chronic myelogenous leukemia cell lines.

BACKGROUND: Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is a therapy target of cancer. We aimed to confirm the effect of dual PI3K/mTOR inhibitor NVP-BEZ235 on proliferation, apoptosis, and autophagy of chronic myelogenous leukemia (CML) cells and sensitivity of tyrosine kinase inhibitor in vitro. METHODS: Two human CML cell lines, K562 and KBM7R (T315I mutant strain), were used. The proliferation of CML cells was detected by MTS (Owen's reagent) assay. Cell cycle and apoptosis assay were examined by flow cytometric analysis. The phosphorylation levels and the expression levels were both evaluated by Western blot analysis. NVP-BEZ235 in combination with imatinib was also used to reveal the effect on proliferation and apoptosis. RESULTS: NVP-BEZ235 significantly inhibited the proliferation in a time- and dose-dependent manner, and the half-maximal inhibitory concentration values of NVP-BEZ235 inhibiting the proliferation of K562 and KBM7R were 0.37±0.21 and 0.43±0.27 µmol/L, respectively, after 48 h. Cell apoptosis assay showed that NVP-BEZ235 significantly increased the late apoptotic cells. Cell cycle analysis indicated that the cells were mostly arrested in G1/G0 phase after treatment by NVP-BEZ235. In addition, results also found that, after treatment by NVP-BEZ235, phosphorylation levels of Akt kinase and S6K kinase significantly reduced, and the expression levels of cleaved caspase-3 significantly increased; meanwhile, the expression levels of caspase-3, B-cell lymphoma-2, cyclin D1, and cyclin D2 significantly decreased, and the ratio of LC3II/LC3I was significantly increased with increased LC3II expression level. Moreover, imatinib in combination with NVP-BEZ235 induced a more pronounced colony growth inhibition than imatinib alone. CONCLUSION: NVP-BEZ235 effectively inhibited cell proliferation by G0/G1 cell cycle arrest and induced apoptosis through deregulating PI3K/Akt/mTOR pathway in CML cells; in addition, NVP-BEZ235 can enhance cell autophagy, and is conducive to raising CML cell sensitivity to imatinib to inhibit the growth of imatinib-resistant cells.

The dual PI3K/mTOR inhibitor NVP-BEZ235 inhibits proliferation and induces apoptosis of burkitt lymphoma cells.

BACKGROUND: Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is a therapy target of cancer. We aimed to confirm the effect of dual PI3K/mTOR inhibitor NVP-BEZ235 on cell proliferation and apoptosis in Burkitt lymphoma (BL) cells. METHODS: Two human BL cell lines, CA46 and RAJI were used in this study. The proliferation of BL cells was detected by manganese tricarbonyl transfer (MTT) assay. Cell cycle and apoptosis assay were examined by flow cytometric analysis. The phosphorylation levels of AKT (Thr308), AKT (Ser473), and RPS6 were evaluated by western blot analysis. RESULTS: NVP-BEZ235 significantly inhibited the proliferation of BL cells (CA46 and RAJI) and the inhibition effect was time and dose-dependent. Cell cycle analysis indicated that the cells (CA46 and RAJI) were mostly arrested in G1/G0 phase. Cell apoptosis assay showed that the late apoptotic cells were significantly increased after 72 h treatment by 100 nmol/L of NVP-BEZ235. In addition, results also found that NVP-BEZ235 reduced the phosphorylation levels of AKT (Thr308), AKT (Ser473), and PRS6 in BL cells (CA46 and RAJI). Moreover, this inhibition effect on phosphorylation was dose-dependent. CONCLUSIONS: NVP-BEZ235 effectively inhibited cell proliferation by G0/G1 cell-cycle arrest and induced apoptosis through deregulating PI3K/Akt/mTOR pathway in BL cells.