[Reversal Effect of NVP-BEZ235 on Doxorubicin-Resistance in Burkitt Lymphoma RAJI Cell Line].

OBJECTIVE: To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. METHODS: The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. RESULTS: The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. CONCLUSION: The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

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.

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.

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.

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.

Seroprevalence and genotype of Toxoplasma gondii in pigs, dogs and cats from Guizhou province, Southwest China.

BACKGROUND: Toxoplasma gondii is an obligate, intracellular protozoan that infects almost all warm-blooded animals, including humans, domesticated and wild animals. Recent studies of Toxoplasma gondii isolates from animals in different regions of China have shown a limited genetic diversity with the dominance of the ToxoDB PCR-RFLP genotype #9 named as "Chinese 1". However, there is not much published information regarding its prevalence in domestic animals from Guizhou province, a subtropical region in Southwest China. The objectives of this study were to determine seroprevalence and genetic diversity of T .gondii in pigs, dogs and cats in Guizhou province, Southwest China. FINDINGS: The anti-T. gondii IgG were detected in 70.0%(49/70) pigs, 20.56%(22/107) dogs and 63.16(12/19) cats. The anti-T. gondii IgM were found in 0.93%(1/107) dogs, 21.53%(4/19) cats, but not in pigs. In addition, the toxoplasma circulating antigen (CAG) were detected in 16.9%18/70)pigs, 13.1% (14/107) dogs and 10.5%(2/19) cats. The T. gondii DNA were detected in 31.5%(22/70) pigs, 3.7%(4/107) dogs and 52.63%(10/19) cats. Five T. gondii isolates were obtained(3 from pigs and 2 from cats). The genotype of these five isolates belonged to the predominant genotype "Chinese 1". CONCLUSIONS: The high prevalence of T. gondii infection in pigs,cats and dogs indicated that the T. gondii infection is common in Guizhou province. Additionally, the T. gondii genotype "Chinese 1" was dominant in Southwest China.