Yiping Zhu

Resveratrol Induces Apoptosis and Autophagy in T-cell Acute Lymphoblastic Leukemia Cells by Inhibiting Akt/mTOR and Activating p38-MAPK

OBJECTIVEnTo explore the effects of resveratrol-induced apoptosis and autophagy in T-cell acute lymphoblastic leukemia (T-ALL) cells and potential molecular mechanisms.nnnMETHODSnThe anti-proliferation effect of resveratrol-induced, apoptosis and autophagy on T-ALL cells were detected by using MTT test, immunofluorescence, electronic microscope, and flow cytometry, respectively. Western blotting was performed for detecting changes of apoptosis-associated proteins, cell cycle regulatory proteins and state of activation of Akt, mTOR, p70S6K, 4E-BP1, and p38-MAPK.nnnRESULTSnResveratrol inhibited the proliferation and induced apoptosis and autophagy in T-ALL cells in a dose and time-dependent manner. It also induced cell cycle arrest at G0/G1 phase via up regulating cyclin-dependent kinase (CDK) inhibitors p21 and p27 and down regulating cyclin A and cyclin D1. Western blotting revealed that resveratrol significantly decreased the expression of antiapoptotic proteins (Mcl-1 and Bcl-2) and increased the expression of proapoptotic proteins (Bax, Bim, and Bad), and induced cleaved-caspase-3 in a time-dependent manner. Significant increase in ratio of LC3-II/LC3-I and Beclin 1 was also detected. Furthermore, resveratrol induced significant dephosphorylation of Akt, mTOR, p70S6K, and 4E-BP1, but enhanced specific phosphorylation of p38-MAPK which could be blocked by SB203580. When autophagy was suppressed by 3-MA, apoptosis in T-ALL cells induced by resveratrol was enhanced.nnnCONCLUSIONnOur findings have suggested that resveratrol induces cell cycle arrest, apoptosis, and autophagy in T-ALL cells through inhibiting Akt/mTOR/p70S6K/4E-BP1 and activating p38-MAPK signaling pathways. Autophagy might play a role as a self-defense mechanism in T-ALL cells treated by resveratrol. Therefore, the reasonable inhibition of autophagy in T-ALL cells may serve as a promising strategy for resveratrol induced apoptosis and can be used as adjuvant chemotherapy for T-ALL.

Rapamycin sensitizes glucocorticoid resistant acute lymphoblastic leukemia CEM-C1 cells to dexamethasone induced apoptosis through both mTOR suppression and up-regulation and activation of glucocorticoid receptor.

OBJECTIVEnTo explore the role of glucocorticoid (GC) receptor (GR) in rapamycins reversion of GC resistance in human GC-resistant T-acute lymphoblastic leukemia (ALL) CEM-C1 cells.nnnMETHODSnCEM-C1 cells were cultured in vitro and treated with rapamycin at different concentrations with or without 1 μmol/L dexamethasone (Dex). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was performed to assess cell proliferation. The cell cycle and cell apoptosis were analyzed by flow cytometry. The expression of GRα mRNA was determined by real-time quantitative RT-PCR. The expression of GR, p-70S6K, Mcl-1, and Bim proteins was detected by Western blot.nnnRESULTSnWhen incubated with rapamycin at different concentrations, CEM-C1 cells showed significant growth inhibition in a time- and concentration-dependent manner. The growth inhibition was synergistically increased when CEM-C1 cells were treated with rapamycin plus 1 μmol/L Dex. CEM-C1 cells treated with rapamycin alone showed no apparent apoptosis, and were arrested at G0/G1 phase. After the treatment with Dex plus rapamycin, CEM-C1 cells demonstrated apparent apoptosis and increased the cell cycle arrested at G0/G1 phase. Rapamycin combined with Dex up-regulated GRα, phosphorylated GR(p-GR), and pro-apoptotic protein Bim-EL in CEM-C1 cells, but inhibited the expression of p-p70S6K, a downstream target protein of mTOR (mammalian target of rapamycin).nnnCONCLUSIONnAfter the treatment with rapamycin plus Dex, Dex resistant CEM-C1 cells induce growth inhibition and apoptosis. The underlying mechanism may involve inhibition of the mTOR signaling pathway and also be associated with up-regulation of GR expression and activation of GC-GR signaling pathway.

Relapsed APL patient with variant NPM–RARα fusion responded to arsenic trioxide-based therapy and achieved long-term survival

The t(5;17)/NPM–RARα is the second variant chromosomal translocation in acute promyelocytic leukemia (APL) to be characterized and also the second most plentiful variant translocation. So far, there is a lack of information on the effectiveness of arsenic trioxide (ATO) in relapsed APL with variant RARα chimera including t(5;17)/NPM–RARα. We report here a long-term survived APL patient with variant NPM–RARα fusion who relapsed four times and each time responded well to ATO or ATO-based re-induction therapy. The patient had received a total of more than 3,500xa0mg of ATO, but showed no obvious arsenic-related toxicities. This case illustrates the long-term efficiency and safety of ATO-based therapy not only in newly diagnosed APL, but also in relapsed APL including those with variant translocations.

Targeting mTOR/p70S6K/glycolysis signaling pathway restores glucocorticoid sensitivity to 4E-BP1 null Burkitt Lymphoma

BackgroundIncreasing evidence indicates that rapamycin could be used as a potential glucocorticoid (GC) sensitizer in lymphoblastic malignancies via genetic prevention of 4E-BP1 phosphorylation. Interestingly, we found that combined rapamycin with dexamethasone can effectively reverse GC resistance in 4E-BP1 null lymphoma cells. In this study, we investigated the potential link between mTOR/p70S6K signaling pathway, glycolysis, autophagy and GC resistance.MethodsAntitumor effects of the combination of rapamycin and dexamethasone were evaluated on cell viability by MTT assay and in vivo studies, on cell cycle and apoptosis by flow cytometry, on autophagy by western blot, MDC staining and transmission electron microscopy and on cell signaling by western blot. Moreover, to test whether inhibiting glycolysis is the core mechanism in rapamycin restoring GC sensitivity, we took glycolysis inhibitor 2-deoxyglucose to replace rapamycin and then evaluated the antitumor effects in vitro.Results Raji cells are resistant to rapamycin (IC50u2009>u20091000 nM) or dexamethasone (IC50u2009>u2009100xa0μM) treatment alone. The combination of rapamycin and dexamethasone synergistically inhibited the viability of Raji cells in vitro and in vivo by inducing caspase-dependent and -independent cell death and G0/G1 cell cycle arrest. These effects were achieved by the inhibition of mTOR/p70S6K signaling pathway, which led to the inhibition of glycolysis and the induction of autophagy. Pretreatment with pan-caspase inhibitor z-VAD-fmk or autophagy inhibitor 3-MA failed to protect the cells from combined treatment-induced death. Glycolysis inhibitor combined with dexamethasone produced a similar antitumor effects in vitro.ConclusionsInhibition of mTOR/p70S6K/glycolysis signaling pathway is the key point of therapy in reversing GC resistant in Burkitt lymphoma patients.

Low-dose anisomycin sensitizes glucocorticoid-resistant T-acute lymphoblastic leukemia CEM-C1 cells to dexamethasone-induced apoptosis through activation of glucocorticoid receptor and p38-MAPK/JNK.

Abstract Glucocorticoid (GC) resistance in children with acute lymphoblastic leukemia (ALL) usually resulted in the failure of treatment. Exploring new agents to overcome GC resistance is important. Here we reported for the first time that low-dose anisomycin has the potential to sensitize GC-resistant T-ALL CEM-C1 cells to dexamethasone (DEX). Compared with the use of DEX or low-dose anisomycin alone, co-treatment with them resulted in a significant increase of growth inhibition, apoptosis and cell cycle arrest in CEM-C1 cells through induction of activated caspase-3 and up-regulation of Bim, p21and p27, and down-regulation of Mcl-1, Bcl-2, c-myc, cyclin A and cyclin D1. Furthermore, co-treatment remarkably activated glucocorticoid receptor (GR), p38-MAPK and JNK, and all of them were canceled only by the GR inhibitor RU486, indicating GR might be an at the upstream of GR–p38-MAPK/JNK pathway. We conclude that low-dose anisomycin sensitizes GC-resistant CEM-C1 cells to DEX and this effect is mediated, at least in part, by activation of the GR–p38-MAPK/JNK signaling pathway.

Impact of NUDT15 polymorphisms on thiopurines-induced myelotoxicity and thiopurines tolerance dose

Thiopurines are widely used as anticancer and immunosuppressive agents. However, life-threatening myelotoxicity has been noticed and largely explained by genetic variations, including NUDT15 polymorphisms (e.g., rs116855232). In this study, we conduct a meta-analysis to investigate the impact of rs116855232 on thiopurines-induced myelotoxicity susceptibility (1752 patients from 7 independent cohorts), as well as on thiopurines intolerance dose (2745 patients from 13 cohorts). Variant allele of rs116855232 contributes 7.86-fold (P < 0.00001, 95% CI: 6.13–10.08) higher risk to develop leucopenia with high specificity (91.74%) and sensitivity (43.19%), and lower thiopurines intolerance dose (P < 0.00001). Through bioinformatics prediction, amino acid changes induced by genetic variants are considered to reduce the stability, and break an α helix of NUDT15, which is part of the thiopurine binding pocket. Additionally, we conduct an expression quantitative trait loci (eQTL) analysis for NUDT15, and find a promoter-located eQTL signal (rs554405994), which may act as a potential marker to predict thiopurines-induced myelotoxicity. In conclusion, genetic polymorphisms in NUDT15 are strongly associated with adverse drug reaction (ADR) of thiopurines, although more evidences are needed to determine values of all functional NUDT15 polymorphisms for clinical regimen, rs116855232 should be considered as a highly credible pharmacogenetic indicator for thiopurines using espcially is Asians.

Prognostic significance and therapeutic potential of the activation of anaplastic lymphoma kinase/protein kinase B/mammalian target of rapamycin signaling pathway in anaplastic large cell lymphoma

BackgroudActivation of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway has been demonstrated to be involved in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated tumorigenesis in anaplastic large cell lymphoma (ALCL) and correlated with unfavorable outcome in certain types of other cancers. However, the prognostic value of AKT/mTOR activation in ALCL remains to be fully elucidated. In the present study, we aim to address this question from a clinical perspective by comparing the expressions of the AKT/mTOR signaling molecules in ALCL patients and exploring the therapeutic significance of targeting the AKT/mTOR pathway in ALCL.MethodsA cohort of 103 patients with ALCL was enrolled in the study. Expression of ALK fusion proteins and the AKT/mTOR signaling phosphoproteins was studied by immunohistochemical (IHC) staining. The pathogenic role of ALK fusion proteins and the therapeutic significance of targeting the ATK/mTOR signaling pathway were further investigated in vitro study with an ALKu2009+u2009ALCL cell line and the NPM-ALK transformed BaF3 cells.ResultsALK expression was detected in 60% of ALCLs, of which 79% exhibited the presence of NPM-ALK, whereas the remaining 21% expressed variant-ALK fusions. Phosphorylation of AKT, mTOR, 4E-binding protein-1 (4E-BP1), and 70xa0kDa ribosomal protein S6 kinase polypeptide 1 (p70S6K1) was detected in 76%, 80%, 91%, and 93% of ALCL patients, respectively. Both phospho-AKT (p-AKT) and p-mTOR were correlated to ALK expression, and p-mTOR was closely correlated to p-AKT. Both p-4E-BP1 and p-p70S6K1 were correlated to p-mTOR, but were not correlated to the expression of ALK and p-AKT. Clinically, ALKu2009+u2009ALCL occurred more commonly in younger patients, and ALKu2009+u2009ALCL patients had a much better prognosis than ALK-ALCL cases. However, expression of p-AKT, p-mTOR, p-4E-BP1, or p-p70S6K1 did not have an impact on the clinical outcome. Overexpression of NPM-ALK in a nonmalignant murine pro-B lymphoid cell line, BaF3, induced the cells to become cytokine-independent and resistant to glucocorticoids (GCs). Targeting AKT/mTOR inhibited growth and triggered the apoptotic cell death of ALKu2009+u2009ALCL cells and NPM-ALK transformed BaF3 cells, and also reversed GC resistance induced by overexpression of NPM-ALK.ConclusionsOverexpression of ALK due to chromosomal translocations is seen in the majority of ALCL patients and endows them with a much better prognosis. The AKT/mTOR signaling pathway is highly activated in ALKu2009+u2009ALCL patients and targeting the AKT/mTOR signaling pathway might confer a great therapeutic potential in ALCL.

Regulatory network of GATA3 in pediatric acute lymphoblastic leukemia

GATA3 polymorphisms were reported to be significantly associated with susceptibility of pediatric B-lineage acute lymphoblastic leukemia (ALL), by impacting on GATA3 expression. We noticed that ALL-related GATA3 polymorphism located around in the tissue-specific enhancer, and significantly associated with GATA3 expression. Although the regulatory network of GATA3 has been well reported in T cells, the functional status of GATA3 is poorly understood in B-ALL. We thus conducted genome-wide gene expression association analyses to reveal expression associated genes and pathways in nine independent B-ALL patient cohorts. In B-ALL patients, 173 candidates were identified to be significantly associated with GATA3 expression, including some reported GATA3-related genes (e.g., ITM2A) and well-known tumor-related genes (e.g., STAT4). Some of the candidates exhibit tissue-specific and subtype-specific association with GATA3. Through overexpression and down-regulation of GATA3 in leukemia cell lines, several reported and novel GATA3 regulated genes were validated. Moreover, association of GATA3 expression and its targets can be impacted by SNPs (e.g., rs4894953), which locate in the potential GATA3 binding motif. Our findings suggest that GATA3 may be involved in multiple tumor-related pathways (e.g., STAT/JAK pathway) in B-ALL to impact leukemogenesis through epigenetic regulation.

Successful allogeneic hematopoietic stem cell transplantation in a boy with X-linked inhibitor of apoptosis deficiency presenting with hemophagocytic lymphohistiocytosis: A case report

X-linked inhibitor of apoptosis (XIAP) deficiency, also known as X-linked lymphoproliferative syndrome type 2 (XLP2), is a rare inherited primary immunodeficiency resulting from the XIAP (also known as BIRC4) mutation. XIAP deficiency is mainly associated with familial hemophagocytic lymphohistiocytosis (HLH) phenotypes, and genetic testing is crucial in diagnosing this syndrome. Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only successful strategy for the treatment of this disease; however, a limited number of studies has been published concerning the outcomes of allogeneic HSCT in patients with XIAP deficiency. The present study reported a successful allogeneic HSCT performed to treat XIAP deficiency in a Chinese boy presenting with HLH. Polymerase chain reaction and DNA sequencing were performed to confirm the diagnosis of XIAP deficiency, and allogeneic HSCT was performed. Genetic tests revealed a two-nucleotide deletion (c.1021_1022delAA) in the patient, which was inherited from his mother, and resulted in frameshift mutation and premature stop codon (p.N341fsX348); this is considered to be a disease-causing mutation. The XIAP deficiency patient underwent allogeneic HSCT, receiving busulfan-containing reduced intensity myeloablative conditioning regimen, with a good intermediate follow-up result obtained. Therefore, genetic testing is essential to confirm the diagnosis of XIAP deficiency and detect the carrier of mutation. The present case study may promote the investigation of allogeneic HSCT in patients with XIAP deficiency.

Low dose of 2-deoxy-D-glucose kills acute lymphoblastic leukemia cells and reverses glucocorticoid resistance via N-linked glycosylation inhibition under normoxia

Recent studies showed that 2-deoxy-D-glucose (2-DG), a glucose analog with dual activity of inhibiting glycolysis and N-linked glycosylation, can be selectively taken up by cancer cells and be used as a potential chemo- and radio-sensitizer. Meanwhile, 2-DG can kill cancer cells under normoxia. However, its efficacy is limited by the high-dose induced systemic toxicity. Here, we showed that low-dose 2-DG could be used as a single agent to kill acute lymphoblastic leukemia (ALL) cells, and as a GC sensitizer to overcome GC resistance under normoxia. Addition of exogenous mannose, a sugar essential for N-linked glycosylation, rescued 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of endoplasmic reticulum stress is the main mechanism for 2-DG to induce cell death and reverse GC resistance in ALL cells. These data provides new insight into the molecular mechanisms involved in GC resistance. More important, it indicates that 2-DG might be the promising drug for designing novel high efficiency and low toxic protocol for ALL patients.