Effects of Monoammonium Glycyrrhizinate on Stem Cell-like Characteristics, Oxidative Stress and Mitochondrial Function of Acute Promyelocytic Leukemia NB4 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4.@*METHODS@#CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2).@*RESULTS@#Compared with the control group, if the concentration of MAG was less than 5 μmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 μmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 μmol/L, MAG 5 μmol/L, MAG 10 μmol/L, and MAG 20 μmol/L; compared with the control group (MAG 0 μmol/L), the cells in MAG 5 μmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05).@*CONCLUSION@#Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.

Clinical and Prognostic analysis of 43 Children with Mature B-cell Non-Hodgkin's Lymphoma/Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical and prognostic features as well as treatment response of childhood B-cell non-Hodgkin's lymphoma/acute lymphoblastic leukemia (B-NHL/B-ALL), so as to better modify the treatment for further improving the prognosis.</p><p><b>METHODS</b>The clinical data of 43 patients with newly-diagnosed childhood B-NHL/B-ALL from July 2005 to December 2013 in West China Second Hospital of Sichuan University were retrospectively analyzed with particular focus on clinical presentations, laboratory findings and histology. Among them 26 patients received B-NHL-2010 protocol and 17 patients received LMB-89 protocol treatment. Kaplan-Meier method was used to compare the survival rates between groups, while multiple factor logistic regression was used to identify the prognostic factors.</p><p><b>RESULTS</b>(1) The median age at diagnosis was 7.58 (2.42-13.67) years. The male-to-female ratio was 2.9 : 1. No significant difference was found in the median age at diagnosis between male and female children with B-NHL/B-ALL (P = 0.837). (2) Burkitt's lymphoma was the most common (34/43, 79.07%), followed by diffuse large B cell lymphoma (4/43, 9.3%), ALL-L3 (3/43, 6.98%) and others (2/43, 4.65%) in decreasing frequency. (3) According to St. Jude staging classification, 4 patients (9.30%) were divided into stage I, 9 patients (20.93%) into stage II, 23 patients (53.49%) into stage III and 7 patients (16.28%) into stage IV; (4) Clinically, the common predilection sites were as following: ileocecus (11/43, 25.58%), nasopharynx (10/43, 23.26%), faciomaxillary (9/43, 20.93%), superficial lymphadenopathy (8/43, 18.60%), other sites such as mediastinum and bone marrow (5/43, 11.63%). (5) With a median follow up of 24 months (0.7-105 months), the 2-year overall survival (OS) rate and event-free survival (EFS) rate were 79.8% ± 6.5%% and 71.0% ± 7.2%, respectively. The 2-year OS and EFS rates in patients treated with B-NHL-2010 protocol were 79.1% ± 8.4% and 74.1% ± 8.4%, while those in patients treated with LMB-89 protocol were 87.5% ± 8.3% and 66.7% ± 12.4%, respectively, but there was no significant difference between them (P > 0.05). The 2-year EFS rate in patients with LDH > 2N and bone marrow infiltration were significantly lower than that of other groups (P < 0.05). (6) 8 patients (18.6%) relapsed. The median relapsed time was 6 months (2-9 months). 1 patient suffered progressive disease. Male, systemic symptom, elevated LDH, bone marrow and CNS infiltration and advanced stage (stage III and stage IV) were associated with relapse /progressive disease. Logistic regression analysis showed that LDH > 2N was an independent unfavorable prognostic factors (OR = 31.129, P = 0.02).</p><p><b>CONCLUSION</b>Outcome of B-NHL/B-ALL is greatly improved by current intensive and short-time chemotherapy regimen. The 2-year event-free survival (EFS) rate is 71.0% ± 7.2%. There is no significant difference in EFS rate between patients treated with B-NHL-2010 protocol and LMB89 protocol. The long-term survival rate in patient with advanced disease need to be further improved.</p>

Efficacy and safety of the WT-2009 chemotherapy protocol in treatment of Wilms' tumor in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of the WT-2009 chemotherapy protocol for Wilms' tumor (WT) in children.</p><p><b>METHODS</b>The clinical data of 34 children with newly-diagnosed WT between July 2009 and December 2013 were retrospectively analyzed. Among the 34 children, 2 died before treatment, 6 children did not accept therapy and 26 accepted the chemotherapy based on the WT-2009 chemotherapy protocol. Kaplan-Meier method was used to estimate the 2-year survival rate.</p><p><b>RESULTS</b>The pathological analysis revealed the favorable histology WT was common (88%, 30/34). The most common first manifestation was abdominal masses (56%, 19/34). Among the 26 patients who accepted the chemotherapy based on the WT-2009 protocol, complete remission was achieved in 24 cases (92%), partial remission was achieved in 1 case (4%), and disease relapse was found in 1 case (4%). Severe pulmonary infection occurred in 1 case in the course of treatment. The 2-year overall survival rate and event-free survival rate were 100% and 89.7% respectively.</p><p><b>CONCLUSIONS</b>Favorable histology is most common pathological type in children with WT. The chemotherapy based on the WT-2009 protocol for WT can produce a favorable prognosis and a high tolerance.</p>

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 / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the role of glucocorticoid (GC) receptor (GR) in rapamycin's reversion of GC resistance in human GC-resistant T-acute lymphoblastic leukemia (ALL) CEM-C1 cells.</p><p><b>METHODS</b>CEM-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.</p><p><b>RESULTS</b>When 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).</p><p><b>CONCLUSION</b>After 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.</p>

Clinical analysis of Epstein-Barr virus-associated hemophagocytic syndrome in children / 中国实验血液学杂志

The primary infection of Epstein-Barr virus (EBV) may results in hemophagocytic syndrome, known as EBV-associated hemophagocytic syndrome (EBV-AHS), but the clinical risk factors complicating this fatal disease in children with infectious mononucleosis (IM) are unknown. The aim of this study was to identify clinical features of EBV-AHS and to evaluate the curative effect of HLH-2004 protocol. The clinical and laboratory data of 644 IM children including 27 children developed into EBV-AHS and 43 HPS children associated with other diseases were retrospectively analyzed and logistic regression was used to identify the clinical risk factors complicating EBV-AHS. The results showed as follows: (1) the prevalence of EBV-AHS in IM children was 4.2% (27/644), and the prevalence in group aged younger than 3 years was higher than in other age groups. The incidence age of EBV-AHS was significantly younger than that of other HPS patients; (2) Liver function damage of group aged older than 7 years was much more severe in HPS patients. (3) Compared with other HPS patients, male patients were more common and liver function damage was severe in EBV-AHS patients, especially in the patients aged at 2 years or younger. (4) The fatality rate in the EBV-AHS patients was 37.0% (10/27). (5)After treatment with HLH-2004 protocol, the fatality rate in patients with EBV-AHS decreased from 50.0% to 18.2%, the overall survival (OS) of 3 years significantly increased (P = 0.032). It is concluded that IM is a benign self-limited disease, of which only about 4.2% patients will develop into EBV-AHS. Clinical risk factors identified in this study may be helpful for early diagnosis of IM children with complicated EBV-ASH, the HLH-2004 protocol can obviously improve prognosis of EBV-HPS.

Visceral leishmaniasis associated hemophagocytic lymphohistiocytosis: report of four childhood cases / 中华儿科杂志

<p><b>OBJECTIVE</b>The clinical features of four cases of visceral leishmaniasis (VL)-associated hemophagocytic lymphohistiocytosis (VL-HLH) were retrospectively analyzed for the purpose of helping the diagnosis of secondary HLH.</p><p><b>METHOD</b>Clinical data of three childhood cases of VL-HLH documented in our hospital and one case diagnosed in the Capital Institute of Pediatrics was reviewed retrospectively, with particular emphasis on peculiar clinical manifestations and on clues to the diagnosis of this relatively rare disease entity.</p><p><b>RESULT</b>Three children were from endemic areas of VL, and the other one had lived in endemic area for one year, which was revealed by detailed history-taking. Clinically, VL-HLH is characterized by persistent fever, hepatosplenomegaly and pancytopenia, which is similar to those of HLH, and is one of the important reasons of delayed diagnosis or misdiagnosis. Based on the HLH-2004 protocol, all the four cases met the diagnostic criteria of HLH. In addition, bone marrow aspirate and immunologic detection of VL-specific antibody via rk39 dipstick test during the early disease course of VL-HLH yielded negative results. Two cases who received HLH-targeted therapy responded reasonably well, with rapid temperature normalization and spleen retraction. Nevertheless, Hb remained lower than normal, which we believed to be related to persistent red cell destruction by the invading parasite Leishmania donovani.</p><p><b>CONCLUSION</b>VL, a parasitic disease caused by Leishmania donovani, which is currently endemic just in 6 provinces in China, shares similar clinical picture of HLH and is an easily ignored underlying cause of secondary HLH. We suggest that VL should be in the list of differential diagnosis for any patients with HLH who lives in or has a definite travel history to endemic areas. Repeated bone marrow studies are highly warranted to make a definite diagnosis of VL, because bone marrow aspirate or rk39 dipstick test during early disease course might yield negative results. Although VL-HLH responds quite well to HLH-tailored chemotherapy, specific therapy against VL must be given to prevent disease recurrence, and HLH-targeted chemotherapy might be discontinued to prevent chemotherapy-related toxicities.</p>

Clinical risk factors for Epstein-Barr virus-associated hemophagocytic syndrome in children with infectious mononucleosis / 中华儿科杂志

<p><b>OBJECTIVE</b>To compare the clinical features of infectious mononucleosis (IM) and Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (EBV-AHS) and identify the clinical risk factors in IM patients complicated with EBV-AHS.</p><p><b>METHOD</b>A retrospective study was carried out to analyze the clinical and laboratory data of 414 IM and 16 EBV-AHS children from January, 2000 to April, 2006. Then Logistic regression was used to identify the risk factors for progression to EBV-ASH.</p><p><b>RESULTS</b>(1) The incidence of EBV-AHS among the IM children was 3.72% (16/430). There were significant differences between EBV-ASH and IM children in duration of fever (20 days vs. 7 days, P < 0.001), the peaks of fever (40.0 degrees C vs. 39.0 degrees C, P < 0.001), the degree of hepatomegaly (3.5 cm vs 2.0 cm below costal arch, P < 0.05) and splenomegaly (2.75 cm vs. 1.0 cm below costal arch, P < 0.05), while the incidence of isthmitis in EBV-AHS patients was markedly lower than that of IM patients (37.5% vs. 91.1%, P < 0.01). (2) Pancytopenia was often observed in EBV-AHS patients and significant differences between two groups were found in median of leukocytes (3.1 x 10(9)/L vs. 12.8 x 10(9)/L, P < 0.001), median of neutrophils (0.53 x 10(9)/L vs. 3.17 x 10(9)/L, P < 0.001), mean of hemoglobin (80 g/L vs. 120 g/L, P < 0.001) and median of platelet (27.5 x 10(9)/L vs. 183 x 10(9)/L, P < 0.001). (3) Hepatic derangement evidenced by elevated serum enzymes, hyperbilirubinemia and hypoalbuminemia in EBV-ASH children was much more severe than that in IM children, especially LDH level (2128.5 U/L vs. 445 U/L, P < 0.001) and AST level (489 U/L vs. 59 U/L, P < 0.001). (4) The clinical risk factors for IM patients progressing to EBV-ASH were lasting fever >/= 10 days (OR = 8.097, P = 0.008), LDH > 1000 U/L (OR = 7.998, P = 0.033), hypo-albuminemia (albumine < 35 g/L, OR = 7.838, P = 0.038), neutrophils < 1.5 x 10(9)/L (OR = 7.587, P = 0.022) and Plt < 100 x 10(9)/L (OR = 7.190, P = 0.027). The mortality of EBV-AHS in the patients was 50.0% (8/16).</p><p><b>CONCLUSION</b>Most of IM children clinically manifest self-limited process, but about 3.72% of whom may progress to fatal EBV-ASH. The clinical risk factors for EBV-AHS are lasting fever > 10 days, LDH > 1000 U/L, hypoalbuminemia, neutropenia and Plt < 100 x 10(9)/L. EBV-ASH is an extremely dangerous state with high mortality. Repeated bone marrow examinations are helpful for diagnosis in time.</p>

Retrospective analysis of 41 childhood hemophagocytic syndrome / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical features of hemophagocytic syndrome (HPS) and to improve its recognition, early diagnosis and to reduce misdiagnosis.</p><p><b>METHODS</b>A retrospective study was carried out to analyze the underlying diseases, clinical characteristics, laboratory findings and outcomes in 41 patients with HPS.</p><p><b>RESULTS</b>HPS was clinically characterized by prolonged fever (100%), hepatomegaly (97.6%), splenomegaly (95.1%), and other features including lymph adenopathy (65.9%), respiratory symptoms (53.7%), hydrops of multiple serous cavity (26.8%), jaundice (26.8%), central nervous system involvement (14.6%), alimentary tract hemorrhage (12.2%) and skin rash (12.2%). Laboratory data indicated that liver dysfunction was the most prominent feature (100%) mainly manifested with elevated liver enzymes and hypoalbuminemia, and the others were hemophagocytosis in bone marrow (92.7%), pancytopenia 70.7%), coagulation abnormalities (52.4%), DIC, hypertriglyceridemia and refractory hyponatremia. The underlying disease of infection (IAHS) was most common (63.4%), in which EBV-AHS was predominant, making up to 69.2%. Fourteen patients died, 11 of them with IAHS (nine were EBV-AHS) and the other 3 non-IAHS (one of them was malignant lymphoma). The case-fatality rate was increased with the elevated levels of LDH and AST, the correlation coefficient was 0.486 and 0.516 (P < 0.05), respectively. Logistic regression analysis showed that age < 3 years old, levels of LDH > 2000 U/L and AST level > 200 U/L were independent prognostic factors (P value was 0.031, 0.002 and 0.001, respectively).</p><p><b>CONCLUSION</b>There are various underlying diseases and clinical manifestations for HPS. EBV-AHS is the extremely dangerous situation with high mortality. Age, levels of LDH and AST are the death-associated risk factors. Repeat bone marrow examinations are helpful for diagnosis in time.</p>

Comparative study on clinical features between TEL-AML1 positive and negative childhood acute lymphoblastic leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the incidence of TEL-AML1 fusion gene in childhood acute lymphoblastic leukemia (ALL) and to compare the clinical features between TEL-AML1 positive and negative patients.</p><p><b>METHODS</b>Samples of bone marrow or peripheral blood were collected from 95 newly diagnosed ALL children and the TEL-AML1 fusion gene was detected using nested reverse transcription-polymerase chain reaction (RT-PCR). The ALL patients were stratified into TEL-AML1 positive and negative groups and the clinical features were compared.</p><p><b>RESULTS</b>Among 95 patients, 20 (21.05%) were TEL-AML1 positive. The median age of TEL-AML1 positive patients was 5.9 years old and M/F ratio was 1.22:1. There were significant differences between TEL-AML1 positive and negative patients in hepatomegaly (2.75 cm vs. 4 cm below costal arch, P=0.006), splenomegaly (0 cm vs. 3 cm below costal arch, P < 0.001), initial white blood cell count (median 7.40 x 10(9)/L vs.18.70 x 10(9)/L, P=0.011), initial peripheral blood blast (median 2.45 x 10(9)/L vs.11.66 x 10(9)/L, P=0.013), hemoglobin level [(61.45 +/- 13.46) g/L vs. (75.89 +/- 23.11) g/L, P=0.003] and serum lactate dehydrogenase [(621.47 +/- 335.85) U/L vs.(1566.64 +/- 1720.45) U/L, P=0.020], while no differences were found between two groups in age, gender ratio, initial platelet count, percentage of blast in bone marrow, immunophenotypes and the expression of myeloid antigen CD13, CD33 and CD34. The prednisone sensitivity test showed that all 12 TEL-AML1 positive patients were good responders, while there were 11 prednisone poor responders among 40 negative patients (27.50%, P < 0.05). Bone marrow examination on day 15 showed no difference in the rate of complete remission between TEL-AML1 positive and negative patients.</p><p><b>CONCLUSION</b>The incidence of TEL-AML1 fusion gene in cases of ALL is 21.05%. The load of leukemia cells in TEL-AML1 positive patients is significantly smaller than its counterparts, and the blast cells in TEL-AML1 positive patients are more sensitive to prednisone, indicating childhood ALL with TEL-AML1 fusion gene has a favorable prognosis.</p>

Expression of mitochondrial ferritin in K562 leukemic cell during TPA-induced cell differentiation / 中国实验血液学杂志

Mitochondrial ferritin (MtF), a new player in iron metabolism, first identified in 2001, is highly homologous to ferritin both structurally and functionally. Preliminary studies have suggested that MtF might play very important roles in the regulation of mitochondrial iron homeostasis. Leukemic cells, just like other malignant cells, demand more iron for their greater proliferation potential. However, little is known about what roles MtF might play in leukemic cell iron metabolism and cell proliferation. The aim of this study was to investigate the expression of MtF, transferrin receptor 1 (TfR1) and ferritin (Fn) mRNAs in K562 leukemic cells during TPA-induced cell differentiation and to explore the interrelationship between the expression levels of these iron metabolism-related molecules. K562 cells cultured with or without TPA (16 nmol/L) were collected at 24, 72 and 120 hours respectively. Cell differentiation toward monocyte lineage was confirmed by microscopic study (Wright's staining) and flow cytometry. Semiquantitative RT-PCR was performed to determine mRNA expression, with house-keeping gene beta-actin as control reference. This study revealed that over 95% of K562 cells showed morphological features of monocyte/macrophage, and the expression of CD64 on cell surface increased significantly at day 5 with TPA treatment. K562 cells could express a certain level of MtF before TPA-induced differentiation. With increase of TPA-induced cell differentiation, MtF mRNA expressions were downregulated progressively. After 5 days of induced cell differentiation, expression levels of MtF and TfR1 mRNA were just 50.3% and 68.2% of that before TPA treatment. While Fn mRNA expression increased to 1.97 folds of that before TPA treatment. It is concluded that MtF expression is downregulated during TPA-induced K562 cell differentiation, with concomitant downregulation of TfR1 and upregulation of Fn. The coordinated expression regulation of these key iron metabolism-related molecules during cell differentiation may in turn inhibit TfR1-mediated iron uptake via endocytosis and thus adversely affect cell proliferation potential.

Antineoplastic effect of valproic acid and trichostatin on HL-60 and K562 cells / 中国实验血液学杂志

The objective of this study was to investigate antineoplastic effects of valproic acid (VPA) and trichostatin (TSA) on HL-60 and K562 cells in vitro, and the synergic effects of VPA or TSA in combination with ATRA. The inhibitory effects of VPA, TSA and ATRA in various concentrations and different combinations on proliferation of HL-60 and K562 cells were observed by cell growth curves, 50% inhibitory concentration (IC(50)), as well as inhibition of leukemia colony growth at different time points. The characteristics of cell differentiation or apoptosis were analyzed by cytochemical staining, differentiation antigen detection, cell cycle assay and A(NBT)/A(MMT) value determination. The results showed that HL-60 cell had a lower IC(50) of VPA and TSA compared with K562 cells. ATRA could significantly enhance the inhibition of VPA, TSA on clonegenicity of HL-60 cells and inhibition of VPA on clonegenicity of K562 cells. HL-60 cells treated with VPA displayed the phenotype of neutrophilic like cells, and showed the increases of NBT reduction rate and CD11b expression. No evidence for K562 differentiation was found. It is concluded that both VPA and TSA inhibit HL-60 cells growth in vitro. VPA induces differentiation of HL-60 cells to granulocyte. VPA and TSA have a moderate anti-proliferative effect on K562 cells. None of these agents induces K562 cell differentiation.