Effects of Decitabine Combined with Bortezomib on the Proliferation of Mantle Cell Lymphoma Cell Lines and Its Underling Mechanisms / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of decitabine combined with bortezomib on the proliferation of mantle cell lymphoma cell lines (Jeko-1 and Grante519) in vitro and explore the underlying mechanisms.@*METHODS@#Jeko-1 and Grante519 cells were treated with different concentrations of decitabine and/or bortezomib alone and their combination.The cell proliferation was determined by CCK-8 assay. the cell apoptosis were detected by flow cytometry, the mRNA and protein expression levels of genes related with the cell cycle and apoptosis were analyzed by RT-PCR and Western blot respactively.@*RESULTS@#Low dose DAC could significantly inhibit the proliferation and induce apoptosis of Jeko-1 and Grante519 cells which shows a dose-and time-dependent manner. After DAC treatment, caspase 3, BAX and PCDH8 expression levels increased, while BCL-2 and CCND1 expression levels decreased in Jeko-1 and Grante519 cells, but there was no significant difference in NF-κB expression. High dose BTZ could significantly inhibit the proliferation and induce apoptosis of Jeko-1 and Grante519 cells which shows a dose-and time-dependent manner; single drug BTZ could increase the expression level of Caspase 3 and BAX, and decrease the expression level of NF-κB, BCL-2 and CCDN1 in Jeko-1 and Grante519 cells, but there was significant difference in PCDH8 expression level. Compared with single-drug treatment group, DAC combined with BTZ significantly increased the inhibitory rate and apoptotic rate of Jeko-1 and Grante519 cells; PCDH8, Caspase 3 and BAX expression levels significantly increased, and the expression levels of NF-κB, BCL-2 and CCND1 significantly decreased in Jeko-1 and Grante519 cells.@*CONCLUSION@#The combination of DAC and BTZ has obviously synergistic effects on the growth inhibition of Jeko-1 and Grante519 cells which maybe relates with enhancing inbibitory effect on NF-κB signal pathway, down-regulating BAX expression, up-regulating BAX expression as well as increasing cospase 3 expression. This study provides a novel therapeutic approach for mantle cell lymphoma.

Effect of Artesunate on Proliferation, Cell Cycle and Apoptosis of SKM-1 Cells and Its Underlying Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of artesunate (ART) on proliferation, cell cycle and apoptosis of SKM-1 cells in vitro and to explore the underlying mechanisms.</p><p><b>METHODS</b>After SKM-1 cells were treated with different concentrations of ART, the cell proliferation was determined by CCK-8 method. Apoptosis and distribution of cell cycle were detected by flow cytometry. Both DCFH-DA fluorescent probe and Fluo-3-Am fluorescent probe were used to detect the changes of intracellular reactive oxygen species (ROS) and calcium ion concentration. Western blot was used to measure the protein levels of BCL-2, BAX, BAD, P-BAD, survivin and XIAP.</p><p><b>RESULTS</b>ART obviously inhibited the growth of SKM-1 cells in time and dose-dependent manners (r = -0.841; r = 0.-786). The antioxidant trolox-pretreatment significantly decreased the growth inhibition effect of ART on SKM-1 cells. Caspase inhibitor Ac-DEVD-CHO partially reduced the growth inhibition effect of ART on SKM-1 cells. After treatment with ART for 24 hours, the apoptosis of SKM-1 cells was found, the cell cycle of SKM-1 was arrested in G0/G1 phase, ART could elevate the levels of calciumion and reactive orygen. ART could significantly down-regulate the protein expression levels of P-BAD and survivin in SKM-1 cells, and showed a highly negative correlation with ART dose (r = -0.909; r = -0.849). On the contrary, ART had no significant effect on expression levels of BAD and XIAP in SKM-1 cells, and after ART treatment, although BCL-2 protein expression was not significantly different when compared with control group, but the BCL-2/BAX ratio significantly decreased and highly negatively correlated with ART dose (r = -0.866).</p><p><b>CONCLUSION</b>The ART significantly suppresses the cell proliferation, induces the apoptosis and promoted cell cycle arrest at G0/G1 phase in SKM-1 cells. The mechanisms of ART anti-MDS is associated with the increase of intracellular calciumion concentration and ROS levels. In addition, the pro-apoptotic activity of ART may be involved in the regulation of BCL-2 /BAX ratio and the expressions of P-bad and survivin.</p>

Efficacy and Safety of Lenalidomide in the Treatment of Multiple Myeloma: A Systematic Review and Meta-analysis of Randomized Controlled Trials / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Lenalidomide has emerged as an important treatment for patients with multiple myeloma (MM). However, its role in the management of MM is still controversial and requires further clarification. The aim of this study was to evaluate efficacy and safety of lenalidomide for MM using a meta-analysis.</p><p><b>METHODS</b>We searched the electronic databases including: PubMed, EMBASE and the Cochrane Center Register of Controlled Trials. Seven randomized clinical trials were identified, which included a total of 2357 patients with MM who received lenalidomide-containing, noncontaining lenalidomide regimens or placebo as induction therapy or maintenance therapy. The outcomes included overall response (OR) rate, complete response (CR) rate, 3-year progression-free survival (PFS) rate, 3-year overall survival (OS) rate, and different types of treatment-related adverse events. We calculated the risk ratios (RRs) as well as their 95% confidence intervals of these outcomes and pooled the results using RevMan 5.2 software.</p><p><b>RESULTS</b>For patients with previously untreated MM, OR rate and CR rate was significantly higher in lenalidomide-containing group than the control group. For relapsed or refractory MM patients, lenalidomide-containing regimens significantly improved the OR rate, CR rate, 3-year PFS rate and 3-year OS rate. With regard to MM patients after autologous stem cell transplantation, lenalidomide maintenance therapy significantly improved 3-year PFS rate but did not result in improved 3-year OS rate. In terms of toxicities, lenalidomide therapy has a higher rate of Grade 3-4 grade cytopenias, infection, deep-vein thrombosis, and diarrhea. Furthermore, the incidence of second primary malignancies was significantly higher in the lenalidomide group.</p><p><b>CONCLUSIONS</b>The lenalidomide-containing regimens as induction therapy clearly increased response rates and improved intervals of survival with acceptable toxicity rates for patients with MM. However, when physicians choose to use the lenalidomide as maintenance therapy, whether the benefits outweigh the risks should be taken into account.</p>

Magnetic Nanoparticles of Fe3O4 Enhance Artesunate-inducing Apoptosis of SKM-1 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of the copolymer of magnetic nanoparticles of Fe(3)O(4) (MNPs-Fe(3)O(4)) and artesunate (ART) on myelodysplastic syndromes (MDS) cell line SKM-1 cells and the potential mechanisms.</p><p><b>METHODS</b>The protein expression levels of BCL-2, BAX, Caspase-3, and Survivin in SKM-1 cells treated with or without the co-polymer were measured by Western blot. The co-polymer-induced apoptosis rate of SKM-1 cells was measured by flow cytometry.</p><p><b>RESULTS</b>The apoptosis rate of SKM-1 cells in the copolymer groups was higher than that in both MNPs-Fe(3)O(4) and artesunate groups alone. The MNPs-Fe(3)O(4) may enhance ART-induced cell apoptosis. Western blot assay showed that the expression of survivin and BCL-2 protein were down-regulated in the ART group, and this down-regulation was even more significant in the group of copolymer of ART with MNPs-Fe(3)O(4). The levels of BAX were increased both in ART group and the copolymer of ART with MNPs-Fe(3)O(4) group, as compared with control group and MNPs-Fe(3)O(4) group. The levels of active-caspase-3 were obviously up-regulated when the ART was combined with the MNPs-Fe(3)O(4). The copolymer of ART with MNPs-Fe(3)O(4) could trigger changes in the expression levels of apoptosis-related genes in SKM-1 cells, among which up-regulation of BAX and down-regulation of survivin and BCL-2 are the 2 major alterations.</p><p><b>CONCLUSION</b>Artesunate can induce the apoptosis of SKM-1 cells, and MNPs-Fe(3)O(4) may enhance the cell apoptosis induced by ART.</p>

Expression of c-fes gene in leukemia cells and its clinical significance / 中国实验血液学杂志

This study was purposed to investigate the expression of c-fes gene in leukemia patients and its clinical significance. The expression of c-fes mRNA in bone marrow cells from 121 cases of acute and chronic leukemia patients, and the expression of c-fes mRNA in peripheral blood mononuclear cells of 20 normal persons were detected by real time-quantitative reverse transcription polymerase chain reaction (RQ-PCR). The results showed that the level of c-fes mRNA in AML patients was higher than that in normal controls [(48.017 +/- 57.170) x 10(-3) vs (0.152 +/- 0.398) x 10(-3)] (p < 0.0001); but there was no significant differences of level of c-fes mRNA between samples of ALL and normal controls(0.047 +/- 0.068) x 10(-3) vs(0.152 +/- 0.398) x 10(-3) (p>0.05); the level of c-fes mRNA in CML patients was higher than that in normal persons (21.605 +/- 24.818) x 10(-3) vs (0.152 +/- 0.398) x 10(-3) (p < 0.0001). The positive expression rate of c-fes gene in CML-CP patients (80%) was higher than that in CML-AP patients (66.7%) and CML-BP (28.6%) patients. In AML patients, c-fes gene was expressed higher in M(2) (80.77%) and M(3) (92.86%) patients. The remission rate of AML (except M(3))patients who had expression of c-fes gene was 81.08%, which was higher than that of patients with no expression of c-fes gene (40.00%). It is concluded that c-fes gene expression was found in myeloid leukemias, whereas low or no expression in lymphocytic leukemias. The differentiation of myelocytic cells may be related to c-fes gene. All AML (except M(3))patients with high level of c-fes mRNA may get good prognosis.

Immunoglobulin D lambda multiple myeloma: a case report / 中国实验血液学杂志

To improve the recognition of immunoglobulin D multiple myeloma and explore its clinical feature and laboratory examination characteristics, so as to reduce the the missed diagnosis and misdiagnosis, a case of IgD multiple myeloma (MM) with myelofibrosis and bone marrow necrosis is reported. The clinical feature, treatment and prognosis of IgDlambda MM were discussed. Immunoglobulin D multiple myeloma is a rare disease and predominantly occurs in young male patients, which shows an aggressive clinical course with poor response to conventional treatment and unfavorable prognosis. Immunoglobulin D multiple myeloma was usually misdiagnosed as a light chain type multiple myeloma by using routine laboratory examination. Immunoglobulin D monoclonal protein is not easy to be detected owing to its low protein level, resulting in missed diagnosis. Immunofixation electrophoresis is highly sensitive and specific for diagnosis of IgD MM, can enhance accuracy of diagnosis for this rare disease.

Angioimmunoblastic T-cell lymphoma with autoimmune hemolytic anemia and pure red cell aplasia / 中国实验血液学杂志

Angioimmunoblastic T-cell lymphoma (AILT) is a peripheral T-cell lymphoma often complicated autoimmune phenomena such as autoimmune cytopenia, and is a truly rare type of NHL. In order to investigate the clinical features, pathological manifestation of this lymphoma, and to explore its therapy protocol, a 37-years old patient with AILT was investigated. The routine blood examination, bone marrow smear, lymphonodus biopsy, Coombs test, flow cytometry for bone marrow mononuclear cells, serological test, immunochemistry method etc were performed for this patient. The results showed that the systemic lymphadenectasis and hepatosplenomegaly were seen in patient, the cervical lymphonode biopsy revealed AITL. The hematoglobin level and number of reticulocytes were very low. Coombs test was positive. Simultaneously, the bone marrow aspirate revealed erythroid aplasia. The warm type autoimmune hemolytic anemia (AIHA) and pure red cell aplasia (PRCA) were co-existed. After one course of chemotherapy with CHOP-E, infiltration sign of AITL patient with AIHA and PRCA disappeared. In conclusion, the AITL patient complicated with AIHA and PRCA was successfully diagnosed, the lymphonode biopsy and bone marrow smear showed more significant, the chemotherapy protocol of CHOP-E can give some effect to cure such angioimmunoblastic T cell lymphoma.

WT1 gene expression lowered by IL-12 In vitro in peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes / 中国实验血液学杂志

Previous studies demonstrated that interleukin-12 (IL-12) enhances the non-MHC-restricted cytotoxic activity of NK cells and facilitate specific allogeneic human cytotoxic T lymphocyte responses against fresh leukemia cells and cell lines. The Wilms' tumor gene, WT1 mRNA, has been used as a marker of minimal residual disease (MRD) for evaluating therapeutic efficacy of patients with leukemia or myelodysplastic syndrome (MDS). This study was aimed to investigate whether in vitro IL-12 can lower WT1 gene expression in peripheral blood monuclear cells (PBMNC) from patients with leukemia or MDS. PBMNC from these 30 patients and 5 healthy volunteers were cultured at 5 x 10(5) cells/ml alone with or without 100 units/ml of IL-12 for 3 days. WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR) since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia and MDS. The results demonstrated that WT1 mRNA in PBMNC of 5 healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in 6 CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in 5 AML patients in CR, but not reduced in 5 of 7 AML in non-CR. It is concluded that IL-12 significantly decrease the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 may be of considerable benefit in the elimination of MRD in patients with hematological malignancies.

Clinical significance of the expression of DNA methyltransferase genes (DNMT) in acute leukemia patients / 中国实验血液学杂志

To investigate the relationship between the expression of DNMT and clinical prognosis in adult patients with acute leukemia (AL), the mRNA expressions of DNMT, p15(INK4B), mdr1 were measured in 72 AL patients and 20 normal controls by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR); the ratio of p15 CpG land methylation was measured in 56 AL patients and 14 normal controls by methylation-specific PCR (MSP-PCR). The results showed that all three DNMT mRNA expressions in AL patients were significantly higher than that in normal controls (P < 0.01). When the internal control was changed into PCNA, a kind of cell proliferation marker gene, the difference still showed a statistic significance. All three DNMT genes were significantly expressed and positively correlated with AL patients, showing high synergistic expression, and there was a negative correlation between the levels of p15, mdr1 gene expression and DNMT. The complete remission (CR) rate in AL patients with the positive expression of all DNMT genes was significantly higher than that of AL patients with partially positive or negative expression (P < 0.01) of DNMT genes. In 56 AL patients, the P15I(NK4B) was completely methylated in 55.4% (31 of 56), partly methylated in 21.4% (12 of 56) and all 14 cases of normal controls were not methylated. It is concluded that DNMT genes are abnormally high expressed in adult AL patients, which lead to methylation-silence of tumor suppressor genes by CpG land hypermethylation, the AL patients with high expression of DNMT are more sensitive to chemotherapy, which may be a good prognostic factor for AL patients.

Expressions of cyclin B1 and p21cipl in adult acute leukemia and their correlation / 中国实验血液学杂志

Cyclin B1, a positive regulator, controls mitosis occurrence, plays an important role in cell proliferation. To investigate the clinical significance of cyclin B1, the expression of cyclin B1 in acute leukemia (AL) patients was measured; the expression of cyclin B1 and p21(cipl), and their cell cycle distribution were assayed by flow cytometry in 136 adult patients with newly diagnosed AL, 10 continuous complete remission (CCR) AL and 17 normal controls; the mRNA of cyclin B1 and p21(cipl), and the proliferation cell nuclear antigen (PCNA) in patients and normal controls were detected with semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The results showed that the expression of cyclin B1 in newly diagnosed AL patients was significantly higher than that in normal controls. For the relapsed AL patients, the cyclin B1 expression was also higher than that in normal controls, but lower than that in newly diagnosed cases, there was no significant difference between the remission cases and normal controls, nor difference between CCR AL patients and normal controls. All patients with high cyclin B1 expression had an unscheduled expression manner, that cyclin B1 protein appeared in G(1) phase, and in some case it even higher than that of G(2) phase. The response rate (partial remission + complete remission) and survival rate in the cyclin B1 high expressed patients were higher than that of cyclin B1 low expressed patients. The relapse rate in cyclin B1 high expressed patients was higher than that in cyclin B1 normally expressed patients. The survival rate in cyclin B1 high expressed patients was higher than that in cyclin B1 low expression patients. A negative correlation between the expression of cyclin B1 and p21(cipl) was observed. Additionally, cyclin B1 protein expression was generally correlated with proliferation index (PI) and proliferation cell nuclear antigen (PCNA). It is concluded that this study demonstrates for the first time cyclin B1 overexpression and abnormally distribution in cell cyclin of newly diagnosed AL patients. It was considered that cyclin B1 may play an important role in leukemic pathogeneses and can be one of the factors influencing the prognosis of AL patients.

Growth and gene expression of leukemia cell after treated with methylation inhibitor 5-aza-2'-deoxycytidine / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of demethylation therapy of leukemia by 5-aza-2'-deoxycytidine (5-aza-CdR).</p><p><b>METHODS</b>By using MTT test, NBT reduction reaction and DNA agarose gel electrophoresis, changes in proliferation, differentiation and apoptosis were observed in K562, HL-60 and fresh leukemia cells after treated with 5-aza-CdR. The mRNA expressions of DNMTs, p15, p53 and bcl-2 were measured by RT-PCR. The status of p15(INK4B) gene methylation was examined by methylation-specific PCR (MSP-PCR).</p><p><b>RESULTS</b>The growth inhibition of K562, HL-60 and fresh leukemia cells displayed a dose and time-dependent manner after treated by 5-aza-CdR. The differentiation-inducing ability on HL-60 cells was obvious at 0.5 micromol/L of 5-aza-CdR. The up-regulation of p15 mRNA and p53 mRNA expression and down-regulation of bcl-2 mRNA expression were obvious as compared with the control, but the DNMTs expression was not significantly different from the control. The methylation status of p15 gene in fresh leukemia cells decreased gradually with increasing concentration of 5-aza-CdR.</p><p><b>CONCLUSION</b>The proliferation of leukemia cells was obviously inhibited by 5-aza-CdR, its mechanism maybe related to the up-regulation of p15 and p53 genes and down-regulation of bcl-2 gene. The decrease of p15 gene methylation was associated with the competitive inhibition of 5-aza-CdR.</p>

Clinical Prognostic Value of Expression of DNA Methyltransferases Genes in Children with Acute Leukemia / 实用儿科临床杂志

Objective To investigate the relationship between the expression of DNA methyltransferases(DNMT)and clinical prognosis in children with acute leukemia(AL).Methods The mRNA expressions of DNMT1,DNMT3A,DNMT3B,p15,mdrl were measured in 56 AL children and 20 normal controls by semi-quantitative reverse transcriptionpolymerse chain reaction.Results In 56 cases of children with AL,the positive rate of DNMT1 was 73.2%(41/56);the positive rate of DNMT3A was 67.9%(38 /56);the positive rate of DNMT3B was 64.3%(36/56).Thirty-one cases showed positive expressions of the 3 DNMT simultaneously,4 cases with negative expressionss imultaneously,21 cases with at least 1 positive expression of the DNMT,positive rate of p15 was 19.6%(11/56);positive rate of mdrl was 28.6%(16/56),all 3 simultaneous expressions of the 3 DNMT in AL children were significantly higher than those in normal controls(P