[Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors].

OBJECTIVE: To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. METHODS: Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). RESULTS: Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). CONCLUSION: Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.

[The Value of T Cell subsets and Cytokine Levels Changes in the Clinical Diagnosis, Treatment and Prognosis Evaluation of Multiple Myeloma].

OBJECTIVE: To explore the correlation between the changes of T lymphocyte subsets and cytokines in patients with MM and immune function status, biochemical indicators, and their relationships with clinical stage and prognosis, which is expected to provide a scientific basis for the prognosis analysis and condition monitoring of MM patients. METHODS: The clinical data of 89 MM patients in two hospitals were collected, and 36 healthy people without tumor or infectious diseases were selected as the control group. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to detect the changes of core members of peripheral blood T lymphocyte subsets and cytokine levels, respectively. At the same time, automatic biochemical analyzer and automatic blood cell analyzer were used to detect serum ß2-microglobulin (ß2-MG), lactate dehydrogenase (LDH), albumin (ALB), creatinine (CRE) and hemoglobin (HGB) levels, and the relationship between T lymphocyte subsets and the above indexes and their clinical significance were analyzed. RESULTS: The proportions of NK cells and CD8+T lymphocytes in the peripheral blood of MM patients were significantly higher than that of the control group (P<0.01), the proportion of CD4+T and the ratio of CD4+/CD8+ were lower than those of the control group (P<0.05); however, there was no significant difference in the numbers CD3+T cells compared with the control group (P>0.05). The proportion of CD4+T and ratios of CD4+/CD8+ in MM patients were lower than those of normal controls, and were negatively correlated with MM staging (r=-0.964, r=-0.653), that is, the later the MM staging, the more obvious their levels were reduced, while CD8+T and NK cells were positively correlated with MM staging (r=0.891, r=0.728), that is, the later the MM staging, the more significant their levels increased. The levels of Treg cells (CD4+CD25highCD127low/-T cells/CD4+T cells) of MM patients in the disease stage Ⅰ, Ⅱ and Ⅲ were (5.87±0.92)%, (7.97±1.32)%, (11.52±4.71)% respectively, the difference was statistically significant compared with control group (P<0.05), and the level of Treg cells in MM patients with stage III was significantly higher than that in controls and patients with other disease stages (P<0.01). The proportion of Treg cells (CD4+CD25highCD127low/-T cells/CD4+T cells) in MM patients was positively correlated with the concentration of ß2-MG and LDH (r=0.793, r=0.536), but had no significant correlation with HGB, ALB and CRE. The serum levels of IL-6, IL-10 and TNF-α in MM patients were significantly higher than those in the control group (P<0.05), which were closely related to MM staging(r=0.839, r=0.917, r=0.746), that is, the later the MM staging, the higher the levels; The serum IFN-γ level was negatively correlated with the stage of MM (r=-0.689), and its level gradually decreased with the increase of the disease stage and degree (P<0.01). There was no significant correlation between the levels of IL-2 and IL-4 and the disease stage, but they were all up-regulated compared with the control group (P<0.05). CONCLUSION: The abnormal regulation of the core members of T lymphocyte subsets and the levels of various cytokines are closely related to the disease progression and poor prognosis of MM patients, which is an effective indicator for the disease monitoring of MM patients.

Tendoscopic versus open release for de Quervain's disease: earlier recovery with 7.21 year follow-up.

PURPOSE: To compare the time return to work and long-term results of tendoscopic versus open technique for de Quervain's disease. METHODS: From 2005 to 2013, either tendoscopic or open decompression was performed on 56 consecutive patients (56 wrists) with symptomatic de Quervain's disease despite a minimum of 3 months non-operative treatment. Of the 50 patients who met the inclusion criteria, 41 patients were followed-up for a mean of 7.21 years postoperatively. Among these 41 wrists, 20 underwent tendoscopic release (group A), and 21 underwent open release (group B). The clinical evaluations were performed preoperatively, 1 month postoperatively and at last follow-up visit, using visual analog scale (VAS); the Disabilities of the Arm, Shoulder and Hand (DASH) Outcome score; and the Finkelstein's test. The Patient and Observer Scar Assessment Scale (POSAS) was used as an esthetic evaluation tool of the scar at last follow-up. RESULTS: No significant baseline differences were found between two groups. The average time return to work in group A was less than in group B (P < 0.05), The mean VAS and DASH scores improved significantly in both groups at 1 month and last follow-up visit (P < 0.001). At 1 month, the scores in group A were significantly better than in group B (P < 0.05 and P < 0.001, respectively). There was no difference between groups at last follow-up. In addition, the improvement of the mean DASH score was significantly greater in group A than in group B (34.74 ± 10.99 in group A and 23.58 ± 12.01 in group B, P < 0.01) at 1 month. For POSAS scale, both the OSAS and PSAS scores were significantly better in group A. One patient in group A had cephalic vein injury and 3 patients in group B was involved with radial sensory nerve injury. All patients showed negative on Finkelstein's test at last follow-up. CONCLUSIONS: The results of this study suggest that tendoscopic technique for de Quervain's disease could provide earlier symptom relief and earlier recovery with fewer complications and more desirable scar, as well as equivalent successful long-term outcome, when compared with traditional open release technique.

[Biological Characteristics and Therapeutic Efficacy of 103 Patients with Acute Erythroleukemia].

OBJECTIVE: To investigate the biological characteristics and therapeutic efficacyt of acute erythroleukemia (AEL,AML-M6). METHODS: Blood cell count, liver function, lactate dehydrogenase level, coagulation, morphology, immunology, cell genetics and molecular biology were retrospectively analyzed in 103 cases of acute erythroleukemia patients admitted in our department from May 2016 to June 2009. The therapeutic efficacy was observed by means of remission rate, relapse rate, relapse-free survival and overall survival. RESULTS: The medians of white blood cells, granulocyte, hemoglobin and platelet were 3.04×109/L, 0.67×109/L, 66 g/L, and 45×109/L,respectively. Nucleated red blood cells were found in the peripheral blood smears from 71.1% of AEL patients. None of the patients showed abnormal coagulation function. Flow cytometry analysis indicated that CD13 (93.5%),CD117(89.1%), HLA-DR(87.0%), and CD34 (80.0%) were highly expressed in AEL, and lymphoid antigens of CD4 (42.9%) and CD7(28.9%) were expressed in partial patients. Karyotype analysis in 82 patients showed 52.4% (43/82) normal karyotype, 41.5% (34/82) abnormal karyotype, and 6.1% (5/82) failed tests. In the 34 cases with abnormal karyotype, there were 14(41.2%) cases with simple chromosomal abnomality and 20(58.8%) cases with complex karyotype. The positive rate of fusion gene accounted for 16.7% in 60 patients, and the gene mutations accounted for 77.8% in 27 patients. Among 103 cases of AEL, 81 cases were treated with chemotherapy, but 66 cases can be used for therapeutic analysis, as a results the total complete remission rate derived from 2 courses of treatment was 45.5% (30/66). The relapse rate was 36.7% (11/30), and the median relapse time was 15.5 months (6.2-50 months). The median survival time of 66 patients for therapeutic analysis was 29 months. The median survival time of CR patients was very significantly longer than that of the non-CR patients(P=0.001). The 5 year survival rate of CR patients was 65%, the median time of relapse-free survival (RFS) was 46.2 months and 3-years RFS was 58%. CONCLUSION: AEL is characterized by the highly expressed CD34 antigen, and complex karyotype. Although AEL has lower CR rate and poor prognosis, CR patients can achieve long-term survival and have good quality of life.

[Knockout of Micro-RNA-21 Gene in DLBCL OCI-Ly3 cells by TALEN Technique].

OBJECTIVE: To explore an efficient way to knockout microRNA genes in hemapoietic cell lines with a very low transfection efficiency, so as to facilitate the study of microRNA function in hematopoietic malignancies. METHODS: TALE-nucleases was utilized to knockout the microRNA-21 gene in human diffuse large B-cell lymphoma cells (OCI-Ly3). The OCI-Ly3 single cell clones without expression of miR-21 were established through eGFP(+) enrichment, PCR screening, and microRNA quantification. Finally, the miR-21 changes of mutant clones were identified by sequencing. RESULTS: Four miR-21-knockouted OCI-Ly3 single-cell-derived clones were established after 2 round transfection and screening. The miR-21 knockout efficiency was around 10/10(6) original cells. Sequencing the mutant clones indicated that miR-21 expression could be drastically reduced by simply altering sequences immediately adjacent to the microRNA duplex. CONCLUSION: This strategy may be applied to knockout any microRNA of interest even in hemapoietic cell lines with very low transfection efficiency.

[Reversing effects of emodin on multidrug resistance in resistant HL-60/ADR cells].

This study was aimed to investigate the reversing effects of emodin on multidrug resistance (MDR) in resistant HL-60/ADR cells, and to explore the underlying mechanisms. The MTT assay was used to assess the chemoresistance of HL-60/ADR cells to emodin and 8 chemotherapeutic agents commonly used in clinic. The reversal effects of emodin on MDR of HL-60/ADR cells were also evaluated by MTT method. DNA ploidy analysis and DNA Ladder assay were used to detect apoptosis-induced effects on HL-60/ADR cells via the adriamycin (ADR) and emodin combination. The expression changes of the drug resistance-associated genes and proteins were detected by RT-PCR and Western Blot respectively. The intracellular accumulation and subcellular distribution of ADR and DNR were measured by flow cytometry and confocal laser scanning microscopy. The results showed that emodin inhibited HL-60/ADR cell proliferation with an average IC50 value of 24.09 ± 1.72 µmol/L, which was similar to that of the parental HL-60 cells (average IC50 = 23.18 ± 0.87 µmol/L). HL-60/ADR cells were resistant to a variety of chemotherapeutic agents, such as ADR, DNR, VP16, VCR,Ara-C, HHT, MTZ and THP. The reversal multiple were between 1.58 and 4.12 after the treatment with low concentration of emodin combined with the above mentioned different agents. The combination of ADR with emodin showed the best reversal effects, and the typical hypodiploid peak (apoptotic peak) and DNA ladder could be detected after the co-treatment.In addition, emodin down-regulated the mRNA and protein expression levels of MRP1, TOPOIIß, GST π and BCL-2. Furthermore, the addition of emodin enhanced ADR and DNR intracellular accumulation and subcellular distribution in HL-60/ADR cells in dose-dependent manner. It is concluded that the emodin shows reversing effects on the multidrug resistant HL-60/ADR cells, possibly via decreasing the expression levels of drug resistance-associated genes, increasing the intracellular accumulation of chemotherapeutic agents and activating the apoptosis pathway.

[Efficacy of CALLG2008 protocol in treatment of adult acute lymphoblastic leukemia: a single center analysis].

CALLG2008 Protocol is sequential chemotherapy for adult acute lymphoblastic leukemia (ALL) established by Collaborative Group of adults acute lymphoblastic leukemia. It is emphasized that comprehensive treatment of adult ALL according to risk stratification is rather important. This study was purposed to evaluate the therapeutic efficacy of CALLG2008 for adult ALL. The clinical data of adult ALL patients of ≥ 14 years old diagnosed and treated by CALLG2008 Protocol were collected from May 1, 2009 to December 31, 2011 in Fujian Medical University Union Hospital, and the efficacy was analyzed. The results showed that 31 out of 33 cases of ALL achieved CR, the CR rate was up to 93.9%, the PR rate was 3.1%, and the total response rate was 97%. There were no uncontrolled severe toxicities, and no early deaths were observed. The overall survival (OS) at 1 year was only 66.7%,the relapse rate was 43.8% and the 1-year mortality was 33.3 %. This may be related with no-enough compliance, no-enough economical support and short follow-up time of the patients. The risk factor analysis showed that WBC level in newly diagnosed patients may influence the OS and relapse-free survival (RFS) of ALL. It is concluded that CALLG2008 protocol applied to adult ALL has a high remission quality and low mortality rate during the induction. The disease free survival (DFS) needs to be observed longer. It is essential to carry out MRD monitoring to determine the early recurrence and improving the long-term efficacy.

[Expression of eIF4E in patients with leukemia and its clinical significance].

This study was aimed to compare the expression level of eIF4E in patients with leukemia and normal controls, and to explore its role in leukemogenesis. White blood cells were collected in 76 leukemia patients and 10 healthy volunteers. The mRNA and protein expressions of eIF4E were detected by QT-PCR and Western blot in 39 cases of acute myeloid leukemia (AML), 15 cases of chronic myeloid leukemia (CML), 22 cases of acute lymphocytic leukemia (ALL) and 10 healthy volunteers as normal controls. The results demonstrated that compared with normal controls, the absolute expression levels of eIF4E mRNA increased in patients with AML, ALL and CML in blastic phase (P < 0.05), but had no significant change between groups of CML in chronic and accelerated phase although some increasing in group of CML in accelerated phase. The relative expression level of eIF4E mRNA had no significant change in AML, ALL, CML groups except the two subtypes of leukemia M4 and M5. Furthermore, the protein expression level in group of CML in accelerated phase and blastic phase and all acute leukemia patients including AML and ALL were higher than that in normal controls (P < 0.05). It is concluded that although its mRNA relative expressions had no significant change in most leukemia patients, the absolute expression level of eIF4E mRNA and its protein expression is up-regulated in most leukemia patients, which may play an important role in leukemogenesis, so the eIF4E may be a promising target for leukemia therapy and eIF4E-targeted therapy may be an option especially for the relapse and refractory leukemia.

[Efficacy of remission induction chemotherapy and prognostic analysis in elderly patients with acute myeloid leukemia].

OBJECTIVE: To explore the outcome of remission induction chemotherapy (IC) and prognostic in elderly patients with acute myeloid leukemia (AML). METHODS: The clinical data of 156 AML patients older than 60 years in the Institute of Hematology, Union Hospital of Fujian Medical University from January 2003 to July 2010 were analyzed retrospectively. 104 patients received cytarabine-based regimens, including protocol DA,IA or CAG,while 52 patients received palliative treatment. The median survival time was compared between patients with and without IC. The prognostic factors were evaluated by using univariate and multivariate analyses. RESULTS: 145 (93%) cases were followed-up. The median survival time was 316 days in 96 IC patients, compared with 37 days in 49 PT patients (P < 0.01). Not receiving induction chemotherapy,high-risk karyotype,hyperleukocytosis (> or = 100 x 10(9)/L), Charlson Comorbidity Index (CCI) > or = 2 were adverse prognostic factors of the survival time with univariate analysis, and all were independent poor factors affecting the survival time with multivariate analysis. CONCLUSIONS: IC can improve outcomes in elderly AML patients. The patients with hyperleukocytosis (> or = 100 x 10(9)/L) , high-risk karyotype, CCI > or = 2 and without receiving IC have poorer prognosis.

[Two novel splicing variants of eIF4E obtained by electronic cloning technique combined with RT-PCR].

In order to clone the full-length cDNA of a novel EST which is probably related to acute leukemia relapse and to analyse the sequences, the electronic cloning technique combined with RT-PCR was used to clone the full-length cDNA, and the sequences were analyzed by bioinformatics. The results showed that the two novel splicing variants of eIF4E named as splicing variant 1 and 2 of eIF4E were obtained. Bioinformatics analysis showed that variant 1 and 2 exhibited 84% and 47% similarity to eIF4E mRNA, and were localized on eIF4E locus on chromosome 4. The lengths of 2 variants are 1 904 bp and 3 393 bp, encoding 245 amino acids and 132 amino acids respectively. BLAST results showed that the both variants mentioned above contains seven exons. Among them, the sequences of the three exons at 5' end of variant 1, variant 2 and eIF4E mRNA were different from each other. Protein BLAST showed that they are partially different from eIF4E protein. It is concluded that the two novel splicing variants of eIF4E were cloned, and their relation to acute leukemia relapse needs to be further investigated.

[Emodin induces leukemic HL-60 cells apoptosis probably by inhibiting Akt signal pathway].

This study is to investigate the effect of emodin on inducing human myeloid leukemia cell line HL-60 apoptosis and the role of Akt signal pathway in the apoptosis. HL-60 cells were exposed to various dosages of emodin. MTT assay was used to detect HL-60 cell proliferation. Distribution of HL-60 cells in cell cycle was analyzed by flow cytometry and cell apoptosis was observed by MitoCapture apoptosis detection. The protein expressions of Akt signal pathway were detected by Western blotting. The result showed that emodin remarkably inhibited the cell proliferation. The IC50 value for 48 h treatment was about 20 micromol x L(-1). Apoptosis in HL-60 cells could be efficiently induced by emodin in a dose dependent manner and cells were arrested at G0/G1. The expressions of Akt, p-Akt, IkappaB-alpha, p-IkappaB-alpha, p65, p-p65, mTOR and p-mTOR in Akt signal pathway were downregulated after emodin treatment. It can be concluded that emodin could efficiently induce growth inhibition and apoptosis in HL-60 cells. Akt signal pathway may be involved in this process.

[Effects of emodin on the proliferation inhibition and apoptosis induction in HL-60 cells and the involvement of c-myc gene].

OBJECTIVE: To investigate the effects of emodin on apoptosis induction and proliferation inhibition in human apoptosis and on c-myc protein and mRNA expression in human myeloid leukemia cell line HL-60 cells. METHODS: HL-60 cells were exposed to emodin at different dosages. Growth inhibition was detected by MTT assay and colony formation assay, and cell apoptosis by flow cytometry, TUNEL labeling method, DNA fragmentation and MitoCapture apoptosis detection. The expression of c-myc was detected by RT-PCR and Western-blot. RESULTS: Emodin remarkably inhibited the cell proliferation, with an IC(50) value of 20 micromol/L. HL-60 cells apoptosis could be efficiently induced by emodin in a dose dependent manner. The c-myc protein and mRNA expressions on HL-60 cells were decreased after emodin treatment. CONCLUSION: Emodin could efficiently induce growth inhibition and apoptosis in HL-60 cells. c-myc may be involved in this process.