[Inhibitory Effect of Resveratrol on the Proliferation of Multiple Myeloma Cells and the Underlying Mechanism].

OBJECTIVE: To investigate the effect of resveratrol (RSV) on the proliferation of multiple myeloma (MM) cells and its molecular mechanism. METHODS: MM cells (MM1.S, RPMI-8226 and U266) were treated with different concentrations of RSV for 24-72 h. The effect of RSV on the proliferation of MM cells was detected by CCK-8 (cell counting kit-8) assay. RPMI-8226 cells were divided into RSV, miR-21 mimic, RSV+miR-21 mimic, miR-21 inhibitor and RSV+miR-21 inhibitor groups, and transfected with corresponding plasmids. The cell cycle distribution of each group was detected by flow cytometry with propidium iodide (PI) single staining. The cell apoptosis of each group was detected by AnnexinV-FITC/PE-PI double staining. The expression of miR-21 in MM cells treated with RSV and the expression of KLF5 mRNA in each group were detected by qRT-PCR. The expression of KLF5 protein in each group was detected by Western blot. RESULTS: RSV inhibited the proliferation and induced apoptosis of MM cells in a time- and dose-dependent manner. After the MM cells were treated with RSV, the number of cells in sub-G1 phase was increased, and that in G2/M phase was decreased. Moreover, RSV significantly downregulated the expression of miR-21 in MM cells, and the inhibitory effect of miR-21 mimic on KLF5 expression in MM cells was counteracted by RSV. CONCLUSION: RSV may inhibit the proliferation and induce apoptosis of MM cells by inhibiting miR-21 and up-regulating KLF5 expression.

[Clinical Analysis of 12 cases of Systemic Lupus Erythematosus Associated with Thrombotic Thrombocytopenic Purpura].

OBJECTIVE: To investigate the clinical manifestations, treatment strategies and outcomes of 12 patients with systemic lupus erythematosus (SLE) associated with thrombotic thrombocytopenic purpura(TTP). METHODS: The clinical data from 12 cases of SLE associated with TTP admitted in the Second Hospital of Hebei Medical University from January 2002 to August 2015 were retrospectively analyzed. RESULTS: 12 cases of SLE associated with TTP included 11 females and 1 male, their median age was 34.5 years old, among them 5 cases of TTP were diagnosed during the treatment of SLE, 7 cases of TTP were comfirmed together with SLE on admission. The hemolytic anemia, thrombocytopenia and neurological deficits appeared in all the patients, the renal impairment was observed in 10 cases, the schistocytes of peripheral blood smears (>1%) were present in 9 cases, a severely reduction of ADAMTS 13 activity (<5%) with inhibitor-positive had been demonstrated in 5 cases, all of the 12 patients were treated with glucocorticoid, and 11 cases were treated in combination with other drug(10 cases combined with cytotoxics, 1 case with intravenous gamma globulin, 1 case with rituximab), plasma exchange were used in 10 cases, and 2 cases died, 2 cases without receiving plasma exchange all died, renal damage was observed in all the dead patients. CONCLUSION: Clinical manifestation and repeated examinations of peripheral blood smears are helpful for early diagnosis of SLE associated with TTP, the plasma exchange combined with glucocortcoids is an effective treatment method, the renal impairment may be a risk factor related with poor prognosis.

Impacts of K562 cells towards activities of Toll-like receptor pathway of human mesenchymal stem cell-bone marrow.

The study aim was to investigate the impacts of K562 cells towards the activities of Toll-like receptor pathway of human mesenchymal stem cell-bone marrow (HMSC-bm). The in vitro co-culture of HMSC-bm and K562 cells was set as the experiment group (HMSC-bm + K562), the HMSC-bm cultured alone was set as the control group (HMSC-bm), the expressions of six interested genes and their proteins, namely MyD88, P38, NF-κB, TAB1, TLR3 and TBK1, of the Toll -like receptor signaling pathway were detected and compared, as well as the secretions of such cytokines as IL-6, IL-8, TNF-α and IFN-α in the cell supernatant, which were regulated by the Toll-like receptor pathway. The expressions of MyD88, P38, TAB1 and TLR3 of the HMSC-bm + K562 group were higher than the HMSC-bm group, while that of TBK1 was lower, and the NF-κB expression showed no significant difference between the two groups (P > 0.05). Compared with the HMSC-bm group, the supernatant of HMSC-bm + K562 group exhibited the higher secretion levels of IL-6 and IL-8, while that of IFN-α was just contrary, and the differences were significant (P < 0.05). The secretion levels of TNF-α within the two groups were not significantly different (P > 0.05). The co-culture of K562 and HMSC-bm could induce the activity changes of Toll-like receptor pathway of HMSC-bm, which was beneficial towards the proliferation of K562 cells.

[SHP-1 gene in the disease progression of chronic myeloid leukemia].

OBJECTIVE: To investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia (CML). METHODS: The expression level of SHP-1 mRNA and protein in bone marrow or peripheral blood mononuclear cells from CML patients were detected by Western blot and SYBR Green-based qRT-PCR. The methylation status of SHP-1 were assessed by methylation-specific polymerase chain reaction (MSP) assay. K562 cells were infected with the lentiviral plasmids pEX-SHP-1-puro-Lv105 (K562-SHP-1) or pEX-EGFP-puro-Lv105 (K562-EGFP). The levels of proteins and phosphorylated proteins were detected by Western blot. qRT-PCR assay was used to test the level of BCR-ABL mRNA. RESULTS: The relative levels of SHP-1 mRNA were sharply decreased in advanced stages CML compared to chronic phase (CP)-CML (0.79±0.37 vs 1.18±0.64, P= 0.009). The level of SHP-1 protein was lower in advanced stages CML compared to CP-CML (0.57±0.02 vs 1.02±0.04, P=0.039). The frequency of SHP-1 gene promoter methylation at selected loci in CP-CML was 23.8% (10/42), and the methylated regions were detected in all advanced CML samples (P<0.01). SHP-1 was stably transfected into K562 cells and selected with puromycin. Overexpression of SHP-1 inhibited the proliferation and induced the apoptosis of K562 cells, meanwhile leaded to G0/G1 phase arrest. After transfection, the level of BCR-ABL mRNA was not affected in K562-SHP-1 cells (1.32±0.34) compared to K562-EGFP cells (1.18±0.20, P=0.644), but overexpression of SHP-1 caused a slight decrease in BCR-ABL protein in K562-SHP-1 cells compared to K562 -EGFP cells (0.78±0.15 vs 1.27±0.24, P=0.040). Overexpression of SHP-1 resulted in a remarkable decrease in MYC protein, phosphorylated forms of JAK2, STAT5, Akt and MAPK. However, the un-phosphorylated forms of these molecules were not significantly affected. CONCLUSION: Decreased expression of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulation of BCR-ABL, Akt, MAPK, MYC, JAK2 and STAT5 signaling.

[BMMSC from blastic phase CML down-regulate leukemia cell apoptosis].

The purpose of this study was to investigate the effect of bone marrow mesenchymal stem cells (BMMSC) from patients with chronic myeloid leukemia (CML) in blastic phase (Bp) on K562 cells and the primary CML-Bp cells, and to explore its potential mechanisms. K562 cells and primary CML-Bp cells were co-cultured with BMMSC of different groups; the cell proliferation was detected by MTT method, the cell apoptosis rate and mitochondrial membrane potential were measured by flow cytometry, the expression levels of Caspase-8, Caspase-9, and activated Caspase-3 in cells were measured by Western blot. The results showed that the CML-Bp BMMSC could enhance the survival rate of K562 cells treated with adviamycin (ADM) and display protective effect on K562 cells and primary CML-Bp mononuctear cells, inhibited ADM-induced leukimia cell apoptosis (P < 0.05); as compared with CML-chronic phase (CML-Cp) BMMSC and normal BMMSC, the CML-Bp BMMSC showed the highest protective effect on leukemic cells, the mitochondrial membrane potential of co-cultured cells slightly droped (P < 0.05). In the CML-Bp BMMSC cultured with K562 cells, the expression level of caspase-3 was more down-regulated than that in K562 alone plus ADM group, while the expression of caspase-9 significantly increased (P < 0.05). It is concluded that the CML-Bp BMMSC down-regulates ADM-induced leukemia cell appoptosis, its mechanism may relate with the inhibition of mitochondrial membrane potential drop, the stabilization of unactive expression of caspase-9 and down-regulation of caspase-3 expression.

Methylation and decreased expression of SHP-1 are related to disease progression in chronic myelogenous leukemia.

Despite the unprecedented success of tyrosine kinase inhibitors (TKIs) in treating chronic myelogenous leukemia (CML), some patients nevertheless progress to advanced stages of the disease. Thus far, the biological basis leading to CML progression remains poorly understood. SH2-containing tyrosine phosphatase 1 (SHP-1) is reported to bind to p210BCR­ABL1 and to function as a tumor suppressor. Furthermore, its substrates have been found to be essential for p210BCR-ABL1 leukemogenesis or CML progression. In the present study, we found that SHP-1 mRNA and protein levels were markedly decreased in patients in the accelerated and blastic phases of CML (AP-CML and BP-CML) compared to those in the chronic phase (CP-CML). In vitro, we demonstrated that overexpression of SHP-1 reduced p210BCR-ABL1 protein expression and activity in the K562 CML cell line and negatively regulated the AKT, MAPK, MYC and JAK2/STAT5 signaling pathways. Moreover, using a methylation-specific polymerase chain reaction (MSP) assay, abnormal methylation of the SHP-1 gene promoter region was found both in K562 cells and bone marrow (BM) or peripheral blood (PB) cells from AP-CML and BP-CML patients. In conclusion, our findings suggest that decreased expression levels of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulating BCR-ABL1, AKT, MAPK, MYC and JAK2/STAT5 signaling.

Effect of bone marrow mesenchymal stem cells from blastic phase chronic myelogenous leukemia on the growth and apoptosis of leukemia cells.

Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a chronic phase to a terminal blast crisis phase. However, the mechanisms that lead to disease progression remain unclear. Bone marrow mesenchymal stem cells (BMMSCs) play important roles in maintaining the bone marrow microenvironment. In the present study, the biological characteristics of BMMSCs were determined including proliferation, apoptosis and secretion of cytokines during blastic phase CML (CML-Bp). The effect of BMMSCs in CML-Bp on K562 human CML cells and the CML-Bp original generation leukemia cells were also explored. Our results showed that CML-Bp BMMSCs protect tumor cells and increase their anti-apoptotic ability through regulating the expression of apoptosis-related proteins and activating the Wnt pathway.

[Inhibitory effects of rapamycin on proliferation of chronic myelogenous leukemia cells and its mechanism].

OBJECTIVE: To explore the inhibitory effects of rapamycin on proliferation of chronic myelogenous leukemia (CML) cells and its possible mechanism. METHODS: The effects of rapamycin at various concentrations on cell proliferation of CML cell line K562 cells were analyzed by MTT. The expressions of mTOR, 4E-BP1 and p70S6K at protein and mRNA level in K562 cells with rapamycin treatment were detected by Western blot and RT-PCR. The protein expressions and phosphorylation of mTOR, 4E-BP1 and p70S6K in primary bone marrow cells from CML patients at chronic phase (CP) were also investigated by Western blot, bone marrow cells from healthy people were used as control. Data were analyzed by the χ(2) test, Fisher's exact test and one-way analysis of variance (ANOVA). RESULTS: The phosphorylation of mTOR, 4E-BP1 and p70S6K were significantly increased in CML bone marrow cells compared with that of normal control (70.6% vs 30.0%, 76.5% vs 40.0%, 73.5% vs 20.0%, respectively, P < 0.05). The proliferation of K562 cells was significantly inhibited with 20 nmol/L and more rapamycin treatment. The phosphorylation of mTOR was decreased after rapamycin treatment, as well as the expressions of 4E-BP1 and p70S6K at protein and mRNA level (P < 0.05). CONCLUSION: mTOR signaling played an important role in CML pathogenesis, and rapamycin could decrease CML cells proliferation by inhibiting the activity of mTOR signaling in vitro.