Targeting cytohesin-1 suppresses acute myeloid leukemia progression and overcomes resistance to ABT-199.

Adhesion molecules play essential roles in the homeostatic regulation and malignant transformation of hematopoietic cells. The dysregulated expression of adhesion molecules in leukemic cells accelerates disease progression and the development of drug resistance. Thus, targeting adhesion molecules represents an attractive anti-leukemic therapeutic strategy. In this study, we investigated the prognostic role and functional significance of cytohesin-1 (CYTH1) in acute myeloid leukemia (AML). Analysis of AML patient data from the GEPIA and BloodSpot databases revealed that CYTH1 was significantly overexpressed in AML and independently correlated with prognosis. Functional assays using AML cell lines and an AML xenograft mouse model confirmed that CYTH1 depletion significantly inhibited the adhesion, migration, homing, and engraftment of leukemic cells, delaying disease progression and prolonging animal survival. The CYTH1 inhibitor SecinH3 exerted in vitro and in vivo anti-leukemic effects by disrupting leukemic adhesion and survival programs. In line with the CYTH1 knockdown results, targeting CYTH1 by SecinH3 suppressed integrin-associated adhesion signaling by reducing ITGB2 expression. SecinH3 treatment efficiently induced the apoptosis and inhibited the growth of a panel of AML cell lines (MOLM-13, MV4-11 and THP-1) with mixed-lineage leukemia gene rearrangement, partly by reducing the expression of the anti-apoptotic protein MCL1. Moreover, we showed that SecinH3 synergized with the BCL2-selective inhibitor ABT-199 (venetoclax) to inhibit the proliferation and promote the apoptosis of ABT-199-resistant leukemic cells. Taken together, our results not only shed light on the role of CYTH1 in cell-adhesion-mediated leukemogenesis but also propose a novel combination treatment strategy for AML.

Lethal pulmonary thromboembolism in mice induced by intravenous human umbilical cord mesenchymal stem cell-derived large extracellular vesicles in a dose- and tissue factor-dependent manner.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have obvious advantages over MSC therapy. But the strong procoagulant properties of MSC-EVs pose a potential risk of thromboembolism, an issue that remains insufficiently explored. In this study, we systematically investigated the procoagulant activity of large EVs derived from human umbilical cord MSCs (UC-EVs) both in vitro and in vivo. UC-EVs were isolated from cell culture supernatants. Mice were injected with UC-EVs (0.125, 0.25, 0.5, 1, 2, 4 µg/g body weight) in 100 µL PBS via the tail vein. Behavior and mortality were monitored for 30 min after injection. We showed that these UC-EVs activated coagulation in a dose- and tissue factor-dependent manner. UC-EVs-induced coagulation in vitro could be inhibited by addition of tissue factor pathway inhibitor. Notably, intravenous administration of high doses of the UC-EVs (1 µg/g body weight or higher) led to rapid mortality due to multiple thrombus formations in lung tissue, platelets, and fibrinogen depletion, and prolonged prothrombin and activated partial thromboplastin times. Importantly, we demonstrated that pulmonary thromboembolism induced by the UC-EVs could be prevented by either reducing the infusion rate or by pre-injection of heparin, a known anticoagulant. In conclusion, this study elucidates the procoagulant characteristics and mechanisms of large UC-EVs, details the associated coagulation risk during intravenous delivery, sets a safe upper limit for intravenous dose, and offers effective strategies to prevent such mortal risks when high doses of large UC-EVs are needed for optimal therapeutic effects, with implications for the development and application of large UC-EV-based as well as other MSC-EV-based therapies.

The transplantation of rapamycin-treated senescent human mesenchymal stem cells with enhanced proangiogenic activity promotes neovascularization and ischemic limb salvage in mice.

Few therapies can reverse the proangiogenic activity of senescent mesenchymal stromal/stem cells (MSCs). In this study, we investigated the effects of rapamycin on the proangiogenic ability of senescent human umbilical cord MSCs (UCMSCs). An in vitro replicative senescent cell model was established in cultured UCMSCs. We found that late passage (P25 or later) UCMSCs (LP-UCMSCs) exhibited impaired proangiogenic abilities. Treatment of P25 UCMSCs with rapamycin (900 nM) reversed the senescent phenotype and notably enhanced the proangiogenic activity of senescent UCMSCs. In a nude mouse model of hindlimb ischemia, intramuscular injection of rapamycin-treated P25 UCMSCs into the ischemic limb significantly promoted neovascularization and ischemic limb salvage. We further analyzed the changes in the expression of angiogenesis-associated genes in rapamycin-primed MSCs and found higher expression of several genes related to angiogenesis, such as VEGFR2 and CTGF/CCN2, in primed cells than in unprimed MSCs. Taken together, our data demonstrate that rapamycin is a potential drug to restore the proangiogenic activity of senescent MSCs, which is of importance in treating ischemic diseases and tissue engineering.

BCR-ABL1-positive microvesicles malignantly transform human bone marrow mesenchymal stem cells in vitro.

The intercellular communication between leukemia cells and bone marrow mesenchymal stem cells (BM-MSCs) plays more important role in chronic myeloid leukemia (CML) than we previously understood. Recently, we found that microvesicles released from human leukemia cell line K562 (K562-MVs) containing BCR-ABL1 mRNA malignantly transformed normal hematopoietic transplants. Here, we investigated whether K562-MVs contribute to the transformation of human bone marrow mesenchymal stem cells (BM-MSCs). We showed that K562-MVs could be integrated into co-cultured normal BM-MSCs and dose-dependently enhanced the proliferation of BM-MSCs. Meanwhile, K562-MVs (400 ng/mL) significantly increased the expression of BCR-ABL1 in these BM-MSCs, accompanied by the enhanced secretion of TGF-ß1. These BM-MSCs in turn could trigger the TGF-ß1-dependent proliferation of K562 cells. Moreover, we confirmed the presence of BCR-ABL1 in circulating MVs from 11 CML patients. Compared to the normal BM-MSCs, the BM-MSCs from CML patients more effectively increased the BCR-ABL1 expression and TGF-ß1 secretion in K562 cells as well as the proliferation of K562 cells. Our findings enrich the mechanisms involved in the interaction between leukemia cells and BM-MSCs and provide novel ways to monitor minimal residual disease and worthwhile approaches to treat CML.

Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro.

AIM: To investigate the effects of serum deprivation (SD) on microvesicles (MVs) secreted from human myeloma cells and the implications for disease progression. METHODS: RPMI 8226, U266, and KM3 human myeloma cells were incubated in medium containing 10% (non-SD) or 1% fetal bovine serum (SD) and MVs were isolated. The levels and size distribution of MVs were analyzed with flow cytometry. The protein profiles of MVs were studied using 2D SDS-PAGE, MALDI-TOF-MS, and Western blotting. NF-κB activation was analyzed using EMSA. Angiogenesis was examined in Eahy926 endothelial cells. RESULTS: Exposure of RPMI 8226 cells to SD for 24 h did not alter the number of apoptotic cells. However, SD increased the number of MVs from RPMI 8226, U266, and KM3 cells to 2.5-, 4.3-, and 3.8-fold, respectively. The size distribution of SD MVs was also significantly different from that of non-SD MVs. Three proteins ZNF224, SARM, and COBL in SD MVs were found to be up-regulated, which were involved in cell cycle regulation, signal transduction and metabolism, respectively. Co-culture of SD MVs and RPMI 8226 cells increased NF-κB activation in the target RPMI 8226 cells. Furthermore, SD MVs from RPMI 8226 cells significantly increased the microtubule formation capacity of Eahy926 endothelial cells compared with non-SD MVs. CONCLUSION: SD elevates the levels of microvesicles with different size distribution and selectively enriched proteins in human myeloma cells in vitro. The selectively enriched proteins, especially ZNF224, may play key roles in regulation of myeloma cells, allowing better adaptation to SD.

Microvesicles secreted from human multiple myeloma cells promote angiogenesis.

AIM: To investigate whether human multiple myeloma (MM) cells secrete microvesicles (MVs) and whether the MVs secreted from MM cells (MM-MVs) promote angiogenesis. METHODS: RPMI8226 human MM cells and EA.hy926 human umbilical vein cells were used. MVs isolated from RPMI8226 cells were characterized under laser confocal microscopy, electron microscopy and with flow cytometry. The fusion of MM-MVs and EA.hy926 cells was studied under confocal microscopy, and the transfer of CD138 to EA.hy926 cells was demonstrated with flow cytometry. The proliferation, invasion and tube formation of EA.hy926 cells in vitro were evaluated using MTT, transwell migration and tube formation assays, respectively. The vasculization of EA.hy926 cells in vivo was studied using Matrigel plug assay. The expression of IL-6 and VEGF was analyzed with PCR and ELISA. RESULTS: MM-MVs from the RPMI 8226 cells had the characteristic cup-shape with diameter of 100-1000 nm. Most of the MM-MVs expressed phosphatidylserine and the myeloma cell marker CD138, confirming that they were derived from myeloma cells. After added to EA.hy926 cells, the MM-MVs transferred CD138 to the endothelial cells and significantly stimulated the endothelial cells to proliferate, invade, secrete IL-6 and VEGF, two key angiogenic factors of myeloma, and form tubes in vitro and in vivo. CONCLUSION: Our results confirm the presence of MVs in MM cells and support the idea that MM-MVs are newfound mediators for myeloma angiogenesis and may serve as a therapeutic target to treat MM.

Immune protection function of multipotent mesenchymal stromal cells: role of transforming growth factor-ß1.

The immunosuppressive functions of multipotent mesenchymal stromal cells (MSCs) may give cancer cells a survival advantage. This study tests the hypothesis that MSCs protect leukemia cells from immune clearance. Our results demonstrate that MSCs are capable of inhibiting peripheral blood mononuclear cells (PBMNCs) proliferation and their migration toward leukemic cells by the reduction of CCL5 and CXCL12. In addition, we find that MSCs can inhibit the cytolytic functions of NK-cells and CTLs. TGF-ß1 secreted by MSCs is responsible for impaired CTLs and NK function by down-modulating surface NKG2D expression. These inhibitory functions of MSCs have negative effects on the CTLs or NK-mediated graft-versus-leukemia (GVL), particularly in the allogeneic hematopoietic stem cells transplantation setting.

Immunomodulatory function of regulatory dendritic cells induced by mesenchymal stem cells.

Mesenchymal stem cells (MSCs) provide an excellent model for development of stem cell therapeutics, and their potential treatment in the immunopathogenic diseases have gained further interest after demonstration of immunomodulatory effects on complicated interactions between T cells and even dendritic cells (DCs). However, the mechanisms underlying these immunoregulatory effects of MSCs are poorly understood. In this study, we show that bone marrow derived MSCs can differentiate mature DCs (mDCs) into a distinct regulatory DC population. Compared with mDCs, they have lower expression of CD1a, CD80, CD86 and CD40, but higher expression of CD11b. MSCs induced DCs (MSC-DCs) can hardly stimulate T-cell proliferation even when MSC-DCs are stimulated by LPS. In addition, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-DCs are also observed. Moreover, MSC-DCs can efficiently generate CD4+CD25+Foxp3+ Treg cells from CD4+CD25-Foxp3-T cells. The inhibitory function of MSC-DCs is mediated not only through TGF-ß1, but also by inducing the production of Treg cells or T-cell anergy. These results demonstrate that the immunomodulatory effects of regulatory DCs induced by MSCs provide efficacious treatment for immunopathogenic diseases.

Imatinib plasma trough concentration and its correlation with characteristics and response in Chinese CML patients.

AIM: To investigate the pharmacokinetics of imatinib in Chinese chronic myelogenous leukemia (CML) patients. METHODS: Fourty-six naïve Chinese CML patients treated with imatinib (400 and 600 mg daily, n=36 and 10, respectively) were recruited. The correlations of imatinib (400 mg) trough plasma concentrations (C(mins)) with the patients' characteristics and responses were analyzed. RESULTS: The overall mean (+/-SD, CV%) steady-state C(mins) for imatinib at 400 mg (n=36) and 600 mg (n=10) daily was 1325.61 ng/mL (+/-583.53 ng/mL; 44%) and 1550.90 ng/mL (+/-462.63 ng/mL; 30%), respectively, and no statistically significant differences were found between them (P=0.267). At 400 mg daily, female patients had significantly higher C(mins) than the male patients (P=0.048), and molecular responses were not correlated with imatinib C(mins), but they were correlated with time elapsed before imatinib therapy. CONCLUSION: The results suggest that Chinese CML patients have higher imatinib C(mins) than their Caucasian counterparts and that the optimal initial imatinib dose for them requires further investigation.

A comparative study of outcomes of idarubicin- and etoposide-intensified conditioning regimens for allogeneic peripheral blood stem cell transplantation in patients with high-risk acute leukemia.

AIM: To analyze the results of idarubicin (IDA)- versus etoposide (VP16)-intensified myeloablative conditioning regimen in allogeneic hematopoietic stem cell transplantation (allo-SCT) for high-risk acute leukemia. METHODS: From January 2005 to June 2008, 48 consecutive patients (male: n=29; median age: 30 years, range 14-51 years) with high-risk acute leukemia underwent allo-SCT following an IDA- or VP16-intensified conditioning regimen. The conditioning regimens were modified BUCY2 (busulfan+cyclophosphamide) consisting of IDA (15 mg/m2 per day, days -12 to -10) or VP16 (25 mg/kg per day, days -3 to -2) and CY/TBI (cyclophosphamide/total body irradiation) intensified with IDA (15 mg/m2 per day, days -6 to -5) or VP16 (25 mg/kg per day, days -3 to -2) for acute myeloid leukemia and acute lymphoblastic leukemia, respectively. RESULTS: Between the two groups, no significant differences in terms of baseline characteristics, incidence of acute or chronic graft-versus-host disease (GVHD) or transplant-related mortality (TRM) (P=0.50) were observed. However, the IDA group demonstrated higher incidences of mucositis and Aspergillus pneumonia (P<0.01 and P=0.03, respectively). For the IDA and VP16 groups, relapse rates were 28% and 50%, respectively (P=0.13). For the same groups, the 2-year probabilities of leukemia-free survival (LFS) and overall survival (OS) were 72% versus 51% (P=0.04) and 74% versus 53% (P=0.04), respectively. CONCLUSION: This retrospective analysis suggests that conditioning regimens intensified with IDA can achieve better outcomes than conditioning regimens with VP16 in patients preparing to undergo allo-SCT for high-risk acute leukemia.

Fludarabine and cytarabine combined chemotherapy followed by transfusion of donor blood stem cells for treating relapse of acute leukaemia after allogeneic haematopoietic stem cell transplantation.

BACKGROUND: Relapse remains an obstacle to successful allogeneic haematopoietic stem cell transplantation (allo-HSCT) for patients with acute leukaemia and no standard treatment is available. We assessed fludarabine and cytarabine with transfusion of donor haematopoietic stem cell in treating the relapse of acute leukaemia after allo-HSCT. METHODS: Seven patients, median age 34 years, with relapse of acute leukaemia after allo-HSCT received combination chemotherapy of fludarabine with cytarabine for 5 days. Five patients suffered from acute myeloid leukaemia (2 refractory) and 2 refractory acute lymphoblastic leukaemia. After the transplantation, the median relapse time was 110 days (range, 38 - 185 days). Two days after chemotherapy, 5 patients received infusion of donor's peripheral blood stem cells, mobilized by granulocyte colony stimulating factor. No prophylactic agents of graft versus host diseases were administered. RESULTS: Six patients achieved haematopoietic reconstitution. DNA sequence analysis at day 30 after treatment identified all as full donor chimera type. The median observation time was 189 days. After the treatment, the median time for neutrophilic granulocyte value = 0.5 x 10(9)/L and for platelet value = 20 x 10(9)/L were 13 days (range, 10 - 18 days) and 15 days (range, 11 - 24 days), respectively. Graft versus host disease occurred in 2 patients (acute) and 3 (chronic). Five patients suffered from pulmonary fungal infection (2 died), 3 haemorrhagic cystitis and 2 cytomegalovirus viraemia. The other patients died of leukaemia related deaths. Three patients with chronic graft versus host disease who had received donor peripheral blood stem cells reinfusion have survived for 375 days, 232 days and 195 days, respectively. CONCLUSIONS: Fludarabine with cytarabine plus the donor haematopoietic stem cell should be considered as an effective therapeutic regimen for relapse of acute leukaemia after allo-HSCT. The disease free state of patients may increase, though with high risk of secondary fungal infection.

Enhanced Response of Acute Monocytic Leukemia Cells to Low-dose Cytarabine by 1,25-dihydroxyvitamin D3.

Low-dose cytarabine combined with differentiating or DNA hypomethylating agents, such as vitamin D compounds, is a potential regimen to treat acute myeloid leukemia (AML) patients who are unfit for high-intensity chemotherapy. The present study aimed to determine which subset of AML would be most responsive to low-dose cytarabine with the differentiating agent 1,25-dihydroxyvitamin D3 (1,25-D3). Here, firstly, cBioPortal database was used and we found out that vitamin D receptor (VDR) was highly expressed in acute monocytic leukemia (M5) and high VDR expression was associated with a poor survival of AML patients. Then, we confirmed that 1,25-D3 at clinical available concentration could induce more significant differentiation in acute monocytic leukemia cell lines (U937, MOLM-13, THP-1) and blasts from M5 patients than in non-monocytic cell lines (KGla and K562) and blasts from M2 patient. Finally, it was shown that the combination of 1,25-D3 and low-dose cytarabine further increased the differentiating rate, growth inhibition and G0/G1 arrest, while mild changes were found in the apoptosis in acute monocytic leukemia cell lines. Our study demonstrates that the enhanced response of acute monocytic leukemia cells to low-dose cytarabine by 1,25-D3 might indicate a novel therapeutic direction for patients with acute monocytic leukemia, especially for elderly and frail ones.

Phenotypic and functional comparison of mesenchymal stem cells derived from the bone marrow of normal adults and patients with hematologic malignant diseases.

Mesenchymal stem cells (MSCs) have received much attention for their ability to differentiate into various cell types under specific conditions and to support the proliferation of hematopoietic stem cells. However, it is unclear whether the characteristics of MSCs are altered in different disease states. In this study, we obtained and expanded MSCs from bone marrow of patients with acute lymphoblastic leukemia (ALL), Hodgkin disease (HD), and non-Hodgkin lymphoma (NHL). Our results showed that MSCs derived from ALL, HD, and NHL were similar to normal adult bone marrow-derived MSCs in morphology, growth properties, surface epitopes, and differentiation ability in vitro. Moreover, MSCs derived from ALL, NHL, and HD had a normal karyotype and ultrastructure. These cells could express hematopoietic cytokines and support hematopoiesis in long-term culture. However, adherent cells isolated from bone marrow of patients with acute myeloid leukemia (AML) showed abnormal biological properties, including heterogeneity in morphology, limited proliferation capacity, and impaired differentiation and hematopoiesis support ability. These results indicate that there are differences in the characteristics of adherent cells derived from different disease states, which may be important for reasonable MSC selection in stem cell-based therapy.

[A clinical study of treating 120 cases of adult chronic myelocytic leukemia with imatinib mesylate].

OBJECTIVE: To analyze and evaluate the clinical efficacy and safety of imatinib mesylate (IM) as a tyrosine kinase inhibitor on Ph-positive or BCR/ABL positive chronic myelogenous leukemia (CML). METHODS: 120 patients diagnosed as CML with positive Ph chromosome were treated with IM 400 mg/d for CML in chronic phase (CP) (n = 90) or 600 mg/d for CML in accelerated or blastic phase (AP or BP) (n = 30) once daily. Hematological, cytogenetic and molecular effects of IM on the disease process of these patients were evaluated with blood and marrow cells morphology examination, G-band conventional cytogenetics analysis for Ph chromosome and PCR assay for BCR/ABL gene. The treatment efficacy and safety were retrospectively studied. RESULTS: (1) In CML-CP patients, after a follow-up of 9 ( range 3-42) months, cumulative complete hematological response (CHR), complete cytogenetic response (CCyR) and complete molecular response (CMR) rates were 73.3%, 66.7% and 54.4%, which was not influenced by prior treatment of interferon. CMR was better when time from diagnosis to treatment with IM was < or = 6 months (P < 0.05). The effect was not related with sex or age (P < 0.05). It is significant that the time to first CHR and time to first CCyR were related with the time to first CCyR and the time to first negative BCR/ ABL, respectively (both P < 0.05), while there was no relation between the time to first CHR and the time to first negative BCR/ABL (P > 0.05). (2) CHR, CCyR and CMR rates of the patients with progressive course (AP and BP) were 43.3%, 25.9% and 25.0%, respectively. The total mortality rate was 30.0%. (3) The mortality rate of the patients with age < or = 25 was higher than those >25 (P < 0.05). (4) Grade 3 leukocytopenia occurred in 16.0% of the patients and grade 3 thrombocytopenia in 18.0% of the patients and they 12 (5-20) weeks and 9 (3-16) weeks after the treatment, respectively. Nonhematological toxicities were common and tolerable. CONCLUSION: IM can lead to considerable hematological, cytogenetic and molecular response rates in CML, especially CML-CP patients, with minor tolerable side effects.

[Role of amifostine against the effect of etoposide on non-Hodgkin lymphoma bone marrow derived mesenchymal stem cells: an in vitro experiment].

OBJECTIVE: To study the protective role of amifostine (WR-2721) in the mesenchymal stem cells (MSC) derived from non-Hodgkin lymphoma (NHL) bone marrow treated with etoposide (VP-16). METHODS: MSC were obtained from non-Hodgkin lymphoma bone marrow, cultured in expanded medium, and then divided into 4 groups: Group A, without treatment by either WR-2721 or VP-16 and used as control group, Group B, treated with WR-2721, Group C, treated with WR-2721 + VP-16, and Group D, treated with VP-16 alone. Inverted microscopy was used to observe the growth of the MSC. Flow cytometry was used to observe the apoptosis and immunophenotypes of the MSC. Mononuclear growth and apoptosis of the MSC Mononuclear bone marrow cells were obtained from 5 healthy volunteers and added into the MSC of the 4 groups, 4 weeks later methylcellulose progenitor assay and then inverted microscopy were used to measure the numbers of colony so as to detect the hematopoiesis. MSC of different groups were put into osteocyte-inducing and adipocyte-inducing media respectively, and 2 weeks later Von Kossa staining and oil-red O staining were used to identify the differentiation into bone and fat. RESULTS: The NHL derived MSC of the 4 groups all showed a typical fibroblast-like morphology and were all positive in CD29, CD44, and CD105, while negative in CD11b, CD31, CD34, CD45, and HLA-DR. The apoptotic rate of Group D was 31.2% +/- 4.3%, significantly higher than those of the other 3 groups (all P < 0.05), and the apoptotic rate of Group C was significantly higher than those of Groups A and B (both P < 0.05), however, still significantly lower than that of Group D (P < 0.05). The ability to support hematopoiesis of Group B was not significantly different from that of Group A, and Groups C and D showed a lower ability to support hematopoiesis in comparison with Groups A and B, However, the ability of Group C was still significantly higher than that of Group D (P < 0.05). Under suitable conditions, all the MSC differentiated into osteocytes or adipocytes. CONCLUSION: Amifostine alone has no effects on proliferation, apoptosis, immunophenotype or ability of hematopoiesis support of the MSC in vitro; however, it can protect NHL patient-derived MSC from injury by VP-16 in vitro.

[Proliferation inhibiting and apoptosis inducing effects of parthenolide on human multiple myeloma cells].

OBJECTIVE: To investigate the effect of parthenolide (PTL) on human multiple myeloma (MM) cells in vitro and its mechanism. METHODS: Human MM cells of the line PRMI8266 were cultured and treated with PTL of the concentrations of 1, 2.5, 5, 7.5, and 10 micromol/L for 24, 48, or 72 hours. MM cells treated with DMSO were used as control group. The optical density was measured so as to draw a growth curve. The cell viability was detected by MTT and trypan-blue exclusion. The apoptosis was detected by flow cytometry. AO/EB staining and Wright-Giemsa staining were used to observe the morphological changes of the cells by fluorescence microscope and light microscope respectively. The caspase-3 activity was evaluated by BD ApoAlert Caspase Colorimetric Assay Kit. RESULTS: PTL significantly inhibited the proliferation and viability of the MM cells time and dose-dependently (all P < 0.01), and significantly induced the cell apoptosis after 48 h in a dose-dependent manner (P < 0.01). The early cell apoptosis rates for PTL of the concentrations of 2, 5 and 10 micromol/L were 17.1% +/- 2.6%, 33.6% +/- 3.8%, and 40.9% +/- 3.1% respectively, all significantly higher than that of the control group (5.6% +/- 1.2%, all P < 0.01). The MM cells treated with PTL of the concentration of 5 micromol/L for 48 h showed typical cell apoptotic features. The caspase-3 activity of the MM cells was enhanced significantly by PTL in a time and dose-dependent manner (all P < 0.01). CONCLUSION: This first report of anti-proliferation and apoptosis induction effects of PTL on MM cells shows that able to significantly inhibit the proliferation and induce the apoptosis of MM cells and enhance the caspase-3 activity, PTL may be a potentially useful drug for treatment of MM.

Adipocytes secreted leptin is a pro-tumor factor for survival of multiple myeloma under chemotherapy.

Accumulating evidences have shown that adipokines secreted from adipocytes contributes to tumor development, especially leptin. However, underlying mechanisms remain unclear. This study aims to explore the effect of leptin on development and chemoresistance in multiple myeloma cells and the potential mechanism. Analysis of levels of adipokines including leptin and adiponectin in 28 multiple myeloma patients identified significantly higher leptin compared with 28 normal controls(P < 0.05), and leptin level was positively correlated with clinical stage, IgG, ER, and ß2MG. Next, by using co-culture system of myeloma and adipocytes, and pharmacologic enhancement of leptin, we found that increased growth of myeloma cells and reduced toxicity of bortezomib were best observed at 50 ng/ml of leptin, along with increased expression of cyclinD1, Bcl-2 and decreased caspase-3 expression. We also found that phosphorylated AKT and STAT3 but not the proteins expression reached peak after 1h and 6h treatment of leptin, respectively. By using AG490, an agent blocking the phosphorylation of AKT and ERK, the proliferation of myeloma cells was inhibited, as well as the phosphorylation of AKT and STAT3, even adding leptin. Taken together, our study demonstrated that up-regulated leptin could stimulate proliferation of myeloma and reduce the anti-tumor effect of chemotherapy possibly via activating AKT and STAT3 pathways, and leptin might be one of the potential therapeutic targets for treating myeloma.

Inhibitory effects of parthenolide on the activity of NF-κB in multiple myeloma via targeting TRAF6.

This study examined the mechanism of the inhibitory effect of parthenolide (PTL) on the activity of NF-κB in multiple myeloma (MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6 (TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.

[siRNA-induced down-regulation of Livin expression increases spontaneous apoptosis in K562 cell line].

This study was aimed to observe the effects of siRNA on Livin expression and function in K562 cells. Livin siRNA were designed and synthesized, then were transfected into K562 cells by using AMAXA nucle transfactor. Expressions of Livin mRNA and protein in transfected K562 cells was detected by RT-PCR and Western blot respectively. Non-transfected cells were used as control. The enhanced green fluorescent protein plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Cell apoptosis was measured by flow cytometry with Annexin V-FITC/PI double staining. The results showed that the transfection efficiency of electroporation method was about 50. The synthesized siRNA inhibited livin expression at both mRNA and protein levels. The rate of K562 cell apoptosis increased from (9.63 ± 0.89) in control group to (12.07 ± 1.39) and (27.41 ± 2.30) at 24 h and 48 h after transfection, respectively (P < 0.05). It is concluded that the siRNA can inhibit anti-apoptosis of livin gene via down-regulating livin gene expression, which may provide the new method for anti-leukemia study.