OBJECTIVETo explore the role of autophagy in doxorubicin (DOX)-induced resistance of human myeloma cell line RPMI8226.

METHODSWe established doxorubicin induced resistant subline of myeloma cell line RPMI8226/DOX by drug concentration step-elevation method. Resistant index of DOX was measured by MTT assay. Autophagy of myeloma cell lines RPMI8226/s and RPMI8226/DOX was detected by transmission electron microscopy, immunofluorescence (LC3-FITC) and western blot respectively. Apoptosis of RPMI8226/DOX cells induced by DOX combined with autophagic inhibitor hydroxychloroquine or 3-MA was identified by AnnexinV-FITC/PI double fluorescence dyeing.

RESULTSResistant index of RPMI8226/DOX was approximately 10.8 fold of that of RPMI8226/S. Electron microscopic studies revealed that most of RPMI8226/DOX cells displayed viable attributes and contained numerous autophagic vacuoles. Fluorescent images of RPMI8226/DOX cells showed a punctuate distribution in LC3 protein. Increased LC3-II protein in RPMI8226/DOX cells was determined by immunoblotting. There were no differences among 8 μmol/L HCQ (3.24±1.08)%, 10 mmol/L 3-MA (2.81±0.80)% or control \[(2.12±1.24)%\] (P>0.05) in terms of AnnexinV-FITC/PI double fluorescence dyeing; Compared with apoptosis of (9.75±2.15)%, (24.36±2.16)% and (40.51±3.14)% of RPMI8226/DOX cells under 2, 4 and 6 μmol/L DOX, apoptosis increased significantly after 24 h incubation under 2, 4 and 6 μmol/L DOX combined with 8 μmol/L HCQ as of \[(16.56±1.89)%, (36.44±2.91)% and (62.68±3.75)%, respectively\], or under 2, 4 and 6 μmol/L DOX combined with 10 mmol/L 3-MA as of \[(15.47±1.85)%, (39.28±3.06)% and (55.46±4.07)%, respectively\] (P<0.05).

CONCLUSIONAutophagy was involved in doxorubicin-induced resistance of myeloma cell line RPMI8226, DOX resistance in myeloma cells was reversed partly by autophagy inhibitor hydroxychloroquine or 3-MA, and autophagy may be one of mechanisms for drug resistance.

Autophagy ; drug effects ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Multiple Myeloma

This study was aimed to evaluate the therapeutic efficacy of bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation (autoPBSCT) for patients with multiple myeloma (MM). 5 patients underwent autologous hematopoietic stem cell transplantation. Bortezomib treatment was supplied for patients before autoPBSCT and in the conditioning of transplantation, it was also used in maintaining treatment. Patients with transplantation adopted bortezomib plus melphalan conditioning regimen. The number of infused MNC and number of CD34(+) cells were 4.06×10(8) (4.09×10(8) - 4.37×10(8))/kg and 3.98×10(6) (2.49×10(6) - 8.2×10(6))/kg respectively. The results showed that hematopoiesis was reconstituted in 5 patients, with a neutrophil cell count more than 0.5×10(9)/L at day 14 (13 - 25 days) after transplantation and platelet count more than 50×10(9)/L at day 28 (21 - 41 days) after transplantation. Transplantation-associated death was not observed. 5 patients were disease-free survival. In conclusion, treatment of bortezomib combined with autologous peripheral hematopoietic stem cell transplantation is an effective method for patients with multiple myeloma. Use of bortezomib after transplantation might still be favourable to MM patients, for survival prolongation and life quality improvement.
Adult ; Boronic Acids ; therapeutic use ; Bortezomib ; Combined Modality Therapy ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; therapy ; Peripheral Blood Stem Cell Transplantation ; Pyrazines ; therapeutic use ; Transplantation Conditioning ; methods

OBJECTIVETo explore the influence of bone marrow (BM) mesenchymal stem cells (MSCs) on macrophage activation after lipopolysaccharide (LPS) stimulation.

METHODSMouse BM MSCs were isolated and purified by adherence screening, and mouse peritoneal macrophages (MPM) were collected by sodium thioglycollate peritoneal injection, and the co-culture system was established by planting macrophages on the MSCs monolayer. The grouping of experiments: group A: MPM; group B: MPM + LPS; group C: MPM + LPS + MSC; group D: MPM + LPS + MSC supernatant. Cell culture supernatants were collected to detect the changes of TNF-alpha/TGF-beta and nitrogen monoxide (NO) after stimulating macrophages with LPS for 18 hours. At the same time Escherichia coli standard strain (ATCC25922) was added into the culture system and incubated for another 24 hours, macrophages were stained and phagocytosis were examined.

RESULTSThe concentrations of TNF-alpha and NO in culture supernatants were increased significantly to (147.4 +/- 37.1) pg/ml and (59.9 +/- 8.7) micromol/L respectively after macrophage activation, however, at the present of MSC, the concentration of TNF-alpha was dramatically decreased [(97.6 +/- 30.3) pg/ml, P = 0.032], and the concentration of NO was decreased to (50.9 +/- 29.5) micromol/L (P > 0.05). The concentrations of TNF-alpha and NO were further decreased after addition of MSC supernatants [(58.3 +/- 31.5) pg/ml and (-3.4 +/- 2.3) micromol/L respectively, P < 0.01]. There was no change in the phagocytic rate and phagoindex of macrophages after activation.

CONCLUSIONSMSCs can inhibit the activation of mouse peritoneal macrophages after stimulating with LPS but has no influence on the phagocytosis.

Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Lipopolysaccharides ; pharmacology ; Macrophage Activation ; drug effects ; Macrophages, Peritoneal ; immunology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Humans ; Immunoglobulin M ; Lung ; pathology ; Multiple Myeloma ; diagnosis ; pathology

OBJECTIVETo investigate the autophagy in human bone marrow mesenchymal stem cells (hBMMSC) exposed to irradiation.

METHODSThe apoptosis and necrosis rate were assessed by Annexin V and propidium (PI) staining in hBMMSC at 4h after irradiated with X-ray at 0, 2, 4, 8 and 10 Gy. The autophagy was observed by transmission electron microscopy. The mRNA expression of Beclin1 and microtubule-associated protein 1 light chain 3 (MAPLC3 or LC3) was analyzed by RT-PCR in hBMMSC at 4h after X-ray irradiation at 0, 8 and 10 Gy.

RESULTSThe apoptosis rate of hBMMSC was markedly decreased while the necrosis and death rate were slowly increased with the increase of irradiation dose when under 8 Gy. The apoptosis rate was significantly increased and reached a peak while the necrosis and whole death rate were obviously increased when irradiated with 10 Gy X-rays. In addition, the change of apoptosis rate was more significant than that of necrosis rate. By electron microscopy, a mass of autophagic vacuoles (autophagosome and autolysosome) were observed in irradiation and positive control groups, but were only occasionally seen in normal control group. The proportion of hBMMSC with autophagic vacuoles in 8 Gy irradiation group was higher than that in 10 Gy one. The mRNA expression of Beclin1 and LC3 in irradiation groups and positive control group was significantly higher than in normal control group, and so did in 8 Gy irradiation group than that in 10 Gy group.

CONCLUSIONIrradiation may induce the autophagy in hBMMSC, and autophagy could protect hBMMSC from irradiation injury in a certain dose range.

Apoptosis ; radiation effects ; Autophagy ; radiation effects ; Bone Marrow Cells ; radiation effects ; Cell Line ; Humans ; Mesenchymal Stromal Cells ; radiation effects ; X-Rays

Recent studies have shown that low dose X-irradiation shows specific effect different from high dose exposures. However, the biologic responses of bone marrow mesenchymal stem cells (BM-MSC) to low dose X-irradiation have rarely been described in the literature. This study was purposed to investigate the biologic responses of human bone marrow-derived MSC to low dose X-irradiation. The proliferation of cells was tested with MTT assay, so that the cell growth curve was drawn at 1 to 7 days. The expression of survivin mRNA was detected by RT-PCR assay; the BM-MSC DNA damage induced by X-irradiation were detected with mononuclear cell gel electrophoresis. The results indicated that the proliferative ability of BM-MSC exposed to low doses of X rays was obviously enhanced as compared with control group. The low dose X-irradiation caused the damage of DNA in X-ray dose dependent manner. X-irradiation enhanced expression of survivin in MSC. It is concluded that the low dose below 20 cGy of X-irradiation has a promoting effect on survivin expression in BM-MSC. Whether the high expression of survivin plays an important role to resist ionizing radiation needs to be further studied.
Bone Marrow Cells ; radiation effects ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dose-Response Relationship, Radiation ; Humans ; Mesenchymal Stromal Cells ; radiation effects ; X-Rays

This study was purposed to investigate the effects of interferon (IFN)-γ on expression of adhesion molecules in mesenchymal stromal cells derived from human umbilical cord tissue (UC-MSC). The UC-MSC were isolated from human umbilical cord by tissue culture. The expressions of specific markers on UC-MSC were detected by flow cytometry in the physiological condition. The adipogenic and osteogenic induction of UC-MSC was detected by alizarin and Oil red O staining. UC-MSC were exposed to IFN-γ (100, 1 000, 10 000 U/ml) for 24 h, the expressions of CD54, CD58, CD44, CD49d, CD62p, CD62L, CD102 and CD106 on cell surface were detected using flow cytometry. The results showed that in physiological condition, UC-MSC extremely low expressed CD102, CD106, CD62P, CD62L, while the expression of CD54 was relatively high (41.58 ± 0.83)%. When stimulated by IFN-γ, the expression of CD102, CD106, CD62P, CD62L increased slightly, but still low (< 5%), while CD54 and CD58 upregulated concentration-dependently up to (59.66 ± 1.36)% and (43.96 ± 0.62)% respectively. The expression of CD49d upregulated to (51.33 ± 0.74)% when UC-MSC exposed to IFN-γ 100 U/ml. CD44 increased to (73.22 ± 1.93)% when UC-MSC exposed to IFN-γ 1 000 U/ml. It is concluded that IFN-γ can elevate significantly the expression of CD54, CD49d, CD44 and CD58, but has no significant effect on CD102, CD106, CD62P and CD62L expression on the surface of UC-MSC.
Cell Adhesion Molecules ; metabolism ; Cells, Cultured ; Humans ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Umbilical Cord ; cytology

The purpose of this study was to investigate the efficacy of treatment with haploidentical donor's lymphocyte infusion(hiDLI) combined with interleukin-2 (IL-2) after transplantation of autologous peripheral blood stem cells mixed with haploidentical allogeneic bone marrow (mix-HSCT) for acute myeloid leukemia (AML). 49 patients diagnosed as AML were enrolled in this study. After preconditioning with TBI plus VEMAC regimen, all patients received mix-HSCT. Autologous peripheral blood hematopoietic stem cells were mobilized with chemotherapy-combined G-CSF, and haploidentical allogeneic bone marrow cells were not mobilized with G-CSF. 33 patients in test group were treated with hiDLI plus IL-2 for 1-8 times after hematopoietic reconstruction, 16 patients in control group received mix-HSCT only. All the patients were followed-up for more than 3 years. The results showed that all the patients obtained hematopoietic reconstruction, and no graft-versus-host disease (GVHD) was found. In two groups, the median time of absolute neutrophil count (ANC) ≥ 0.5×10(9)/L was 14 (12 - 18) and 14 (11 - 16) days, and WBC count ≥ 4.0×10(9)/L was 17 (16 - 22) and 18(17 - 20) days, Plt count ≥ 50×10(8)/L were 25 (24 - 29) and 25 (23 - 26) days. 9 patients in test group formed mixed chimerism (46XX/46XY) and sustained about 3 - 12 months; disease-free survival (DFS) was 63.6%, 3 patients in control group formed mixed chimerism (46XX/46XY), persistent about 3-6 months; DFS was 50.0%. It is concluded that treatment with hiDLI plus IL-2 after mix-HSCT for AML patients may increase DFS efficiently.
Adolescent ; Adult ; Female ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cell Transplantation ; Humans ; Immunotherapy, Adoptive ; Leukemia, Myeloid, Acute ; therapy ; Male ; Transplantation, Homologous ; Young Adult

This study was aimed to investigate the expression of radioresistant genes survivin and HO-1 in mesenchymal stem cells (MSC). Human bone marrow MSC were isolated and enriched by Fircoll density gradient centrifugation, then identified by flow cytometry. MSC were induced with dexamethasone, insulin, 3-isobutyl-1-methyl-xanthine (IBMX) and indomethacin to differentiate into adipocytes. Then the expression of survivin and HO-1 in MSC was detected by RT-PCR. The results indicated that the expressions of surface antigen CD34 and HLA-DR in MSC in vitro were negative while the expressions of CD44 and CD71 were positive. MSC could be differentiated into adipocytes by inductor. RT-PCR showed the expression of radioresistant genes survivin and HO-1 in MSC. It is concluded that MSC have lower sensitivity to radiation, which may associate with the expression of radioresistant genes survivin and HO-1 in MSC.
Bone Marrow Cells ; metabolism ; radiation effects ; Cell Differentiation ; Cells, Cultured ; Flow Cytometry ; Gene Expression ; Heme Oxygenase-1 ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; Mesenchymal Stromal Cells ; metabolism ; radiation effects