Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM.@*METHODS@#Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor.@*RESULTS@#The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells.@*CONCLUSION@#The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Effect of the Connexin 43 Coupling to the Biobehavior of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of gap junction intercellular communication (GJIC) combined by connexin43 (Cx43) and its signal to the biobehavior of multiple myeloma (MM) cells, and its possible mechanism.@*METHODS@#The mesenchymal stem cell (MSC) cells were isolated and cultured from patients with MM and normal donors. The expression of connexin43 (Cx43) in MSC cells from different sources was detected by RT-PCR and Western blot. The side population (SP) cells were sorted by flow cytometry (FCM). The effect of MSC cells from different sources to the cell cycle, Cx43 expression, colony formation in vitro, stem cell related genes expression, cytokines secretion and chemoresistance in MM SP cells as well as with or without Cx43 inhibitor 18α-glycyrrhetinic acid (18α-GA) was observed.@*RESULTS@#There was no significantly difference between the MSC isolated from normal donor and MM patients. Western blot showed that Cx43 expression in SP cells was up-regulated when the cells were incubated with MSC, and medium containing 18α-GA could partially inhibit it, moreover, it was more significant in MSC cells of MM patients. The ability of colony formation of SP cells in vitro was higher than those of MM cells and MM-MSC could promote the colony formation in a co-culture manner. The effect of MM-MSC to SP cells was down-regulated after 18α-GA was added. RT-PCR showed that there was several important stem cell-related genes including c-myc, Oct-4 Klf-4, and Sox-2 were found in RPMI 8226 cells, but those cells were up-regulated in SP cells (P0.05). Cytometry bead array assays showed that MM-MSCs could secrete high level of IL-6, but the levels of IL-6, IL-10 and TGF-β increased significantly when the MM-MSCs were co-cultured with SP cells (P<0.05), especially the levels of IL-6 and IL-10 were significantly higher than cultured alone. There was no significant change in the levels of bFGF and IL-17 before and after co-cultured. The levels of IL-6, IL-10 and TGF-β in supernatant decreased significantly after GJ inhibitor 18α-GA was added. PI/Annexin V assay showed that MM cells were sensitive to bortezomib (BTZ)-induced apoptosis, but the sensitivity for SP cells was weaker. The ratio of cell apoptosis was 75.2%±0.77% and 8.12%±0.86% (P<0.001), respectively. MM-MSC could down-regulate the cell apoptosis induced by BTZ, while the sensitivity of MM cells to BTZ could be partially recovered after GJ inhibitor was added.@*CONCLUSION@#MSC derived from MM patients can enhance GJIC to maintain its "hematopoiesis" by up-regulating the expression of Cx43 in MM cells, and at the same time promote cell proliferation and drug recistance by secreting multiple cytokines, which finally contributes to the relapse of MM.

Effects of Bone Marrow Mesenchymal Stem Cells on the Biological Characteristics of Migrating and Homing in Multiple Myeloma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct a co-culture system for bone marrow mesenchymal stem cells (BMMSC) and multiple myeloma (MM) cells, and to investigate the effects of co-cultured BMMSC on the migrating and homing of multiple myeloma cells.</p><p><b>METHODS</b>The BMMSC from the transgenic mice with green fluorescent protein (GFP) fetal bone were cultured by adherent screening. A co-culture system of BMMSC and MM cell line XG-7 cells was constracted, the proliferation and apoptosis of cells were determined by trypan blue exclusion and Annexin V/PI, respectively, MDC staining was employed to detect the autophagy. The moving direction distribution of molecule in BMMSC and XG-7 cells labeled with PE-CD138 in co-culture process were observed dinamically by confocal microscopy.</p><p><b>RESULTS</b>After co-culture with GFP-BMMSC, the resistance of XG-7 cells to apoptosis and autophagy were enhanced; at the same time, their proliferation increased. Apoptosis rates of XG-7 cells directly and indirectly co-cultured with BMMSC were (6.23 ± 0.12)% and (6.97 ± 0.03)% respectively, which were lower than that of XG-7 cells cultured alone (17.90 ± 1.46)% (P < 0. 01). There was low level of autophagy in XG-7 cells co-cultured with BMMSC. XG-7 cells are highly polarized and contained a specialized membrane domain with specific protein and lipid components to contact with BMMSC under confocal microscope. After methyl-β-cyclodextrin treatment, the molecules were normally enriched in the specialized domain.</p><p><b>CONCLUSION</b>BMMSC can protect XG-7 cells from apoptosis and autophagy, and obviously promote the proliferation of XG-7 cells, and can influence the migrating and homing of multiple myeloma cells.</p>

Alterations of connexin 43 expression and SDF-1α secretion of bone marrow mesenchymal stem cells co-cultured with myeloma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To construct a co- culture system of mesenchymal stem cells (MSC) and multiple myeloma (MM) cells and investigate the alterations of connexin 43 (CX43) expression and stromal derived growth factor (SDF)- 1α secretion of MSC.</p><p><b>METHODS</b>CX43 expression and SDF- 1α secretion of MM cell lines (RPMI8226) and human primary MM cells were analyzed by western blot and immunofluorescence. Western blot, RT- PCR and immunofluorescence were employed to detect the alterations of CX43 expression and distribution in MSC directly and indirectly co-cultured with myeloma cells. Lucifer yellow dye spread was utilized to evaluate gap junctional intercellular communication (GJIC) between co- cultured MSC. Transwell was applied to study the transmigration of RPMI8266 induced by MSC under the condition of 18α- glycyrrhetinic acid (18α-GA). The level of SDF- 1α was detected by EILSA.</p><p><b>RESULTS</b>RPMI8266, U266 and one-third primary MM cells expressed CX43 at low or moderate levels. CX43 wasn't expressed in XG- 4 and XG- 7 cells but highly expressed in MSC. The expressions of CX43 mRNA of MSC were up- regulated after directly and indirectly co- cultured with RPMI8226, 1.36 and 2.10 times that of MSC cultured alone respectively. Western blot analysis showed that CX43 protein expression of MSC was also up-regulated, mainly distributed in cytoplasm. Lucifer yellow dye spread showed that GJIC was up-regulated in MSC. SDF-1α concentration in supernatant of MSC directly and indirectly co-cultured with RPMI8226 were (373.02±10.11)pg/ml and (309.71±10.71)pg/ml respectively, which were higher than that of MSC cultured alone (237.84±9.23)pg/ml (P<0.01), and could be inhibited by 18α-GA [(237.84±9.23)pg/ml and (94.31±6.44)pg/ml] respectively (P<0.01). 18α-GA could inhibit the transmigration of RPMI8226 induced by MSC, decrease from (8.00±0.67)% to (4.82±0.19)%.</p><p><b>CONCLUSION</b>CX43 expression of MSC was up-regulated after directly and indirectly co-cultured with MM cells, which could improve the level of SDF-1α secretion of MSC. GJ inhibitor could downregulate SDF-1α secretion of MSC and inhibit the transmigration of MM cells induced by MSC.</p>

Sensitivity to bortezomib of RPIM8226 cells after co-cultured with down-regulated Cav-1 expression HUVECs / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the sensitivity to bortezomib of RPMI8226 cells after co-cultured with down-regulated Caveolin (Cav)-1 expression of HUVECs by transfection with Cav-1 shRNA (HUVECs(Cav-1 low)).</p><p><b>METHODS</b>Exposure to bortezomib with or without 50 nmol/L dexamethasone at different concentration, the proliferation of RPMI8226 was analyzed by MTT assay when it was cultured alone or co-cultured with HUVECs(Cav-1 low). Cav-1 expression was detected by using of Western blot and cell cycle, apoptosis and the level of reactive oxygen species (ROS) were analyzed by flow cytometry.</p><p><b>RESULTS</b>Cav-1 expression was notably down-regulated in HUVECs(Cav-1 low) (0.2199±0.0288 vs 1.3195±0.2393) (P<0.01). The IC(50) of bortezomib for RPMI8226 cultured alone, co-cultured with HUVECs orHUVECCav- 1 low were 20 nmol/L, 50 nmol/L and 65 nmol/L, respectively. The percentages of G₀/G₁ phase in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) were 28.49%, 30.41%, and 36.15% respectively. The protection of RPMI 8226 against apoptosis by HUVECs was demonstrated that the apoptosis/death rates were 66.8%, 10.7% and 8.6% in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) after exposure to 20 nmol/L bortezomib for 24 h. RPMI8226 could induce the oxidative stress of HUVECs before and after co-culture. The ROS level was raised from 15.0% to 35.2% in RPMI8226, from 80.4% to 91.0% in HUVECs, and from 84.6% to 96.8% in HUVECs(Cav-1 low).</p><p><b>CONCLUSION</b>The down-regulated Cav-1 expression of HUVECs could promote proliferation and induce apoptosis of RMPI8226 cells, lead to G₀/G₁ phase arrest, and reduce the sensitivity to bortezomib.</p>

The influence of induced autophagy in vitro on proliferation of multiple myeloma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the influence of autophagy on the survival and proliferation of multiple myeloma (MM) cells.</p><p><b>METHODS</b>Multiple myeloma (MM) cell line U266 cell autophagy was induced by serum-free culture condition, and adding rapamycin or 3-MA respectively. The cells proliferation was observed. U266 cells, lymphoma cell Jurket under normal culture condition, and serum-free cultured Jurket cell were used as control group. The proliferation and apoptosis of cells were determined by CCK8 and flow cytometry, respectively. MDC staining were employed to detect the autophagy. The mRNA expression of Mtor and Beclin1 gene of U266 cells were assayed by RT-PCR. Protein LC3I/LCII and LAMP1 was analyzed by western blot.</p><p><b>RESULTS</b>There was low level of autophagy in U266 cells, sera starvation increased the level of autophagy. Rapamycin upregulated autophagy of the U266 cells and stimulated their proliferation. But the autophagy level of sera starvation and rapamycin group declined when culture for 96h.3-MA had the same effects on U266 cells, although it was on 24 h. But rapamycin and 3-MA could inhibit cell proliferation under normal culture condition. Compared with normal culture condition, apoptosis of U266 cells increased significantly after 24h incubation in medium without sera \[(1.33 ± 0.09)% and (17.90 ± 1.46)%, respectively\] (P < 0.01). Rapamycin and 3-MA could inhibit the serum-free induced apoptosis \[(6.23 ± 0.12)% and (6.97 ± 0.03)%, respectively\](P < 0.01), but cell apoptosis was at the same level after 72 hour incubation \[(30.37 ± 0.27)%, (30.13 ± 1.93)% and (28.57 ± 2.83)%, respectively\] (P > 0.05). However, apoptosis of U266 cells decreased to 18.7% and 12.6% after removal of rapamycin and 3-MA.</p><p><b>CONCLUSION</b>There is basically level of autophagy in MM cells which is higher than those in the Jurkat cells. Both Rapamycin and 3-MA can inhibit the cells proliferation under normal culture condition. Up-regulated autophagy promotes survival and proliferation of MM cells under sera deletion. Rapamycin strengthens this effect with limited duration. 3-MA has dual effects on cell autophagy.</p>

Influence of rosiglitazone and all-trans-retinoic acid on angiogenesis and growth of myeloma xenograft in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the effect of rosiglitazone (RGZ) and all-trans-retinoic acid (ATRA) on the growth of myeloma xenograft in nude mice and to explore the influence of RGZ and ATRA on VEGF expression and angiogenesis in the tumor.</p><p><b>METHODS</b>VEGF gene expression in myeloma cell line U266 cells was analyzed by semi-quantitative RT-PCR after incubation with RGZ, ATRA, or RGZ + ATRA for 24 h. Myeloma xenograft was established by subcutaneous injection of 10(7) U266 cells in the scapula area of 4-week old nude mice. 7 days later, the nude mice were administered with RGZ, ATRA or RGZ + ATRA, respectively, by intraperitoneal injection once every day for 21 days. The control mice were given equal volume of normal saline instead of the drug. On the 21(st) day of treatment, the mice were sacrificed and the tumors were taken off, and the tumor volume and weight were measured. The tumors were examined by histopathology with HE staining, and microvessel density (MVD), CD34 and VEGF expression in the tumors were analyzed by immunohistochemical staining.</p><p><b>RESULTS</b>VEGF mRNA was highly expressed in U266 cells and was decreased in a dose-dependent manner after incubation with RGZ. The VEGF mRNA level was further more decreased after RGZ + ATRA treatment. Xenografts of U266 cells were developed in all nude mice. The volume and weight of xenografts in the RGZ group were (785 ± 262) mm(3) and (1748 ± 365) mg, respectively, significantly lower than those of the control group (both P < 0.01). More significant inhibition was in the RGZ + ATRA group, (154 ± 89) mm(3) and (626 ± 102) mg, respectively, both were P < 0.05 vs. the RGZ group. RGZ inhibited the angiogenesis in U266 xenografts and immunohistochemical staining showed that the tumor MVD and VEGF expression were significantly decreased by RGZ treatment, and further more inhibited in the RGZ + ATRA group. VEGF protein was expressed in all xenografts in the nude mice. Its immunohistochemical staining intensity was 2.20 ± 0.40 in the control group, significantly higher than that of 1.48 ± 0.37 in the RGZ group (P < 0.01), and that of RGZ + ATRA group was 0.58 ± 0.26, further significantly lower than that of the RGZ group (P < 0.01). CD34 was expressed in all xenografts, most highly in the control group and lowest in the RGZ + ATRA group. The microvessel density (MVD) was highest in the control group (56.4 ± 15.2), significantly lower in the RGZ group (44.6 ± 11.2) (P < 0.05), and lowest in the RGZ + ATRA group (21.5 ± 8.6, P < 0.01).</p><p><b>CONCLUSIONS</b>The growth of myeloma cells can also be inhibited by RGZ and ATRA in nude mice in vivo. In addition to differentiation and apoptosis induction, RGZ can inhibit the formation of myeloma xenograft probably also through the downregulation of VEGF expression and subsequent angiogenesis.</p>

Inhibition of hypoxia-inducible factor-1 alpha in RPMI8226 myeloma cells results in reduced tumor growth in nude mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the influence of inhibition of hypoxia-inducible factor-1 alpha (HIF-1 alpha) by RNA interference (RNAi) on tumorigenesis of human myeloma cell line (HMCL) RPMI8226 cells in nude mice.</p><p><b>METHOD</b>RNAi vector of HIF-1 alpha was constructed with commercial shRNA expression vector pSilencer 2. 1-U6 hygro. RT-PCR and western blot were used to detect HIF-1 alpha mRNA and protein expression respectively. Vascular endothelial growth factor (VEGF) secretion and cell cycle changes were detected by ELISA and flow cytometry respectively. Expression of target gene of HIF-1 alpha, VEGF and Glut-1 were tested under hypoxia condition. Tumorigenesis was observed after transfected cells were injected subcutaneously in nude mice.</p><p><b>RESULTS</b>After interference, expression of HIF-1 alpha decreased significantly at both mRNA and protein level. Under normoxia condition, VEGF concentrations in HIF-la inhibited cells (RPMI8226-il and RPMI8226-i2) and non-inhibited cells (RPMI8226-c and RPMI8226) showed no differences. While under hypoxia condition, VEGF concentration in the above four cells was (506.0 +/- 53.2), (494.7 +/- 63.1), (984.4 +/- 61.9) and (938.2 +/- 62.2) pg/ml, respectively, being significantly lower in RPMI8226-il and RPMI8226-i2 cells than in RPMI8226-c and RPMI8226 cells (P <0.05). HIF-1 alpha interference was found to suppress the cells shift from S-phase to G1 induced by hypoxia. VEGF and Glut-1 expressions were markedly attenuated (P <0.05). The growth rate of HIF-1 alpha inhibition tumors in subcutaneous xenograft model decreased drastically.</p><p><b>CONCLUSIONS</b>RNAi inhibits HIF-1 alpha expression. Reduced tumor growth by HIF-1 alpha inhibition may partly through inhibiton of angiogenesis and glycolysis metabolism.</p>

Effects of the osteoclast in pathogenesis of multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of myeloma cells on the differentiation of osteoclast precursors (pOCs) into OCs in different culture systems in vitro and the interaction between OCs and myeloma cells.</p><p><b>METHODS</b>Myeloma cell lines 8226, XG1 and XG7 and pOCs were cocultured in different culture system. OCs was examined by TRAP staining. RT-PCR was used to evaluate the expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) of myeloma cells and the effects of myeloma cells on RANKL/OPG expression in coculture. The role of OCs in myeloma cells cycle was measured by FCM with PI staining. The supportive effects of OCs on myeloma cells survival were determined by FCM with double staining for annexin V and PI.</p><p><b>RESULTS</b>8226 and XG1 cells could directly stimulate the differentiation of pOCs into TRAP+ multinuclear mature OCs. Myeloma cells, which expressed neither RANKL nor OPG, upregulated RANKL expression and decreased OPG expression in mouse primary bone marrow stromal cells (pBMSC). When OCs were co-cultured with myeloma cells, all OCs apparently remained alive after 7 days while devoid of sRANKL and M-CSF. OCs stimulated the proliferation of myeloma cells in co-culture systems,the cell number increased to (3.8 +/- 0.1) x 10(5)/well, (3.9 +/- 0.1) x 10(5)/well, (4.0 +/- 0.1) x 10(5)/well, and to (8.7 +/- 0.1) x 10(5)/well, (9.1 +/- 0.1) x 10(5)/well, (9.0 +/- 0.1 ) x 10(5)/well after co-culture for 3 days and 7 days for XG1 cells, XG7 cells and 8226 cells, respectively (P <0.01). However, OCs could counteract cytotoxic effects of dexamethasone. The proportion of Annexin V-/PI- cells were 57.71%, 82.18% and 90.92% for 8226 cells, XG1 and XG7 cells after co-culture with OCs (P <0.01).</p><p><b>CONCLUSION</b>Myeloma cells stimulated the differentiation of pOCs into TRAP+ multinuclear mature OCs by directly and/or indirectly disrupting the balance of RANKL/OPG, OCs promoted MM cells growth and survival, thus maintaining a vicious circle between myeloma cells and osteoclasts.</p>

Study of biological behavior of stromal cell-derived factor-1 on multiple myeloma cell migration and adhesion / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological behavior of stromal cell-derived factor-1 (SDF-1) on multiple myeloma (MM) cell migration and adhesion and it related signaling pathways.</p><p><b>METHODS</b>Expression of adhesion molecules on MM cells of RPMI8226, XG-1 and XG-7 cells was analysed by flow cytometry, the influence of SDF-1 on CD29 and CD49e distribution by immunofluorescence, the effect of SDF-1 on chemotaxis of MM cells by transwell assay. Activation of phosphoinositide-3 kinase (PI3K) in MM cells treated with SDF-1 and by immunoblotting.</p><p><b>RESULTS</b>3 strains of MM cell line expressed many adhesion molecule. RPMI8226, XG-7 cells were all high level of expression of CD29 (> 70%). XG-1, XG-7 cells were all high level of expression of CD44 (> 80%), and XG-7 cells was of CD49d (> 90%). In all of 3 strains, the levels of expression of CD49e were low (< 30%). SDF-1 could not upregulate their expression, but could trigger the establishment of polarized morphology of MM cells and the redistribution of CD29 and CD49e. SDF-1 promoted MM cells adhesion to endothelial cells, stimulated phosphorylation of P85 subunit of PI3K in MM cells and induced MM cells migration, which were inhibited by G protein inhibitor PTX and PI3K inhibitor wortmannin.</p><p><b>CONCLUSION</b>SDF-1 can promote MM cell adhesion to endothelial cells, trigger establishment of a polarized morphology of MM cells and redistribution of adhesion molecules and induce MM cells migration via PI3K signaling pathway.</p>