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BackgroundMultiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-β, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-β and its inhibition in bone formation and tumor growth in MM.Methodology/Principal FindingsTGF-β suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-β. Inhibitors for a TGF-β type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-β inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-β inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-β inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models.Conclusions/SignificanceThe present study demonstrates that TGF-β inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-β appears to be an important therapeutic target in MM bone lesions.

Bone marrow stromal cells (BMSCs) and osteoclasts (OCs) confer multiple myeloma (MM) cell survival through elaborating factors. We demonstrate herein that IL-6 and TNF family cytokines, TNFa, BAFF and APRIL, but not IGF-1 cooperatively enhance the expression of the serine/threonine kinase Pim-2 in MM cells. BMSCs and OCs upregulate Pim-2 expression in MM cells largely via the IL-6/STAT3 and NF-jB pathway, respectively. Pim-2 short interfering RNA reduces MM cell viability in cocultures with BMSCs or OCs. Thus, upregulation of Pim-2 appears to be a novel anti-apoptotic mechanism for MM cell survival. Interestingly, the mammalian target of rapamycin inhibitor rapamycin further suppresses the MM cell viability in combination with the Pim-2 silencing. The Pim inhibitor (Z)-5-(4-propoxybenzylidene) thiazolidine-2, 4-dione and the PI3K inhibitor LY294002 cooperatively enhance MM cell death. The Pim inhibitor suppresses 4E-BP1 phosphorylation along with the reduction of Mcl-1 and c-Myc. Pim-2 may therefore become a new target for MM treatment.

Purpose: Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression.We showed previously that myeloma cells and osteoclasts are mutually stimulated to form a vicious cycle to lead to enhance both osteoclastogenesis and tumor growth. The present study was undertaken to clarify whether myeloma cell-osteoclast interaction enhances angiogenesis and whether there is any mutual stimulation between osteoclastogenesis and angiogenesis. Experimental Design: Myeloma cells and monocyte-derived osteoclasts were cocultured, and angiogenic activity produced by the cocultures was assessed with in vitro vascular tubule formation assays and human umbilical vascular endothelial cell (HUVEC) migration and survival. Osteoclastogenic activity was determined with rabbit bone cell cultures on dentine slices. Results: Myeloma cells and osteoclasts constitutively secrete proangiogenic factors, vascular endothelial growth factor (VEGF) and osteopontin, respectively. A cell-to-cell interaction between myeloma cells and osteoclasts potently enhanced vascular tubule formation. Blockade of both VEGF and osteopontin actions almost completely abrogated such vascular tubule formation as well as migration and survival of HUVECs enhanced by conditioned medium from cocultures of myeloma cells and osteoclasts. Furthermore, these factors in combination triggered the production of osteoclastogenic activity by HUVEC. Conclusions: Osteoclast-derived osteopontin and VEGF from myeloma cells cooperatively enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. These observations suggest the presence of a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion.

The preparations, X-ray structures, and detailed physical characterizations are presented for two new mixed-valence tetranuclear manganese complexes that function as single-molecule magnets (SMM's): [Mn4(hmp)6Br2(H2O)2]Br2-4H2O (2) and [Mn4(6-me-hmp)6Cl4]-4H2O (3), where hmp(-) is the anion of 2-hydroxymethylpyridine and 6-me-hmp(-) is the anion of 6-methyl-2-hydroxymethylpyridine. Complex 2-4H2O crystallizes in the space group P2(1)/c, with cell dimensions at -160 degrees C of a = 10.907(0) A, b = 15.788(0) A, c = 13.941(0) A, beta = 101.21(0) degrees, and Z = 2. The cation lies on an inversion center and consists of a planar Mn4 rhombus that is mixed-valence, Mn2(III)Mn2(II). The hmp(-) ligands function as bidentate ligands and as the only bridging ligands in 2-4H2O. Complex 3-4H2O crystallizes in the monoclinic space group C2/c, with cell dimensions at -160 degrees C of a = 17.0852(4) A, b = 20.8781(5) A, c = 14.835(3) A, beta = 90.5485(8) degrees, and Z = 4. This neutral complex also has a mixed-valence Mn2(III)Mn2(II) composition and is best described as having four manganese ions arranged in a bent chain. A mu2-oxygen atom of the 6-me-hmp(-) anion bridges between the manganese ions; the Cl(-) ligands are terminal. Variable-field magnetization and high-frequency and -field EPR (HFEPR) data indicate that complex 2-4H2O has a S = 9 ground state whereas complex 3.4H(2)O has S = 0 ground state. Fine structure patterns are seen in the HFEPR spectra, and in the case of 2.4H(2)O it was possible to simulate the fine structure assuming S = 9 with the parameters g = 1.999, axial zero-field splitting of D/k(B) = -0.498 K, quartic longitudinal zero-field splitting of B4(omicron)/k(B) = 1.72 x 10(-5) K, and rhombic zero-field splitting of E/k(B) = 0.124 K. Complex 2-4H2O exhibits a frequency-dependent out-of-phase AC magnetic susceptibility signal, clearly indicating that this complex functions as a SMM. The AC susceptibility data for complex 2-4H2O were measured in the 0.05-4.0 K range and when fit to the Arrhenius law, gave an activation energy of DeltaE = 15.8 K for the reversal of magnetization. This DeltaE value is to be compared to the potential-energy barrier height of U/k(B) = absolute value DSz(2) = 40.3 K calculated for 2-4H2O.

Transforming growth factor-␤ (TGF-␤) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-␤ signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitinproteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-␤ and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitinmediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-␤-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-␤ signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC) transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA) suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2), KG-1 (ABCB1) and HepG2 cells (ABCB1 and ABCG2). Interestingly, although side population (SP) cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

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