scholarly journals Reelin depletion alleviates multiple myeloma bone disease by promoting osteogenesis and inhibiting osteolysis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Aixia Dou ◽  
Ying Zhang ◽  
Yongjing Wang ◽  
Xiaoli Liu ◽  
Yanan Guo
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Immune Cell ◽  
Morphological Changes ◽  
Osteoclast Differentiation ◽  
Myeloma Bone Disease ◽  
Osteoblast Activity ◽  
Specific Mrnas ◽  
Regulatory Effects

AbstractExtracellular matrix glycoprotein Reelin is associated with tumor metastasis and prognosis in various malignancies. However, its effects on multiple myeloma (MM) are not fully understood. Here, we investigated the regulatory effects of Reelin on MM and its underlying pathogenic mechanisms. Lentivirus plasmid containing short hairpin RNA targeting Reelin (LV3-Reln) was transfected into SP2/0 cells to knockdown Reelin expression. Flow cytometry assay analyzed cell cycle and apoptosis while Transwell assay evaluated invasiveness. BALB/c mice were inoculated with LV3-Reln-transfected SP2/0 cells to establish MM model. Primary myeloma cells and osteoblasts/osteoclast were isolated from tumor tissue and limb long bones respectively. ELISA examined serum biomarkers and immunohistochemistry detected immunoglobulin light chain expression. Morphological changes and osteoclast/osteoblast differentiation were observed by histological staining. mRNA and proteins expression were determined by qPCR and WB. In vitro studies showed that Reelin depletion regulated osteolysis and osteogenesis balance, cell cycle, invasiveness, and apoptosis in SP2/0 cells. In LV3-Reln mice, tumor growth and invasiveness were suppressed, meanwhile, reduced osteoclast activation and enhanced osteoblast activity were observed. Reelin knockdown alleviated extramedullary morbidity and inhibited spleen immune cell apoptosis by down-regulating CDK5, IL-10, and Cyto-C expression. Furthermore, reduced Reelin expression restrained osteoclast differentiation while promoted osteogenesis in the bone of LV3-Reln mice. This was further supported by down-regulation of osteolytic specific mRNAs and proteins (Trap, Mmp9, Ctsk, Clcn7) and up-regulation of osteogenic specific ones (COL-1, Runx2, β-Catenin). Reelin exerted important impacts on myeloma development through rebalancing osteolysis and osteogenesis, thus might be a potential therapeutic target for MM.

scholarly journals GDF15 Promotes Osteoclast Differentiation and High Serum GDF15 Levels Are Associated with Multiple Myeloma Bone Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2065-2065
Author(s):  
Marita Westhrin ◽  
Siv Helen Moen ◽  
Toril Holien ◽  
Oddrun Elise Olsen ◽  
Anne Kærsgaard Mylin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Disease ◽  
Osteoclast Differentiation ◽  
Serum Levels ◽  
High Serum ◽  
Healthy Controls ◽  
Serum Samples ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

Abstract Introduction Growth differentiation factor 15 (GDF15) is a multifunctional growth factor of the transforming growth factor beta (TGFbeta) family that plays a complex role in several types of cancers. In multiple myeloma, GDF15 was recently shown to enhance the tumor-initiating and self-renewal potential of the cancer cells (Tanno et al, Blood 2014). Moreover, blood and bone marrow plasma levels of GDF15 are elevated in myeloma patients compared with healthy persons, and high serum levels are associated with a poor prognosis (Corre et al, Cancer Research 2012). GDF15 seems important for bone remodeling during hypoxia (Hino et al, JBMR 2012), and one study proposed GDF15 to increase osteoclast activation in prostate cancer metastasizing to bone (Wakchoure et al, Prostate 2009). Whether GDF15 plays a role in the bone disease of multiple myeloma is not well characterized. Aim Our aim was to investigate whether high GDF15 serum levels are associated with multiple myeloma bone disease and to characterize the effect of GDF15 on osteoclast differentiation in vitro. Methods GDF15 was measured in serum samples obtained at diagnosis from 138 myeloma patients and 58 age and sex-matched healthy controls. The patient serum samples were collected for the Nordic Myeloma Study Group during a randomized phase 3 clinical trial which compared the effect of two different doses of pamidronate on bone. The bone disease was therefore particularly well-characterized in this study (Gimsing et al, Lancet Oncol 2010). Peripheral blood mononuclear cells (PBMC) isolated from buffy coats were cultured in osteoclast medium (a-MEM with human serum (20%), M-CSF (30ng/ml) and RANKL (50ng/ml)) for up to 14 days with or without GDF15. Purchased pre-osteoclasts (Lonza Inc.) were cultured in purchased bullet kit (OC medium with M-CSF (33ng/ml) and RANKL (66ng/ml)) for 7 days with or without GDF15. Cells positive for tartrate resistant acidic phosphatase (TRAP) staining and with more than two nuclei were counted as osteoclasts. Results GDF15 was significantly higher in serum obtained from myeloma patients (median 1.08 ng/ml, range 27.91) compared with healthy controls (median 0.46 ng/ml, range 1.66, Independent samples Kruskal-Wallis test p< 0.0001). Moreover, serum GDF15 was elevated in patients with a more advanced osteolytic bone disease (n= 51, median 1.44 ng/ml, range 6.48) as compared to patients without osteolytic lesions (n= 16, median 0.84 ng/ml, range 10.62) at inclusion (p<0.05). The difference between serum GDF15 in patients with limited bone disease at inclusion (n=51, median 1.07 ng/ml, range 6.84) and patients with no bone disease at baseline was not significantly different. In vitro, addition of GDF15 (0-100 ng/ml) to osteoclast precursors or PBMC increased numbers of multi-nucleated TRAP positive cells in a dose dependent manner (n=3, O ng/ml GDF15 mean 25.7, SEM 5.9, 2 ng/ml GDF15 mean 29.7, SEM 4.3, 20 ng/ml GDF15 mean 38.7, SEM 4.9, 50 ng/ml GDF15 mean 53.3, SEM 11.6, 100 ng/ml GDF15 mean 78.7 SEM 7.8). OPG inhibited the pro-osteoclastogenic activity of GDF15 inferring that the effect is mediated by RANKL. Hence, GDF15 increases osteoclast differentiation. Conclusion Serum GDF15 is elevated in myeloma patients with advanced osteolytic bone disease compared to patients with no lesions. GDF15 increases osteoclast differentiation in vitro. Hence, GDF15 could play a role in regulating bone remodeling in myeloma patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Xuxing Shen ◽  
Chao Wu ◽  
Meng Lei ◽  
Qing Yan ◽  
Haoyang Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Serum Enzyme ◽  
Herg Channels ◽  
Anti Tumor Activity ◽  
Dose Dependent

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

scholarly journals A Study of Late Ventral Body Wall Degeneration in the Embryonic Chick, with Special Reference to the Cell Cycle

2021 ◽  
Author(s):  
◽  
Peter Barwell
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Body Wall ◽  
Morphological Changes ◽  
Cell Loss ◽  
Tissue Growth ◽  
Embryonic Chick ◽  
Cell Death Pathway ◽  
Medial Migration

<p>The cell kinetics and morphological changes during late ventral body wall development of the embryonic chick were studied, particularly midline degeneration and the medial migration of lateral tissues. An histological examination of these events was undertaken, along with autoradiography to determine the duration of the cell cycle, followed by teratological studies involving the prevention of differentiative events in the cell death pathway, using BrDU and Janus B Green as agents. The effects of cell cycle blockade on rates of cell death were also examined, as was the tissues ability to express differentiative features in vitro. Ventral body wall (VBW) cell death was classified as apoptosis, and was involved in two distinct events. Medial migration of lateral tissues began at day 5 of development, with widespread VBW apoptosis being seen by day 6, limited to the original mesoderm of the region. A later precise line of apoptosis (the VBL), involving both ectodermal cells of the midline ectodermal ruffle and the underlying mesodermal cells, was observed at day 7, spreading in a rostral to caudal fashion down the embryo, appearing as the migratory lateral tissues fused in the midline body wall. Increases in the amount of cell death are matched by decreases in the MI, such that at its peak (day 7.5 of development) the cell death rate is sufficiently greater than both the cell proliferation and immigration rates that a state of negative tissue growth ensues. The histological half-life of the apoptotic bodies approximates 3.8 hours. The ability to undergo apoptosis at day 7 is dependent upon a differentiative event around day 4 of incubation, and involves signal mechanisms intrinsic to the VBW tissues. BrDU application was found to inhibit apoptotic differentiation, in contrast to Janus B Green, which had a more generalised teratogenic effect on the region as a whole. Tissue culturing experiments revealed that an ectodermal-mesodermal interaction is important in regulating the extent of mesodermal apoptosis, the ectoderm playing a maintenance role for the mesoderm. Dead cells derive from the cycling cell population, as shown by the occurrence of labelled dead cells after autoradiography, and by the prevention of apoptosis by a cell cycle blockade, and by the production of a semi-synchronised wave of apoptoses after release of this blockade. These cell blockading results further suggest that entry into the apoptotic death program requires cells to be in a particular cell cycle stage, and it seems most likely that the decision to die was made in early G1. Tissue and cell growth rates, cell loss and death rates, cell birth rates and cell immigration rates were all determined for the VBW region throughout the time period studied.</p>

scholarly journals A role for IL-34 in osteolytic disease of multiple myeloma

2019 ◽  
Vol 3 (4) ◽  
pp. 541-551 ◽  
Author(s):  
Muhammad Baghdadi ◽  
Kozo Ishikawa ◽  
Sayaka Nakanishi ◽  
Tomoki Murata ◽  
Yui Umeyama ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Osteoclast Differentiation ◽  
Neutralizing Antibody ◽  
Axial Skeleton ◽  
Osteoclast Formation ◽  
Interfering Rna ◽  
First Time ◽  
Stimulating Factor

AbstractMultiple myeloma (MM) is a hematological malignancy that grows in multiple sites of the axial skeleton and causes debilitating osteolytic disease. Interleukin-34 (IL-34) is a newly discovered cytokine that acts as a ligand of colony-stimulating factor-1 (CSF-1) receptor and can replace CSF-1 for osteoclast differentiation. In this study, we identify IL-34 as an osteoclastogenic cytokine that accelerates osteolytic disease in MM. IL-34 was found to be expressed in the murine MM cell line MOPC315.BM, and the expression of IL-34 was enhanced by stimulation with proinflammatory cytokines or by bone marrow (BM) stromal cells. MM-cell–derived IL-34 promoted osteoclast formation from mouse BM cells in vitro. Targeting Il34 by specific small interfering RNA impaired osteoclast formation in vitro and attenuated osteolytic disease in vivo. In BM aspirates from MM patients, the expression levels of IL-34 in CD138+ populations vary among patients from high to weak to absent. MM cell–derived IL-34 promoted osteoclast formation from human CD14+ monocytes, which was reduced by a neutralizing antibody against IL-34. Taken together, this study describes for the first time the expression of IL-34 in MM cells, indicating that it may enhance osteolysis and suggesting IL-34 as a potential therapeutic target to control pathological osteoclastogenesis in MM patients.

ANKHD1 Silencing Reduces In Vitro Clonogenicity and In Vivo Tumorogenicity Of Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3168-3168
Author(s):  
Anamika Dhyani ◽  
João Agostinho Machado-Neto ◽  
Patricia Favaro ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Tumor Size ◽  
Control Group ◽  
Mice Model ◽  
And Migration

Abstract Introduction ANKHD1 is a multiple ankyrin repeats containing protein, highly expressed in cancers, such as acute leukemia. Earlier studies showed that ANKHD1 is highly expressed and plays important role in proliferation and cell cycle progression of multiple myeloma (MM) cells. It was also observed that ANKHD1 downregulation modulates cell cycle gene expression and upregulates p21 irresepective of TP53 mutational status of MM cell lines. Objective The present study aimed to study the effect ofANKHD1 silencing on MM growth both in vitro (clonogenicity, migration) and in vivo (xenograft tumor mice model). The purpose was to investigate the feasibility of ANKHD1 gene therapy for MM. Methods In the present study, ANKHD1 expression was silenced using short hairpin RNA (shRNA)-lentiviral delivery vector in MM cell lines (U266 and MM1S). For control MM cells were tranduced by lentiviral shRNA against LacZ. Downregulation of ANKHD1 expression was confirmed by qPCR and Western blot. Colony formation capacity and migration of control and ANKHD1 silenced MM cells was determined by methylcellulose and transwell migration assays, respectively. For in vivo MM growth, NOD-SCID mice were divided in two groups injected with control and ANKHD1 silenced cells, separately. Mice were observed daily for tumor growth. Once the tumor size reached 1 mm3, mice in both groups were sacrificed and tumor was excised to measure tumor volume and weight. Results Corroborating the results obtained in our earlier studies, in the present study also inhibition of ANKHD1 expression suppressed growth of MM cells in vitro. MM cell lines tranduced with ANKHD1 shRNA showed significantly low number of colonies ten days after plating in methylcellulose medium as compared to control (p<0.05). Similarly, in transwell migration assay, cell lines transduced with ANKHD1 showed significantly less migration as in response to 10% FBS at lower chamber as compared to control group (p<0.05) in both the cell lines analyzed. Further in xenograft MM mice model, the growth of tumor was visibly suppressed in mice injected with ANKHD1 silenced cells compared to control group. There was significant difference in tumor size (volume) between these 2 groups (P< 0.006). The tumor weight of the inhibition group was 0.71 ±0.2 g, significantly lighter than those of the control group (1.211 ± 0.5 g, P =0.02) Conclusion Our data indicates ANKHD1 downregulation significantly inhibits colony-forming ability and migration of both glucocorticoid resistant (U266) and sensitive (MM1S) MM cells. Further, gene silencing of ANKHD1 also resulted in reduced in vivo tumor growth in NOD/SCID mice. Collectively, the result obtained indicates that ANKHD1 may be a target for gene therapy in MM. Disclosures: No relevant conflicts of interest to declare.

HGF inhibits BMP-induced osteoblastogenesis: possible implications for the bone disease of multiple myeloma

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 3024-3030 ◽  
Author(s):  
Therese Standal ◽  
Niels Abildgaard ◽  
Unn-Merete Fagerli ◽  
Berit Stordal ◽  
Øyvind Hjertner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Alkaline Phosphatase ◽  
Bone Formation ◽  
Bone Disease ◽  
Nuclear Translocation ◽  
Osteolytic Lesion ◽  
Bmp Signaling ◽  
Myoid Cell ◽  
Osteoblast Activity

AbstractThe bone disease in multiple myeloma is caused by an uncoupling of bone formation from bone resorption. A key difference between patients with and patients without osteolytic lesion is that the latter have fewer and less active osteoblasts. Hepatocyte growth factor (HGF) is often produced by myeloma cells and is found at high concentrations in the bone marrow of patients with multiple myeloma. Here we show that HGF inhibited bone morphogenetic protein (BMP)–induced in vitro osteoblastogenesis. Thus, HGF inhibited BMP-induced expression of alkaline phosphatase in human mesenchymal stem cells (hMSCs) and the murine myoid cell line C2C12, as well as mineralization by hMSCs. Furthermore, the expression of the osteoblast-specific transcription factors Runx2 and Osterix was reduced by HGF treatment. HGF promoted proliferation of hMSCs, and the BMP-induced halt in proliferation was overridden by HGF, keeping the cells in a proliferative, undifferentiating state. BMP-induced nuclear translocation of receptor-activated Smads was inhibited by HGF, providing a possible explanation of how HGF inhibits BMP signaling. The in vitro data were supported by the observation of a negative correlation between HGF and a marker of osteoblast activity, bone-specific alkaline phosphatase (rho = −0.45, P = .008), in sera from 34 patients with myeloma. These observations suggest that HGF inhibits bone formation in multiple myeloma.

Immature dendritic cells from patients with multiple myeloma are prone to osteoclast differentiation in vitro

2011 ◽  
Vol 39 (7) ◽  
pp. 773-783.e1 ◽  
Author(s):  
Marco Tucci ◽  
Sabino Ciavarella ◽  
Sabino Strippoli ◽  
Oronzo Brunetti ◽  
Franco Dammacco ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dendritic Cells ◽  
Osteoclast Differentiation ◽  
Immature Dendritic Cells

scholarly journals Monoclonal immunoglobulins promote bone loss in multiple myeloma

Blood ◽  
2020 ◽  
Vol 136 (23) ◽  
pp. 2656-2666
Author(s):  
Marita Westhrin ◽  
Vlado Kovcic ◽  
Zejian Zhang ◽  
Siv H. Moen ◽  
Tonje Marie Vikene Nedal ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Sialic Acid ◽  
Bone Loss ◽  
Bone Disease ◽  
Osteoclast Differentiation ◽  
Osteolytic Bone Disease ◽  
Monoclonal Immunoglobulins ◽  
Diagnostic Criterion ◽  
Acid Precursor

Abstract Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient’s sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.

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