Bone Marrow Stromal Cell-Derived Exosomes Facilitate Multiple Myeloma Cell Survival Through Inhibition Of The JNK Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 679-679
Author(s):  
Jinheng Wang ◽  
An Hendrix ◽  
Elke De Bruyne ◽  
Els Van Valckenborgh ◽  
Eddy Himpe ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Viability ◽  
Cell Survival ◽  
Caspase 3 ◽  
Annexin V ◽  
Jnk Pathway ◽  
Functional Components

Abstract Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a crucial role in MM pathogenesis by secreting growth factors, cytokines, and other functional components. Exosomes are 30-100nm diameter membranous vesicles constitutively released by several cell types including reticulocytes, cytotoxic T lymphocytes, B lymphocytes, epithelial and endothelial cells. Exosomes mediate local cell-cell communication by transferring mRNAs, miRNAs and proteins. Due to their ability to transfer functional components, exosomes play multiple roles by stimulating target cells, transferring membrane receptors, delivering proteins, and inducing epigenetic changes in recipient cells. Although the promotion of MM growth and survival induced by BMSCs has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here, we investigated the effect and mechanisms of BMSC-derived exosomes on the proliferation and survival of MM cells using the murine 5T33MM model. This model mimics the human disease closely and of this model two lines exist: the 5T33MMvv model which is propagated in vivo and the 5T33MMvt line which is derived from 5T33MMvv cells but which can grow stroma-independently. Exosomes were isolated from conditioned medium using the ExoQuick-TC Exosome Precipitation Solution (System Biosciences) after culture of primary BMSCs obtained from naïve C57BL/KaLwRij mice or 5T33MM diseased mice. The size of exosomes derived from naïve BMSCs, 5T33 BMSCs and 5T33MMvt cells were confirmed using a NanoSight LM10. Several exosomal markers such as CD63, Flotillin-1, heat shock protein 90 (HSP90), and HSP70 were detected using Western blot. We co-cultured the BMSCs or MM cells with fluorescent dye-labeled exosomes to examine whether exosomes could be transferred into cells. The results showed that both naïve and 5T33 BMSC-derived exosomes could fuse with 5T33MMvt cells and that the uptake of 5T33MMvt cell-derived exosomes by BMSCs was also observed. As several cytokines were found to be present in BMSC- and MMvt cell-derived exosomes, this suggests that BMSCs and MM cells could exchange cytokines with each other through exosomes secretion and uptake. Furthermore, the cytokine composition of 5T33BMSC-derived exosomes compared to naïve BMSC-derived exosomes was different. We next performed luminescent cell viability assays, BrdU cell proliferation assays and 7-AAD/annexin-V stainings to examine the effects of BMSC-derived exosomes on MM cell viability, proliferation and survival, respectively. Both naïve and 5T33 BMSC-derived exosomes increased 5T33MMvt and MMvv cell viability in a dose- and time-dependently manner. BrdU uptake in 5T33MMvt and MMvv cells was also increased after treatment with BMSC-derived exosomes. Significantly reduced apoptosis of 5T33 MMvt and MMvv cells was observed when they were treated with BMSC-derived exosomes as judged by 7-AAD/annexin-V staining. 5T33MMvt and MMvv cells were treated with different amounts of BMSC-derived exosomes and apoptosis-related proteins Bcl-2, Bax, and caspase-3 were determined using western blot. Bcl-2 was increased slightly and activated (cleaved) caspase-3 was reduced after co-culture with exosomes, coinciding with the results of 7-AAD/annexin-V staining. To elucidate the mechanisms responsible for exosome-induced MM cell survival, we examined the activation of several proteins involved. Reduced phosphorylation of p53, p38MAPK and JNK were detected when 5T33MMvt were treated with naïve-BMSC-derived exosomes for 24h, whereas phosphorylated Erk1/2, Akt, and IGF1Rβ were not changed. Surprisingly, activation of p53 and p38MAPK were not changed after the treatment with 5T33 BMSC-derived exosomes. 5T33 BMSC-derived exosomes further decreased the activation of JNK, Bim expression and phosphorylated Bim compared to naïve BMSC-derived exosomes. As Bim is a pro-apoptosis protein, mainly regulated by the JNK pathway; promotion of MM cell survival likely results from the inhibition of the JNK pathway by BMSC-derived exosomes. In summary, our results demonstrate a positive role for BMSC-derived exosomes in induction of MM cell proliferation and survival. BMSC-derived exosomes could inhibit the JNK pathway, thereby reducing caspase-3 activation and protecting MM cells from apoptosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD28-mediated regulation of multiple myeloma cell proliferation and survival

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 5002-5010 ◽  
Author(s):  
Nizar J. Bahlis ◽  
Anne M. King ◽  
Despina Kolonias ◽  
Louise M. Carlson ◽  
Hong Yu Liu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Cell Survival ◽  
Plasma Cells ◽  
Cell Interaction ◽  
Myeloma Cell ◽  
Serum Starvation ◽  
Costimulatory Receptor ◽  
Multiple Myeloma Cell

Abstract Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown, due in large part to the complexity of the bone marrow microenvironment itself. The T-cell costimulatory receptor CD28 is also expressed on normal and malignant plasma cells, and CD28 expression in MM correlates significantly with poor prognosis and disease progression. In contrast to T cells, activation and function of CD28 in myeloma cells is largely undefined. We have found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induces activation of PI3K and NFκB, suppresses MM cell proliferation, and protects against serum starvation and dexamethasone (dex)–induced cell death. Coculture with dendritic cells (DCs) expressing the CD28 ligands CD80 and CD86 also elicits CD28-mediated effects on MM survival and proliferation, and DCs appear to preferentially localize within myeloma infiltrates in primary patient samples. Our findings suggest a previously undescribed myeloma/DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also point to CD28 as a potential therapeutic target in the treatment of MM.

CD28-Mediated Regulation of Multiple Myeloma Cell Proliferation and Survival.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 355-355
Author(s):  
Kelvin P. Lee ◽  
Nizar J. Bahlis ◽  
Anne M. King ◽  
Despina Kolonias ◽  
Louise M. Carlson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
Cell Survival ◽  
Plasma Cells ◽  
Survival Function ◽  
Myeloma Cell ◽  
Serum Starvation ◽  
Bone Marrow Biopsies

Abstract Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown due to the complexity of the bone marrow microenvironment. The CD28 receptor, which costimulates survival signals in T cells, is also expressed on normal plasma cells and myeloma cells. In MM, CD28 expression correlates significantly with disease progression, also suggesting a pro-survival function. In contrast to T cells however, activation and function of CD28 in myeloma and plasma cells is almost entirely undefined. We found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induced activation of NFkappaB, suppressed MM cell proliferation and protected against serum starvation and dexamethasone-induced cell death. We hypothesized that the specific CD80/CD86 expressing stromal cell partner of this interaction is a professional antigen presenting cells, in particular dendritic cells. Histological studies demonstrated DC were extensively interdigitated throughout the myeloma infiltrates in patient bone marrow biopsies. In vitro coculture with DC also elicited CD28-mediated effects on MM survival and proliferation, and could be blocked by CD28Ig. Our findings suggest a previously undescribed myeloma:DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also suggest that CD28 may represent a therapeutic target in the treatment of multiple myeloma.

IL-6, IL-8 and VEGF Neutralisation Restores Drug Sensitivity to Conventional and Novel Treatment Combinations in a Multiple Myeloma Bone Marrow Micro-Environment Model.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2949-2949 ◽  
Author(s):  
Lenushka Maharaj ◽  
Rakesh Popat ◽  
John G Gribben ◽  
Simon Joel
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Stromal Cells ◽  
Direct Contact ◽  
Neutralizing Antibodies ◽  
Drug Sensitivity ◽  
Chemotherapeutic Agents ◽  
Humoral Factors

Abstract Abstract 2949 Stromal cells within the bone marrow microenvironment support the survival and proliferation of multiple myeloma (MM) cells, and confer resistance against chemotherapeutic agents. Although drug combinations (such as velcade and melphalan) are used to overcome such resistance; outcomes for myeloma patients remains sub-optimal. The microenvironment therefore represents a target to sensitise cells to chemotherapy. To further investigate this, we established a co-culture platform to culture CD138–positive MM cells and stromal cells directly or indirectly (separated by a 0.4μm micropore membrane). Co-culture with the human bone marrow stromal cell line (BMSC), HS-5, increased cell proliferation and cell viability of both MM cell lines (MMCLs) and 17 primary MM samples (Cell viability 53.6±11.2 (mean±s.d.) compared with 35.3±13.9 in media alone, (p<0.0001)). The stimulation of primary MM cell proliferation (p<0.01), confirmed the survival effects of BMSCs. Such antiapoptotic effects were markedly pronounced when the BMSCs were in direct contact with MM cells, however importantly conditioned media from BMSC cultures also demonstrated a significant increase in MM cell viability compared to control media alone (p<0.01). Exposure to standard chemotherapy (melphalan and dexamethasone) and novel therapies (bortezomib and the HDAC inhibitor UCL67022) resulted in a marked inhibition of MMCL and primary MM cell growth. However, these effects were attenuated when cells were either co-cultured in direct contact or in non-contact co-culture assays with HS-5 cells using transwell inserts. Such attenuation was accompanied by upregulation of cytokine-mediated STAT3 signaling, and PI3K/AKT, P38MAPK and ERK cell survival pathways within the MM cell. Cytokine profiling of supernatants from co-culture assays detected high levels of IL-6, IL-8, and VEGF. Levels of IL-8 correlated directly with resistance to velcade, and levels of both IL-8 and VEGF correlated with resistance to melphalan, whilst levels of IL-6 correlated with resistance to both agents in combination (Pearson's correlation coefficient p<0.05). Furthermore, neutralizing antibodies against these cytokines removed the protective effect conferred by the BMSC co-culture and restored drug sensitivity in primary MM cells. Combination of the IL-8 and VEGF antibodies gave an additive effect whereas all three antibodies in combination had the greatest sensitising effect. In conclusion, we demonstrate that humoral factors secreted by stromal cells protect MM cells from both standard, novel chemotherapy and the highly clinically active combination of velcade and melphalan. Whilst IL-6 neutralising antibodies are being evaluated in the clinic, this data provides compelling evidence towards the further evaluation of IL-8 and VEGF antibodies both alone and in combination. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Hedgehog Pathway as a Potential Therapeutic Target in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 672-672
Author(s):  
Simona Blotta ◽  
Joeseph Negri ◽  
Purushothama Nanjappa ◽  
Anne-Sophie Moreau ◽  
Rao Prabhala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Lines ◽  
Small Molecule ◽  
Plasma Cells ◽  
Annexin V ◽  
Inhibition Of Proliferation ◽  
Ic50 Values ◽  
Gli 1

Abstract We have previously demonstrated that a consistent feature of malignant plasma cells of multiple myeloma (MM) is the aberrant expression of genes important in patterning and development, such as members of Hedghehog (Hh) pathway (FE Davies et al, Blood 2003). These findings suggest that overexpression of genes of this pathway, already involved in many solid tumors and recently implicated in maintaining a proposed MM stem cell compartment (CD Peacock et al, PNAS 2007), might be one of the mechanism through which Hh-signaling contributes to tumorigenesis in MM. Therefore, several small molecule modulators of Hh-pathway, which work as agonists and antagonists, are currently under development. We evaluated, by microarray analysis, the expression of Hh pathway genes in MM cell lines and primary MM cells vs. plasma cells from healthy donors. We found that primary MM cells overexpress Sonic (Shh), Smoothened (Smo), Patched (Ptc), Gli-1 and Gli-3 (relative expression ratios ranging from +1.8 to +5.0). Overexpression of Patched was also observed in most of the MM cell lines analyzed (+5.0 ratio in 5 of 6 MM cell lines). Additionally, we confirmed the expression of Shh and of Gli-1, by flow cytometry and western blotting respectively, in a large panel of MM cell lines. These data suggest an activation of the Hh-pathway in MM that, in some cell lines, is Shh-dependent. Therefore, we investigated the therapeutic potential of Hh-inhibitors in MM. We assayed the cell viability and proliferation, by MTT and Thymidine uptake respectively, in 8 MM cell lines after 72 hours of treatment with the small molecule Smo-inhibitor CUR-0199691 (Genentech). We observed a reduction in MM cell viability, with IC50 values ranging between 4.5–9.5 μM in these 8 cell lines and an inhibition of MM cell proliferation with IC50 values ranging between 0.5 and 2.5μM in the same cell lines. MM cell sensitivity to this compound appears to be related to the level of expression of Gli-1, since the cell lines with lower level of expression of Gli-1 were more sensitive. The treatment of these MM cell lines with Cyclopamine, another Hh-inhibitor, showed an IC50 between 7.5μM and 10μM after at least 96 hours of treatment in 4 of the MM cell lines tested. CUR-0199691 is also active in primary MM cells, triggering inhibition of proliferation by 50% at 5μM after only 24h of treatment, while cyclopamine reduces MM cell proliferation (normalized to the effect of tomatidine, its inactive analog) by 30% at 20μM after a 48 hour treatment. Annexin V-PI staining of Hh inhibitor-treated KMS11 cells, which are one of the most sensitive MM cell lines, showed induction of apoptosis, evidenced by detection of 12 and 15% of MM cells being Annexin V+/PI- after 48h and 72h respectively with 5μM of CUR-0199691. These results, taken together, show that the Hh-pathway is fuctionally active in MM and that the novel Hh pathway inhibitor CUR-0199691 is 4–5 times more effective than cyclopamine in both MM cell lines and primary MM cells. These studies provide the framework for further preclinical evaluation of CUR-0199691 in MM models towards possible future clinical trials.

TRPV2 Expression and Its Role in Proliferation of Human Multiple Myeloma Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5003-5003
Author(s):  
Massimo Nabissi ◽  
Massimo Offidani ◽  
Maria Beatrice Morelli ◽  
Manila Pettinari ◽  
Patrizia Caraffa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Lines ◽  
Annexin V ◽  
Cation Channel ◽  
Brdu Incorporation ◽  
Signal Pathways ◽  
Transient Receptor Potential Vanilloid

Abstract Abstract 5003 Multiple myeloma (MM) is a disease typically characterized by repeated relapse that needs several new-drugs throughout its clinical course. Therefore, increasing knowledge of the pathways involved in the pathophysiology of MM will contribute to identified new therapeutic strategies. Specific signaling pathways as: nuclear factor kappa B (NFkB), farnelsyl-transferase (FTI), mitogenen-activated protein kinase (MAPK), proteosoma and others have been found to be disregulated in MM patients and cell lines. These findings have allowed the design of specific inhibitors that have been assessed in preclinical and clinical studies. Recent data have highlighted the contribute of ion channels (K+, Cl−, Na+ and Ca2+) in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-2 (TRPV2) is a cation channel, member of the TRPV family, and its expression in human cancer cells and tissues has been reported for gliomas, prostate, bladder and pancreas, while no data were available in MM or in other hematological malignancies. TRPV2 expression has been found to influence cancer cell responses to chemo-therapeutic drugs as well as proliferation, migration and apoptosis, in part by increasing intracellular Ca2+ influx and/or by regulating specific signal pathways, like MAPK associated pathways. TRPV2 responds to noxious heat with an activation threshold of >52°C as well as to changes in osmolarity and membrane stretch; in addition, TRPV2 is activated by agonists such as 2-aminoethoxydiphenyl borate, as well as cannabinoids. The aim of this study was, firstly, to evaluate if TRPV2 was expressed in three human MM cell lines established models (RPMI, SKO-007, U266). Secondly we investigated on the role of TRPV2 activation in regulating the proteosoma inhibitor Bortezomib activity, in MM cell lines. By Real-Time PCR analysis we demonstrated that TRPV2 gene was expressed, with comparable relative levels, in the MM cell lines studied, and similar results were obtained at protein levels by Western blot analysis. Moreover, by immunofluorescence and FACS analysis we found that the percentage of TRPV2+ cells was 11% in RPMI, 3% in U266 and 2.6% in SKO-007. These data indicate that TRPV2 was expressed at low levels in MM cell lines. Since lost of TRPV2 expression was found to be associated with higher cell proliferation rate, in other tumor cell lines, and increasing of TRPV2 transcription and/or activity by specific agonists induced cell death and anti-proliferative effects, we evaluated the biological effects of TRPV2 activation in MM cell lines by MTT assay. The results showed that, after three incubation days, TRPV2 activation induced a decrease of cell viability of 70% in RPMI, 60% in U266 and 55% in SKO-007, compared to control. To evaluate if the reduced cell viability was dependent by an anti-proliferative and/or apoptotic process, the three cell lines treated with the TRPV2 agonist were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay for proliferation and by Annexin-V apoptosis assays. Data shown a decrease of BrdU+ cells in TRPV2 agonist treated cells (40% in RPMI, 30% in U266 and 28% in SKO-007) compared to control, while no significant differences were observed by Annexin-V analysis. Moreover, to evaluate a putative role of TRPV2 in influencing Bortezomib cytotoxicity, RPMI, SKO-007 and U266, were co-treated with the TRPV2 agonist in combination or not with Bortezomib, for three days. By a dose-response MTT analysis we determined that TRPV2 activation reduced MM cell lines viability more than 40% in RPMI, 20 % in U266 and 15% in SKO-007, compared to Bortezomib (5 ng/ml) alone. Summarizing, these preliminary data demonstrated that the TRPV2 cation channel was expressed in human MM cell lines and that activation of TRPV2 could play a role in increasing Bortezomib cytotoxicity, by inhibiting cell proliferation rather than increasing apoptosis of MM cells. These data may open new perspectives in combination therapy, albeit the molecular mechanisms undergone TRPV2 activation needs to be clarified. Disclosures: No relevant conflicts of interest to declare.

Disruption of the Crosstalk CXCL12/CXCR4 Chemotactic Pathway To Enhance Melphalan-Induced Apoptosis in Multiple Myeloma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3445-3445
Author(s):  
Ayman A. Saad ◽  
James Fortney ◽  
Lin Wang ◽  
Heather O’Leary ◽  
Laura Gibson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Western Blot ◽  
Cell Lines ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Apoptotic Markers ◽  
Induced Apoptosis ◽  
B1 Cells

Abstract INTRODUCTION: Multiple myeloma cells display functional CXCR4 chemokine receptor that stimulates the migration of these cells toward their natural ligand, CXCL12 (stromal-derived factor, SDF-1a). CXCL12 is secreted by bone marrow stroma. Consistent with their CXCR4 expression, myeloma cells home to the marrow microenvironment, where adhesive interactions promote growth, survival, and confer cell adhesion-mediated drug resistance. METHODS U266-B1 cells (ATCC myeloma cell line) were pre-treated with recombinant CXCL12 for 30 minutes prior to the addition of melphalan for up to 72 hours. Both melphalan and CXCL12 were added at 24 hour intervals. Cell lines alone and cell lines with only melphalan or only CXCL12 were used as controls. We have also tested the influence of adding AMD3100, a reversible inhibitor of CXCR4, on myeloma cell survival. U266-B1 cells in media alone were pre-treated with AMD3100 for 24 hours prior to treatment with melphalan for 16 hours. Cell viability following treatment was quantified by flow cytometry assay using Annexin-V-FITC staining. Western blot analysis was used to quantify the apoptotic activity of the cell lines using 4 apoptotic markers: PARP (poly ADP-ribose polymerase), caspase-3, Bcl-2, and Mcl-1. RESULTS: Recombinant CXCL12 conferred a protective effect to myeloma cell lines during melphalan treatment. This effect was more pronounced at 72 hours of treatment. Western blot analysis showed diminished expression of the apoptotic markers, cleaved PARP (poly ADP-ribose polymerase) and active caspase-3 in the melphalan-treated cell lines with prior exposure to CXCL12. Additionally, pretreatment with AMD3100 resulted in enhanced apoptosis following melphalan treatment. CONCLUSION: Our data showed that CXCL12, a naturally occurring cytokine secreted by bone marrow stromal cells confers a protective effect on myeloma cells against apoptosis. Disruption of this effect by AMD3100 resulted in enhanced melphalan-induced apoptosis of myeloma cells. We are continuing to study this effect with the potential future utility of AMD3100 as a melphalan chemo-sensitizer in the treatment of multiple myeloma.

Daratumumab Directly Induces Human Multiple Myeloma Cell Death and Acts Synergistically with Conventional and Novel Anti-Myeloma Drugs

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3013-3013
Author(s):  
Sun-Young Kong ◽  
Xian-Feng Li ◽  
Sabikun Nahar ◽  
Weihua Song ◽  
Michel de Weers ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Viability ◽  
Caspase 3 ◽  
Annexin V ◽  
Dependent Manner ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Synergistic Cytotoxicity ◽  
Dose Dependent

Abstract Abstract 3013 Daratumumab is a novel fully human therapeutic CD38-specific monoclonal antibody (mAb) that is currently in phase I/II safety and dose finding clinical studies in MM. We recently demonstrated that daratumumab induces antibody dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) against multiple myeloma (MM) cells (ASH Abstract #608, 2009). Significantly, daratumumab induces ADCC-mediated autologous lysis against MM patient cells. In addition, when cross-linked, daratumumab directly induces Ramos lymphoma cell death. We here studied whether daratumumab directly kills MM cells and whether daratumumab could be combined with other anti-MM drugs to further enhance its direct cytotoxicity. Direct daratumumab-induced MM cell death was determined using CellTiter-Glo luminescent cell viability assay and Annexin V/PI flow cytometry analysis, with or without goat anti-human IgG crosslinking. Following 48h incubation, daratumumab (0.1-10 μg/ml), when cross-linked, directly induced cytoxicity against dexamethasone (dex)-sensitive MM1S and dex-resistant MM1R cells, as evidenced by decreased cell viability in a dose-dependent manner. Importantly, cross-linked daratumumab increased caspase 3/7 activities in a dose-dependent fashion, as assessed by the Caspase-Glo® 3/7 luminescence assay. Furthermore, daratumumab upregulated Annexin V+ and Annexin V+/PI+ cells in freshly isolated CD138+ MM patient cells, from 7.7% to 20.6% and 10.9% to 15.4 %. Therefore, cross-linked daratumumab can directly trigger apoptosis of patient myeloma cells. Cell viability assay was further performed on MM1S cells when daratumumab (0.1, 1, 10 μg/ml) was combined with dex (0.5 and 1 μM) or bortezomib (2.5, 5, and 10 nM). Following 48–72h incubation with daratumumab, both dex and bortezomib synergistically inhibited MM cell viability, as determined by combination index (CI) < 0.5 at given combined concentrations of these drugs. Enhanced caspase 3/7 activation was also seen when daratumumab was combined with dex. To evaluate combination cytotoxicity induced by lenalidomide with daratumumab, peripheral blood mononuclear effector cells (PBMCs) from normal donors (n=2) were pretreated with lenalidomide (2 μM) for 3 days followed by daratumumab-mediated ADCC assays against MM1S cells. Using calcein-AM release measurements, lenalidomide-pretreated PBMCs further augmented daratumumab-induced MM1S cell lysis, whereas daratumumab-pretreated PBMCs did not alter ADCC. Taken together, our studies show that daratumumab directly induces MM cell death via activation of caspase 3/7 and daratumumab induced synergistic cytotoxicity with dex or bortezomib. Moreover, lenalidomide augments daratumumab-induced ADCC against MM cells. These results further support combination clinical trials of conventional and novel anti-MM drugs with daratumumab in MM. Disclosures: Weers: Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Autocrine and Paracrine Regulatory Mechanisms of Growth Arrest-Specific Gene 6 Contribute to Disease Progression of Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4179-4179
Author(s):  
Miki Furukawa ◽  
Hiroshi Ohkawara ◽  
Kazuhiko Ikeda ◽  
Emi Ito ◽  
Jun-ichi Imai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Disease Progression ◽  
Growth Arrest ◽  
Annexin V ◽  
Neutralizing Antibody ◽  
Specific Gene ◽  
Proliferation Assay ◽  
Apoptosis Inhibition

Abstract Multiple myeloma (MM) is a lethal hematologic neoplasm characterized by proliferation of malignant plasma cells in part due to abnormal surrounding microenvironment, but the mechanism of disease progression remains largely unknown in MM. Thus, we screened molecules differentially expressed in bone marrow (BM) cells of MM patients using microarray analysis, which revealed significant upregulation of Gas6 (growth arrest-specific gene 6), a vitamin K-dependent protein with a structural homology with protein S. Since upregulation of Gas6 and its tyrosine kinase receptors have been reported to associate with various malignancies, we hypothesized that Gas6 contributed to the pathogenesis of MM. Gas6 was overexpressed in the BM of MM patients and in various MM cell lines. We found that the serum levels of Gas6 protein were markedly increased in 18 symptomatic MM patients compared to 16 healthy volunteers using human Gas6 ELISA. High levels of soluble Gas6 were detected in the supernatants of in vitro cultured MM cell lines RPMI-8226 and AMO-1 using Gas6 ELIZA. Analysis of apoptosis by flow-cytometry (FCM) with annexin V showed that exogenous Gas6 (100-200 ng/mL) inhibited the apoptosis of MM cells. MTT cell proliferation assay showed that exogenous Gas6 induced cell proliferation of MM cells with increased extracellular signal regulated kinase (ERK) phosphorylation. These results suggest that the presence of an autocrine loop of Gas6 and its receptor with ERK signaling enhanced the proliferative and anti-apoptotic effects on MM cells. Bone marrow stromal cell (BMSC) line HS-5 was found to secrete high levels of soluble Gas6 into culture supernatants using Gas6 ELIZA. Reduction of MM cell apoptosis by HS-5 cell-conditioned medium (CM) was suppressed by Gas6 neutralizing antibody (20 microg/mL) using FCM with annexin V. HS-5 cell-CM induced a marked increase in cell proliferation of MM cells, which was completely inhibited by Gas6 neutralizing antibody (0.2-20 microg/mL) using MTT cell proliferation assay. Our results showed a critical role of Gas6 in the pathology of apoptosis inhibition and cell proliferation of MM cells through paracrine mechanisms. Interleukin (IL)-6 is identified as a major growth and anti-apoptotic factor of MM cells through autocrine mechanisms. Conversely, BMSCs provide efficient support for MM cell survival by paracrine IL-6 stimulation. As determined by western blotting and real-time PCR, HS-5 cell-CM induced Gas6 upregulation, which was suppressed by IL-6 neutralizing antibody (1-10 microg/mL in a dose-dependent manner in MM cells. Meanwhile, HS-5 cell-CM induced the increased IL-6 expression, which was reversed by Gas6 neutralizing antibody in MM cells. Our findings indicate that Gas6 possessed both autocrine and paracrine functions similar to IL-6 between MM cells and BMSCs. Intercellular cell adhesion molecule-1 (ICAM-1) enhanced adhesion of MM cells to BMSCs and subsequently resulted in MM disease progression. Our immnoblotts showed that HS-5 cell-CM induced ICAM-1upregulation, and the ICAM-1 upregulation was reversed by Gas6 neutralization antibody, indicating that Gas6 stimulation increased ICAM-1 synthesis and subsequently accelerated the proliferation of MM cells. The present study offers new insights into autocrine and paracrine actions of Gas6 in concert with IL-6 between MM cells and BMSCs on apoptosis inhibition and cell proliferation of MM cells. We suggest that Gas6-related signaling pathways may be an attractive therapeutic target for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circ-DONSON Knockdown Inhibits Cell Proliferation and Radioresistance of Breast Cancer Cells via Regulating SOX4

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Xiufang Zhu ◽  
Lei Li
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Survival ◽  
Cell Lines ◽  
Caspase 3 ◽  
Biological Role ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay

Background. Circular RNAs have been validated as critical regulators in the development of breast cancer (BC). Circ-DONSON is involved in the progression of glioma and gastric cancer. However, the biological role of circ-DONSON in BC remains unclear, and the aim of this study was to explore the biological role of circ-DONSON in BC. Methods. Human tissue samples and BC cell lines were collected in this study. siRNAs against circ-DONSON were transfected into BC cell lines for silencing of circ-DONSON. Quantitative real-time PCR was used to test the circ-DONSON expression. Cell counting kit-8 (CCK-8), 5-bromo-2′ deoxyuridine enzyme-linked immunosorbent assay (BrdU-ELISA), colony formation, and caspase-3 activity assays were used to assess cell proliferation, cell survival, and cell viability. Western blotting analysis was used to detect the protein expression levels. Results. Our findings showed that circ-DONSON showed high expression in BC tissues and cell lines. CCK-8 and BrdU-ELISA assays showed that circ-DONSON knockdown inhibited BC cell proliferation. Moreover, cell survival, cell viability, and caspase-3 activity assays showed that circ-DONSON knockdown reduced the radioresistance of BC cells. Mechanistically, circ-DONSON regulated BC cell proliferation and radioresistance via SRY-box transcription factor 4 (SOX4). SOX4 overexpression significantly rescued the effect of circ-DONSON knockdown on BC cell proliferation and radioresistance. Moreover, circ-DONSON activated the Wnt/β-catenin pathway in BC cells via SOX4. Conclusion. Our study concluded that circ-DONSON knockdown hindered cell proliferation and radioresistance through the SOX4/Wnt/β-catenin pathway in BC.

Overexpression of Guanine Nucleotide Binding Protein, Alpha Stimulating (GNAS) Regulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in High Glucose Environment by Regulating ERK/P38 Signaling Pathway

2020 ◽  
Vol 10 (2) ◽  
pp. 259-264
Author(s):  
Wei Zhang ◽  
Yuanbo Wang ◽  
Song Jin ◽  
Hui Xin ◽  
Changxin Wang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Signaling Pathway ◽  
High Glucose ◽  
Caspase 3 ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) can treat osteoporosis. Whether GNAS affects BMSCs osteogenic differentiation under high glucose condition is unknown. Rat BMSCs were isolated and randomly divided into control group, high glucose group and GNAS group. The BMSCs were transfected with GNAS plasmid in high glucose environment followed by analysis of GNAS expression by Real time PCR and Western blot, BMSCs proliferation by MTT assay, Caspase 3 activity, ALP activity, formation of calcified nodules by alizarin red staining, OC and BMP-2 expression by Real time PCR and expression of ERK/P38 signaling pathway protein by Western blot. In high glucose environment, GNAS expression was significantly decreased, cell proliferation was inhibited, Caspase 3 activity was increased, along with decreased ALP activity, calcified nodules formation and expression of OC, BMP-2, p-ERK1/2 and p-P38 (P < 0.05). GNAS plasmid transfected into high glucose environment BMSCs can significantly promote GNAS expression and cell proliferation, decrease Caspase 3 activity, increase p-ERK1/2 and p-P38 expression, ALP activity and calcified nodules formation as well as increase OC and BMP-2 expression (P < 0.05). GNAS1 expression is decreased in BMSCs cells in a high glucose environment. Overexpression of GNAS1 can inhibit the apoptosis of BMSCs by regulating the ERK/P38 signaling pathway, promote its proliferation and differentiation into osteogenic direction.

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