Fibroblast Activation Protein Protects Bortezomib Induced Apoptosis In Multiple Myeloma Cells Through β-Catenin Signaling Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3083-3083
Author(s):  
Fuming Zi ◽  
Jingsong He ◽  
Donghua He ◽  
Yi Li ◽  
Li Yang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Fibroblast Activation Protein ◽  
Myeloma Cells ◽  
Fibroblast Activation ◽  
Qrt Pcr ◽  
Induced Apoptosis

Abstract Background Multiple myeloma(MM) is a malignant plasma cells proliferative disease which is characterized by increased blood calcium level, renal insufficiency, anemia, and bone lesions(CRAB). The intricate cross-talk with the bone marrow microenvironment plays an important role in facilitating the growth and survival of myeloma cells. Fibroblast activation protein(FAP) is a vital transmembrane protein expressed in 90% epithelial tumor stroma, which is reported to be involved in mediating drug resistance, tumorigenesis, neoplastic progression, angiogenesis, invasion and metastasis of tumor cells. The present study is aimed to investigate the roles of FAP may play in regulation of apoptosis in MM cells induced by bortezomib and the potential signaling pathway that FAP may be participated in. Methods Bone marrow mesenchymal stem cells(hBMMSCs) from MM patients and normal donors and the cell line of hBMMSCs were analyzed for expression of the FAP protein by semiquantitative real time-polymerase chain(qRT-PCR), flow cytometry(FCM) and immunofluorescence(IF). Tumor cell-conditioned medium (TCCM) from supernatant of MM cell lines were added to hBMMSCs to observe the effect of TCCM for the expression of FAP. We further studied the function and mechanism of FAP in bortezomib induced apoptosis of myeloma cells by silencing FAP with small interfering RNA(siRNA). Apoptotic cells of MM cells were detected by APC-CD138/annexin V-FITC using flow cytometry analysis. Western blotting was used to elucidate the signaling pathway that FAP may be involved in mediating apoptosis of MM cells induced by bortezomib. Results There was no significant difference in the expression of FAP in hBMMSCs isolated from MM patients and normal donors(p>0.05) as determined by qRT-PCR, FCM and immunofluorescence. hBMMSCs stimulated by TCCM for 7 days displayed an elevated expression of FAP as detected by qRT-PCR(p<0.05). In the presence of 30nM bortezomib, MM cell lines RPMI8226 or CAG cells co-cultured with hBMMSCs in which FAP was knockdown or not by siRNA for 48h demonstrated that FAP is capable of protecting RPMI8226 and CAG cells induced by bortezomib from apoptosis as determined by FCM(NC siRNA vs FAP siRNA, p<0.05). Further study showed that the activity of β-catenin was significantly elevated in RPMI8226 cells after co-cultured with hBMMSC in the presence of bortezomib. Knockdown FAP can reduce the expression of β-catenin and its downstream target proteins, such as c-myc, survivin, cyclin D1 in RPMI8226 cells detected by western blot. Conclusions Taken together, our data indicated that the expression level of FAP was no difference between the hBMMSCs isolated from MM patients and normal donors. The expression of FAP can be increased by TCCM stimulation. Further study demonstrated that FAP can protect MM cells from apoptosis induced by bortizomib, which is likely through β-catenin signaling pathway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1

Blood ◽  
2014 ◽  
Vol 124 (25) ◽  
pp. 3748-3757 ◽  
Author(s):  
Tomohiro Umezu ◽  
Hiroko Tadokoro ◽  
Kenko Azuma ◽  
Seiichiro Yoshizawa ◽  
Kazuma Ohyashiki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Tube Formation ◽  
Myeloma Cells ◽  
Endothelial Tube Formation ◽  
Key Points ◽  
Resistant Cells

Key Points We established hypoxia-resistant cells that can mimic in vivo conditions of hypoxic bone marrow. Exosomal miR-135b derived from these cell lines enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway.

A Novel Mouse Model of Multiple Myeloma Representative of Human Disease and Its Use in Preclinical Therapeutic Assessment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2907-2907
Author(s):  
Rosemary A Fryer ◽  
Timothy J Graham ◽  
Emma M Smith ◽  
Brian A Walker ◽  
Gareth J Morgan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Human Condition ◽  
Myeloma Cells ◽  
Treatment Groups ◽  
Hyperintense Signal

Abstract Abstract 2907 In order to aid the pre-clinical development of novel therapeutics for multiple myeloma, an in vivo model which recapitulates the human condition in particular tumor growth patterns and response to treatment is required. An important feature of such a model is the interaction of the myeloma cells with the bone marrow microenvironment as this is known to modulate tumor activity and protect against drug-induced apoptosis. We have developed a model with myeloma restricted to the bone marrow, which proceeds rapidly from initial inoculation to disease progression, and possesses a range of chemo-sensitive markers with which to monitor anti-tumor response. Female NOD/SCID γcnull mice were injected inta-osseously with luciferase-tagged myeloma cell lines. Disease progression was monitored weekly by bioluminescent imaging (BLI) and measurement of paraprotein levels (ELISA). These methods were compared to histological assessment of tumor infiltration and MRI which provided a quantitative measurement of progression. On T2-weighted images tumor was identified as a hyperintense signal enclosed within cortical bone. Tumor burden was quantified from regions of interest drawn on the periphery of the hyperintense signal. Luciferase-tagged cells engrafted by 3 weeks at the injection site and progressed to the femurs, spine and pelvis from week 4. BLI showed a significant increase in radiance from 5.6×105 to 43.0×105p/s/cm2/sr between weeks 5 and 7 (p<0.05). Quantification of tumor volume by MRI showed a significant increase from 6.4mm3 to 27.6mm3 between weeks 4 and 8 (p<0.05) and μCT demonstrated lytic disease. Serum levels of Igλ increased from 860ng/ml to 4325ng/ml during this period (p<0.05), which mirrored the changes seen with BLI and MRI. Flow cytometry and histology confirmed the confinement of CD138 positive myeloma cells within the bone. These results indicate successful engraftment of human myeloma cell lines with induction of myeloma in a pattern similar to the human condition. We have adapted this model to study primary patient material. 10 mice were implanted with samples from 3 cases of plasma cell leukemia with complex cytogenetics. 5 of these developed myeloma confined to the bone marrow, 2 with additional plasmacytoma localized at the injection site, over a period of 1–5months. We have characterized the original patient cells with gene expression, SNP based gene mapping and have characterized the nature of the engrafted cells using similar technology. We have also shown the model is suitable for preclinical assessment of anti-myeloma agents using bortezomib and a novel aminopeptidase inhibitor, tosedostat (CHR-2797). Non-treated mice displayed a significant increase in radiance from 16.13×105 to 69.00×105p/s/cm2/sr (p<0.01). In comparison, in the bortezomib and tosedostat treated groups no significant increase in radiance was seen (bortezomib: 5.22×105 to 1.12×105 p/s/cm2/sr; tosedostat: 9.92×105 to 13.78×105p/s/cm2/sr). Paraprotein levels mimicked these changes in BLI. At the end of treatment Igλ levels in control, bortezomib and tosedostat treated mice were 2473.7, 132.5 and 923.0ng/ml, respectively. Igλ levels in both treatment groups were significantly different from control (p<0.001). Average tumor volumes derived from MRI were significantly different in bortezomib (14.7mm3) and tosedostat treated (23.4mm3) groups compared to non-treatment (33.0mm3). The volumes for the bortezomib treated group showed no significant difference from control mice. In addition, there was a decrease in CD138 expression by flow cytometry in bone aspirates from treatment groups compared to control which was mirrored in histological samples. In conclusion using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma with secretion of paraprotein, disease confined to the bone marrow, lytic bone lesions and spinal compression. In addition, this model is suitable for assessing the efficacy of novel therapeutics in vivo, using a number of non-invasive tumor markers such as BLI and MRI. Disclosures: Morgan: J&J: Honoraria, Speakers Bureau. Davies:J&J: Honoraria, Speakers Bureau.

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.

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 610-618 ◽  
Author(s):  
Inge Tinhofer ◽  
Ingrid Marschitz ◽  
Traudl Henn ◽  
Alexander Egle ◽  
Richard Greil
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Constitutive Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cytotoxic Treatment ◽  
Interleukin 15 ◽  
Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

Reduced Response of IRE1α/Xbp-1 Signaling Pathway to Bortezomib Contributes to Drug Resistance in Multiple Myeloma Cells

2016 ◽  
Vol 103 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Xiaoxuan Xu ◽  
Junru Liu ◽  
Beihui Huang ◽  
Meilan Chen ◽  
Shiwen Yuan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Resistance Mechanism ◽  
Proteasome Inhibition ◽  
Myeloma Cells ◽  
Potential Marker ◽  
Rpmi 8226 ◽  
Basic Level

Purpose Proteasome inhibition with bortezomib eliminates multiple myeloma (MM) cells by partly disrupting unfolded protein response (UPR). However, the development of drug resistance limits its utility and resistance mechanism remains controversial. We aimed to investigate the role of IRE1α/Xbp-1 mediated branch of the UPR in bortezomib resistance. Methods The expression level of Xbp-1s was measured in 4 MM cell lines and correlated with sensitivity to bortezomib. LP1 and MY5 cells with different Xbp-1s level were treated with bortezomib; then pivotal UPR regulators were compared by immunoblotting. RPMI 8226 cells were transfected with plasmid pEX4-Xbp-1s and exposed to bortezomib; then apoptosis was determined by immunoblotting and flow cytometry. Bortezomib-resistant myeloma cells JJN3.BR were developed and the effect on UPR signaling pathway was determined. Results By analyzing 4 MM cell lines, we found little correlation between Xbp-1s basic level and bortezomib sensitivity. Bortezomib induced endoplasmic reticulum stress-initiated apoptosis via inhibiting IRE1α/Xbp-1 pathway regardless of Xbp-1s basic level. Exogenous Xbp-1s reduced cellular sensitivity to bortezomib, suggesting the change of Xbp-1s expression, not its basic level, is a potential marker of response to bortezomib in MM cells. Furthermore, sustained activation of IRE1α/Xbp-1 signaling pathway in JJN3.BR cells was identified. Conclusions Our data indicate that reduced response of IRE1α/Xbp-1 signaling pathway to bortezomib may contribute to drug resistance in myeloma cells.

Multiple Myeloma: Interleukin-15 Antagonizes Bone Morphogenetic Protein-Induced Apoptosis and Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3444-3444
Author(s):  
Magne Rekvig ◽  
Anne-Tove Brenne ◽  
Torstein Baade Ro ◽  
Anders Waage ◽  
Magne Borset ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Inhibition ◽  
Cell Line ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Stimulus Figure ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract Multiple myeloma has two distinct features: Expansion of malignant plasma cells within the bone marrow accompanied by skeletal destruction. Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and inhibit growth in myeloma cells. BMPs are members of the TGF-β superfamily of proteins capable of inducing bone formation, and regulate proliferation, differentiation and apoptosis. We have investigated myeloma cell apoptosis and proliferation with BMP-4 and −6 in concert with the myeloma cell growth factors interleukin (IL)-2, IL-6, IL-10, IL-15, IL-21, tumor necrosis factor (TNF)-α and insulin-like growth factor (IGF)-1. Eight samples of highly purified myeloma cells from patients and a human myeloma cell line, IH-1 (Brenne AT et al. Blood. 2002 May 15;99(10):3756–62.), were used in this study. Cytokine concentrations used in the referred experiments were for BMP-4 20ng/ml, BMP-6 250ng/ml, IL-15 20ng/ml and IL-6 0,1ng/ml, respectively. Growth inhibition was measured in a proliferation assay by methyl-[3H]-thymidine incorporation and apoptosis by annexin V- FITC-binding/PI-uptake on flow cytometry. IL-15 antagonized growth inhibition (Figure A) and prevented apoptosis induced by BMP-4 (Figure B) and BMP-6 in the myeloma cell line IH-1. IL-15 also antagonized the growth inhibition induced by BMP-4 and/or BMP-6 in three out of eight patient samples. Neither IL-6, nor any of the other investigated cytokines were able to rescue the myeloma cells from growth inhibition and apoptosis induced by BMP-4 and -6. Among the investigated cytokines, we found that IL-15 has a unique capability to antagonize BMP- induced apoptosis and growth inhibition in myeloma cells. We examined cleavage of the proapoptotic protein caspase-3 and found that BMP-4 activated caspase-3 in the IH-1 cell line. This activation of caspase-3 was blocked by IL-15 but not by IL-6. We have demonstrated a possible mechanism for myeloma cells to escape apoptosis and growth-inhibition within the bone marrow. Intramedullar levels of IL-15 and BMPs may play a role in the pathogenesis of multiple myeloma. Figure A. Proliferation in response to BMP-4 stimulus Figure A. Proliferation in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus

CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3457-3457 ◽  
Author(s):  
Eric D. Hsi ◽  
Roxanne Steinle ◽  
Balaji Balasa ◽  
Aparna Draksharapu ◽  
Benny Shum ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

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