interleukin-8 and Multiple-Myeloma

Peripheral blood cell counts and cytokines can be used as predictors of multiple myeloma (MM) patients' outcomes. 313 newly diagnosed MM patients treated with novel agents were divided into training and validation cohorts. We selected the common peripheral blood cell counts, including the lymphocyte/monocyte ratio (LMR), neutrophil/lymphocyte ratio (NLR), and platelet/lymphocyte ratio (PLR), systemic inflammation response index (SIRI), and serum cytokines which contained tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-2 receptor (IL-2R), interleukin-8 (IL-8), interleukin-6 (IL-6), and interleukin-10 (IL-10) as related variables. The least absolute shrinkage and selection operator (LASSO) regression was conducted to sort the predictor variables in the training cohort, and then the developed nomogram was assessed in the training and validation cohort. Our study showed that SIRI, PLR, and IL-8 were independent prognostic factors for the survival of MM patients. Patients with lower SIRI (≤ 0.87) had superior survival than patients with higher SIRI (> 0.87). Further, according to the LASSO regression, a nomogram embracing LMR (> 3.78), SIRI (> 0.87), PLR (≤ 106.44), and IL-8 was established. The nomogram demonstrated a better correlation with the outcomes of MM patients in the training cohort than International Staging System (ISS) and Revised-International Staging System (R-ISS). The same results were verified in the validation cohort. The nomogram incorporating inflammatory cells and cytokines could be a helpful tool to stratify MM patients in the era of novel agents.

Topics: Cytokines; Humans; Interleukin-8; Multiple Myeloma; Nomograms; Prognosis

Angiogenesis is considered important in the pathogenesis of multiple myeloma (MM), as well as in the targeted treatment of the disease. Leucine-rich α2-glycoprotein 1 (LRG1) is a protein that participates in angiogenesis and its effect on solid organ tumors has been investigated recently. This study aimed to investigate the relationship between MM and LRG1.. The MM patients who admitted to Hatay Mustafa Kemal University Hematology Clinic between September 2021 and October 2022 were included in the study. The study consists of a total of 4 groups: newly diagnosed MM (NDMM), relapsed refractory MM (RRMM), MM in remission (Rem-MM), and control group. Demographic data were retrieved from hospital records. Blood samples of our study groups were centrifuged at 1,500 × g for 10 min and serum was collected. LRG1, IL-6, IL-8, TGF-β1, HIF-1α, FGF-2, and VEGF levels were analyzed in all groups by ELISA method, and statistical analysis was performed.. A total of 112 individuals, including NDMM (n: 27), RRMM (n: 18), Rem-MM (n: 42), and control group (n: 25), were enrolled in the study. Based on the analyses, the NDMM group exhibited significantly elevated levels of LRG1 (p < 0.001), TGF-1 (p < 0.001), and HIF-1α (p = 0.046, p < 0.001, and p = 0.003 compared to the RRMM, Rem-MM, and control groups, respectively) compared to the other groups. LRG1 levels were positively correlated with creatinine (r: 0.363, p = 0.001), calcium (r: 0.344, p = 0.001), total protein (r: 0.473, p < 0.001), erythrocyte sedimentation rate (r: 0.547, p < 0.001), lactate dehydrogenase (r: 0.321, p = 0.003), beta-2-microglobulin (r: 0.312, p = 0.017), IL-6 (r: 0.478, p < 0.001), IL-8 (r: 0.240, p = 0.03), TGF-β1 (r: 0.521, p < 0.001), and HIF-1α (r: 0.321, p = 0.003) levels and were negatively correlated with hemoglobin (r: -0.512, p < 0.001) and albumin (r: -0.549, p < 0.001) levels. Receiver operating characteristics (ROC) analysis revealed the association of LRG1 with the highest AUC value of 0.959 (95% CI: 0.904-1, p < 0.001) and the optimal cut-off value of 534.95 ng/mL (sensitivity: 93% and specificity: 99%) in the NDMM group compared to the control group.. In this study, providing data for the first time on LRG1 levels in the setting of MM. LRG1 levels were found to be significantly higher in NDMM patients and in our study discriminate this patient population from RRMM, Rem-MM, and normal controls. Therefore, LRG1 seems to a potential biomarker that should be evaluated in future studies addressing the diagnosis, staging, follow-up, prognosis, and treatment target of MM.

Topics: Glycoproteins; Humans; Interleukin-6; Interleukin-8; Leucine; Multiple Myeloma; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Multiple myeloma (MM) remains an incurable disease that needs better recognition and further research. Previous studies elucidated the interaction between myeloma cells and showed the necessity of bone marrow stromal cells for the initiation and progression of MM. Many chemokines and their receptors including interleukin-8 (IL-8) and soluble interleukin-6 receptor (sIL-6R) play important roles in this interaction. The main purpose of this study is evaluating the serum level of IL-8 and sIL-6R on stage-I of MM patients and healthy controls.. Serum samples from 30 stage-I MM  patients (13 males and 17 females) and 30 healthy subjects as controls (13 males and 17 females) were examined in this study. The protein concentrations of serum IL-8 and sIL-6R were assessed by enzyme-linked immunosorbent assay (ELISA).. The mean level of IL-8 and sIL-6R were significantly elevated in stage-I MM. The mean levels of IL-8 were 1246.57±279.22 ng/ml in stage-I MM and 902.53± 294.61 ng/ml in controls (P<0.001). The mean levels of sIL-6R were 5.39±1.38 ng/ml and 4.1±1.14 ng/ml in stage-I MM and controls, respectively (P<0.001). The mean levels of IL-8 were 1342.18±193.4 ng/ml in patient females and 859± 278.2ng/ml in control females (P <0.001). The mean levels of sIL-6R were 5.21±1.55 ng/ml and 3.91±1.22 ng/ml in patient females and control females, respectively (P=0.01). The mean level of sIL-6R in patient males and control males were 5.63±1.43 ng/ml and 4.34±1.04 ng/ml, respectively (P=0.01). A significant correlation (Pearson's correlation = 0.45, P=0.008) was observed in the population of females (patients and controls).. The results of study suggest the possible involvement of IL-8 and the sIL-6R at stage-I MM and can better characterize the role of chemokines and their receptors in the disease process, especially in the early stages.

Topics: Aged; Biomarkers, Tumor; Case-Control Studies; Female; Follow-Up Studies; Humans; Interleukin-8; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Receptors, Interleukin-6

Although numerous cytokines influence proliferation and progression of multiple myeloma (MM), a relevant action in the onset of the disease also seems to be played by the oxidative state.. In the present study we evaluated the concentrations of interleukin-8 (IL-8) and soluble receptor of advanced glycation end products (sRAGE) in patients with MM, assessing the existing variations with respect to a control group and the possible existence of correlations between these molecules and the biological variables or the presence of a correlation between IL-8 and sRAGE. The study was conducted on 33 patients affected by MM compared to 39 healthy subjects.. IL-8 and sRAGE levels were significantly higher in MM patients compared to healthy subjects. sRAGE and IL-8 evidence no significant linear correlation. Furthermore, IL-8 was significantly increased in both sexes, but we found a slight variation for females compared to males.. IL-8 could play an important role in the onset of MM and the progression of bone disease, while the increased sRAGE values would seem to have a protective action in MM patients. Further studies on animal models may clarify the real impact of introducing modulation of IL-8 and sRAGE levels.

Topics: Aged; Antigens, Neoplasm; Case-Control Studies; Disease Progression; Female; Humans; Interleukin-8; Male; Mitogen-Activated Protein Kinases; Multiple Myeloma

Topics: Antineoplastic Agents; Bone Marrow; Bortezomib; Carcinogenesis; Cell Line, Tumor; Cell Survival; Chemokine CXCL10; Humans; Interleukin-6; Interleukin-8; Multiple Myeloma; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteomics; Recurrence; Signal Transduction; STAT3 Transcription Factor; STAT6 Transcription Factor; Tumor Microenvironment

Multiple myeloma (MM) is a clonal plasma cell malignancy associated with osteolytic bone disease. Recently, the role of MM-derived exosomes in the osteoclastogenesis has been demonstrated although the underlying mechanism is still unknown. Since exosomes-derived epidermal growth factor receptor ligands (EGFR) are involved in tumor-associated osteolysis, we hypothesize that the EGFR ligand amphiregulin (AREG) can be delivered by MM-derived exosomes and participate in MM-induced osteoclastogenesis.. Exosomes were isolated from the conditioned medium of MM1.S cell line and from bone marrow (BM) plasma samples of MM patients. The murine cell line RAW264.7 and primary human CD14. We found that AREG was specifically enriched in exosomes from MM samples and that exosomes-derived AREG led to the activation of EGFR in pre-OC, as showed by the increase of mRNA expression of its downstream SNAIL in both RAW264.7 and CD14. In conclusion, our data indicate that AREG is packed into MM-derived exosomes and implicated in OC differentiation through an indirect mechanism mediated by OBs.

Topics: Amphiregulin; Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Exosomes; Humans; Interleukin-8; Ligands; Mesenchymal Stem Cells; Mice; Multiple Myeloma; Osteoblasts; Osteoclasts; Osteogenesis; RAW 264.7 Cells; Tumor Microenvironment

DNA damage-induced NF-κB activation and the secretion of inflammatory cytokines play crucial roles in carcinogenesis and cellular senescence. However, the underlying mechanisms, especially the initial sensors and transducers connecting the nuclear DNA damage signal with cytoplasmic NF-κB activation remain incompletely understood. Here, we report that TRAF-interacting protein with forkhead-associated domain (TIFA), an established NF-κB activator in the cytosol, unexpectedly exhibited nuclear translocation and accumulation on damaged chromatin following genotoxic stress. Accordingly, we also found that DNA damage-induced transcriptional activation and the resulting secretion of classic NF-κB targets, including interleukin (IL)-6 and IL-8, was greatly enhanced in TIFA-overexpressing cells compared with control cells. Mechanistically, DNA damage-induced TIFA phosphorylation at threonine 9 (pThr-9), and this phosphorylation event, involving the pThr-binding forkhead-associated domain, was crucial for its enrichment on damaged chromatin and subsequent NF-κB activation. Moreover, in conjunction with its partner protein, the E3 ligase TNF receptor-associated factor 2 (TRAF2), TIFA relayed the DNA damage signals by stimulating ubiquitination of NF-κB essential modulator (NEMO), whose sumoylation, phosphorylation, and ubiquitination were critical for NF-κB's response to DNA damage. Consistently, TRAF2 knockdown suppressed TIFA overexpression-enhanced NEMO ubiquitination under genotoxic stress, and a unphosphorylatable Thr-9-mutated TIFA variant had only minor effects on NEMO poly-ubiquitination. Finally, in agreement with the model of DNA damage-associated secretory senescence barrier against carcinogenesis, ectopic TIFA expression limited proliferation of multiple myeloma cancer cells. In conclusion our results indicate that TIFA functions as a key transducer in DNA damage-induced NF-κB activation.

Topics: Adaptor Proteins, Signal Transducing; Carcinogenesis; Cell Proliferation; Chromatin; DNA Damage; HEK293 Cells; HeLa Cells; Humans; I-kappa B Kinase; Interleukin-6; Interleukin-8; Multiple Myeloma; Mutagens; NF-kappa B; Phosphorylation; Protein Binding; Signal Transduction; TNF Receptor-Associated Factor 2; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; Ubiquitination

Multiple myeloma (MM) remains an incurable malignancy despite the recent advancements in its treatment. The protective effects of the niche in which it develops has been well documented; however, little has been done to investigate the MM cell's ability to 're-program' cells within its environment to benefit disease progression. Here, we show that MM-derived macrophage migratory inhibitory factor (MIF) stimulates bone marrow stromal cells to produce the disease critical cytokines IL-6 and IL-8, prior to any cell-cell contact. Furthermore, we provide evidence that this IL-6/8 production is mediated by the transcription factor cMYC. Pharmacological inhibition of cMYC in vivo using JQ1 led to significantly decreased levels of serum IL-6-a highly positive prognostic marker in MM patients.. Our presented findings show that MM-derived MIF causes BMSC secretion of IL-6 and IL-8 via BMSC cMYC. Furthermore, we show that the cMYC inhibitor JQ1 can reduce BMSC secreted IL-6 in vivo, irrespective of tumor burden. These data provide evidence for the clinical evaluation of both MIF and cMYC inhibitors in the treatment of MM.

Topics: Bone Marrow Cells; Humans; Interleukin-6; Interleukin-8; Intramolecular Oxidoreductases; Macrophage Migration-Inhibitory Factors; Multiple Myeloma; Proto-Oncogene Proteins c-myc; Stromal Cells

IL-8 promotes cancer cell growth, survival, angiogenesis, and metastasis in several tumors. Herein, we investigated the sources of IL-8 production in multiple myeloma (MM) and its potential roles in MM pathogenesis. We found that bone marrow cells from patients with MM secreted higher amounts of IL-8 than healthy donors. IL-8 production was detected in cultures of CD138(+) plasma cells and CD138(-) cells isolated from bone marrows of MM patients, and in three of seven human myeloma cell lines (HMCLs) analyzed. Interactions between MM and stromal cells increased IL-8 secretion by stromal cells through cell-cell adhesion and soluble factors. Interestingly, IL8 expression also increased in HMCLs, stromal cells, and osteoclasts after treatment with the antimyeloma drugs melphalan and bortezomib. In fact, the effect of bortezomib on IL-8 production was higher than that exerted by stromal-MM cell interactions. Addition of exogenous IL-8 did not affect growth of HMCLs, although it protected cells from death induced by serum starvation through a caspase-independent mechanism. Furthermore, IL-8 induced by stromal-MM cell interactions strongly contributed to osteoclast formation in vitro, because osteoclastogenesis was markedly reduced by IL-8-specific neutralizing antibodies. In conclusion, our results implicate IL-8 in myeloma bone disease and point to the potential utility of an anti-IL-8 therapy to prevent unwanted effects of IL-8 up-regulation on survival, angiogenesis, and osteolysis in MM.

Topics: Cell Separation; Cell Survival; Coculture Techniques; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-8; Multiple Myeloma; Osteoclasts; Osteogenesis; Polymerase Chain Reaction; Stromal Cells; Up-Regulation

Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70-80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance.

Topics: Antineoplastic Agents, Alkylating; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Interleukin-8; Melphalan; Metabolic Networks and Pathways; Multiple Myeloma; Oxidative Stress; Proteome; Proteomics; Signal Transduction; Transcriptome; Up-Regulation; Vascular Endothelial Growth Factor A

Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase β (IKKβ) and an increased recruitment of the nuclear IKKβ, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKβ and with p65, with preferential binding to S536P-p65. Both IKKβ activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKβ and EGR-1 as potential new targets in ovarian cancer combination therapies.

Topics: Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Line, Tumor; Cell Nucleus; Chemokine CCL2; Chemokine CXCL5; Early Growth Response Protein 1; Female; Gene Expression Regulation, Leukemic; Humans; I-kappa B Kinase; Interleukin-8; Multiple Myeloma; Ovarian Neoplasms; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Transcription Factor RelA

In multiple myeloma (MM), angiogenesis plays a substantial role in disease progression. Interleukin-8 (IL-8), a pro-inflammatory chemokine with potent pro-angiogenic properties, has been implicated in the pathophysiology of MM. The aim of the study is to measure serum levels of IL-8 in MM patients and to correlate them with markers of angiogenesis, such as circulating levels of platelet derived growth factor-AB (PDGF-AB) and angiogenin (Ang), and bone marrow microvascular density (MVD). Fifty-three newly diagnosed MM patients, 23 of them, who reached plateau phase after effective treatment and 20 healthy controls, were studied. Serum levels of PDGF-AB, Ang and IL-8 were measured by ELISA, whereas bone marrow MVD was estimated by immunohistochemical staining of vessels with anti-CD31. All measured parameters were higher in MM patients compared to controls and in increased disease stages. They all also significantly decreased in plateau phase. IL-8 correlated positively with Ang and PDGF-AB, but not with MVD. The circulating levels of IL-8, PDGF-AB and Ang are elevated in patients with MM. The lack of correlation between IL-8 with MVD suggests that its levels represent the inflammatory element of MM disease and the participation in angiogenesis process is rather complex with multifactorial mechanisms.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Bone Marrow; Female; Humans; Interleukin-8; Linear Models; Male; Microvessels; Middle Aged; Multiple Myeloma; Neovascularization, Pathologic; Ribonuclease, Pancreatic; Statistics, Nonparametric

Lenalidomide is an active immunomodulatory and antiproliferative agent in multiple myeloma. However, the molecular mechanisms driving these activities are not yet fully elucidated. Therefore, we investigated the modulation of the cytokine/growth factor patterns of myeloma cells under LEN treatment.. Lenalidomide effect on myeloma cell proliferation was investigated in a myeloma cell line collection (n=23) by (3)H-thymidine incorporation. Modulation of the cytokine/growth factor patterns of myeloma cells under LEN treatment was analysed by real-time quantitative PCR.. Lenalidomide inhibits the proliferation of two-thirds of myeloma cell lines independently of their genetic background. We demonstrated that LEN increased TNF-α and IL-8 inflammatory cytokines and insulin-like growth factor-1 (IGF-1) growth factor in both sensitive and resistant myeloma cells to LEN.. Lenalidomide favours a uniform TNF-α and IL-8 inflammatory and IGF-1 secretory profile of myeloma cells, an observation that raises important questions for therapeutic approaches incorporating the agent.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Bone Marrow Cells; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Immunologic Factors; Insulin-Like Growth Factor I; Interleukin-8; Lenalidomide; Multiple Myeloma; Thalidomide; Tumor Necrosis Factor-alpha

Free light chains (LCs) are among the many ligands that bind to cubilin/megalin for endocytosis via the clathrin-dependent endosomal/lysosomal pathway. Receptor associated protein (RAP), is a 39 kDA high-affinity, chaperone-like ligand for megalin that assists in the proper folding and functioning of megalin/cubilin. Although RAP is known to inhibit ligand binding to megalin/cubilin, its effect on LC endocytosis has not been shown directly.. We investigated whether RAP can block the endocytosis of LC in cultured human proximal tubule cells and whether this can prevent LC cytotoxicity. Immunofluorescence microscopy and flow cytometry showed that fluorescently labeled LC endocytosis was markedly inhibited in HK-2 cells pretreated with human RAP. The effect of RAP was dose-dependent, and was predominantly on endocytosis as it had no effect on the small acid-washable fraction of LC bound to cell membrane. RAP significantly inhibited LC induced cytokine production and phosphorylation of ERK1/2 and p38 MAPK. Prolonged exposure to LC for 48 h resulted in epithelial-to-mesenchymal transformation in HK-2 cells as evidenced by marked reduction in the expression of the epithelial cell marker E-cadherin, and increased the expression of the mesenchymal marker α-SMA, which was also prevented by RAP in the endocytosis medium.. RAP inhibited LC endocytosis by ∼88% and ameliorated LC-induced cytokine responses and EMT in human PTCs. The results not only provide additional evidence that LCs endocytosis occurs via the megalin/cubilin endocytic receptor system, but also show that blocking LC endocytosis by RAP can protect proximal tubule cells from LC cytotoxicity.

Topics: Cell Line; Endocytosis; Epithelial-Mesenchymal Transition; Humans; Immunoglobulin Light Chains; Interleukin-6; Interleukin-8; Kidney Tubules, Proximal; LDL-Receptor Related Protein-Associated Protein; Low Density Lipoprotein Receptor-Related Protein-2; Multiple Myeloma; Renal Insufficiency

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.

Topics: Cell Movement; DNA Methylation; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; MicroRNAs; Multiple Myeloma; NF-kappa B; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

In human multiple myeloma (MM), the tumor cells exhibit strict dependence on bone marrow (BM) stromal elements. It has been suggested that, in turn, MM cells modify multipotent stromal cells (MSCs), diverting them to support the myeloma. We investigated MM-derived MSCs by comparing their toll-like receptor (TLR) responses to those of MSCs derived from healthy controls. We now report that MM-derived MSCs manifested intact proliferation responses and IL-6 secretion and their adipose and osteogenic differentiation responses to TLR ligands were also similar to those of healthy controls, ranging from augmentation to inhibition. However, MM-derived MSCs were found to be defective in IL-8 secretion and ERK1/2 phosphorylation following TLR-2 activation. Moreover, MM-derived MSCs failed to respond to EGF by elevation of ERK1/2 phosphorylation. The persistence of these changes in extensively cultured MM-derived MSCs, suggests that these cells are stably, if not irreversibly modified.

Topics: Adult; Biomarkers; Cell Differentiation; Cell Membrane; Cell Proliferation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Interleukin-8; Kinetics; Ligands; Lipoproteins; Male; Mesenchymal Stem Cells; Middle Aged; Multiple Myeloma; Phosphorylation; Toll-Like Receptors

This study was aimed to investigate the mRNA expression levels of hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in bone marrow mesenchymal stem cells (MSC) from multiple myeloma (MM) patients. The mRNA expression levels of HGF, SDF-1, MCP-1 and IL-8 in bone marrow MSC from 20 newly diagnosed MM patients were detected by real time quantitative RT-PCR and were compared with that in 9 controls. The results indicated that the mean mRNA expression level of HGF was up-regulated in MM patients, as compared with controls (p < 0.01). However, the mean mRNA expression level of SDF-1 mRNA was down-regulated in MM patients, as compared with controls (p < 0.05). There was no significant difference in the mRNA expression levels of MCP-1 and IL-8 between MM and control cohorts (p > 0.05). It is concluded that BM-MSC from MM patients express HGF, SDF-1, MCP-1, IL-8, but these chemotaxis-related factors expression of bone marrow microenvironment cellular component are dysregulated in MM patients, which may result from the interplay between MM cells and MSC.

Topics: Bone Marrow Cells; Cells, Cultured; Chemokine CCL2; Chemokine CXCL12; Chemotactic Factors; Hepatocyte Growth Factor; Humans; Interleukin-8; Mesenchymal Stem Cells; Multiple Myeloma; RNA, Messenger

Components of the microenvironment such as bone marrow stromal cells (BMSCs) are well known to support multiple myeloma (MM) disease progression and resistance to chemotherapy including the proteasome inhibitor bortezomib. However, functional distinctions between BMSCs in MM patients and those in disease-free marrow are not completely understood. We and other investigators have recently reported that NF-kappaB activity in primary MM cells is largely resistant to the proteasome inhibitor bortezomib, and that further enhancement of NF-kappaB by BMSCs is similarly resistant to bortezomib and may mediate resistance to this therapy. The mediating factor(s) of this bortezomib-resistant NF-kappaB activity is induced by BMSCs is not currently understood.. Here we report that BMSCs specifically derived from MM patients are capable of further activating bortezomib-resistant NF-kappaB activity in MM cells. This induced activity is mediated by soluble proteinaceous factors secreted by MM BMSCs. Among the multiple factors evaluated, interleukin-8 was secreted by BMSCs from MM patients at significantly higher levels compared to those from non-MM sources, and we found that IL-8 contributes to BMSC-induced NF-kappaB activity.. BMSCs from MM patients uniquely enhance constitutive NF-kappaB activity in MM cells via a proteinaceous secreted factor in part in conjunction with IL-8. Since NF-kappaB is known to potentiate MM cell survival and confer resistance to drugs including bortezomib, further identification of the NF-kappaB activating factors produced specifically by MM-derived BMSCs may provide a novel biomarker and/or drug target for the treatment of this commonly fatal disease.

Topics: Antineoplastic Agents; Bone Marrow Cells; Boronic Acids; Bortezomib; Humans; Interleukin-8; Multiple Myeloma; NF-kappa B; Pyrazines; Stromal Cells

A number of growth factors secreted by bone marrow stromal cells (BMSCs), including interleukin-6 and -8 (IL-6/8), are important for the initiation and progression of multiple myeloma (MM). However, the mechanisms that regulate the production of IL-6/8 by BMSC have not yet been well characterized. Human dual functional protein apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is essential for cell survival and proliferation. Previous studies showed that APE1/Ref-1 was overexpressed in tumor cells, but few studies showed its expression in supportive cells in the tumor microenvironment. We first detected APE1/Ref-1 expression in BMSCs of normal, initial, and recurrent MM patients, and then explore the correlation between APE1/Ref-1 level and IL-6/8 secretion of BMSCs. A marked increase of APE1/Ref-1 expression and abnormal subcellular distribution were observed in MM BMSCs. APE1/Ref-1 overexpression was related to higher secretary level of IL-6/8 by MM BMSCs and the IL-6/8 secretion was blocked significantly by adenovirus-mediated APE1/Ref-1-specific (small interfering RNA) siRNA. Our results also demonstrated that APE1/Ref-1-specific siRNA significantly inhibited DNA binding activity of AP-1 and nuclear factor-κB (NF-κB), 2 important transcription factors in the regulation IL-6/8 secretion in MM BMSCs. The results provided by the present study indicate APE1/Ref-1, which plays a regulatory role in IL-6/8 production by BMSCs, may be a potential therapeutic target of MM.

Topics: Adult; Aged; Bone Marrow Cells; DNA-(Apurinic or Apyrimidinic Site) Lyase; Female; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Multiple Myeloma; RNA, Small Interfering; Stromal Cells

Multiple myeloma (MM) is a product of interactions between tumor plasma cells and multiple cell types native to the bone marrow (BM). We have used antibody array technology to examine the proteins produced by BM stromal cells in response to stimulation by BM taken from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. We observed increased production of the chemokine IL-8 by stromal cells co-cultured with supernatants from bone marrow cells of patients with active myeloma. IL-8 production is correlated with active disease and is dependent upon IL-1beta and NF-kappaB signaling. Consistent with the pro-angiogenic activity of IL-8, increased BM microvessel density (MVD) correlated with stimulation of stromal cell IL-8 production. In addition, the majority of MM cell lines and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1beta induced, and correlates with bone marrow angiogenesis.

Topics: Bone Marrow; Chemokines; Disease Progression; Flow Cytometry; Humans; Interleukin-1beta; Interleukin-8; Microarray Analysis; Multiple Myeloma; Neovascularization, Pathologic; NF-kappa B; Paraproteinemias; Prognosis; Receptors, Interleukin-8; Stromal Cells

Angiogenesis has a critical role in the pathophysiology of multiple myeloma (MM); however, the molecular mechanisms underlying this process are not completely elucidated. The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of angiogenesis. In this study, we found that ING4 expression in MM cells was correlated with the expression of the proangiogenic molecules interleukin-8 (IL-8) and osteopontin (OPN). Moreover, we demonstrate that ING4 suppression in MM cells up-regulated IL-8 and OPN, increasing the hypoxia inducible factor-1alpha (HIF-1alpha) activity and its target gene NIP-3 expression in hypoxic condition. In turn, we show that the inhibition of HIF-1alpha by siRNA suppressed IL-8 and OPN production by MM cells under hypoxia. A direct interaction between ING4 and the HIF prolyl hydroxylase 2 (HPH-2) was also demonstrated. Finally, we show that ING4 suppression in MM cells significantly increased vessel formation in vitro, blunted by blocking IL-8 or OPN. These in vitro observations were confirmed in vivo by finding that MM patients with high IL-8 production and microvascular density (MVD) have significantly lower ING4 levels compared with those with low IL-8 and MVD. Our data indicate that ING4 exerts an inhibitory effect on the production of proangiogenic molecules and consequently on MM-induced angiogenesis.

Topics: Aged; Angiogenic Proteins; Bone Marrow Examination; Cell Cycle Proteins; Cell Line, Tumor; Homeodomain Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; Middle Aged; Multiple Myeloma; Neovascularization, Pathologic; Osteopontin; Tumor Suppressor Proteins

Bone marrow endothelial cells (EC) from patients with multiple myeloma (MM) were found to express and secrete higher amounts of the CXC-chemokines CXCL8/interleukin (IL)-8, CXCL11/interferon-inducible T-cell alpha chemoattractant (I-TAC), CXCL12/stromal cell-derived factor (SDF)-1alpha, and CCL2/monocyte chemotactic protein(MCP)-1 than EC from human umbilical vein (HUVEC), considered as a healthy counterpart. Paired plasma cells and several MM cell lines expressed cognate receptors of each chemokine to a variable extent. When cells were exposed to chemokines, CXCL8/IL-8 and CXCL12/SDF-1alpha stimulated their proliferation and all chemokines stimulated cell chemotaxis. It is suggested that angiogenesis also favours MM progression through the release of CXC-chemokines.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Bone Marrow Cells; Case-Control Studies; Cell Communication; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Chemokine CXCL11; Chemokine CXCL12; Chemokines, CXC; Chemotaxis, Leukocyte; Endothelial Cells; Female; Flow Cytometry; Humans; Interleukin-8; Male; Middle Aged; Multiple Myeloma; Plasma Cells; Receptors, Chemokine; Reverse Transcriptase Polymerase Chain Reaction; Stimulation, Chemical

Multiple myeloma (MM) is a malignant disease characterized by the clonal proliferation of plasma cells within the bone marrow. Several cytokines have been demonstrated to be involved in the control of growth, progression, and dissemination of MM. We determined serum levels of interleukin-1beta (IL-1beta), soluble interleukin-2 receptor (sIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP) in 14 newly diagnosed MM patients. The median age of the patients was 63.4 +/- 10.8 years and all of the patients were stage III (classified according to the Durie-Salmon classification). The same parameters were measured in 15 healthy controls. In addition, we also examined the effects of vincristine-adriamycin-dexamethasone (VAD) therapy on the same parameters and mediators as well as the relationship among the parameters in the same patient groups. The serum concentrations of TNF-alpha, IL-1beta, sIL-2R, IL-6, IL-8, and CRP (18.6 +/- 3.7 pg/mL, 10.1 +/- 2.8 pg/mL, 730 +/- 220 U/mL, 11.4 +/- 3.3 pg/mL, 23.9 +/- 8.3 pg/mL, and 49.9 +/- 19.5 mg/dL, resp) were significantly higher in newly diagnosed MM patients than in healthy controls (P < .0001). All of the parameters were found to be significantly reduced after chemotherapy. In conclusion, we found that after the VAD therapy, the level of these cytokines which are thought to play an important role in the pathogenesis of MM was significantly suppressed. This is the first study demonstrating strong impact of VAD treatment on circulating mediators of sIL-2R and IL-8 levels parameters.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; C-Reactive Protein; Dexamethasone; Doxorubicin; Female; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Male; Middle Aged; Multiple Myeloma; Receptors, Interleukin-2; Tumor Necrosis Factor-alpha; Vincristine

CD40 ligand (CD154) expressed on activated T helper cells is a key costimulatory molecule for antigen-presenting cells expressing the corresponding receptor CD40. Moreover, CD40 stimulation in nonimmune cells, such as endothelial cells, may play an important role in atherogenesis. One gene product that is induced in endothelial cells on exposure to CD154 is CD154 itself.. In human primary cultured endothelial cells, constitutive CD154 expression was virtually absent and insensitive to proinflammatory cytokines such as tumor necrosis factor alpha and/or interferon-gamma. However, CD154 expression was markedly induced, both on the mRNA and protein level, after CD40 stimulation. Moreover, CD40-positive human monocytes (THP-1 cell line) transmigrating through CD154-expressing endothelial cells responded with an increased expression of interleukin-1beta (IL-1beta) mRNA, indicative of their activation. This increase in IL-1beta expression was confirmed on the protein level and could be abrogated by prior treatment of the endothelial cells with a neutralizing anti-CD154 antibody.. By way of CD154-induced CD154 expression, human endothelial cells thus seem capable of influencing the progression of proinflammatory reactions, including atherogenesis through activation of extravasating monocytes.

Topics: Animals; Antibodies, Monoclonal; CD40 Antigens; CD40 Ligand; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cell Line, Tumor; Cell Movement; Cells, Cultured; Chemokine CCL2; Chemotaxis, Leukocyte; Coculture Techniques; Culture Media, Conditioned; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Humans; Interleukin-1; Interleukin-8; Mice; Monocytes; Multiple Myeloma; Recombinant Fusion Proteins; RNA, Messenger; T-Lymphocytes, Helper-Inducer; Transfection

In proteinuric states increased cytokine production through endocytosis of filtered proteins by proximal tubule cells (PTCs) has been proposed as a major mechanism mediating tubulointerstitial injury and progressive kidney disease. We studied the effects of six different light chains (LCs) on the production of cytokines in cultured human PTCs.. LCs were isolated and purified from the urine of patients with myeloma and human PTCs were exposed to either LC or human serum albumin (HSA) for up to 24 hours. LC endocytosis was monitored by immunocytochemistry. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) in the supernatants and activation of nuclear factor-kappa B (NF-kappaB) was detected by electrophoretic mobility shift assays (EMSA) and immunocytochemistry.. Endocytosis of LCs induced the release of interleukins (IL) IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1); however, there was considerable variability among the six different LCs. In contrast, HSA had no effect on cytokine production even at very high concentrations. Removal of LC-containing media resulted in cessation of IL-6 release. LC-induced cytokine release was associated with nuclear translocation of NF-kappaB subunits p50 and p65, as demonstrated by both EMSA and immunocytochemistry. Inhibitors of NF-kappaB, aspirin and pyrrolidineditiocarbamate (PDTC) markedly suppressed LC-induced cytokine production.. LC endocytosis leads to production of inflammatory cytokines through activation of NF-kappaB. This may be an important mechanism of chronic tubulointerstitial inflammation process commonly seen in multiple myeloma. These findings also point out a potential role by filterable low-molecular-weight proteins, like LCs, in PTC injury during all proteinuric diseases.

Topics: Cells, Cultured; Chemokine CCL2; Electrophoretic Mobility Shift Assay; Endocytosis; Fluorescent Antibody Technique, Indirect; Humans; Immunoglobulin Light Chains; Interleukin-6; Interleukin-8; Kidney Tubules, Proximal; Multiple Myeloma; NF-kappa B; Serum Albumin

CD28 is the major costimulatory molecule on T cells. CD28 activation, in conjunction with T-cell receptor engagement, up-regulates transcription of several cytokines, including interleukin-2 (IL-2), through transcriptional activation of the RE/AP composite element. Although CD28 is not normally expressed on B cells or plasma cells, more than 90% of extramedullary myelomas (a late stage B-cell neoplasm) express CD28. The functional significance of this is unknown. The results of this study demonstrate that CD28 stimulates transcriptional activation of RE/AP-based reporters in B cells and myeloma cells. However, CD28 stimulation does not up-regulate IL-2 production in myeloma cell lines, demonstrating that the IL-2 promoter may not be a relevant RE/AP-containing target of CD28 in myelomas. Instead, an RE/AP composite element has been identified within the promoter of the IL-8 gene, a chemokine that promotes angiogenesis. Furthermore, stimulation of endogenous CD28 expressed by 3 myeloma cell lines increased IL-8 production. Therefore, the study demonstrates that CD28 is functional in myelomas to up-regulate transcription of endogenous genes, including IL-8. The proposal is made that aberrant expression of CD28 may play a role in the progression of multiple myeloma.

Topics: Animals; B-Lymphocytes; CD28 Antigens; Disease Progression; Humans; Interleukin-2; Interleukin-8; Mice; Multiple Myeloma; Promoter Regions, Genetic; Signal Transduction; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Up-Regulation

Monocyte chemoattractant protein-1 (MCP-1) belongs to the newly recognized "chemokine" superfamily of activation-inducible cytokines. We report here that MCP-1 gene-transferred mouse myeloma cells modulate tumor necrosis in myeloma-bearing nude mice. We established an MCP-1-producing myeloma cell line (X63-MCP-1) by transfection with human MCP-1 cDNA as well as interleukin-8-producing X63 cells (X63 IL-8). Each cell line showed the same growth characteristics in vitro, and 1 x 10(7) cells per mouse were injected into the peritoneal cavity resulting in the formation of tumors. Hematologic studies, including peripheral white blood cell counts and differentiation, showed no differences among the groups. They formed tumors in the same manner, which we observed from weeks 2.5 to 9. MCP-1 mice showed more tumor necrosis and infiltration of the macrophages into the tissue surrounding the tumor. In situ hybridization, using a partial cDNA as a probe, showed that macrophages contained MCP-1 mRNA. Bone marrow cell colony-forming assay showed a greater number of both granulocyte and macrophage colonies in MCP-1 mouse femur than in those of controls or interleukin-8 mice. MCP-1 has no direct stimulatory activity on stem cells, but longer exposure to MCP-1 in vivo might stimulate both granulocyte and macrophage progenitors and recruitment of macrophages into tumors, and it might explain the antitumor activity of macrophages in tumor-bearing nude mice.

Topics: Animals; Ascites; Bone Marrow; Cell Line; Chemokine CCL2; Chemotactic Factors; Cytokines; Female; Granulocytes; Hematopoietic Stem Cells; In Situ Hybridization; Interleukin-8; Leukocyte Count; Macrophages; Mice; Mice, Nude; Multiple Myeloma; Necrosis; Neoplasm Transplantation; Recombinant Proteins; RNA, Messenger; Transfection; Tumor Cells, Cultured