RESUMEN
Iron plays a major role in multiple processes involved in cell homeostasis such as metabolism, respiration and DNA synthesis. Cancer cells exhibit pronounced iron retention as compared to healthy counterpart. This phenomenon also occurs in multiple myeloma (MM), a hematological malignancy characterized by terminally differentiated plasma cells (PCs), in which serum ferritin levels have been reported as a negative prognostic marker. The aim of current study is to evaluate the potential role of iron metabolism in promoting drug resistance in myeloma cancer cells with particular regard to the interactions between PCs and tumor-associated macrophages (TAMs) as a source of iron. Our data showed that myeloma cell lines are able to intake and accumulate iron and thus, increasing their scavenger antioxidant-related genes and mitochondrial mass. We further demonstrated that PCs pre-treated with ferric ammonium citrate (FAC) decreased bortezomib (BTZ)-induced apoptosis in vitro and successfully engrafted in zebrafish larvae treated with BTZ. Treating human macrophages with FAC, we observed a switch toward a M2-like phenotype associated with an increased expression of anti-inflammatory markers such as ARG1, suggesting the establishment of an iron-mediated immune suppressive tumor microenvironment favouring myeloma growth. Using mfap4:tomato mutant zebrafish larvae, we further confirmed the increase of PCs-monocytes interactions after FAC treatment which favour BTZ-resistance. Taken together our data support the hypothesis that targeting iron trafficking in myeloma microenvironment may represent a promising strategy to counteract a tumor-supporting milieu and drug resistance.
Asunto(s)
Antineoplásicos , Mieloma Múltiple , Animales , Antineoplásicos/farmacología , Apoptosis , Bortezomib/farmacología , Proteínas Portadoras , Línea Celular Tumoral , Resistencia a Antineoplásicos , Proteínas de la Matriz Extracelular , Glicoproteínas/farmacología , Glicoproteínas/uso terapéutico , Humanos , Hierro/farmacología , Macrófagos , Mieloma Múltiple/tratamiento farmacológico , Microambiente Tumoral , Pez CebraRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM-MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM-MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.
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Iron toxicity is associated with organ injury and has been reported in various clinical conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasmaâoptical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.
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Compuestos Férricos/efectos adversos , Quelantes del Hierro/administración & dosificación , Sobrecarga de Hierro/tratamiento farmacológico , Compuestos de Amonio Cuaternario/efectos adversos , Ácido Tióctico/administración & dosificación , Animales , Autofagia/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Glutatión/metabolismo , Hemo-Oxigenasa 1/genética , Humanos , Quelantes del Hierro/farmacología , Sobrecarga de Hierro/inducido químicamente , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Ácido Tióctico/farmacología , Pez CebraRESUMEN
Hodgkin Lymphoma (HL) is associated with deep microenvironment re-shaping and myeloid dysfunction. Given that only limited data are available regarding the role of Brentuximab Vedotin (BV) as single agent in transplant-naive relapsed/refractory (R/R) patients and its off-target effects on immune system, we evaluated the amount of regulatory T-cells (T-regs), myeloid-derived suppressor cells (MDSC) subpopulations, and their functional marker, serum arginase-1 (s-Arg-1), in peripheral blood of 15 consecutive R/R HL patients. After a median of four BV cycles, the overall response rate (complete response + partial response) was 47%, with 4 (27%) complete metabolic remissions. BV reduced the absolute number of three MDSC subtypes and s-Arg-1 levels. Patients with baseline s-Arg-1 ≥200 ng/ml had inferior progression-free survival at 36 months compared to those with low s-Arg-1. T-regs dysfunction was recovered by BV: absolute T-regs count was increased after treatment with BV, independently of metabolic response achieved, with a significant reduction of CD30+ T-regs. Our data disclose off-target effects of BV in the microenvironment that could explain its deep and durable clinical efficacy.
Asunto(s)
Arginasa , Biomarcadores de Tumor , Brentuximab Vedotina/administración & dosificación , Enfermedad de Hodgkin , Células Supresoras de Origen Mieloide , Proteínas de Neoplasias , Linfocitos T Reguladores , Adolescente , Adulto , Arginasa/sangre , Arginasa/inmunología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/inmunología , Supervivencia sin Enfermedad , Femenino , Enfermedad de Hodgkin/sangre , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Proteínas de Neoplasias/sangre , Proteínas de Neoplasias/inmunología , Tasa de Supervivencia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in patients with chronic myeloid leukaemia (CML) inducing T cell tolerance. In this study, we found that therapy with tyrosine kinase inhibitors (TKI) decreased the percentage of granulocytic MDSC, but only patients treated with dasatinib showed a significant reduction in the monocytic subset (M-MDSC). Moreover, a positive correlation was observed between number of persistent M-MDSC and the value of major molecular response in dasatinib-treated patients. Serum and exosomes from patients with CML induced conversion of monocytes from healthy volunteers into immunosuppressive M-MDSC, suggesting a bidirectional crosstalk between CML cells and MDSC. Overall, we identified M-MDSC as prognostic factors in patients treated with dasatinib. It might be of interest to understand whether MDSC may be a candidate predictive markers of relapse risk following TKI discontinuation, suggesting their potential significance as practice of precision medicine.
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Dasatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Monocitos/patología , Células Supresoras de Origen Mieloide/patología , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular/efectos de los fármacos , Dasatinib/farmacología , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Exosomas/ultraestructura , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Células Supresoras de Origen Mieloide/efectos de los fármacos , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Adulto JovenRESUMEN
Azacytidine (5-AZA) is the standard first-choice treatment for high-risk myelodysplasia (MDS) patients. However, the clinical outcome for those patients who interrupt treatment or whose disease failed to respond is very poor. In order to identify the cellular pathways that are modified by long-term exposure to 5-AZA, we evaluated key proteins associated with the autophagy pathway by reverse-phase microarray (RPPA). Comparing bone marrow mononucleated cells (BMMCs) obtained from 20 newly-diagnosed patients and after four 5-AZA cycles we found an increased autophagy signaling. We then evaluated ex-vivo the effect of the combination of 5-AZA with autophagy inhibitors chloroquine (CQ) and leupeptin. Since 5-AZA and CQ showed synergism due to an increase of basal autophagy after 5-AZA exposure, we adopted a sequential treatment treating BMMCs with 5 µM 5-AZA for 72 h followed by 10 µM CQ for 24 h and found increased apoptosis, associated to a reduction of G2M phase and increase in G0-G1 phase. Long-term exposure to 5-AZA induced the reduction of the autophagic marker SQSTM1/p62, reversible by CQ or leupeptin exposure. In conclusion, we identified autophagy as a compensatory pathway occurring in MDS-BM after long-term exposure to 5-AZA and we provided evidences that a sequential treatment of 5-AZA followed by CQ could improve 5-AZA efficacy, providing novel insight for tailored therapy in MDS patients progressing after 5-AZA therapy.
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Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Despite recent advances in pharmacological therapy, MM remains a largely incurable pathology. Therefore, novel effective and less toxic agents are urgently necessary. In the last few years, pomegranate has been studied for its potential therapeutic properties including treatment and prevention of cancer. Pomegranate juice (PGJ) contains a number of potential active compounds including organic acids, vitamins, sugars, and phenolic components that are all responsible of the pro-apoptotic effects observed in tumor cell line. The aim of present investigation is to assess the antiproliferative and antiangiogenic potential of the PGJ in human multiple myeloma cell lines. Our data demonstrate the anti-proliferative potential of PGJ in MM cells; its ability to induce G0/G1 cell cycle block and its anti-angiogenic effects. Interestingly, sequential combination of bortezomib/PGJ improved the cytotoxic effect of the proteosome inhibitor. We investigated the effect of PGJ on angiogenesis and cell migration/invasion. Interestingly, we observed an inhibitory effect on the tube formation, microvessel outgrowth aorting ring and decreased cell migration and invasion as showed by wound-healing and transwell assays, respectively. Analysis of angiogenic genes expression in endothelial cells confirmed the anti-angiogenic properties of pomegranate. Therefore, PGJ administration could represent a good tool in order to identify novel therapeutic strategies for MM treatment, exploiting its anti-proliferative and anti-angiogenic effects. Finally, the present research supports the evidence that PGJ could play a key role of a future therapeutic approach for treatment of MM in order to optimize the pharmacological effect of bortezomib, especially as adjuvant after treatment.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Bebidas , Proliferación Celular/efectos de los fármacos , Frutas/química , Lythraceae , Mieloma Múltiple/tratamiento farmacológico , Antineoplásicos Fitogénicos , Antioxidantes/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Flavonoides , Humanos , Mieloma Múltiple/irrigación sanguínea , Mieloma Múltiple/patología , Invasividad Neoplásica/prevención & control , Fitoterapia , Taninos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Granulocytic-Myeloid-derived suppressor cells (G-MDSC) are increased in Multiple Myeloma (MM) patients but the mechanisms of G-MDSC generation are still unknown. There are many evidences of the role of mesenchymal stem cells (MSC) in promoting MM cell growth, survival and drug-resistance. We here used a specific experimental model in vitro to evaluate the ability of MSC to induce G-MDSC. We found that although MSC derived from healthy donors (HD), MGUS and MM were able to generate the same amount of MDSC, only MM-MSC-educated G-MDSC exhibited suppressive ability. In addition, in comparison with MSC derived from HD, MM-MSC produce higher amount of immune-modulatory factors that could be involved in MDSC induction. Compared to G-MDSC obtained from co-culture models with MSC from healthy subjects, both MGUS and MM-MSC-educated G-MDSC showed increase of immune-modulatory factors. However, only MM-MSC educated G-MDSC 1) up-regulated immune-suppressive factors as ARG1 and TNFα, 2) expressed higher levels of PROK2, important in angiogenesis and inflammatory process, and 3) showed ability to digest bone matrix.Our data demonstrate that MM-MSC are functionally different from healthy subjects and MGUS-MSC, supporting an evolving concept regarding the contribution of MM-MSC to tumor development and progression.
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Granulocitos/fisiología , Células Madre Mesenquimatosas/fisiología , Mieloma Múltiple/inmunología , Células Supresoras de Origen Mieloide/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/etiología , Microambiente TumoralRESUMEN
Osteolytic bone disease is a common manifestation of multiple myeloma (MM) that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases chitotriosidase (CHIT1) and YKL40 is associated with osteoclast (OCs) differentiation and bone digestion. In the current study, we investigated the effect of two Bortezomib's concentration (2.5 and 5 nM) on osteoclastogenesis by analyzing regulation of chitinase expression. OCs exposition to bortezomib (BO) was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disk assay resorption pits. Silencing chitinase proteins in U266 cell line with specific small interfering RNA, resulted in pits number reduction on dentine disks. In conclusion, we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients.
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The better definition of the mutual sustainment between neoplastic cells and immune system has been translated from the bench to the bedside acquiring value as prognostic factor. Additionally, it represents a promising tool for improving therapeutic strategies. In this context, myeloid-derived suppressor cells (MDSCs) have gained a central role in tumor developing with consequent therapeutic implications. In this review, we will focus on the biological and clinical impact of the study of MDSCs in the settings of lymphoid malignancies.