RESUMEN

Subsequently to the publication of the above article and a Corrigendum that was published to indicate corrections made to Fig. 7 (DOI: 10.3892/or.2021.7922; published online on January 5, 2021), a concerned reader drew the Editor's attention to the fact that, comparing between a pair of panels in the Figure, there was an overlapping section of data; moreover, this overlapping section contained apparent anomalies that could not be easily accounted for through a straightforward re­use of one of the data panels. The authors conceded that there was partial duplication between the images shown in Fig. 7B and F, although they were unable to access the related raw data as the experiments had been performed almost 10 years ago. Secondly, the authors informed the Editor that the corresponding author did not know he was on the author list at the time of submission. Although the authors' were granted permission to publish the Corrigendum, the Editor now considers that the paper should be retracted on account of the uncertainties in the presented revised data and the authors' admission concerning the corresponding author. Therefore, this paper has been retracted from the Journal. The authors are in agreement with the decision to retract the article. The Editor apologizes for any inconvenience caused. [Oncology Reports 37: 2751­2760, 2017; DOI: 10.3892/or.2017.5569].

RESUMEN

Following the publication of this article, an interested reader drew to the authors' attention that, in Fig. 7 on p. 2757, sections of the data panels in Fig. 7A and B, showing the results of the non­transfected HeLa cell (Pnon group) and pGenesil­1­transfected HeLa cell (P0 group) experiments respectively, were strikingly similar. Both the Pnon and the P0 groups were control groups; upon re­examining their original data, the authors have realized that, when uploading the images, the data shown in Fig. 7B for the pGenesil­1­transfected HeLa cells (P0 group) were selected incorrectly. The authors were able to locate the data that were intended to have been shown in Fig. 7B; moreover, the text describing the number of migrated cells in the Results section also requires a correction. In the 'Downregulation of BDNF expression suppresses the migratory and invasive capabilities of HeLa cells' subsection, the text on lines 9­11 of this paragraph should be changed to the following (changed text is highlighted in bold): 'Migrated cells/field in the PBDNF1 group (37±17) were significantly less than those in the Pnon (105±31) and P0 (86±27) groups'. Likewise, the same correction to the text has been made to the Figure legend, as shown opposite. The revised version of Fig. 7, showing the correct data for Fig. 7B, is shown opposite. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum, and all of the authors agree to the publication of this Corrigendum. The authors sincerely apologize for this mistake, and regret any inconvenience this mistake has caused. [the original article was published on Oncology Reports 37: 2751-2760, 2017; DOI: 10.3892/or.2017.5569].

RESUMEN

In recent years, emerging evidence has suggested that noncoding RNAs (ncRNAs) participate in nearly every aspect of biological processes and play a crucial role in the genesis and progression of numerous tumors, including B-cell lymphoma. The exploration of ncRNA dysregulations and their functions in B-cell lymphoma provides new insights into lymphoma pathogenesis and is essential for indicating future clinical trials and optimizing the diagnostic and therapeutic strategies. In this review, we summarize the role of ncRNAs in B-cell lymphoma and discuss their potential in clinical applications.

RESUMEN

Despite the administration of new effective drugs in recent years, relapse and drug resistance are still the main obstacles in multiple myeloma (MM) treatment, making MM an incurable disease. To overcome drug resistance in MM, it is critical to understand the underlying mechanisms of malfunctioning gene expression and develop novel targeted therapies. During the past few decades, with the discovery and characterization of noncoding RNAs (ncRNAs), the landscape of dysregulated ncRNAs of cancers as well as their biological and pathobiological functions in tumorigenesis and drug resistance have been recognized. Studies about ncRNAs improved the understanding of variations of drug response among individuals at a level distinguished from genetic polymorphism, and provided with new orientations for targeted therapies. In this review, we will summarize the emerging impact and underlying molecular mechanisms of the most relevant classes of ncRNAs in drug resistance of MM, and discuss the potential as well as strategies of treating ncRNAs as therapeutic targets.

Asunto(s)

Resistencia a Antineoplásicos/genética , Mieloma Múltiple/genética , ARN no Traducido/genética , Animales , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Mieloma Múltiple/tratamiento farmacológico

RESUMEN

Inherent or acquired resistance to chemotherapeutic drugs is still an obstacle for the treatment of multiple myeloma (MM). MicroRNA dysregulation is related to the development of chemoresistance in cancers. However, its role in chemoresistance of MM is largely unknown. Here we demonstrated that miR-221/222 were upregulated in plasma cells from patients with MM, especially those with relapsed or refractory disease. Moreover, expression levels of miR-221/222 were inversely correlated with dexamethasone (Dex) sensitivity of human MM cell lines. Importantly, we found that Dex induced pro-death autophagy in MM cells and the inhibition of autophagy significantly decreased Dex-induced cell death. Mechanistically, autophagy-related gene 12 (ATG12) was identified as a novel target gene of miR-221/222, and miR-221/222 overexpression inhibited autophagy by directly targeting ATG12 and the p27kip (p27)-mammalian target of rapamycin (mTOR) pathway. Indeed, Dex treatment decreased the expression of miR-221/222, thereby activating the ATG12/p27-mTOR autophagy-regulatory axis and inducing cell death in Dex-sensitive MM cells. Furthermore, both in vitro and in vivo results showed that the inhibitions of miR-221/222 increased the expression of ATG12 and p27 and functionally induced extended autophagy and cell death of MM cells. In conclusion, our findings demonstrated the crucial role of the miR-221/222-ATG12/p27-mTOR autophagy-regulatory axis in Dex resistance of MM, and they suggest potential prediction and treatment strategies for glucocorticoid resistance.

Asunto(s)

Autofagia/fisiología , Dexametasona/uso terapéutico , MicroARNs/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Autofagia/genética , Proteína 12 Relacionada con la Autofagia/genética , Proteína 12 Relacionada con la Autofagia/metabolismo , Línea Celular Tumoral , Humanos , MicroARNs/genética , Mieloma Múltiple/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo

RESUMEN

Multiple myeloma (MM) is a B-cell neoplasm with a high incidence of relapse. Bortezomib has been extensively studied for the maintenance treatment of MM. Here, we carried out a meta-analysis to determine the efficacy and safety of maintenance therapy with bortezomib. We searched for clinical trials in PubMed (Medline), Embase (OVID), and the Cochrane Library. Two randomized controlled trials (RCTs) enrolling a total of 1338 patients were included. Bortezomib maintenance statistically significantly improved both progression-free survival (PFS) (hazard ratio (HR) 0.67, 95% confidence interval (CI) = 0.51 to 0.87, P=0.003) and overall survival (OS) (HR = 0.75 therapy, 95% CI = 0.63 to 0.89, P=0.001) more than did non-bortezomib maintenance therapy. Our analysis revealed higher incidence of neutropenia (risks ratios (RR) = 1.39; 95% CI = 1.08 to 1.79), peripheral neuropathy (PN) (RR = 2.23; 95% CI = 1.38 to 3.61, P=0.001), and cardiologic events (RR = 1.91; 95% CI = 1.12 to 3.28, P=0.02) in patients with bortezomib maintenance therapy. Our meta-analysis demonstrates OS and PFS benefits of bortezomib maintenance therapy in patients with newly diagnosed MM. However, the therapy is associated with increased risk of adverse events. Additionally, more RCTs are needed for better understanding and determination of optimal bortezomib maintenance therapy in MM.

Asunto(s)

Bortezomib/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Bortezomib/efectos adversos , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Mieloma Múltiple/mortalidad , Tasa de Supervivencia

RESUMEN

Brain-derived neurotrophic factor (BDNF) is expressed in a number of neural and non-neuronal tumors. The present study investigated the effect of endogenous BDNF on the biological behavior of cervix cancer cells using small interfering RNA (siRNA). HeLa, a cervix cancer cell line with high expression of BDNF, was used as a living model to screen out the effective sequences of short hairpin RNA of the BDNF gene, and the effects of RNA interference on proliferation, apoptosis, migration and invasion of these cells were evaluated. Among the 4 siRNAs examined, siRNA1 caused a 99% reduction in the relative BDNF mRNA level, while a 58% decrease in the relative BDNF protein level (p<0.01) was noted, and thus this siRNA was selected as the most efficient for use in the present study. In subsequent experiments, MTT assay revealed that BDNF silencing caused marked inhibition of HeLa cell proliferation while Hoechst 33258 staining assay demonstrated apoptosis-related changes in cell morphology. Downregulation of BDNF expression induced cell cycle arrest in the G1 phase as shown by flow cytometry. As indicated by Transwell migration and invasion assays, downregulation of BDNF expression suppressed the migratory and invasive capabilities of the HeLa cells. Together, our data revealed that BDNF modulates the proliferation, apoptosis, migratory and invasive capabilities of HeLa cells. BDNF siRNA may represent a novel therapy or drug target for preventing the tumorigenesis of cervical cancer.

Asunto(s)

Antineoplásicos/farmacología , Factor Neurotrófico Derivado del Encéfalo/genética , ARN Interferente Pequeño/farmacología , Neoplasias del Cuello Uterino/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Puntos de Control del Ciclo Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos

RESUMEN

Despite recent therapeutic advances that have doubled the median survival time of patients with multiple myeloma, intratumor genetic heterogeneity contributes to disease progression and emergence of drug resistance. miRNAs are noncoding small RNAs that play important roles in the regulation of gene expression and have been implicated in cancer progression and drug resistance. We investigated the role of the miR-221-222 family in dexamethasone-induced drug resistance in multiple myeloma using the isogenic cell lines MM1R and MM1S, which represent models of resistance and sensitivity, respectively. Analysis of array comparative genome hybridization data revealed gain of chromosome X regions at band p11.3, wherein the miR-221-222 resides, in resistant MM1R cells but not in sensitive MM1S cells. DNA copy number gains in MM1R cells were associated with increased miR-221-222 expression and downregulation of p53-upregulated modulator of apoptosis (PUMA) as a likely proapoptotic target. We confirmed PUMA mRNA as a direct target of miR-221-222 in MM1S and MM1R cells by both gain-of-function and loss-of-function studies. In addition, miR-221-222 treatment rendered MM1S cells resistant to dexamethasone, whereas anti-miR-221-222 partially restored the dexamethasone sensitivity of MM1R cells. These studies have uncovered a role for miR-221-222 in multiple myeloma drug resistance and suggest a potential therapeutic role for inhibitors of miR-221-222 binding to PUMA mRNA as a means of overcoming dexamethasone resistance in patients. The clinical utility of this approach is predicated on the ability of antisense miR-221-222 to increase survival while reducing tumor burden and is strongly supported by the metastatic propensity of MM1R cells in preclinical mouse xenograft models of multiple myeloma. Moreover, our observation of increased levels of miR-221-222 with decreased PUMA expression in multiple myeloma cells from patients at relapse versus untreated controls suggests an even broader role for miR-221-222 in drug resistance and provides a rationale for the targeting of miR-221-222 as a means of improving patient outcomes.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Dexametasona/farmacología , Resistencia a Antineoplásicos/genética , MicroARNs/genética , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Regiones no Traducidas 3' , Animales , Proteínas Reguladoras de la Apoptosis/genética , Sitios de Unión , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Mieloma Múltiple/mortalidad , Mieloma Múltiple/patología , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Transducción de Señal , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genética

RESUMEN

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) of human multiple myeloma (MM) cell line RPMI8226 regulated by brain-derived neurotrophic factor (BDNF), and preliminarily approach the close relationship between BDNF and angiogenesis of MM. METHODS: The recombinant eukaryotic BDNF siRNA expression vector was designed and constructed. The empty vector pGenesil-1, and the recombinant plasmid, pGenesil-shRNA-BDNF were transfected into RPMI8226 cells using Lipofectamine™ 2000 (groups P0 and P1, respectively). BDNF mRNA and protein level in RPMI8226 cells were detected by RT-PCR and Western blotting, respectively; the cellular proliferation activity was determined by MTT assay, while the cell apoptosis was measured by flow cytometry; the variation of VEGF mRNA level in RPMI8226 cells via transfection was determined by RT-PCR, the secretion of VEGF was detected by ELISA. RESULTS: (1)The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. BDNF mRNA expression and protein level in P1 group were significantly inhibited compared to those in non-transfected group (Pn) and P0 groups (P<0.05); (2)MTT tests demonstrated that the cellular proliferation activities were obviously decreased in Pn (0.42 ± 0.06) vs P0 (0.56 ± 0.06) and P1 (0.50 ± 0.04) groups (P<0.05); (3)The early cell apoptosis rates were statistically increased in P1 [(53.84 ± 9.95)%] vs Pn [(5.23 ± 2.46)%] and P0 [(9.10 ± 3.46)%] groups (P<0.01); (4)The silence of endogenous BDNF significantly decreased the expression of VEGF in RPMI8226 cells:the relative expression level of VEGF121, VEGF145 and VEGF165 in P1 group were (0.62 ± 0.07), (0.47 ± 0.09) and (0.57 ± 0.02) folds compared to Pn group (P<0.05); (5)ELISA demonstrated that secretion of VEGF in P1 group were (0.36 ± 0.05) and (0.44 ± 0.06) folds compared to Pn and P0 group, respectively (P<0.05). CONCLUSION: BDNF gene silence can obviously increase apoptosis of RPMI8226 cells, inhibit their proliferation and decrease the expression of VEGF. BDNF might mediate the expression of VEGF in MM cells, which may be involved in MM angiogenesis.

Asunto(s)

Silenciador del Gen , Apoptosis , Factor Neurotrófico Derivado del Encéfalo , Línea Celular Tumoral , Vectores Genéticos , Humanos , Mieloma Múltiple , Neovascularización Patológica , ARN Mensajero , ARN Interferente Pequeño , Transfección , Factor A de Crecimiento Endotelial Vascular

RESUMEN

Wnt/ß-catenin signaling underlies the pathogenesis of a broad range of human cancers, including the deadly plasma cell cancer multiple myeloma. In this study, we report that downregulation of the tumor suppressor microRNA miR-30-5p is a frequent pathogenetic event in multiple myeloma. Evidence was developed that miR-30-5p downregulation occurs as a result of interaction between multiple myeloma cells and bone marrow stromal cells, which in turn enhances expression of BCL9, a transcriptional coactivator of the Wnt signaling pathway known to promote multiple myeloma cell proliferation, survival, migration, drug resistance, and formation of multiple myeloma cancer stem cells. The potential for clinical translation of strategies to re-express miR-30-5p as a therapeutic approach was further encouraged by the capacity of miR-30c and miR-30 mix to reduce tumor burden and metastatic potential in vivo in three murine xenograft models of human multiple myeloma without adversely affecting associated bone disease. Together, our findings offer a preclinical rationale to explore miR-30-5p delivery as an effective therapeutic strategy to eradicate multiple myeloma cells in vivo.

Asunto(s)

MicroARNs/genética , Mieloma Múltiple/genética , Proteínas de Neoplasias/genética , Vía de Señalización Wnt , Regiones no Traducidas 3' , Animales , Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Células HEK293 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Interferencia de ARN , Factores de Transcripción , Carga Tumoral

RESUMEN

This study examined the expression of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM) and its role in bone marrow angiogenesis. The peripheral blood plasma was harvested from 71 MM patients and 63 patients without hematological malignancy. The BDNF level in the blood plasma was determined by ELISA. Human bone marrow endothelial cells (HBMECs) were cultured. The mRNA and protein expression levels of the BDNF receptor TrkB in HBMECs were detected by using RT-PCR and flow cytometry, respectively. The viability of HBMECs treated with recombinant human (rh) BDNF or not was measured by using MTT assay. The migration of HBMECs in the presence of rhBDNF or not was determined by modified Boyden chamber assay. In vitro tube formation assay was used to assess the effect of rhBDNF on HBMECs differentiation. The results of ELISA revealed that the BDNF level was significantly higher in peripheral blood plasma of MM patients than in that of control patients (4.39±0.67 vs. 1.96±0.39 ng/mL, P<0.05). The BDNF receptor TrkB was expressed in HBMECs at mRNA and protein level. MTT assay manifested that rhBDNF could significantly concentration-dependently promote the HBMECs proliferation. The number of HBMECs treated with 160 ng/mL rhBDNF for 48 h was 1.57±0.10 folds higher than that in control group (P<0.05). Moreover, rhBDNF could enhance HBMECs migration in a concentration-dependent manner and the maximal migration was reached in the presence of 100 ng/mL rhBDNF. The migration indexes were 1.40±0.11, 1.64±0.16, 2.06±0.25 and 2.18±0.21 in 25, 50, 100 ng/mL rhBDNF groups and 25 ng/mL rhVEGF group, respectively. In vitro tube formation assay demonstrated that the area of the formed tubular structure was increased with the rhBDNF concentration. In control group, there was no formation of intact tubular structure and the HBMECs on the matrigel were irregularly dispersed. HBMECs treated with 100 ng/mL rhBDNF could form intact tubular structure and the area and the diameter of tubes were significantly greater than those in control group (P<0.05). There was no significant difference in the formed tubular area between 25 ng/mL VEGF group and 100 ng/mL rhBDNF group. It was concluded that BDNF plays an important role in myeloma cell-induced angiogenesis, and it may become a new target of anti-angiogenesis treatment for MM.

Asunto(s)

Médula Ósea/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Mieloma Múltiple/metabolismo , Neovascularización Patológica/metabolismo , Adulto , Anciano , Médula Ósea/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/patología , Neovascularización Patológica/patología

RESUMEN

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF-κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as a MM-derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma-osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM-derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time- and dose-dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID-RPMI8226 mice model was constructed using lentiviral short hairpin RNA-transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma-myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/fisiología , Mieloma Múltiple/terapia , Receptor trkB/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/antagonistas & inhibidores , Factor Neurotrófico Derivado del Encéfalo/genética , Línea Celular Tumoral , Femenino , Silenciador del Gen , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Osteoclastos/fisiología , Osteólisis , Osteoprotegerina/genética , Ligando RANK/genética , ARN Interferente Pequeño/genética , Receptor trkB/antagonistas & inhibidores , Receptor trkB/genética , Carga Tumoral

RESUMEN

Deregulated microRNAs (miRNAs) and their roles in cancer development have attracted much attention. Two miRNAs, miR-15a and miR-16, which act as putative tumor suppressor by targeting the oncogene BCL2, have been implicated in cell cycle, apoptosis and proliferation. In this study, we investigated the possible role of miR-15a/16 in the angiogenesis of multiple myeloma (MM). Using a stem-loop quantitative reverse transcription-PCR, we analyzed miR-15a/16 expressions in bone marrow samples from newly diagnosed MM patients and a panel of MM cell lines. miRNA transfection, western blotting analysis and assay of luciferase activity were used to examine whether vascular endothelial growth factor (VEGF) is the target of miR-15a/16. The functional roles of miR-15a/16 on tumorigenesis and angiogenesis were examined by in vitro angiogenesis models and in vivo tumor xenograft model. We showed that miR-15a and miR-16 were significantly underexpressed in primary MM cells as well as in MM cell lines. The aberrant expression of miR-15a/16 was detected especially in advanced stage MM. In human MM cell lines and normal plasma cells, expression of miR-15a/16 inversely correlated with the expression of VEGF-A. Western blotting combined with the luciferase reporter assay demonstrated that VEGF-A was a direct target of miR-15a/16. Ectopic overexpression of miR-15a/16 led to decreased pro-angiogenic activity of MM cells. Finally, infection of lentivirus-miR-15a or lentivirus-miR-16 resulted in significant inhibition of tumor growth and angiogenesis in nude mice. This study suggest that miR-15a/16 could play a role in the tumorigenesis of MM at least in part by modulation of angiogenesis through targeting VEGF-A.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Gammopatía Monoclonal de Relevancia Indeterminada/patología , Mieloma Múltiple/irrigación sanguínea , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Regiones no Traducidas 3'/genética , Animales , Apoptosis , Western Blotting , Estudios de Casos y Controles , Adhesión Celular , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Gammopatía Monoclonal de Relevancia Indeterminada/genética , Gammopatía Monoclonal de Relevancia Indeterminada/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Estadificación de Neoplasias , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

Brain-derived neurotrophic factor (BDNF) was recently identified as a factor produced by multiple myeloma (MM) cells, which may contribute to bone resorption and disease progression in MM, though the molecular mechanism of this process is not well understood. The purpose of this study was to test the effect of BDNF on bone disease and growth of MM cells both in vitro and in vivo. Co- and triple-culture systems were implemented. The in vitro results demonstrate that BDNF augmented receptor activator of nuclear factor kappa B ligand (RANKL) expression in human bone marrow stromal cells, thus contributing to osteoclast formation. To further clarify the effect of BDNF on myeloma bone disease in vivo, ARH-77 cells were stably transfected with an antisense construct to BDNF (AS-ARH) or empty vector (EV-ARH) to test their capacity to induce MM bone disease in SCID-rab mice. Mice treated with AS-ARH cells were preserved, exhibited no radiologically identifiable lytic lesions and, unlike the controls treated with EV-ARH cells, lived longer and showed reduced tumor burden. Consistently, bones harboring AS-ARH cells showed marked reductions of RANKL expression and osteoclast density compared to the controls harboring EV-ARH cells. These results provide further support for the potential osteoclastogenic effects of BDNF, which may mediate stromal-MM cell interactions to upregulate RANKL secretion, in myeloma bone diseases.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/antagonistas & inhibidores , Regulación hacia Abajo , Mieloma Múltiple/patología , Osteoclastos/patología , Ligando RANK/metabolismo , Células del Estroma/patología , Animales , Secuencia de Bases , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Quinasas/metabolismo , ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal

RESUMEN

Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In our study, we investigated whether BDNF was possibly involved in MM cell-induced osteolysis. We showed that BDNF was elevated in MM patients and the bone marrow plasma levels of BDNF positively correlated with extent of bone disease. In osteoclast formation assay, bone marrow plasma from patients with MM increased osteoclast formation and the effect was significantly blocked by neutralizing antibody to BDNF, suggesting a critical role for BDNF in osteoclast activation. Furthermore, the direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF, demonstrating that BDNF used TrkB for its effects on osteoclast. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein-1α and receptor activator of nuclear factor-κB ligand, two major osteoclast stimulatory factors in MM. These results support an important role for BDNF in the development of myeloma bone disease.

Asunto(s)

Resorción Ósea/etiología , Factor Neurotrófico Derivado del Encéfalo/fisiología , Mieloma Múltiple/complicaciones , Péptidos/fisiología , Adulto , Anciano , Factor Neurotrófico Derivado del Encéfalo/análisis , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Persona de Mediana Edad , Osteoclastos/fisiología , Receptor trkB/fisiología

RESUMEN

Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. Here, we functionally validate the role of the microRNAs-15a/16-1 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using "sponge" lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Importantly, miR-16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load and host angiogenesis. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes including FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. We validated designated genes showing binding sites within the conserved 3'-UTR and also within the mRNA coding region as direct miR-16 targets, thus indicating that the miRNAs may have many more targets than anticipated by conventional prediction methods. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.

RESUMEN

This study investigated the signaling pathways involved in the different biological effects of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM). The effects of BDNF on proliferation of MM cell lines and primary myeloma cells were examined by [(3)H]thymidine incorporation assay. The effects of BDNF on MM cells migration were studied by transwell migration assay. Stimulation by BDNF of vascular endothelial growth factor (VEGF) production was analyzed by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The signal-transduction pathways that are activated in response to BDNF were determined by Western blots. VEGF is induced by BDNF in a dose-dependent manner in MM cells. Stimulation of MM cells with BDNF led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the MEK-extracellular signal-regulated protein kinase pathways. Using specific signal-transduction inhibitors, we demonstrated that MEK is required for BDNF-induced proliferation, whereas activation of PI3K is required for BDNF-stimulated migration and VEGF production. BDNF affects different cell signaling pathways mediating growth, migration, and VEGF secretion in MM cells. Our observations provided the framework for novel therapeutic strategies targeting BDNF signaling cascades in MM.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/farmacología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , MAP Quinasa Quinasa 1/fisiología , Mieloma Múltiple/patología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia

RESUMEN

Bone marrow (BM) neovascularization and vascular endothelial growth factor (VEGF) expression in multiple myeloma (MM) correlate with disease progression. Brain derived neurotrophic factor (BDNF) is highly expressed by malignant plasma cells isolated from the majority of MM patients. Recently, BDNF was identified as a potential proangiogenic factor for the promotion of endothelial cell survival, induction of neoangiogenesis in ischemic tissues, and increase of VEGF expression in neuroblastoma. Since tropomyosin receptor kinase B (TrkB), the receptor of BDNF, is expressed by stromal cells within the BM milieu, here we sought to evaluate the involvement of BDNF/TrkB in myeloma-marrow stroma interaction and its effects on BM angiogenesis. TrkB was abundantly expressed by bone marrow stromal cells (BMSCs) isolated from healthy donors. Stimulation of BMSCs with BDNF induced a time- and dose- dependent increase in VEGF secretion, which was completely abolished by K252alpha, an inhibitor of TrkB. BDNF triggered activation of signal transducer and activator of transcription 3 (STAT3) and activator protein-1 (AP-1), whereas STAT3 was involved in mediating VEGF expression. We further delineated the biological significance of BDNF in MM by using lentiviral short-interfering RNA (shRNA). When myeloma cells were cocultured with BMSCs in a noncontact Transwell system, VEGF levels in supernatants were significantly decreased when BDNF expression was knocked down. Furthermore, silencing of BDNF expression significantly inhibited xenograft tumor growth and angiogenesis, and prolonged survival in mouse model. Our studies demonstrate that BDNF, as a potential stimulator of angiogenesis, contributes to MM tumorgenesis; it mediates stromal-MM cell interactions via selective activation of specific receptor TrkB and downstream signal transducer STAT3, regulating VEGF secretion.

Asunto(s)

Médula Ósea/metabolismo , Factor Neurotrófico Derivado del Encéfalo/fisiología , Mieloma Múltiple/etiología , Neovascularización Patológica/etiología , ARN Interferente Pequeño/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Factor Neurotrófico Derivado del Encéfalo/antagonistas & inhibidores , Factor Neurotrófico Derivado del Encéfalo/genética , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Lentivirus/genética , Ratones , Mieloma Múltiple/patología , Factor de Transcripción STAT3/fisiología , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

To investigate the in vitro and in vivo proangiogenic effects of brain-derived neurotrophic factor (BDNF), human umbilical vein endothelial cells (HUVECs) were isolated and cultured in primary culture. The effect of BDNF on the proliferation of HUVECs was examined by MTT assay. The effects of BDNF on HUVEC migration and tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively. Matrigel plug assay and chorioallantoic membrane assay were used to evaluate the effects of BDNF on angiogenesis in vivo. Our results showed that BDNF substantially stimulated the migration and tube formation of HUVECs in vitro, although it did not induce HUVEC proliferation. BDNF also induced angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. In conclusion, BDNF can promote angiogenesis both in vitro and in vivo, and may be a proangiogenic factor.

Asunto(s)

Inductores de la Angiogénesis/farmacología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Células Endoteliales/citología , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Venas Umbilicales/citología

RESUMEN

OBJECTIVE: To screen effective sequences of short hairpin RNA on brain-derived neurotrophic factor (BDNF) gene and the effect of RNA interference on the proliferation and apoptosis of HeLa cells, a cervix carcinoma cell line with high expression of BDNF. METHODS: Two recombinant eukaryotic human-BDNF siRNA expression vectors were designed and constructed. Sequences were confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector pGenesil-1 and two recombinant plasmids, pGenesil-shRNA-BDNF1 and pGenesil-shRNA-BDNF2, were transfected into HeLa cells using Lipofectamine 2000 (groups: P(0), P(1) and P(2), respectively). The mRNA and protein levels of BDNF in HeLa cells were detected by RT-PCR and Western blot, respectively. The cellular proliferation rates were determined by MTT assay and the apoptotic rates were measured by flow cytometry and Hoechest 33258. RESULTS: The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. The expression of mRNA and protein of BDNF in P(1) group were significantly decreased, comparing with non-transfected group, P(0) and P(2) groups (F = 48.19, P < 0.01). P(2) group failed to meet the expected results (P > 0.05). In addition, the proliferation activity was reduced in P(1) group and the peak point of proliferation curve was prolonged. Moreover, the early cell apoptotic rates were statistically increased in P(1)[(53.4 +/- 4.2)%] VS. non-transfected [(0.8 +/- 0.4)%], P(0) [(5.1 +/- 1.8)%] and P(2)[(7.9 +/- 2.4)%] groups (F = 269.77, P < 0.01). CONCLUSION: HeLa cells express a high level of BDNF. BDNF gene silencing by RNA interference increases the apoptosis of HeLa cells and inhibits cell proliferation, offering a possible target for efficient tumor therapy.

Asunto(s)

Apoptosis , Factor Neurotrófico Derivado del Encéfalo/genética , Proliferación Celular , Interferencia de ARN , ARN Interferente Pequeño/genética , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Células HeLa , Humanos , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección