RESUMEN
Despite the recent advances in the clinical management of melanoma, there remains a need for new pharmacological approaches to treat this cancer. 2-methoxyestradiol (2ME) is a metabolite of estrogen that has shown anti-tumor effects in many cancer types. In this study we show that 2ME treatment leads to growth inhibition in melanoma cells, an effect associated with entry into senescence, decreased pRb and Cyclin B1 expression, increased p21/Cip1 expression and G2/M cell cycle arrest. 2ME treatment also inhibits melanoma cell growth in colony formation assay, including cell lines with acquired resistance to BRAF and BRAF+MEK inhibitors. We further show that 2ME is effective against melanoma with different BRAF and NRAS mutational status. Moreover, 2ME induced the retraction of cytoplasmic projections in a 3D spheroid model and significantly decreased cell proliferation in a 3D skin reconstruct model. Together our studies bring new insights into the mechanism of action of 2ME allowing melanoma targeted therapy to be further refined. Continued progress in this area is expected to lead to improved anti-cancer treatments and the development of new and more effective clinical analogues.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Estradiol/análogos & derivados , Melanoma/tratamiento farmacológico , Invasividad Neoplásica/prevención & control , Neoplasias Cutáneas/tratamiento farmacológico , Piel/efectos de los fármacos , 2-Metoxiestradiol , Apoptosis/efectos de los fármacos , Ciclo Celular , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Estradiol/farmacología , Humanos , Melanoma/patología , Invasividad Neoplásica/patología , Piel/patología , Neoplasias Cutáneas/patologíaRESUMEN
BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFß/SMAD (transforming growth factor-ß/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Indoles/farmacología , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Melanoma/metabolismo , Sulfonamidas/farmacología , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Vemurafenib , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína Gli2 con Dedos de ZincRESUMEN
BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [³H-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-³H]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9±0.54%) compared with the control cells (39.8±2.2%, (***) P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPARγ2 (peroxisome proliferator-activated receptor γ 2), C/EBPß (CCAAT/enhancer-binding protein ß) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-³H]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor α, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.
Asunto(s)
Células 3T3-L1/metabolismo , Adipocitos/metabolismo , Desoxiglucosa/metabolismo , Resistencia a la Insulina , ARN Mensajero/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animales , Diferenciación Celular/genética , Proliferación Celular , Citometría de Flujo , Humanos , Resistencia a la Insulina/genética , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Obesos , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Amiloide A Sérica/genética , Regulación hacia ArribaRESUMEN
The objective was to evaluate the in vitro and in vivo phosphate binding properties of cross-linked chitosan iron (III) (CH-Fe(III)-CL), a potential oral phosphate binder for treating hyperphosphatemia. At equilibrium, the in vitro phosphate binding of CH-Fe(III)-CL was 23.6 mg g(-1) for simulated gastrointestinal conditions. In hyperphosphatemic rats, CH-Fe(III)-CL was similar to iron sulfate in reducing serum phosphate by about 35%.