ABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Estradiol/analogs & derivatives , Melanoma/drug therapy , Neoplasm Invasiveness/prevention & control , Skin Neoplasms/drug therapy , Skin/drug effects , 2-Methoxyestradiol , Apoptosis/drug effects , Cell Cycle , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Estradiol/pharmacology , Humans , Melanoma/pathology , Neoplasm Invasiveness/pathology , Skin/pathology , Skin Neoplasms/pathologyABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Indoles/pharmacology , Kruppel-Like Transcription Factors/antagonists & inhibitors , Melanoma/metabolism , Sulfonamides/pharmacology , Zinc Finger Protein GLI1/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cellular Senescence/genetics , Gene Expression , Gene Knockdown Techniques , Hedgehog Proteins/metabolism , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Melanoma/drug therapy , Melanoma/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Signal Transduction/drug effects , Vemurafenib , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein Gli2ABSTRACT
The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system.B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed.BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.
Subject(s)
Rats , Cell Cycle/radiation effects , Melanoma, Experimental/chemically induced , Melanoma, Experimental/radiotherapy , Boron Neutron Capture Therapy/methods , /isolation & purification , Mitochondria/radiation effects , Cell Cycle Checkpoints/radiation effects , Cell Culture TechniquesABSTRACT
Antiproliferative and oxidative damage effects occurring in Boron Neutron Capture Therapy (BNCT) in normal fibroblasts and melanoma cell lines were analyzed. Melanoma cells and normal fibroblasts were treated with different concentrations of Boronophe- nylalanine and irradiated with thermal neutron flux. The cellular viability and the oxidative stress were determined. BNCT induced free radicals production and proliferative potential inhibition in melanoma cells. Therefore, this therapeutic technique could be considered efficient to inhibit growth of melanoma with minimal effects on normal tissues.
Subject(s)
Cells, Cultured/radiation effects , Melanoma/radiotherapy , Free Radicals/radiation effects , Free Radicals/supply & distributionABSTRACT
Antiproliferative and oxidative damage effects occurring in Boron Neutron Capture Therapy (BNCT) in normal fibroblasts and melanoma cell lines were analyzed. Melanoma cells and normal fibroblasts were treated with different concentrations of Boronophenylalanine and irradiated with thermal neutron flux. The cellular viability and the oxidative stress were determined. BNCT induced free radicals production and proliferative potential inhibition in melanoma cells. Therefore, this therapeutic technique could be considered efficient to inhibit growth of melanoma with minimal effects on normal tissues.
Subject(s)
Boron Neutron Capture Therapy , Free Radicals/metabolism , Melanoma, Experimental/radiotherapy , Animals , Cell Line , Cell Line, Tumor , Humans , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , MiceABSTRACT
The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system. B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed. BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.
