RESUMEN
Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ceramidas/farmacología , Lisosomas/metabolismo , Neoplasias/patología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dronabinol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Modelos Biológicos , Permeabilidad , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Esfingolípidos/biosíntesisRESUMEN
Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain <10%. Novel treatment strategies are therefore urgently required, particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ(9)-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.
Asunto(s)
Autofagia , Cannabinoides/química , Melanoma/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Cannabidiol , Cannabinol/química , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Dacarbazina/análogos & derivados , Dacarbazina/química , Dronabinol/química , Combinación de Medicamentos , Humanos , Masculino , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Microscopía Confocal , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias/metabolismo , Extractos Vegetales/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/metabolismo , Temozolomida , Proteínas ras/metabolismoRESUMEN
The Bcl-2 family member Mcl-1 is essential for melanoma survival; however, the influence of oncogenic BRAF signalling remains elusive. In this study, Mcl-1 splice variant expression was determined in a panel of melanoma cell lines in relation to BRAF mutational status. Mcl-1L mRNA expression was increased in melanoma cells compared with primary melanocytes with significantly increased mRNA and protein expression observed in BRAF(V600E) mutant melanoma cells. Although no change in Mcl-1S mRNA was observed, Mcl-1S protein expression also increased in BRAF mutant melanoma cells. Additionally, while over-expression of mutant BRAF(V600E) increased both Mcl-1L and Mcl-1S expression, inhibition of hyperactive BRAF signalling resulted in decreased Mcl-1L expression. These studies suggest that the regulation of Mcl-1 expression by BRAF signalling is increased by oncogenic activation of BRAF, revealing a mechanism of apoptotic resistance which may be overcome by the use of more specifically targeted Mcl-1 inhibitors.