RESUMEN
The growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the 'two-in-one' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy.
Asunto(s)
Autofagia/efectos de los fármacos , Azepinas/farmacología , Lisosomas/efectos de los fármacos , Neoplasias de la Próstata/patología , Calcio/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Lisosomas/ultraestructura , Masculino , Modelos Biológicos , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/ultraestructura , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Chinese hamster ovary (CHO-K1) cells were stably transfected with prolactin (PRL) receptor cDNA. These cells (CHO-E32) expressed the long form of functional PRL receptor. Using microfluorimetric and patch-clamp techniques, we have investigated the effects of PRL on intracellular Ca2+ concentration ([Ca2+]i) and membrane ion conductances. Exposure of CHO-E32 cells to 5 nM PRL resulted in an increase in [Ca2+]i. Two types of response were observed: 1) a stimulation of Ca2+ entry and 2) an intracellular Ca2+ mobilization. As PRL inhibited voltage-activated Ca2+ current, the PRL-induced Ca2+ increase does not involve voltage-activated Ca2+ channels. PRL also increased a charybdotoxin-sensitive Ca(2+)-dependent K+ conductance. Simultaneous measurements showed that PRL hyperpolarized the membrane potential before increasing intracellular Ca2+ levels. In voltage clamp, hyperpolarizing voltage steps were associated with increased Ca2+ concentrations, whereas depolarizing voltage steps decreased [Ca2+]i. Cell-free patch-clamp experiments showed that PRL directly stimulates K+ channel activity. Our results suggest the existence of a regulatory complex involving a protein kinase tightly associated with the Ca(2+)-activated K+ channels and that PRL stimulates these channels by means of the activation of protein kinase. The resulting hyperpolarization stimulates Ca2+ entry, probably through voltage-insensitive nonspecific channels.