RESUMEN
We previously reported that methiothepin, a small molecule known as a nonselective serotonin 5-HT receptor antagonist, inhibited the doxorubicin efflux activity of the Hedgehog receptor Ptch1 and enhanced the cytotoxic, pro-apoptotic, anti-proliferative, and anti-clonogenic effects of doxorubicin on adrenocortical carcinoma cells. Here, we show that methiothepin also inhibits doxorubicin efflux and increases doxorubicin cytotoxicity in melanoma cells which endogenously overexpress Ptch1. Melanoma patients having the BRAFV600E mutation are treated with vemurafenib, an inhibitor of BRAFV600E, often in combination with trametinib, an inhibitor of MEK. Almost all patients ultimately acquire resistance to the treatment leading to disease progression. Here, we report that methiothepin overcomes the resistance of BRAFV600E melanoma cells by enhancing the cytotoxicity of vemurafenib and trametinib on these cells leading to melanoma cells death. We observe that the addition of methiothepin to vemurafenib prevents migration of resistant melanoma cells more efficiently than vemurafenib alone. Our results provide an additional proof that Ptch1 drug efflux inhibition increases the effectiveness of anti-cancer treatments and overcomes resistance of melanoma cells expressing Ptch1.
Asunto(s)
Antineoplásicos , Resistencia a Antineoplásicos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Metiotepina , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Humanos , Metiotepina/farmacología , Metiotepina/uso terapéutico , Receptor Patched-1/metabolismo , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Vemurafenib/administración & dosificaciónRESUMEN
Melanoma patients harboring the BRAFV600E mutation are treated with vemurafenib. Almost all of them ultimately acquire resistance, leading to disease progression. Here, we find that a small molecule from a marine sponge, panicein A hydroquinone (PAH), overcomes resistance of BRAFV600E melanoma cells to vemurafenib, leading to tumor elimination in corresponding human xenograft models in mice. We report the synthesis of PAH and demonstrate that this compound inhibits the drug efflux activity of the Hedgehog receptor, Patched. Our SAR study allowed identifying a key pharmacophore responsible for this activity. We showed that Patched is strongly expressed in metastatic samples from a cohort of melanoma patients and is correlated with decreased overall survival. Patched is a multidrug transporter that uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that PAH is a highly promising lead for the treatment of vemurafenib resistant BRAFV600E melanoma.
RESUMEN
One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug-like molecule enhances the cytotoxic, pro-apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched-expressing cancers.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos , Metiotepina/administración & dosificación , Receptor Patched-1/metabolismo , Neoplasias de la Corteza Suprarrenal/metabolismo , Carcinoma Corticosuprarrenal/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metiotepina/farmacología , Ratones , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.
Asunto(s)
Apoptosis/genética , Proteína 11 Similar a Bcl2/fisiología , Familia-src Quinasas/fisiología , Animales , Proteína 11 Similar a Bcl2/antagonistas & inhibidores , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Resistencia a Antineoplásicos/genética , Células HEK293 , Células HeLa , Humanos , Células K562 , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Oncogenes/fisiología , Transducción de Señal/genética , Familia-src Quinasas/genéticaRESUMEN
Perturbation of thyroid iodide uptake is a well-documented side effect of the use of iodinated contrast media (ICM) administered intravenously. This side effect is thought to be mediated by free iodide in ICM formulations, but this hypothesis has never been formally proven. The aim of the present study was to assess the validity of this hypothesis. Methods: We used mass spectrometry analysis to quantify free-iodide contamination in ICM. Established cell lines expressing the Na/I symporter (NIS) were used to quantify the effect of ICM on iodide uptake. SPECT/CT was used to measure the in vivo uptake of 99mTc-pertechnetate and 123I in 2 NIS-expressing mouse tissues, thyroid and salivary glands. Scintiscans of ICM-naïve and ICM-administered patients were compared. Immunohistologic and Western blot analyses were performed to evaluate NIS protein expression in these organs. Results: Although free iodide was present in ICM formulations, in vitro uptake of iodide by NIS-expressing cells was not significantly affected by ICM. In mice, intravenous or sublingual administration of ICM led to a reduction in radiotracer uptake by the thyroid, accompanied by a dramatic reduction in NIS protein expression in this tissue. In the salivary glands, neither radiotracer uptake nor NIS protein expression was affected by ICM. The thyroid-selective effect of ICM was also observed in humans. Administration of potassium iodide as a source of free iodide led to a diminution of 99mTc-pertechnetate uptake in both mouse thyroid and mouse salivary glands. Altogether, these data rule out a direct intervention of free iodide in the perturbation of thyroid uptake and suggest a direct and selective effect of ICM on the thyroid. Conclusion: We demonstrated that ICM reduce thyroid uptake of iodide independently of free iodide. This effect is due to a specific and dramatic decrease in NIS expression in thyrocytes. These data cast serious doubt on the relevance of measuring urinary iodide concentration to evaluate the delay between ICM administration and radioiodine therapy in patients with differentiated thyroid carcinoma. Finally, the ability of ICM to perturb iodide uptake in the thyroid may be used in radioprotection.
Asunto(s)
Medios de Contraste/química , Medios de Contraste/farmacología , Halogenación , Yoduros/metabolismo , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células HT29 , Humanos , Ratones , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Glándula Tiroides/diagnóstico por imagenRESUMEN
Extracellular vesicles (EVs) have been shown to play an important role in intercellular communication as carriers of DNA, RNA and proteins. While the intercellular transfer of miRNA through EVs has been extensively studied, the stability of extracellular miRNA (ex-miRNA) once engulfed by a recipient cell remains to be determined. Here, we identify the ex-miRNA-directed phenotype to be transient due to the rapid decay of ex-miRNA. We demonstrate that the ex-miR-223-3p transferred from polymorphonuclear leukocytes to cancer cells were functional, as demonstrated by the decreased expression of its target FOXO1 and the occurrence of epithelial-mesenchymal transition reprogramming. We showed that the engulfed ex-miRNA, unlike endogenous miRNA, was unstable, enabling dynamic regulation and a return to a non-invasive phenotype within 8 h. This transient phenotype could be modulated by targeting XRN1/PACMAN exonuclease. Indeed, its silencing was associated with slower decay of ex-miR-223-3p and subsequently prolonged the invasive properties. In conclusion, we showed that the 'steady step' level of engulfed miRNA and its subsequent activity was dependent on the presence of a donor cell in the surroundings to constantly fuel the recipient cell with ex-miRNAs and of XRN1 exonuclease, which is involved in the decay of these imported miRNA.