RESUMO
Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf-κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal-myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.
Assuntos
Epitélio/fisiologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/embriologia , Progesterona/metabolismo , Regeneração/fisiologia , Células-Tronco/metabolismo , Proteína Wnt4/metabolismo , Animais , Primers do DNA/genética , Feminino , Deleção de Genes , Técnicas Histológicas , Processamento de Imagem Assistida por Computador , Glândulas Mamárias Animais/fisiologia , Camundongos , Microscopia de Fluorescência , Receptor Cross-Talk/fisiologia , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Transplante de Células-TroncoRESUMO
Previously, we described alkoxyamines bearing a pyridine ring as new pro-drugs with low molecular weights and theranostic activity. Upon chemical stimulus, alkoxyamines undergo homolysis and release free radicals, which can, reportedly, enhance magnetic resonance imaging and trigger cancer cell death. In the present study, we describe the synthesis and the anti-cancer activity of sixteen novel alkoxyamines that contain an imidazole ring. Activation of the homolysis was conducted by protonation and/or methylation. These new molecules displayed cytotoxic activities towards human glioblastoma cell lines, including the U251-MG cells that are highly resistant to the conventional chemotherapeutic agent Temozolomide. We further showed that the biological activities of the alkoxyamines were not only related to their half-life times of homolysis. We lastly identified the alkoxyamine (RS/SR)-4a, with both a high antitumour activity and favourable logD7.4 and pKa values, which make it a robust candidate for blood-brain barrier penetrating therapeutics against brain neoplasia.
Assuntos
Aminas/química , Antineoplásicos/química , Imidazóis/química , Pró-Fármacos/química , Aminas/metabolismo , Aminas/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Carbono/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Meia-Vida , Humanos , Nitrogênio/química , Oxigênio/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , EstereoisomerismoRESUMO
Brain tumors are an important cause of suffering and death. Glioblastoma are the most frequent primary tumors of the central nervous system in adults. They are associated with a very poor prognosis, since only 10% of GBM patients survive 5 years after diagnosis. Medulloblastoma are the most frequent brain malignancies in childhood; they affect the cerebellum in children under 10 years of age in 75% of cases. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Herein, we propose the synthesis of a library of novel alkoxyamines as anticancer drug candidates. The most efficient molecule, ALK4, was selected based on its ability to inhibit both survival and migration of GBM and MB cells in 2D cultures and in 3D tumor spheroids. A fluorescent derivative was used to show the early cytosolic accumulation of ALK4 in tumor cells. Spontaneous homolysis of ALK4 led to the release of alkyl radicals, which triggered the generation of reactive oxygen species, fragmentation of the mitochondrial network and ultimately apoptosis. To control its homolytic process, the selected alkoxyamine was bioconjugated to a peptide selectively recognized by matrix metalloproteases. This bioconjugate, named ALK4-MMPp, successfully inhibited survival, proliferation, and invasion of GBM and MB tumor micromasses. We further developed innovative brain and cerebellum organotypic models to monitor treatment response over time. It confirmed that ALK4-MMPp significantly impaired tumor progression, while no significant damage was observed on normal brain tissue. Lastly, we showed that ALK4-MMPp was well-tolerated in vivo by zebrafish embryos. This study provides a new strategy to control the activation of alkoxyamines, and revealed the bioconjugate ALK4-MMPp bioconjugate as a good anticancer drug candidate.
RESUMO
BACKGROUND: Medulloblastoma is the most frequent brain malignancy of childhood. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Drug repurposing is a strategy to fast-track anti-cancer therapy with low toxicity. Here, we showed the ability of ß-blockers to potentiate radiotherapy in medulloblastoma with bad prognosis. METHODS: Medulloblastoma cell lines, patient-derived xenograft cells, 3D spheroids and an innovative cerebellar organotypic model were used to identify synergistic interactions between ß-blockers and ionising radiations. Gene expression profiles of ß-adrenergic receptors were analysed in medulloblastoma samples from 240 patients. Signaling pathways were explored by RT-qPCR, RNA interference, western blotting and RNA sequencing. Medulloblastoma cell bioenergetics were evaluated by measuring the oxygen consumption rate, the extracellular acidification rate and superoxide production. FINDINGS: Low concentrations of ß-blockers significantly potentiated clinically relevant radiation protocols. Although patient biopsies showed detectable expression of ß-adrenergic receptors, the ability of the repurposed drugs to potentiate ionising radiations did not result from the inhibition of the canonical signaling pathway. We highlighted that the efficacy of the combinatorial treatment relied on a metabolic catastrophe that deprives medulloblastoma cells of their adaptive bioenergetics capacities. This led to an overproduction of superoxide radicals and ultimately to an increase in ionising radiations-mediated DNA damages. INTERPRETATION: These data provide the evidence of the efficacy of ß-blockers as potentiators of radiotherapy in medulloblastoma, which may help improve the treatment and quality of life of children with high-risk brain tumours. FUNDING: This study was funded by institutional grants and charities.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Criança , Metabolismo Energético , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/radioterapia , Qualidade de Vida , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta/uso terapêutico , SuperóxidosRESUMO
The mammary gland is the only organ to undergo most of its development after birth and therefore particularly attractive for studying developmental processes. In the mouse, powerful tissue recombination techniques are available that can be elegantly combined with the use of different genetically engineered mouse models to study development and differentiation in vivo.In this chapter, we describe how epithelial intrinsic gene function can by discerned by grafting mammary epithelial cells of different genotypes to wild-type recipients. Either pieces of mammary epithelial tissue or dissociated mammary epithelial cells are isolated from donor mice and subsequently transplanted into recipients whose mammary fat pads were divested of their endogenous epithelium. This is followed by phenotypic characterization of the epithelial outgrowth either by fluorescence stereomicroscopy for the fluorescently marked grafts or carmine alum whole mount for the unmarked epithelia.
Assuntos
Células Epiteliais/fisiologia , Epitélio/fisiologia , Glândulas Mamárias Animais/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Camundongos , FenótipoRESUMO
Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo, we further revealed that the chronic administration of low-doses of paclitaxel - i.e. metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors.