The cannabinoid WIN 55,212-2 prevents neuroendocrine differentiation of LNCaP prostate cancer cells

BACKGROUND: Neuroendocrine (NE) differentiation represents a common feature of prostate cancer and is associated with accelerated disease progression and poor clinical outcome. Nowadays, there is no treatment for this aggressive form of prostate cancer. The aim of this study was to determine the influence of the cannabinoid WIN 55,212-2 (WIN, a non-selective cannabinoid CB1 and CB2 receptor agonist) on the NE differentiation of prostate cancer cells. METHODS: NE differentiation of prostate cancer LNCaP cells was induced by serum deprivation or by incubation with interleukin-6, for 6 days. Levels of NE markers and signaling proteins were determined by western blotting. Levels of cannabinoid receptors were determined by quantitative PCR. The involvement of signaling cascades was investigated by pharmacological inhibition and small interfering RNA. RESULTS: The differentiated LNCaP cells exhibited neurite outgrowth, and increased the expression of the typical NE markers neuron-specific enolase and ?III tubulin (?III Tub). Treatment with 3??M WIN inhibited NK differentiation of LNCaP cells. The cannabinoid WIN downregulated the PI3K/Akt/mTOR signaling pathway, resulting in NE differentiation inhibition. In addition, an activation of AMP-activated protein kinase (AMPK) was observed in WIN-treated cells, which correlated with a decrease in the NE markers expression. Our results also show that during NE differentiation the expression of cannabinoid receptors CB1 and CB2 dramatically decreases. CONCLUSIONS: Taken together, we demonstrate that PI3K/Akt/AMPK might be an important axis modulating NE differentiation of prostate cancer that is blocked by the cannabinoid WIN, pointing to a therapeutic potential of cannabinoids against NE prostate cancer.Ministerio de Economía y CompetitividadJunta de Comunidades de Castilla-La ManchaFundación Tatiana Pérez de Guzmán el BuenoFondo Europeo de Desarrollo RegionalComunidad de Madri

Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and ?III tubulin (?III tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1?M AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer

Efecto de la capsaicina en el metabolismo de células de hepatocarcinoma

Los fitoquímicos son compuestos presentes en los vegetales, que pueden ser beneficiosos para el ser humano. En los últimos años han ganado relevancia gracias a su poder terapéutico en el tratamiento de determinadas enfermedades humanas. Algunos poseen propiedades antitumorales como es el caso de la capsaicina (CAP), el agente responsable de las propiedades picantes de los pimientos. El objetivo de este trabajo ha sido valorar el efecto de la capsaicina sobre la viabilidad de la línea celular de cáncer de hígado HepG2 y su relación con el metabolismo celular. Los resultados obtenidos muestran que CAP disminuye la viabilidad celular en HepG2 de manera dosis dependiente, y regula negativamente la proteína AMPK, enzima reguladora del metabolismo, activando ACC. Los resultados obtenidos en los ensayos de tinción de lípidos neutros muestran que CAP produce un acúmulo, lo cual podría estar correlacionado con la activación de ACC y el aumento de expresión de PPAR[gamma

A BODIPY-Based Fluorescent Sensor for Amino Acids Bearing Thiol

Herein, we describe the synthetic route to access a red-emitting BODIPY from its α-diformylated precursor. The photophysical signatures of this dye are sensitive to the presence of thiol-containing amino acids (like cysteine, homocysteine, and glutathione) in the surrounding environment. This sensor provides up to three detection channels to monitor and quantify these biomolecules, even at low concentrations (down to micromolar). Moreover, owing to the pronounced splitting of the spectral band profile induced by these amino acids, the detection can be visualized following just the evolution of the fluorescence color by the naked eye

Up-regulated expression of LAMP2 and autophagy activity during neuroendocrine differentiation of prostate cancer LNCaP cells

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer

Derivados de indolin-2-ona y su uso terapéutico

[ES] Derivados de lndolin-2-ona y su uso terapéutico. Derivados de indolin-2-ona de fórmula (I), donde el significado para R1, R2, R3 y X es el indicado en la descripción. Estos compuestos son útiles como moduladores de la enzima AMPK.[EN] The invention relates to the use of a series of indazole compounds and the derivatives thereof in the production of a drug to be administered alone or combined with another suitable active ingredient in the treatment of cancer and, more specifically, the treatment of haematological cancer.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de AlcaláA1 Solicitud de patente con informe sobre el estado de la técnic

Derivados de indolin-2-ona y su uso terapéutico

[ES] Derivados de lndolin-2-ona y su uso terapéutico. Derivados de indolin-2-ona de fórmula (I), donde el significado para R1, R2, R3 y X es el indicado en la descripción. Estos compuestos son útiles como moduladores de la enzima AMPK.[EN] The invention relates to the use of a series of indazole compounds and the derivatives thereof in the production of a drug to be administered alone or combined with another suitable active ingredient in the treatment of cancer and, more specifically, the treatment of haematological cancer.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de AlcaláB1 Patente sin examen previ

FormylBODIPYs by PCC-Promoted Selective Oxidation of α-MethylBODIPYs. Synthetic Versatility and Applications

An efficient synthesis of formylBODIPYs has been established based on an oxidation with PCC of 3-methylBODIPYs. It has been demonstrated that this reagent can oxidize methyl groups at such position of the BODIPY core, regardless of its substitution pattern. Moreover, through this procedure it is possible to synthesize 8-aryl-3,5-diformylBODIPYs, which are otherwise difficult to obtain. These precursors have been functionalized to develop fluorescent sensors of amino acids or photosensitizers for singlet oxygen generation

Hierarchical Self‐Assembly of BODIPY Dyes as a Tool to Improve the Antitumor Activity of Capsaicin in Prostate Cancer

Capsaicin (CAP) has been long known for its analgesic properties and more recently for its antitumor activity in various cell types. However, its pungency and the high doses needed to achieve a significant activity have precluded its application in cancer therapy. Herein, we propose a straightforward novel strategy to improve the antitumor effect of CAP based on the enhancement of its aggregation propensity in aqueous media by covalent attachment of a BODIPY (BDP) dye. The target CAP-BDP 1 self-assembles in aqueous solutions into weakly fluorescent globular assemblies that become highly emissive upon cell uptake-induced disassembly. Remarkably, due to the improved delivery to the tumour tissue upon aggregation, we have succeeded in reducing the doses of CAP-based drugs in vivo in prostate cancer by two orders of magnitude while maintaining a substantial antitumor activity.Guzman el Bueno Foundatio

Autophagy inhibition decrease LAMP2 levels and survival of neuroendocrine differentiated LNCaP cells.

<p>LNCaP cells were transfected with negative control siRNA (siC) or siRNA targeting Beclin1 (siBeclin1) or si RNA targeting Atg5 (SiATG5) and grown in serum containing medium (C, control cells) or serum-free medium (NE, neuroendocrine cells) during 6 days. <b>(A)</b> Cells were lysed and Beclin1 (left), Atg5 (right) and LAMP2 were measured in whole lysates by Western blot. GAPDH was used as a loading control. Densitometric analysis of the Western blot bands are shown below. <b>(B)</b> Cell viability of control and NE cells treated with siC, siBeclin1 (left) or SiATG5 (right) was monitored by MTT assay. Results are the mean ± S.D. of three independent experiments (* p<0.05 versus siC transfected control cells and # p<0.05 and ## p<0.01 versus siC transfected neuroendocrine cells, compared by the Student’s t test).</p