New insights into sphingolipid metabolism and functions by using chemical tools

Abstract

Sphingolipids (SLs), essential components of cell membranes, are bioactive molecules involved in the regulation of a variety of cellular processes. Ceramide and sphingosine generally mediate anti-proliferative stimuli, while sphingosine-1-phospate plays an opposite role. The bioactive role of dihydroceramide has also been recently described, showing its involvement in processes like apoptosis and autophagy. Alteration of SLs levels is involved in different disease, including cancer. Sphingolipid metabolism is a complex network of anabolic and catabolic reactions catalyzed by specific enzymes. Consistent with the key role of sphingolipids in cell biology, the modulation of key­enzyme of the sphingolipid metabolism such as dihydroceramide desaturase (Des1), ceramide synthase (CerS), ceramidases (CDases) and sphingosine kinase (SK) represent a strategy to better understand the role of SLs in physiophatological processes. During this PhD thesis three different studies that show the use of SL analogues and SL metabolism inhibitors as pharmacological tools for biomedical investigation in cancer cells were performed.

The first study was focused on the alteration of sphingolipid metabolism and cytotoxic effect induced by Jaspine B (JB) in cancer cells. JB, a natural sphingolipid analogue from marine origin, is cytotoxic in a variety of cancer cell lines, including HGC-27 gastric cancer cells. JB induces changes in the sphingolipidome in this cell line, mainly the accumulation of dihydrosphingosine (dhSo) and sphingosine (So), and their phosphorylated forms. Moreover lipidomic analysis show the formation of acyl-JB. These effects appear to be due to inhibitory action of JB on CerS that use the natural sphingolipid as substrate. The hallmark of JB cytotoxicity in HGC-27 cells is the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Apoptosis is not involved in cytoplasmic vacuolation and JB induced cytotoxicity. In addition, despite the accumulation of the autophagic marker LC3-II, autophagy is probably not activated by JB treatment. The micropinocytic nature of vacuoles suggest the involvement of methuosis in JB-induced cell death.

The second study was focused on SKI II is a well-known SK inhibitor. In a study on HGC-27 cells we show that SKI II is also an uncompetitive inhibitor of Des1. Des1 inhibition in intact cells is not connected to protein degradation. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF-543, reduced cell proliferation, with accumulation of cells in the G0/G1 phase. Similarly SKI II, but not PF543 increases LC3-II levels in HGC-27 cells, suggesting autophagy induction. These overall findings indicate that some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.

The use of ceramide analogues as chemical tool for ceramidases investigation is the topic of the third study. RBM14 are coumarinic ceramide analogues that differ from the acyl chain length. Among them RBM14C12 was reported as the best substrate for acid ceramidase. In this study it was shown that RBM14 molecules are also hydrolyzed by neutral and alkaline ceramidase. Neutral ceramidase from Pseudomonas aeuriginosa prefers C12 and C14 analogues, while human recombinant NC better hydrolyze RBM14C14 and RBM14C16 molecules. RBM14 substrates are hydrolyzed by MEF cells lacking NC, whit higher activity at basic pH, suggesting alkaline ceramidase activity. SABRAC and RBM1-12 have been designed to inhibit the cysteine protease AC. In this study it was shown that papain, a protein belonging to the same protein family, is not inhibited by SABRAC and RBM1-12. Moreover preliminary studies suggest an irreversible inhibition of AC. These molecules can be used in combination with RBM14C12 substrate as a suitable tool for AC activity determination in intact cancer cells.

Los esfingolípidos (SLs), componentes esenciales de las membranas celulares, son moléculas bioactivas que regulan distintos procesos celulares. Ceramida y esfingosina (So) median estímulos anti-proliferativos, mientras que la esfingosina-1-fosfato (S1P) juega un papel opuesto. La alteración del metabolismo de los SLs está relacionada al desarrollo de muchas enfermedades, entre ellas el cáncer. El metabolismo de los SLs representa una red compleja de reacciones anabólicas y catabólicas catalizadas por enzimas específicas, cuya modulación permite investigar el papel de los SL en los procesos fisiopatológicos.

En esta tesis doctoral se han realizado tres estudios que muestran el uso de análogos de SLs y de inhibidores del metabolismo de SLs como herramientas farmacológicas para la investigación biomédica en las células cancerosas.

El primer estudio se centró en los efectos de la Jaspina B, un esfingolípido natural, en células de cáncer humano HGC-27. La JB induce alteración del metabolismo de los SLs con aumento de dihidroesfingosina y So y sus formas fosforiladas, como efecto de la inhibición de la ceramida sintasa. Además la JB lleva la célula a muerte celular tras un mecanismo de vacuolización del citoplasma que no implica activación de apoptosis o autofagia.

En el segundo estudio se caracterizo el SKI II, un inhibidor de la esfingosina quinasa, como inhibidor no competitivo de la Des1. El SKI II induce acumulación de dihidroceramida (dhCer) y sus metabolitos y a nivel biológico induce parada de ciclo celular y autofagia. Con este estudio se propone que los efectos biológicos del SKI II se deben a aumento de dhCer y sus metabolitos, más que a la reducción de la S1P. El tercer estudio se centró en el utilizo de análogos a ceramida como herramienta para el estudio de las ceramidas. Los análogos RBM14s, descritos como substrato de la ceramidasa acida (AC), también se hidrolizan por las ceramidasas neutra y alcalinas. SABRAC y RBM1-12 son inhibidores específicos de la AC y se pueden utilizar en ensayos bioquímico para identificar selectivamente la actividad de la AC en células cancerosas.