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Signalling pathways involved in the apoptotic action of ouabain in two different cancer cell lines
No full textCardiac glycosides, like ouabain, are specific inhibitors of plasma membrane Na+/K+-ATPase,
enzyme responsible for translocating Na+and K+ ions across cell membrane using ATP as energy
source. Recent findings suggest for Na+/K+-ATPase a role as signal transducer, involved in the
control of cell proliferation and growth or in the protection against apoptotic stimuli. In fact, the
binding of ouabain to Na+/K+-ATPase (at concentrations that do not inhibit the pump activity)
triggers a complex signaling cascade that is initiated by interacting with neighboring membrane
proteins and organized cytosolic cascades of signaling molecules. These signaling complexes send
messages to intracellular organelles via the activation of the protein tyrosine kinase Src,
transactivation of epidermal growth factor receptor (EGFR) by Src, activation of Ras and the
extracellular signal-regulated kinase (ERK). Also a ROS dependent c-Jun N-terminal kinase (JNK)
activation has been shown to be involved in the pathways activated by ouabain (1). In agreement
with these findings, we have previously showed that ouabain has an antiapoptotic effect on HUVEC
through the activation of phosphoinositide-3 kinase (PI3K) and ERK (2). On the other hand, several
studies have suggested that cardiac glycosides may have an anticancer utilization. Evidences of the
antineoplastic potential of cardiac glycosides have been obtained with in vitro studies, but the most
relevant evidences of the beneficial effects of cardiac glycosides in cancer treatment were drawn
from epidemiological data. The death rate and cancer recurrence turned out to be lower in women
with breast cancer treated with digitalis than in non-treated patients. Moreover, it was observed a
reduced incidence of leukemia/lymphoma and kidney/urinary tract tumours in subjects with
elevated plasmatic concentrations of digitoxin (3). The aim of my doctoral work is to characterize
the effect of nM concentrations of ouabain on two cancer cell lines: Jurkat (immortalized cell line
of T lymphocytes) and A549 (carcinomic human alveolar basal epithelial cells) with particular
attention to the signaling pathway involved. Cell treatment with ouabain (1-100 nM) for 24 h
induced a concentration-dependent decrease in cell viability measured by the MTT reduction assay
both in Jurkat and A549 cells. The decrease in cell viability at 100 nM was 67.0±2.2 e 70.0±2.1 in
Jurkat and A549, respectively. Incubation of both cell lines with 100 nM ouabain for 24 h induced a
significant raise in the number of apoptotic cells, as indicated by flow cytometric analysis of
annexin V/propidium iodide binding: ouabain increased the proportion of annexin V positive
(apoptotic) cells from 7.0±0.7% to 33.0±6.0% in Jurkat cells and from 7.0±0.6% to 22.0±2.0% in
A549 cells. However, an increase in caspase-3 activation induced by ouabain treatment was
observed only in Jurkat cells. In order to clarify the signaling pathways involved in the mechanism
of action of ouabain, the role of ERK, Src kinase and JNK was investigated. A transient increase in
ERK1/2 phosphorylation (determined by Western blotting analysis) was observed in Jurkat cells
treated with 100 nM ouabain for 30 min. Nevertheless, incubation of the cells with a specific MEK
inhibitor, PD98059 (25 μM) or U0126 (10 μM) did not abolish the apoptotic effect of ouabain, as
shown by MTT test and flow cytometric analysis of annexin V/PI binding. Similar results were
obtained with A549 cells. Furthermore, treatment of both tumour cell lines with the inhibitor of
JNK, SP600125, did not affect the apoptotic action of ouabain. To ascertain whether the activation
of Src kinase is required for the ouabain-effect on cancer cells we used the Src-kinase inhibitor PP2
(50 μM). This compound did not affect the ouabain-induced apoptosis in both Jurkat and A549
cells. Our results show that nM concentrations of ouabain are pro-apoptotic for Jurkat and A549
cells through a mechanism that does not involve the classical pathways of signal transduction
activated by ouabain in non-tumoral cells.
References
Schoner W et al. (2007) Am J Physiol Cell Physiol 293:509
Trevisi L et al. (2004) Biochem Biophys Res Commun 321:716
Newman RA et al. (2008) Mol Interv 8:3
Differential post-transcriptional regulation of COX-2 expression in human umbilical vein endothelial cells derived from diabetic and healthy women: role of microR-As
No full textCOX-2 is one of the vasoprotective genes up-regulated by steady laminar shear stress (Topper et al. 1996; Di
Francesco et al. 2009) and produces the vasoprotective prostacyclin (Grosser et al. 2006; Di Francesco et al.
2009). Early investigations have shown that diabetes markedly alters prostanoid synthesis in the vasculature
(Bagi et al. 2006) but studies on the potential involvement of COX-2 in diabetic vascular complications have
given controversial results. The aim of this study was to investigate whether the exposure to a diabetic
environment in vivo could affect the regulation of endothelial COX-2 expression through post-transcriptional
mechanisms. Thus, we compared COX-2 expression in human umbilical vein endothelial cells derived from
normal (nHUVEC) and type I diabetic mothers (dHUVEC). Confluent monolayers of cells at passage level 3
were treated with or without IL-1β 5 ng/ml for 6 or 24 h in medium 199/DMEM (50:50) supplemented with
ECGF, pen-strep, glutamine, and 5% FCS. The medium was assayed for 6-keto-PGF1α, PGE2, PGF2α, PGD2
by radioimmunoassay or ELISA, cell lysates for COX-1, COX-2, microsomal PGES-1 (mPGES-1),
prostacyclin synthase (PGIS), and heme oxygenase (HO)-1 by specific Western blot techniques. RNAs were
extracted and analysed for COX-2, microRNA(miR)542-3p, and miR16 by real time-PCR. In dHUVEC there
was a statistically significant increase in the biosynthesis of 6-keto-PGF1α, PGE2, and PGD2, but not PGF2α,
in response to IL-1β vs nHUVEC. The values of prostanoid generation are reported in the table.
Prostanoid levels measured in nHUVEC and dHUVEC after IL-1β (5 ng/ml) for 6 and 24 h (values are
reported as ng, mean±SEM, n=5-7; *P<0.05 vs nHUVEC)
6-keto-PGF1α PGE2 PGF2α PGD2
6 h 24 h 6 h 24 h 6 h 24 h 6 h 24 h
nHUVEC 2.8 ±0.5 13.7±2.1 3.0±0.7 9.2±3.0 22.7±6.9 122.4±29.8 0.6±0.1 1.1±0.3
dHUVEC 9.9±3.6* 37.7±10.5* 5.4±1.0 23.2±5.2* 35.2±14.7 132.1±54.6 0.8±0.3 2.4±0.7*
In both types of HUVEC, prostanoids were generated by COX-2; in fact, pretreatment with 1 μM NS-398 (a
selective inhibitor of COX-2) caused an almost complete inhibition of prostanoid biosynthesis. After IL-1β
treatment, Western blot analysis showed that COX-1 and PGIS protein levels were comparable in nHUVEC
and dHUVEC while mPGES-1 levels were undetectable both in nHUVEC and dHUVEC. The protein levels
of COX-2 were significantly (P<0.05) higher in dHUVEC vs nHUVEC at 24 h (COX-2/β-actin optical
density 2.30±0.50 vs 0.88±0.12, respectively). mRNA levels of COX-2 in response to IL-1β were
significantly (P<0.01) higher in dHUVEC than in nHUVEC (17.98±1.50 vs 5.91±0.60 at 6 h; 9.16±0.60 vs
3.46±0.70 at 24 h, respectively). Experiments of mRNA stability performed in the presence of actynomycin
D (0.65 μg/ml), to inhibit transcription, demonstrated that COX-2 mRNA was more stable in dHUVEC than
in nHUVEC after IL-1β stimulation; in fact, at 3h there was 30% vs 13% of COX-2 mRNA remaining in
dHUVEC and nHUVEC, respectively (P<0.01). We analysed the levels of two different miRNAs involved in
the destabilization of COX-2 mRNA through their binding in the 3’UTR: miR542-3p and miR16. In
nHUVEC at 24 h with IL-1β, miR542-3p and miR16 levels were significantly (P<0.05) increased vs
unstimulated cells (relative miRNA levels: miR542-3p, 2.2±0.8 vs 1.00±0.02; miRNA 16, 1.71±0.26 vs
1.04±0.02, respectively). On the contrary, in dHUVEC stimulated with IL-1β for 24 h, miR542-3p and
miR16 levels were lower than in unstimulated cells. Interestingly, miR542-3p and miR16 levels were lower
in dHUVEC vs nHUVEC, at 24 h with IL1β (0.53±0.20 and 0.74±0.20, respectively; P<0.05). HO-1
expression was higher (P<0.01) in HUVEC from diabetic than healthy women, in response to IL-1β and NS-
398 reduced HO-1 levels both in nHUVEC and dHUVEC. In summary, IL-1β induces COX-2-dependent
prostanoids in HUVEC. Prostacyclin is a dominant autocrine prostanoid. HUVEC exposed to a diabetic
environment express higher levels of COX-2 through post-transcriptional mechanisms. Loss of IL-1β-
dependent inducibility of miRNA-542-3p and miR-16 in dHUVECs is associated with enhanced COX-2
whose expression was associated with higher levels of HO-1. In conclusion, targeting of miRNAs in
endothelial cells may represent a new therapeutic strategy to modulate COX-2-dependent prostacyclin.
References
Topper JN et al. (1996) Proc Natl Acad Sci U.S.A. 93:10417
Di Francesco L et al. (2009) Circ Res 104:506
Grosser T et al. (2006) J Clin Invest 116:4
Bagi Z et al. (2006) Pharmacol Rep 58 Suppl:5
Analysis of the molecular mechanisms of the antineoplastic effect of ouabain
Full text linkOuabain is a cardiac glycoside whose primary action is inhibition of Na/K ATPase activity, a ubiquitous enzyme responsible for translocating Na and K ions across the cell membrane using ATP as the driving force. It has been demonstrated that the Na/K ATPase also functions as a classical receptor, capable of converting cardiac glycodise binding into activation of various protein kinase cascades (Liu and Xie, 2010). Also, recent studies have shown that cardiac glycosides selectively inhibit cell proliferation and/or induce apoptosis in several cancer cell lines (Schoner and Sheiner-Bobis, 2007). These in vitro studies are supported by epidemiological data reporting a protection from several types of cancer in patients who were on cardiac glycoside treatment (Stenkvist, 1999; Haux 1999; Haux et al, 2001).
To elucidate the cellular and molecular mechanisms underlying cardiac glycoside effect against cancer cells, we studied the effect of ouabain (1-100 nM) on two cancer cell lines, Jurkat (human T cell lymphoblast-like cell line) and A549 (human lung adenocarcinoma epithelial cell line). Ouabain (1-20 nM) induced a concentration-dependent decrease in proliferation of both cell lines. At higher concentrations (50-100 nM) ouabain was cytotoxic for the two cancer cell lines, as demonstrated by the increase in Annexin V/propidium iodide binding. At the same concentrations, ouabain did not affect cell viability of peripheral blood mononuclear cells (PBMC) and, as previously shown in our laboratory, protected from apoptosis human umbilical vein endothelial cells (HUVEC) (Trevisi L et al., 2004).
Ouabain increased Src and ERK1/2 phosphorylation in A549 and Jurkat thus demonstrating the activation of the known signaling pathways triggered by Na/K ATPase signalosome. However, pharmacological inhibition of Src or MEK did not abolish the cytotoxic activity of ouabain. In both cell lines ouabain caused a decrease of phospho-Akt levels.
Decrease of Bcl-2 protein levels and mitochondrial membrane potential were early events of ouabain treatment in both cell lines. However, only in Jurkat cells were observed caspase 3/7 activation, DNA ladder fragmentation and inhibition of cell death by the pan caspase inhibitor Z-VAD-fmk, hallmarks of caspase-dependent apoptosis. On the other hand, a marker of autophagy, i.e. conversion of LC3-I to LC3-II isoform, was observed in ouabain-treated A549. Moreover, ouabain enhanced the autophagic flux in A549 as demonstrated by increase of p62 degradation and increase of punctate pattern of LC3 after co-incubation with chloroquine, a drug that blocks fusion of autophagosomes with lysosomes. Furthermore, cell death induced by ouabain was completely blocked by treatment with an inhibitor of autophagy such as 3-methyladenine. It has been suggested that decrease of Bcl-2 levels could induce autophagy since Bcl-2 interacts with the autophagic protein Beclin 1, inhibiting the formation of the autophagy initiation complex (Pattingre S et al., 2005). This interaction is regulated by JNK because JNK-dependent phosphorylation of Bcl-2 causes Bcl-2 degradation and disruption of Bcl-2/Beclin1 complex. In A549 treated with ouabain the levels of Beclin 1 were manteined costant. However, inhibition of JNK with SP600125 blocked cell death induced by ouabain only in A549. We speculate that JNK activation by ouabain in A549 reduces Bcl-2 levels thus releasing Beclin 1 and inducing autophagy. Further studies will be required to confirm this hypothesis.
Finally, to ascertain whether differences in sensitivity to ouabain among normal and cancer cells are related to a specific pattern of Na/K ATPase α subunit isoform expression, western blotting analysis of α isoforms was performed in Jurkat and A549 cells and compared to the expression pattern of two normal cell lines: HUVEC and PBMC. We found that α1 isoform is ubiquitous, α2 isoform is not expressed only in PBMC and that α3 isoform is expressed exclusively in the two cancer cell lines. These data suggest that α3 subunit could be critical for ouabain cytotoxic effect.L’ouabaina é un glicoside cardioattivo la cui azione più nota è l’inibizione della Na/K ATPasi, enzima ubiquitario della membrana plasmatica responsabile del trasporto di ioni Na e K attraveso le membrane utilizzando ATP come forza motrice. È stato dimostrato che la Na/K ATPasi, oltre alla sua funzione di pompa, é in grado di agire come recettore, attivando varie cascate di segnale (Liu and Xie, 2010). Studi recenti hanno dimostrato che i glicosidi cardioattivi inibiscono in maniera selettiva la proliferazione e/o inducono apoptosi in diverse linee di cellule tumorali (Schoner and Sheiner-Bobis, 2007). Questi studi in vitro sono supportati da dati epidemiologici che evidenziano una protezione da vari tipi di cancro in pazienti trattati con glicosidi cardioattivi (Stenkvist, 1999; Haux 1999; Haux et al, 2001).
Al fine di chiarire il meccanismo molecolare e cellulare alla base dell’effetto citotossico dei glicosidi cardioattivi sulle cellule tumorali, abbiamo studiato gli effetti dell’ouabaina (1-100 nM) nei confronti di due linee cellulari tumorali, le Jurkat (una linea proveniente da un linfoma a cellule T) e le A549 (linea ottenuta da carcinoma bronchiolo-alveolare). Il trattamento con ouabaina (1-20 nM) provocava una diminuzione della proliferazione cellulare concentrazione dipendente in entrambe le linee cellulari. Alle concentrazioni più elevate (50-100 nM) l’ouabaina era citotossica per le due linee cellulari testate, come dimostrato dall’aumento di cellule Annessina V/propidio positive. Alle stesse concentrazioni l’ouabaina non alterava la vitalità delle cellule mononucleate estratte da sangue periferico umano (PBMC) e, come già dimostrato nel nostro laboratorio, proteggeva dall’apoptosi le cellule endoteliali della vena ombelicale umana (HUVEC) (Trevisi L et al., 2004).
Il trattamento con ouabaina induceva l’attivazione delle chinasi Src ed ERK1/2 in entrambe le linee cellulari, confermando così la stimolazione della nota cascata di segnale innescata dala Na/K ATPasi. Tuttavia, l’inibizione farmacologica della Src o della MEK non contrastava l’effetto citotossico dell’ouabaina. In entrambe le linee cellulari il trattamento con ouabaina induceva una diminuzione della fosforilazione di Akt.
In entrambe le linee cellulari, la diminuzione della proteina antiapoptotica Bcl-2 e del potenziale di membrana mitocondriale costituivano gli eventi iniziali a seguito del trattamento con ouabaina. Tuttavia, solo nelle Jurkat sono stati riscontrati alcune marcatori dell’apoptosi caspasi-dipendente quali l’attivazione delle caspasi 3/7, la tipica frammentazione del DNA (DNA ladder) e l’inibizione della morte cellulare indotta dall’inibitore pan caspasico Z-VAD-fmk. Al contrario nelle A549 sono stati riscontrati i classici marcatori di autofagia come la conversione di LC3 dalla forma LC3-I alla isoforma LC3-II. Inoltre l’ouabaina aumentava il flusso autofagico nelle A549, come dimostrato dall’aumento della degradazione di p62 e dall’aumento delle forme puntate di LC3 in caso di co-incubazione con clorochina, un inibitore della fusione lisosomi-autofagosomi. Infine la morte cellulare indotta da ouabaina era bloccata dal trattamento con l’inibitore dell’autofagia 3-metiladenina. È stato ipotizzato che la diminuzione della Bcl-2 induca autofagia in quanto la Bcl-2 interagisce con Beclin 1 prevenendo così la formazione del complesso di iniziazione (Pattingre S et al., 2005). Tale interazione è regolata da JNK, la quale fosforilando Bcl-2, causa la sua degradazione e la rottura del complesso Bcl-2/Beclin 1. Nelle A549 trattate con ouabaina i livelli di Beclin 1 erano costanti, ma l’inibizione di JNK con SP600125 bloccava la morte cellulare. Noi ipotizziamo che l’attivazione di JNK da parte dell’ouabaina nelle A549 riduca i livelli di Bcl-2 permettendo il rilascio di Beclin 1 e di conseguenza attivi il processo di autofagia. Ulteriori studi saranno necessari per chiarire questo punto.
Infine, attraverso l’analisi western blotting è stata studiata l’espressione delle varie isoforme della subunità α della Na/K ATPase nelle due linee tumorali studiate (A549 e Jurkat) e paragonata a quella di due linee normali (HUVEC e PBMC), allo scopo di verificare se le differenze nella sensibilità all’ouabaina fra cellule normali e tumorali possano essere ascritte ad uno specifico tipo di espressione della varie isoforme della subunità α. I nostri risultati indicano che l’isoforma α1 è ubiquitaria, l’isoforma α2 non è espressa solo nei PBMC mentre l’isoforma α3 è espressa solo nelle due linee cellulari tumorali. Questo dato suggerisce che la subunità α3 possa essere determinante per gli effetti citotossici dei glicosidi cardioattivi nei confronti delle cellule tumorali
SIGNALING PATHWAYS INVOLVED IN THE APOPTOTIC ACTION OF OUABAIN IN TWO DIFFERENT CANCER CELL LINES
No full textOuabain induces autophagic cell death in A549 lung cancer cells through a mechanism dependent on JNK activation
No full textAutophagy is a cellular self-digestive process that ensures degradation of long-lived or damaged proteins and organelles; it
is also induced under certain stress conditions, such as nutrient deprivation, to ensure energy balance [1]. Although
autophagy was initially described as a protective mechanism, studies have demonstrated that persistent stress can promote
autophagic (programmed type II) cell death. The regulation of autophagy in cancer cells is complex since it can enhance
cell survival in response to stress, but it can also suppress the initiation of tumor growth [2]. Cardiac glycosides such as
ouabain, digoxin and digitoxin, are a class of naturally derived compounds that bind and inhibit Na/K-ATPase and are
clinically used for congestive heart failure and atrial arrhythmia. Recent findings have demonstrated that, besides its
pumping function, Na/K-ATPase acts as a signal transducer, converting cardiac glycosides binding into signalling cascades
involved in regulation of cell proliferation, differentiation and death [3]. Interestingly, in vitro studies have demonstrated
that cardiac glycosides show a selective cytotoxicity against cancer cells. These studies are consistent with epidemiological
data reporting protection from some types of cancer (i.e. breast. lymphoma/leukemia, prostate/urinary) in patients who are
on cardiac glycoside treatment [4]. We studied the anticancer effect of ouabain in A549 lung cancer cells and the signalling
pathways involved. Ouabain inhibited cell proliferation at concentrations below 50 nM as shown by MTT test, trypan blue
exclusion assay and clonogenic assay. Treatment with 100 nM ouabain for 24 h induced cell death, confirmed by flow
cytometric analysis of annexin V and propidium iodide binding. Cell death was caspase-independent and showed classical
patterns of autophagy: conversion of LC3-I to LC3-II, increase of LC3 puncta and increase of autophagic flux. Moreover,
cell death was completely blocked by class III phosphatidylinositol-3 kinase inhibitor 3-methyladenine. Western blotting analysis showed that ouabain caused the stimulation of AMP activated protein kinase (AMPK). Accordingly, ouabain
induced the activation of the autophagy-initianting kinase Ulk1 (evidenced by increase of Ser 555 phosphorylation and the
decrease of Ser 757 phosphorylation of Ulk1 protein). Furthermore, ouabain reduced Bcl-2 protein levels and did not
change the expression of the autophagic protein Beclin 1. Early signalling events were ERK and JNK activation, however,
their role on ouabain-induced cell death were opposite. In fact, MEK inhibitor U0126 did not antagonize cell death and, on
the contrary, enhanced the cytotoxic effect of the glycoside indicating a pro-survival role of ERK. On the other hand,
inhibition of JNK with SP600125 was able to prevent conversion of LC3-I to LC3-II, Bcl-2 decrease and cell death. We
hypothesize that JNK activation by ouabain leads to decrease of Bcl-2 levels and disruption of the inhibitory interaction of
Bcl-2 with Beclin 1 thus promoting autophagy.
[1] Klionsky DJ, Emr SD (2000). Science. 290, 1717-1721.
[2] Turcotte S, Giaccia AJ (2010) Curr Opin Cell Biol. 22, 246-251.
[3] Schoner W, Scheiner-Bobis G. (2007) Am J Physiol Cell Physiol. 293, C509-536.
[4] Newman RA et al (2008) Mol Interv. 8, 36-49
Cardiac glycoside ouabain induces autophagic cell death in non-small cell lung cancer cells via a JNK-dependent decrease of Bcl-2
No full textCardiac glycosides are Na/K-ATPase inhibitors, clinically used for congestive heart failure and cardiac arrhythmias. Epidemiological studies have reported that patients on cardiac glycosides treatment are protected from some types of cancers. This evidence together with the demonstration that cardiac glycosides show selective cytotoxicity against cancer cells has raised new interest on the anticancer properties of these drugs. This study examines the mechanism involved in the anticancer effect of ouabain in non-small cell lung cancer cells lines (A549 and H1975). Ouabain inhibited cell proliferation and induced cell death in a concentration-dependent manner. Cell death was caspase-independent and showed classical patterns of autophagic cell death: conversion of LC3-I to LC3-II, increase of LC3 puncta and increase of autophagic flux. Moreover, cell death was completely blocked by the class III phosphatidylinositol-3 kinase inhibitor 3-methyladenine. Here we show that ouabain caused the reduction of Bcl-2 protein levels, with no change in the expression of the autophagic protein Beclin 1. Early signalling events of ouabain exposure were ERK1/2 and JNK activation, however only JNK inhibition with SP600125 or JNK knockdown by shRNA were able to prevent Bcl-2 decrease, conversion of LC3-I to LC3-II and cell death. We propose that JNK activation by ouabain leads to a decrease of Bcl-2 levels, resulting in disruption of the inhibitory interaction of Bcl-2 with Beclin 1, that promotes autophagy. These findings indicate that pharmacological modulation of autophagy by cardiac glycosides could be exploited for anticancer therapy
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