Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway

Abstract

Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key “executioner” caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression.

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways

“…DNA damage has been recognized as one of the factors for cell death . However, herein, we just examined the integrity of DNA by TUNEL assay in ouabain‐treated cells and results showed that ouabain induced DNA damage in DU 145 cells (Figure B).…”

“…46,47 DNA damage has been recognized as one of the factors for cell death. 48 However, herein, we just examined the integrity of DNA by TUNEL assay in ouabain-treated cells and results showed that ouabain induced DNA damage in DU 145 cells ( Figure 3B). Furthermore, ouabain increased the expression of DNA damage and repair-associated proteins including p-ATM Ser1981 , p-H2A.X Ser139 , and p-p53 Ser15 at 48 hours treatment but decreased that of p-MDM2 Ser166 , MDM2, p53, BRCA1, DNA-PK, MGMT at 48 hours treatment ( Figure 6A).…”

Specialized Functional Diversity and Interactions of the Na,K-ATPase

“…In addition, upon ouabain binding the Na,K-ATPase interacts with neighboring molecular environment to downstream a number of signaling intracellular pathways (Xie and Askari, 2002 ; Aperia, 2007 ; Li and Xie, 2009 ; Fontana et al, 2013 ; Reinhard et al, 2013 ). In different cell types, ouabain has dual effects to promote programmed cell death (Xiao et al, 2002 ; Kulikov et al, 2007 ; Blanco and Venugopal, 2016 ) and to protect against apoptosis (Isaev et al, 2000 ; Dvela et al, 2012 ). Nanomolar ouabain concentrations were also shown to stimulate viability and proliferation of NT2 cells, precursors for human neuronal cells, by a mechanism involving Erk1/2 activation (Dvela et al, 2012 ).…”

“…It remains uncertain whether it affects numerous cellular functions by inhibiting enzymatic activity that leads to modulation of ion homeostasis or by conformational changes of the α subunit and initiation of a signal transduction. The role of the Na,K-ATPase CTS binding site suggests its involvement in regulation of diverse cellular functions, including synaptic and neural processes (Lichtstein and Rosen, 2001 ; Goldstein et al, 2006 , 2011 ; Song et al, 2013 ), cell survival and neuroprotection (Golden and Martin, 2006 ; Dvela et al, 2012 ; Sibarov et al, 2012 ; Dvela-Levitt et al, 2014 ), muscle contraction (Dostanic-Larson et al, 2005 ; Radzyukevich et al, 2009 ), intercellular communications (Matchkov et al, 2007 , 2012 ), and gene expression (Xiao et al, 2002 ; Kulikov et al, 2007 ; Orlov and Hamet, 2015 ; Blanco and Venugopal, 2016 ). Knowledge on the role of Na,K-ATPase and endogenous CTS in intracellular signaling opens new perspectives for modulation of cell function under normal and pathological conditions.…”

The Anticancer Effects of Radachlorin-mediated Photodynamic Therapy in the Human Endometrial Adenocarcinoma Cell Line HEC-1-A

“…Radachlorin has better light penetration of target tissue, as compared to first-generation photosensitizers (18). The anticancer effects of PDT using radachlorin in some in vitro and in vivo cancer models have been reported (19)(20)(21)(22); however, the potential of radachlorin-mediated PDT for the treatment of endometrial adenocarcinoma remains unclear.…”