Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation.

Belinda Halling Sørensen,Dorthe Nielsen,Else Kay Hoffmann,Ian Henry Lambert,Unnur Arna Thorsteinsdottir

doi:10.1152/ajpcell.00256.2015

Belinda Halling Sørensen, Dorthe Nielsen + Show 3 more

Open Access

https://doi.org/10.1152/ajpcell.00256.2015

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Abstract

The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21Waf1/Cip1, Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21Waf1/Cip1 as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21Waf1/Cip1 and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A.

Highlights

UNDER PHYSIOLOGICAL and pathophysiological conditions and following chemotherapeutic treatment, outdated/damaged cells become eliminated by apoptosis
Several groups have recently shown that LRRC8A significantly contributes to VRAC/VSOAC channel activity either by being a regulatory subunit or a critical channel pore-forming component [36, 39, 49]
With respect to drug resistance, data from our group have indicated that development of resistance in human ovarian A2780 carcinoma cells correlates with a reduced LRRC8A protein expression, and an inability to activate VSOAC and to volume-regulate in response to hyposmotic cell swelling [44]

Summary

EXPERIMENTAL PROCEDURES

9284) antibodies were from Cell Signaling (Danvers, MA) and used in a dilution of 1:250 (Noxa, phosphor-MDM2, Bax, Caspase-9, Histone H3, ␣-tubulin and phosphor-p53) or 1:100 (ATM, phosphor-ATM and p53), respectively. SDS-PAGE and Western blotting were used to analyze the amount of LRRC8A, Naϩ/KϩATPase (positive plasma membrane control/loading control), and Histone 3 (nuclear control) in both whole cell lysate and purified samples. Wild-type, resistant, and transiently transfected A2780 cells were grown to 80% confluency in T25 culture flasks and incubated in the presence or absence of 10 ␮M NS3728 or 400 ␮M DIDS in combination with 10 ␮M Cisplatin, 20 ng/ml TNF␣, or 150 mM NaCl (600 mOsM) for 18 or 4.5 h. Swelling-induced taurine efflux was estimated at room temperature on cells grown to 80% confluence in 6 well polyethylene culture plates as previously described [26]. In bar- and scatterplots the error bars signify standard error of the mean (SE)

RESULTS

1.20 A549CisR10

DISCUSSION

10 Caspase 9

DISCLOSURES