title: Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation.
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 p21(Waf1/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 p21(Waf1/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 p21(Waf1/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.
authors.affiliation: Department of Biology, Section of Cell Biology and Physiology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Belinda Halling
authors.initials: BH
authors.lastname: Sørensen
authors.affiliation: Department of Biology, Section of Cell Biology and Physiology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Dorthe
authors.initials: D
authors.lastname: Nielsen
authors.affiliation: Department of Biology, Section of Cell Biology and Physiology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Unnur Arna
authors.initials: UA
authors.lastname: Thorsteinsdottir
authors.affiliation: Department of Biology, Section of Cell Biology and Physiology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Else Kay
authors.initials: EK
authors.lastname: Hoffmann
authors.affiliation: Department of Biology, Section of Cell Biology and Physiology, The August Krogh Building, University of Copenhagen, Copenhagen, Denmark ihlambert@bio.ku.dk.
authors.forename: Ian Henry
authors.initials: IH
authors.lastname: Lambert
mesh_terms: D000072283:A549 Cells; D002282:Adenocarcinoma, Bronchiolo-Alveolar / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D027321:Anion Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D048429:Cell Size; D002945:Cisplatin / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D049990:Membrane Transport Modulators / Q000494:pharmacology; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D027321:Anion Transport Proteins; D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; D027682:Cation Transport Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C485976:LRRC8A protein, human; D008565:Membrane Proteins; D049990:Membrane Transport Modulators; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D002945:Cisplatin
keywords: apoptosis; multidrug resistance; organic anion channels; platinum drugs; taurine; transcription factors