Sørensen BH, Thorsteinsdottir UA, Lambert IH. Acquired cisplatin resistance in human ovarian A2780 cancer cells correlates with shift in taurine homeostasis and ability to volume regulate. Am J Physiol Cell Physiol 307: C1071-C1080, 2014. First published September 24, 2014; doi:10.1152/ajpcell.00274.2014.-Cisplatin resistance is a major challenge in the treatment of cancer and develops through reduced drug accumulation and an increased ability to avoid drug-induced cell damage, cell shrinkage, and hence initiation of apoptosis. Uptake and release of the semiessential amino acid taurine contribute to cell volume homeostasis, and taurine has been reported to have antiapoptotic effects. Here we find that volume-sensitive taurine release in cisplatin-sensitive [wild-type (WT)] human ovarian cancer A2780 cells is reduced in the presence of the phospholipase A2 inhibitor bromenol lactone, the 5-lipoxygenase (5-LO) inhibitor ETH 615-139, and the cysteine leukotriene receptor 1 (CysLT1) antagonist zafirlukast and impaired by the anion channel blocker DIDS (4,4=-diisothiocyanatostilbene-2,2=-disulfonate). Comparing WT and cisplatin-resistant (RES) A2780 cells we also find that evasion of cisplatin-induced cell death in RES A2780 cells correlates with an increased accumulation of taurine, due to an increased taurine uptake and a concomitant impairment of the volume-sensitive taurine release pathway, as well an inability to reduce cell volume after osmotic cell swelling. Downregulation of volume-sensitive taurine release in RES A2780 cells correlates with reduced expression of the leucine-rich repeat-containing protein 8A (LRRC8A). Furthermore, acute (18 h) exposure to cisplatin (5-10 M) increases taurine release and LRRC8A expression in WT A2780 cells whereas cisplatin has no effect on LRRC8A expression in RES A2780 cells. It is suggested that shift in LRRC8A activity can be used as biomarker for apoptotic progress and acquirement of drug resistance. key word: platinum drugs; drug resistance; cell volume regulation; LRRC8A; TauT OVARIAN CANCER IS A LEADING cause of death among gynecological cancer patients, and the 5-yr survival rate has been estimated to 30% (64). The main reasons for the failure in treatment outcome are that 70% of the patients have already reached advanced stages of cancer progression at the time of diagnosis, i.e., the cancer has already spread within the pelvis and abdomen (68) and that most ovarian cancer cells rapidly develop resistance or multidrug resistance (MDR) against the chemotherapy.Cisplatin and chemotherapeutic resistance. Cisplatin was the first member of a class of platinum-based anticancer drugs, which also includes carboplatin and oxaliplatin (7,25), and today cisplatin is frequently used in chemotherapeutic treatment of ovarian cancer, lung cancer, and lymphomas (60). Cisplatin is administered to cancer patients intravenously as a sterile saline solution. Once circulating in the bloodstream, cisplatin is taken up by the cells and accumulated in the cell nucleus, where it causes DNA l...

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.

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Background/Aims:Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin β₁, in cell volume regulation and drug-induced apoptosis in adherent and non-adherent Ehrlich ascites cell lines. Methods:Adhesion phenotypes were verified by colorimetric cell-adhesion-assay. Quantitative real-time PCR and western blot were used to compare expression levels of integrin subunits. Small interfering RNA was used to silence integrin β₁ expression. Regulatory volume decrease (RVD) after cell swelling was studied with calcein-fluorescence-self-quenching and Coulter counter analysis. Taurine efflux was estimated with tracer technique. Caspase assay was used to determine apoptosis. Results:We show that adherent cells have stronger fibronectin binding and a significantly increased expression of integrin α₅, α_v, and β₁ at mRNA and protein level, compared to non-adherent cells. Knockdown of integrin β₁ reduced RVD of the adherent but not of the non-adherent cells. Efflux of taurine was unaffected. In contrast to non-adherent, adherent cells exhibited chemoresistance to chemotherapeutic drugs (cisplatin and gemcitabine). However, knockdown of integrin β₁ promoted cisplatin-induced caspase activity in adherent cells. Conclusion:Our data identifies integrin β₁ as a part of the osmosensing machinery and regulator of cisplatin resistance in adherent Ehrlich cells.