Characterization of a Threonine-Ligated Molybdenyl-Sulfide Cluster as a Putative Cyanide Poisoning Antidote; Intracellular Distribution, Effects on Organic Osmolyte Homeostasis, and Induction of Cell Death.

Binuclear molybdenum sulfur complexes are effective for the catalytic conversion of cyanide into thiocyanate. The complexes themselves exhibit low toxicity and high aqueous solubility, which render them suitable as antidotes for cyanide poisoning. The binuclear molybdenum sulfur complex [(thr)MoO(μ-S)(S)] (thr - threonine) was subjected to biological studies to evaluate its cellular accumulation and mechanism of action.

Impact of the histone deacetylase inhibitor trichostatin A on active uptake, volume-sensitive release of taurine, and cell fate in human ovarian cancer cells.

The histone deacetylase inhibitor trichostatin A (TSA) reduces cell viability in cisplatin-sensitive (A2780WT) and cisplatin-resistant (A2780RES) human ovarian cancer cells due to progression of apoptosis (increased caspase-9 activity), autophagy (increased LC3-II expression), and cell cycle arrest (increased p21 expression). The TSA-mediated effect on p21 and caspase-9 is mainly p53 independent. Cisplatin increases DNA-damage (histone H2AX phosphorylation) in A2780WT cells, whereas cisplatin, due to reduced uptake [inductively coupled-plasma-mass spectrometry (Pt) analysis], has no DNA-damaging effect in A2780RES cells.

Distribution of platinum between nuclear and cytosolic fractions - Can subcellular fractionation be performed quantitatively?

The feasibility of quantitatively tracking platinum, derived from platinum-based compounds, during subcellular fractionation was studied. Cisplatin-exposed murine Ehrlich Lettré Ascites cells were fractionated into cytosolic and crude nuclear fractions. The latter was subsequently purified.

Stress-induced modulation of volume-regulated anions channels in human alveolar carcinoma cells.

Shift in the cellular homeostasis of the organic osmolyte taurine has been associated with dysregulation of the volume-regulated anion channel (VRAC) complex, which comprises leucine-rich repeat-containing family 8 members (LRRC8A-E). Using SDS-PAGE, western blotting, qRT-PCR, and tracer technique ([ H]taurine) we demonstrate that reactive oxygen species (ROS) and the cell growth-associated kinases Akt/mTOR, play a role in the regulation of VRAC in human alveolar cancer (A549) cells. LRRC8A is indispensable for VRAC activity and long-term exposure to hypoosmotic challenges and/or ROS impairs VRAC activity, not through reduction in total LRRC8A expression or LRRC8A availability in the plasma membrane, but through oxidation/inactivation of kinases/phosphatases that control VRAC activity once it has been instigated.

Facilitating the Cellular Accumulation of Pt-Based Chemotherapeutic Drugs.

Cisplatin, carboplatin, and oxaliplatin are Pt-based drugs used in the chemotherapeutic eradication of cancer cells. Although most cancer patient cells initially respond well to the treatment, the clinical effectiveness declines over time as the cancer cells develop resistance to the drugs. The Pt-based drugs are accumulated via membrane-bound transporters, translocated to the nucleus, where they trigger various intracellular cell death programs through DNA interaction.

Development and validation of an ICP-MS method for quantification of total carbon and platinum in cell samples and comparison of open-vessel and microwave-assisted acid digestion methods.

Cisplatin is a widely used chemotherapeutic drug. Due to severe side effects and intrinsic or acquired resistance, there is a great interest in developing new platinum-based anticancer agents and a need for robust and validated analytical methods for determination of platinum accumulation in biological samples. A validated ICP-MS method for quantification of total carbon and platinum in cell samples is presented, applicable for cellular drug accumulation studies of platinum-based drugs, enabling estimation of drug accumulation while simultaneously determining carbon to monitor the sample digestion efficiency.

Downregulation of Leucine-Rich Repeat-Containing 8A Limits Proliferation and Increases Sensitivity of Glioblastoma to Temozolomide and Carmustine.

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Ubiquitously expressed volume-regulated anion channels (VRAC) are thought to play a role in cell proliferation, migration, and apoptosis. VRAC are heteromeric channel complexes assembled from proteins belonging to the leucine-rich repeat-containing 8A (LRRC8A through E), among which LRRC8A plays an indispensable role.

In vitro evaluation of the enantiomeric R- and S-1,1'-binaphthyl-2,2'-diaminodichlorido-Pt(ii) complexes in human Burkitt lymphoma cells: emphasis on cellular accumulation, cytotoxicity, DNA binding, and ability to induce apoptosis.

The aim of this project is to gain insights into the uptake and cellular actions of the enantiomeric R- and S-1,1'-binaphthyl-2,2'-diaminodichlorido-Pt(ii) complexes (R- and S-[Pt(DABN)Cl]) in the cisplatin-sensitive human Burkitt lymphoma cell line (Gumbus, IC: 1.3 ± 0.2 μM) and its cisplatin-resistant sub-line (CDDPrGB, IC: 6.

Dual role of LRRC8A-containing transporters on cisplatin resistance in human ovarian cancer cells.

Acquired resistance to chemotherapeutic drugs in cancer cells can reflect an ability to limit cellular drug availability, to repair drug induced DNA damage, and to limit initiation/progression of cell death (apoptosis). The leucine-rich-repeat-containing 8A (LRRC8A) protein is an essential component of volume sensitive channels for organic osmolytes (VSOAC) and volume regulated anion channels (VRAC), which are activated during the apoptotic process. Here we illustrate that cisplatin resistance in human ovarian cancer cells (A2780) correlates with a reduced expression of LRRC8A and copper transporter receptor 1 (CTR1), as well as a concomitant increased expression of copper-transporting P-type ATPases (ATP7A/ATP7B).

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

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.

Cellular uptake of metallated cobalamins.

Cellular uptake of vitamin B12-cisplatin conjugates was estimated via detection of their metal constituents (Co, Pt, and Re) by inductively coupled plasma mass spectrometry (ICP-MS). Vitamin B12 (cyano-cob(iii)alamin) and aquo-cob(iii)alamin [Cbl-OH2](+), which differ in the β-axial ligands (CN(-) and H2O, respectively), were included as control samples. The results indicated that B12 derivatives delivered cisplatin to both cellular cytosol and nuclei with an efficiency of one third compared to the uptake of free cisplatin cis-[Pt(II)Cl2(NH3)2].

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance.

Volume-regulated channels for anions (VRAC) / organic osmolytes (VSOAC) play essential roles in cell volume regulation and other cellular functions, e.g. proliferation, cell migration and apoptosis.

Protolichesterinic Acid, Isolated from the Lichen Cetraria islandica, Reduces LRRC8A Expression and Volume-Sensitive Release of Organic Osmolytes in Human Lung Epithelial Cancer Cells.

We have tested the effect of protolichesterinic acid (PA) on the activity of the volume-sensitive release pathway for the organic osmolyte taurine (VSOAC) and the expression of the leucine-rich-repeat-channel 8A (LRRC8A) protein, which constitutes an essential VSOAC component. Exposing human lung cancer cells (A549) to PA (20 µg/mL, 24 h) reduces LRRC8A protein expression by 25% and taurine release following osmotic cell swelling (320 → 200 mOsm) by 60%. C75 (20 µg/mL, 24 h), a γ-lactone with a C8 carbon fatty acid chain, reduces VSOAC activity by 30%, i.

Integrin β1, Osmosensing, and Chemoresistance in Mouse Ehrlich Carcinoma Cells.

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.

In vitro characterization of a novel C,N-cyclometalated benzimidazole Ru(II) arene complex: stability, intracellular distribution and binding, effects on organic osmolyte homeostasis and induction of apoptosis.

In the present work a novel C,N-cyclometalated benzimidazole Ru(ii) arene complex (GY34) was characterized by applying an alternative, diverse approach considering both chemical and biological aspects. RP-HPLC-ICP-MS and RP-HPLC-ESI-MS analysis proved that GY34 in both RPMI-1640 cell medium and ammonium acetate buffer was transformed into several subspecies and the importance of evaluating and controlling analyte stability throughout experiments was demonstrated. Applying a novel cell fractionation protocol GY34 was found to target cell nuclei and mitochondria in Ehrlich Lettré Ascites (ELA) cells, with the intracellular distribution depending on GY34 concentration in the cell medium during incubation.

Acquired cisplatin resistance in human ovarian A2780 cancer cells correlates with shift in taurine homeostasis and ability to volume regulate.

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).

Physiological role of taurine--from organism to organelle.

Taurine is often referred to as a semi-essential amino acid as newborn mammals have a limited ability to synthesize taurine and have to rely on dietary supply. Taurine is not thought to be incorporated into proteins as no aminoacyl tRNA synthetase has yet been identified and is not oxidized in mammalian cells. However, taurine contributes significantly to the cellular pool of organic osmolytes and has accordingly been acknowledged for its role in cell volume restoration following osmotic perturbation.

Functions of volume-sensitive and calcium-activated chloride channels.

The review describes molecular and functional properties of the volume regulated anion channel and Ca(2+)-dependent Cl(-) channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl(-) channels in various diseases.

mTOR ensures increased release and reduced uptake of the organic osmolyte taurine under hypoosmotic conditions in mouse fibroblasts.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that modulates translation in response to growth factors and alterations in nutrient availability following hypoxia and DNA damage. Here we demonstrate that mTOR activity in Ehrlich Lettré ascites (ELA) cells is transiently increased within minutes following osmotic cell swelling and that inhibition of phosphatidylinositol-3-phosphatase (PTEN) counteracts the upstream phosphatidylinositol kinase and potentiates mTOR activity. PTEN inhibition concomitantly potentiates swelling-induced taurine release via the volume-sensitive transporter for organic osmolytes and anion channels (VSOAC) and enhances swelling-induced inhibition of taurine uptake via the taurine-specific transporter (TauT).

Ion channels and transporters in the development of drug resistance in cancer cells.

Multi-drug resistance (MDR) to chemotherapy is the major challenge in the treatment of cancer. MDR can develop by numerous mechanisms including decreased drug uptake, increased drug efflux and the failure to undergo drug-induced apoptosis. Evasion of drug-induced apoptosis through modulation of ion transporters is the main focus of this paper and we demonstrate how pro-apoptotic ion channels are downregulated, while anti-apoptotic ion transporters are upregulated in MDR.

Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca2+.

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6's physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling.

Double mutation at the putative protein kinase C phosphorylation sites Thr151 plus Thr323 in the mouse leukotrieneD4 receptor eliminates homologous desensitization.

Background/aims: Signalling via CysLT1 is involved in activation of volume sensitive K(+) channels and homologous desensitization of the LTD4 receptor impairs regulatory volume decrease (RVD). The aim is to illustrate the effect of mutation of putative PKC consensus phosphorylation sites in the CysLT1R on desensitization and RVD.

Methods: mCysLT1 contains 4 putative PKC consensus phosphorylation sites, and four mutants were created: Thr151Gly, Thr323Gly, Thr151Gly plus Thr323Gly, and Thr236Gly plus Ser243Gly.