Potassium Ionophore Research Articles

Plasticized Poly(vinyl chloride)-Based Photonic Crystal for Ion Sensing

In this study, we, for the first time, developed a plasticized poly(vinyl chloride) (PVC)-based two-dimensional photonic crystal (2D-PhC) optical sensor using nanoimprint lithography (NIL), which can perform highly sensitive, fast, and selective ion sensing based on ion extraction. Concerning the principle of response, present plasticized PVC-based PhC works as a waveguide and a grating. Incident light was guided in the bulk of plasticized PVC and, then, guided light of a specific wavelength was diffracted by a periodic nanostructure. The guided and diffracted light intensity changes of PVC-based PhCs possessing various thicknesses were monitored at 580 nm; then, we found that the 0.35 μm-thick PhC film exhibited the highest diffraction intensity. For the ion-sensing application, potassium-selective sensing elements involving potassium ionophore and lipophilic dye were dissolved in a plasticized PVC-based PhC, and the K(+)-selective response was successfully observed by monitoring the diffracted peak intensity change. The present 2D-PhC optical sensor exhibited a fast response within 5 s (95% response time) due to the use of thin film, and sensitivity was 20 times higher than that of a PVC plane-film optical sensor, due to efficient collection of diffracted light by employing a periodic nanostructure of the photonic crystal.

ET-66 * ER-STRESS INDUCING DRUGS SENSITIZES GBM TO TEMOZOLOMIDE THROUGH DOWNREGULATION OF MGMT AND INDUCTION OF REGULATED NECROSIS

Termozolamide (TMZ) is the standard treatment against GBM, unfortunately its therapeutic effect is limited due to the expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Therefore, approaches that overcome the resistance to TMZ could be feasible therapeutic alternatives for this deadly disease. Endoplasmic reticulum (ER) stress suppresses several DNA damage proteins through the unfolding protein response. In this work we sought to evaluate whether ER-stress inducing drugs were able to downmodulate MGMT and sensitize GBM cells to TMZ treatment. Salinomycin (SLM) is a potassium ionophore that has proven effective against cancer stem cells and a possible candidate to induce ER stress. Our data showed that SLM triggered ER stress that was accompanied by the downregulation of MGMT. We obtained the same results with other ER stress inducing drugs (thapsigergin, tunicamycin) suggesting that this is a general mechanism. Chemical inhibition of ER stress reverted the abrogation of MGMT downregulation. Of importance, SLM induced an aberrant autophagic flux that led to regulated necrosis cell death mediated by the action of AIF protein, which induces DNA damage when localized in the nucleus. Combination of TMZ and SLM displayed a potent antitumor effect in vitro and in vivo in mice bearing a GBM stem cell model. Combination treatment induced a significant increase in DNA damage as shown by H2AX activation and PARP. Moreover, we observed AIF in the nucleus, as a result of the regulated necrosis, furthering favoring the DNA damage. Combination treatment showed an increment of the median survival and of long term survivors. Moreover tissue analysis confirmed a dramatic increase in the level of DNA damage. Altogether our results showed that combination treatment induces a potent antiglioma effect in vitro and in vivo. Our data uncover the possibility to exploit ER stress and regulated necrosis as therapeutic strategies for GBM treatment.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1β is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5. Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1β secretion6. Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin. Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and measurement of NLRP3 inflammasome activity using IL-1β in human monocyte-derived dendritic cells.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control(1) (,) (2) . Interleukin-1β is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection(1-5). Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1β secretion(6). Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin. Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Salinomycin increases chemosensitivity to the effects of doxorubicin in soft tissue sarcomas

BackgroundChemotherapy for soft tissue sarcomas remains unsatisfactory due to their low chemosensitivity. Even the first line chemotherapeutic agent doxorubicin only yields a response rate of 18-29%. The antibiotic salinomycin, a potassium ionophore, has recently been shown to be a potent compound to deplete chemoresistant cells like cancer stem like cells (CSC) in adenocarcinomas. Here, we evaluated the effect of salinomycin on sarcoma cell lines, whereby salinomycin mono- and combination treatment with doxorubicin regimens were analyzed.MethodsTo evaluate the effect of salinomycin on fibrosarcoma, rhabdomyosarcoma and liposarcoma cell lines, cells were drug exposed in single and combined treatments, respectively. The effects of the corresponding treatments were monitored by cell viability assays, cell cycle analysis, caspase 3/7 and 9 activity assays. Further we analyzed NF-κB activity; p53, p21 and PUMA transcription levels, together with p53 expression and serine 15 phosphorylation.ResultsThe combination of salinomycin with doxorubicin enhanced caspase activation and increased the sub-G1 fraction. The combined treatment yielded higher NF-κB activity, and p53, p21 and PUMA transcription, whereas the salinomycin monotreatment did not cause any significant changes.ConclusionsSalinomycin increases the chemosensitivity of sarcoma cell lines - even at sub-lethal concentrations - to the cytostatic drug doxorubicin. These findings support a strategy to decrease the doxorubicin concentration in combination with salinomycin in order to reduce toxic side effects.

Respiratory uncoupling by increased H+ or K+ flux is beneficial for heart mitochondrial turnover of reactive oxygen species but not for permeability transition

BackgroundIschemic preconditioning has been proposed to involve changes in mitochondrial H+ and K+ fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K+ channels (MitoKATP). The objectives of the present study were to explore how increased H+ and K+ fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT).ResultsIsolated rat heart mitochondria were exposed to a wide concentration range of the protonophore CCCP or the potassium ionophore valinomycin to induce increased H+ and K+ conductance, respectively. Simultaneous monitoring of mitochondrial respiration and calcium retention capacity (CRC) demonstrated that the relative increase in respiration caused by valinomycin or CCCP correlated with a decrease in CRC, and that no level of respiratory uncoupling was associated with enhanced resistance to mPT. Mitochondria suspended in hyperosmolar buffer demonstrated a dose-dependent reduction in CRC with increasing osmolarity. However, mitochondria in hypoosmolar buffer to increase matrix volume did not display increased CRC. ROS generation was reduced by both K+- and H+-mediated respiratory uncoupling. The ability of heart mitochondria to detoxify H2O2 was substantially greater than the production rate. The H2O2 detoxification was dependent on respiratory substrates and was dramatically decreased following calcium-induced mPT, but was unaffected by uncoupling via increased K+ and H+ conductance.ConclusionIt is concluded that respiratory uncoupling is not directly beneficial to rat heart mitochondrial resistance to calcium overload irrespective of whether H+ or K+ conductance is increased. The negative effects of respiratory uncoupling thus probably outweigh the reduction in ROS generation and a potential positive effect by increased matrix volume, resulting in a net sensitization of heart mitochondria to mPT activation.

The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders

Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacity of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP+ accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [14C]-palmitate oxidation (measured as [14C]O2 release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.

Cry1Ab Treatment Has No Effects on Viability of Cultured Porcine Intestinal Cells, but Triggers Hsp70 Expression

In vitro testing can contribute to reduce the risk that the use of genetically modified (GM) crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2) as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment.

Nontypeable Haemophilus Influenzae Infection Upregulates the NLRP3 Inflammasome and Leads to Caspase-1-Dependent Secretion of Interleukin-1β — A Possible Pathway of Exacerbations in COPD

Rationale Nontypeable Haemophilus influenzae (NTHi) is the most common cause for bacterial exacerbations in chronic obstructive pulmonary disease (COPD). Recent investigations suggest the participation of the inflammasome in the pathomechanism of airway inflammation. The inflammasome is a cytosolic protein complex important for early inflammatory responses, by processing Interleukin-1β (IL-1β) to its active form.ObjectivesSince inflammasome activation has been described for a variety of inflammatory diseases, we investigated whether this pathway plays a role in NTHi infection of the airways.MethodsA murine macrophage cell line (RAW 264.7), human alveolar macrophages and human lung tissue (HLT) were stimulated with viable or non-viable NTHi and/or nigericin, a potassium ionophore. Secreted cytokines were measured with ELISA and participating proteins detected via Western Blot or immunohistochemistry.Measurements and Main ResultsWestern Blot analysis of cells and immunohistochemistry of lung tissue detected the inflammasome key components NLRP3 and caspase-1 after stimulation, leading to a significant induction of IL-1β expression (RAW: control at the lower detection limit vs. NTHi 505±111pg/ml, p<0.01). Inhibition of caspase-1 in human lung tissue led to a significant reduction of IL-1β and IL-18 levels (IL-1β: NTHi 24 h 17423±3198pg/ml vs. NTHi+Z-YVAD-FMK 6961±1751pg/ml, p<0.01).ConclusionOur data demonstrate the upregulation of the NRLP3-inflammasome during NTHi-induced inflammation in respiratory cells and tissues. Our findings concerning caspase-1 dependent IL-1β release suggest a role for the inflammasome in respiratory tract infections with NTHi which may be relevant for the pathogenesis of bacterial exacerbations in COPD.

Kinetics of NLRP3 inflammasome independent and dependent mitochondrial damage during inflammasome activation (P1276)

Abstract The NLRP3 inflammasome is a protein complex that plays a central role in innate immunity and is involved with caspase-1 activation. In murine bone marrow derived macrophages (BMDM), NLRP3 inflammasome activation can lead to the processing and secretion of IL-1 beta, as well as pyroptotic cell death. Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. In response to treatment with nigericin, we have observed that mitochondrial depolarization occurs independent of the presence of the NLRP3 inflammasome, however the loss of mitochondrial membrane integrity is NLRP3 dependent. This loss of mitochondrial integrity is preceded by NLRP3 dependent lysosomal membrane permeability. Hence, we have shown kinetically that NLRP3 inflammasome independent damage to the mitochondria precedes NLRP3 inflammasome dependent organelle damage and eventual cell death. The kinetic information provided via these data suggests a multi-step process involving both NLRP3 dependent and independent events during inflammasome activation.

Abstract 3724: Salinomycin reduced breast cancer stem cell properties by reducing SOX2 and OCT4.

Abstract Salinomycin isolated from Streptomyces albus and a potassium ionophore. It has been used to eliminate bacteria, fungi and parasites. Also, Cancer stem cells (CSCs) are a subpopulation of cells within the tumor that has ability to self-renew and undergo differentiation to phenotypically diverse cell populations. So, CSCs are involved in metastasis, chemoresistance and even cancer relapse. Recently, there are many evidences to CSCs subpopulations enhance capability of DNA repair and reduce the apoptosis. Furthermore, salinomycin has been reported to act as selective breast cancer stem cell inhibitor based on its property of reducing the cancer stem cell and to induce the apoptosis in cancer cell. Therefore, we investigated the effect of salinomycin on breast cancer stem cells. First, Mammosphere formation assay was performed to observe stem cell-like property after salinomycin treat in anchorage-independent state. We confirmed salinomycin reduces number and size of mammosphere at 3, 6, 9 day. Also, we compared mRNA expressing level of stem cell marker such as SOX2 and OCT4 by RT-PCR. Consequently, salinomycin was the down-regulated stem cell marker mRNA level as well as protein level. To confirm the reduction of the side population level, we performed FACS analysis using CD44+ and CD24- markers. Results by FACS analysis revealed that salinomycin treated with breast cancer stem cell reduced stemness by downregulating CD44+/CD24- population. Consistently, when mammosphere implanted into the mammary fat pads of BALB/c female nude mice cleared of endogenous epithelium, the tumor size was decreased. In conclusion, we concluded that effect of salinomycin to reduce stemness is mediated by downregulation of SOX2 and OCT4 in human breast cancer cell. Citation Format: Hyun Sook An, Han Na Kang, Myoung Hee Kang, Jung Lim Kim, Jun Suk Kim, Jae Hong Seo. Salinomycin reduced breast cancer stem cell properties by reducing SOX2 and OCT4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3724. doi:10.1158/1538-7445.AM2013-3724

Abstract LB-100: Exploring the mechanisms of resistance to Salinomycin, a cancer stem cell-specific agent, in budding yeast as a model.

Abstract Introduction The antibiotic salinomycin (SM) represents a selective potassium ionophore. It has recently attracted considerable attention due to its stem-cell specific mode of action that has been elucidated in a breast cancer stem cell model [1]. Further studies revealed additional benefits, e.g. the triggering of apoptosis in resistant cancer cell lines and reversal of multidrug resistance [2,3]. In budding yeast, we discovered that this agent prominently causes mitochondrial damage as revealed by the emergence of respiration-defective cells (“petites”), similar to nigericin. We also noted the frequent occurrence of SM-resistant clones - a possible obstacle in therapy. Methods We used the collection of systematic gene deletions in haploid Saccharomyces cerevisiae to characterize genes whose deletion confers SM sensitivity or resistance if a carbon source is provided (glycerol) that requires active respiration for growth. In parallel, the emergence of reactive oxygen species (ROS) was measured by flow cytometry. With this toxicogenomics approach in a model organism, we attempt to elucidate cancer-stem cell specific therapy targets and suggest strategies to avoid the development of salinomycin resistance. Results and Conclusions Among SM-sensitive deletion mutants, mutants that compromise vacuole/Golgi function and autophagy dominate. Novel genes which confer SM sensitivity if deleted were also uncovered, such as CAJ1, a heat shock protein of the dnaJ family. In almost all cases, a strong correlation between SM and nigericin sensitivity was observed. Among the most SM-resistant mutants were deletions of the E2-ubiquitin conjugating enzymes Ubc13 and Mms2 as well as deletions of TOR pathway components Sch9 and Tco89. In the presence of SM, Sch9 (homolog of human Akt) underwent dephosphorylation; a deletion of Sch9 was shown to improve growth in glycerol media and to counteract petite induction in glucose media in SM-treated cells. The observed mitochondria-dependent ROS production by SM, however, was unaltered and we suggest that, while initial damage is not affected, cells acquire resistance to the consequences of such damage. [1] Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, et al. (2009) Cell 138: 645-659. [2] Fuchs D, Heinold A, Opelz G, Daniel V, Naujokat C (2009) Biochem Biophys Res Commun 390: 743-749. [3] Fuchs D, Daniel V, Sadeghi M, Opelz G, Naujokat C (2010) Biochem Biophys Res Commun 394: 1098-1104. Supported by Cancer Prevention and Research Institute of Texas. Citation Format: Donald Rozario, Sammer Zeglam, Wolfram Siede. Exploring the mechanisms of resistance to Salinomycin, a cancer stem cell-specific agent, in budding yeast as a model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-100. doi:10.1158/1538-7445.AM2013-LB-100

Abstract 3735: Preclinical studies of salinomycin as a therapeutic agent for pediatric high-grade glioma cancer stem cells.

Abstract Brain tumors are the most prevalent childhood solid tumors. In the low-grade astrocytomas complete surgical resection has achieved over 90% five-year survival rate. In contrast, the aggressive anaplastic astrocytoma high-grade 3 and the fast growing glioblastoma multiforme high-grade 4 are often refractory to standard therapies; the majority of these patients at presentation are beyond cure by surgery and or radiotherapy. Emerging data substantiate the idea that a small pool of post-therapy residual cells within a tumor displaying stem-like properties is responsible for relapse. Therefore, selectively targeting these cancer stem-like cells (CSCs) may lead to advances in the treatment of these tumors. Salinomycin, an antibiotic potassium ionophore, has been shown to be highly effective against breast cancer stem cells and other malignancies; however, its potential use has not been studied in childhood astrocytomas. Recent studies indicate that salinomycin exerts its effect on components of the Wnt signaling cascade. In this study, we undertook in vitro and in vivo analyses on the effect of salinomycin on four pediatric high-grade astrocytoma cell lines, UW479 (provided by Dr. John Silber, University of Washington), SF188 (from Dr. Daphne Haas-Kogan, UCSF), SJ-GBM2, and CHLA-200 (COG Cell Culture and Xenograft Repository). Our results indicated that salinomycin was able to inhibit cellular viability in a dose-dependent manner. The soft-agar anchorage independent growth assay showed salinomycin inhibition of colony formation in 3 of the 4 cell lines tested. A decrease in the number of cycling cells (S-phase) was observed in cells treated with salinomycin. Notably, a 95% reduction in the glioma stem cells, read-out as CD133+, and impairment in the ability to form spheres (read-out as self-renewal) was observed. A xenograft mouse model was used to determine the effect of salinomycin on tumor growth in vivo and is ongoing. Salinomycin's modulation of Wnt targets measured by qRT-PCR, western blot analysis, and Bio-Plex protein assays, is in progress. Our data suggest that salinomycin could be exploited for the treatment of the aggressive form of pediatric gliomas. Citation Format: Emily S. Moses, Judy C. Chang, Jaclyn Y. Hung. Preclinical studies of salinomycin as a therapeutic agent for pediatric high-grade glioma cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3735. doi:10.1158/1538-7445.AM2013-3735

Abstract 489: Mechanisms of Carbon Monoxide Attenuation of Tubuloglomerular Feedback

Tubuloglomerular feedback (TGF) is an autoregulatory mechanism of the renal microcirculation in which the macula densa (MD) senses NaCl concentration in the lumen of the nephron and sends a signal that controls glomerular filtration rate by constricting the afferent arteriole (Af-Art). We have shown that MD depolarization is sufficient for inducing TGF. Carbon monoxide (CO), either endogenous or exogenous, is known to inhibit TGF, at least in part via cGMP. However, whether cGMP-independent mechanisms are involved, and where in the TGF cascade CO exerts its inhibitory effect, remain unknown. Thus we hypothesize that CO, acting via both cGMP-dependent and -independent mechanisms, attenuates TGF by acting downstream from MD cell depolarization. In vitro , microdissected rabbit Af-Arts and their attached MD were simultaneously perfused and TGF was measured as the decrease in Af-Art diameter. Depolarization of the MD was induced by switching luminal KCl from 4 to 50 mM in the presence of the potassium ionophore valinomycin, while adding the CO-releasing molecule CORM-3 to the MD perfusate at non-toxic concentrations. CORM-3 blunted depolarization-induced TGF at a concentration of 50 μM, from 3.6±0.4 to 2.5±0.4 μm (P&lt;0.01), and completely abolished it at a concentration of 100 μM, to 0.1±0.1 μm (P&lt;0.001, n=6). Similar results were found with 100 μM CORM-3 when depolarization was induced by nystatin (3.0±0.2 vs. 0.4±0.2 μm, P &lt;0.001, n=6). This indicates that CO inhibits TGF acting downstream from depolarization. When cGMP generation was blocked with the guanylate cyclase inhibitor LY-83583 (1 μM) added to the MD, CORM-3 no longer had an effect on depolarization-induced TGF at 50 μM (2.9±0.4 vs. 3.0±0.4 μm), but retained partial inhibitory effect on TGF at 100 μM (1.3±0.2 μm, P =0.02, n=9). This suggests that CO acts via cGMP at low concentrations, but additional mechanisms of action may be involved at higher concentrations. Finally, we confirmed that cGMP inhibits TGF downstream from MD depolarization by adding the degradation-resistant cGMP analog dibutyryl-cGMP (500 μM), which attenuated depolarization-induced TGF (from 3.9±0.5 to 0.6±0.2 μm, P &lt;0.01, n=6). Our results could help explain the physiological role of CO in controlling the renal microcirculation.

Osmotic tolerance and intracellular ion concentrations of bovine sperm are affected by cryopreservation

In this study, the effects of cryopreservation on osmoregulation and ion homeostasis in bovine sperm were studied. We determined: (1) the osmotic tolerance limits and cell volume response upon exposure to anisotonic conditions, (2) the intracellular pH and potassium concentration, and (3) expression and localization of proteins encoding for potassium and chloride ion channels. A flow cytometric approach was used for simultaneous assessment of cell volume and viability of propidium iodide stained sperm in anisotonic media. Osmotic tolerance was found to be decreased after cryopreservation, especially in the 120 to 60 mOsm/kg osmotic range. The critical osmolality at which half of the sperm population survived increased from 55 to 89 mOsm/kg. The osmotic cell volume response for viable sperm was similar before and after cryopreservation, with an osmotic inactive volume of about 70%. The intracellular pH, determined by recording changes in carboxyfluorescein fluorescence of sperm in media with different pH before and after addition of digitonin, decreased from 6.28 in diluted sperm to 6.16 after cryopreservation. The intracellular potassium concentration, determined using the potassium ionophore nigericin and incubation in media with various potassium concentrations, increased from 154 mM to 183 mM before and after cryopreservation, respectively. The levels of the chloride and potassium ion channel proteins chloride channel 3 protein (CLC-3) and two pore domain potassium channel 2 protein (TASK-2), as detected using Western blot analysis, were not affected by cryopreservation. Immunolocalization studies showed that CLC-3 is present in the acrosome and midpiece as well as in the upper and lower tail. In conclusion, cryopreserved sperm exhibit reduced tolerance to hypotonic stress, a decreased intracellular pH, and increased intracellular potassium level.

NLRP3 inflammasome dependent loss of mitochondrial and cell membrane integrity in bone marrow derived macrophages (114.3)

Abstract The NLRP3 inflammasome is a multiprotein complex that includes the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which binds with caspase-1 via CARD-CARD interactions and is involved with caspase-1 activation. Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. We have observed that NLRP3 knockout macrophages are more resistant to the loss of membrane integrity compared to wild type macrophages exposed to nigericin or TLO, as assessed by a dye uptake assay. Furthermore, this NLRP3 dependent loss of membrane integrity corresponds to the loss of mitochondrial membrane integrity as demonstrated via live cell microscopy. The loss of mitochondrial membrane integrity, but not mitochondrial depolarization was found to be dependent on the presence of NLRP3. Furthermore, the loss of mitochondrial integrity and the loss of cell membrane integrity suggest a potential mechanistic pathway for the effects of NLRP3 inflammasome activation in BMDM. These results further our understanding of how NLRP3 functions in macrophages exposed to stressful stimuli such as bacterial toxins.

Abstract 879: Developing a predictive in vitro assay for evaluating therapies against metastatic breast cancer

Abstract The major challenge in breast cancer research pertains to tumor heterogeneity being a constant characteristic in the disease process. Although it is recognized that rare cancer cells drive the disease, there is a lack of suitable function-based assays to identify and focus on such cells for therapy development. Based on the hypothesis that only rare but highly adaptable cancer cells succeed in metastasizing, we have recently isolated rare (&amp;lt; 0.01% of population) highly metastatic cells present in an aggressive inflammatory breast cancer cell line SUM149. Using a variety of long-term adaptability assays we have found that such cells have adaptability to survive lack of glutamine, glucose, oxygen, and serum. Unlike the majority of cells, such cells have a high ability to dissociate their metabolic state from regulatory state, thus making them more resistant to chemotherapy drugs. These properties of cancer cells make them suitable for survival as resistant cells in the body, which often use quiescence as a way of surviving unfavorable conditions. As an initial step to optimize our in vitro system for evaluating potential personalized therapies, we compared the efficacy of several experimental targeted therapy drugs on metastatic Gln-independent variants and on parental SUM149 cell line. Since cell proliferation and apoptosis assays after a short exposure to experimental therapies are not good at predicting clinical response, we chose to determine therapeutic efficacy by analyzing residual/resistant cells after a relatively long exposure to therapy. Celecoxib, a COX-2 inhibitor (2-10 micromolar range), failed to inhibit resistant cells in both Gln-ind and parental SUM149 cell lines. At a 50 micromolar concentration, celecoxib eradicated all cells with a proliferative potential in the parental cell line but not in the Gln-ind subpopulation. Metformin, an anti-diabetes drug that activates AMPK and also inhibits “cancer stem cells,” was less effective in inhibiting Gln-ind cells than the parental cell line. BEZ235, a dual PI3K/mTOR inhibitor, sensitized Gln-ind and parental cells to the chemotherapy drugs doxorubicin and paclitaxel. However, Gln-ind cells yielded significantly more clonogenic (10-fold) cells after they were treated sequentially with BAZ235 and doxorubicin/paclitaxel. Salinomycin, a potassium ionophore and an inhibitor of “cancer stem cells,” was also less effective against Gln-ind cells. HDAC inhibitors, valproic acid, and butyrate, which are being evaluated as sensitizers to other therapies in various cancers, sensitized both Gln-ind and parental cell line to doxorubicin and paclitaxel. We conclude that the pair of cell lines used in this study will be useful in discovering strategies (e.g., novel therapies and ways to administer them) for eradicating metastatic breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 879. doi:1538-7445.AM2012-879

Abstract 3359: Salinomycin reduces stem cell properties and induces apoptosis in mammary epithelial cell

Abstract Tumor initiating cells or cancer stem cells (CSCs) were characterized as a unique subpopulation with stem cell features in many types of cancer. Although there has been a number of studies on breast cancer stem cells, inhibitory mechanisms of tumor initiation, progression, and resistance to therapy are unknown. Salinomycin is a chemical isolated from Streptomyces albus and a potassium ionophore. It has been used for many years to control coccidiosis. Recently, salinomycin has been reported to act as selective breast cancer stem cell inhibitor because of its property of reducing the cancer stem cell and inducing the apoptosis in cancer cell. Thus, we investigated the anti-cancer property of salinomycin in mouse mammary epithelial cell. With CDμgeo (mouse mammary epithelial cell), MTT assay was used to confirm cell viability after salinomycin treatment (0, 0.5, 1, 2, 5, 10 μm/ml) upon anchorage-dependent state. We observed that salinomycin reduced the cell proliferation dose-dependently. Also, mammosphere formation assay was performed to observe stem cell-like property after salinomycin treatment in anchorage-independent state. We confirmed salinomycin reduced the number and the size of mammosphere in 72, 144, and 216 hours. We performed salinomycin pre-treatment (0, 0.5, 1, 2, 5 μm/ml) for 12-48 hours upon anchorage-dependent state. Results by western blotting revealed that salinomycin promoted apoptosis of mouse mammary epithelial cells by activating both caspase-3 and -9 and inducing PARP cleavage. To confirm the reduction of the side population level, we performed FACS analysis using CD49+, CD29+, and CD24+ markers. Based on our findings, we concluded that anti-cancer effect of salinomycin reduced stemness and induced apoptosis in mouse mammary epithelial cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3359. doi:1538-7445.AM2012-3359

Abstract B47: Exploring the mechanism of cancer stem cell-specific action by salinomycin in yeast as a model

Abstract The antibiotic salinomycin represents a selective potassium ionophore. It has recently attracted considerable attention due to its stem-cell specific mode of action as elucidated in a breast cancer stem cell model [1]. Further studies revealed additional benefits, e.g. the triggering of apoptosis in resistant cancer cell lines and reversal of multidrug resistance [2,3]. In budding yeast, we discovered that this agent is active and, similar to nigericin, prominently causes mitochondrial damage as revealed by the emergence of respiration-defective cells. We also noted the frequent occurrence of salinomycin-resistant clones which may prove to be an obstacle in chemotherapy. We used the collection of systematic gene deletions in haploid yeast to characterize genes whose deletion confers sensitivity or resistance to salinomycin if a carbon source is provided (glycerol) that requires active respiration for growth. With this toxicogenomics approach in a model organism, we attempt to elucidate cancer stem cell-specific therapy targets and suggest strategies to avoid the development of salinomycin resistance. Among salinomycin-sensitive deletion mutants, significant overlap was found with known mutants sensitive to the ionophore monensin. Within this group, mutants that compromise vacuole/Golgi function and autophagy dominate. Novel genes were also uncovered which confer salinomycin sensitivity if deleted, such as CAJ1, encoding a heat shock protein of the dnaJ family, and certain arginine synthesis genes. On the other hand, deletions of TOR pathway components as well as deletions of the E2-ubiquitin conjugating enzymes UBC13 and MMS2 were among the most salinomycin-resistant mutants we found. In almost all cases, a strong correlation between salinomycin and nigericin sensitivity was observed. Since salinomycin appears to be the more efficient anti-cancer stem cell agent [1], the few cases of differential sensitivity will be discussed. [1] Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, et al. (2009) Cell 138:645–659. [2] Fuchs D, Heinold A, Opelz G, Daniel V, Naujokat C (2009) Biochem Biophys Res Commun 390:743–749. [3]Fuchs D, Daniel V, Sadeghi M, Opelz G, Naujokat C (2010) Biochem Biophys Res Commun 394:1098–1104. Supported by Cancer Prevention and Research Institute of Texas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B47.

Inhibition of Wnt signaling and cancer stem cells

Cancer stem cells represent a small number of pluripotent and self-renewing cells within a tumor, which are resistant to conventional chemotherapy and are responsible for tumor initiation and maintenance. The Wnt/β-catenin signaling pathway drives stem cell self-renewal and is involved in the pathogenesis of various types of cancer. Aberrant activation of the Wnt signaling pathway in normal stem cells can promote their transformation into cancer stem cells [1,2]. Thus, drugs targeted to the Wnt/β-catenin pathway may potentially be effective in eliminating cancer stem cells. Recently, several small molecule Wnt signaling inhibitors have been identified, that can have therapeutic potential against cancers associated with aberrant Wnt signaling [3]. However, most reported Wnt inhibitors are still in the developmental stage, due to lack of specificity and a poor understanding of their precise mechanisms of action. Furthermore, there is limited evidence that these Wnt signaling inhibitors can eradicate cancer stem cells, without depleting normal stem cells in the bone marrow and the gastrointestinal tract. Salinomycin, an antibiotic potassium ionophore, is the first drug reported to act as a selective breast cancer stem cell inhibitor. It kills breast cancer stem cells at least 100 times more effectively than paclitaxel in mice, but is relatively non-toxic to normal stem cells [4]. The mechanism of salinomycin action in the cancer stem cells remains unclear. Our recent studies have shown that salinomycin potently inhibits proximal Wnt/β-catenin signaling. It blocks the phosphorylation of the Wnt co-receptor LRP6, and induces its degradation [5]. Considering the importance of Wnt signaling in stem cell biology, the Wnt antagonistic action of salinomycin may contribute to its selective toxicity toward breast cancer stem cells. It will be interesting to test whether other Wnt signaling inhibitors with targets downstream of LRP6 are able to kill selectively breast cancer stem cells. A successful anticancer therapeutic regime should eliminate both the differentiated cancer cells and the cancer stem cell population. Classical cytotoxic agents may deplete the bulk of a cancer but not the inherently chemo-resistant cancer stem cells, which ultimately recur and metastasize. The molecularly targeted agents act on aberrant molecular pathways associated with tumor development and progression, and have been successfully introduced to treat patients with various cancers [6]. These drugs include Bcr-abl inhibitors, epidermal growth factor receptor (EGFR) inhibitors, vascular endothelial growth factor (VEGF) antagonists, and recently Raf kinase and Alk kinase inhibitors. These single-target agents have spectacularly initial clinical efficacy when used alone, but none are able to cure a cancer. Future experiments need to determine if the combination of a single-target agent with salinomycin or other Wnt signaling inhibitors might have the ability to induce curative response in some tumors.