Sonali P. Barwe

Dexamethasone-loaded block copolymer nanoparticles induce leukemia cell death and enhance therapeutic efficacy: a novel application in pediatric nanomedicine.

Nanotechnology approaches have tremendous potential for enhancing treatment efficacy with lower doses of chemotherapeutics. Nanoparticle (NP)-based drug delivery approaches are poorly developed for childhood leukemia. Dexamethasone (Dex) is one of the most common chemotherapeutic drugs used in the treatment of childhood leukemia. In this study, we encapsulated Dex in polymeric NPs and validated their antileukemic potential in vitro and in vivo. NPs with an average diameter of 110 nm were assembled from an amphiphilic block copolymer of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals (ECT2). The blank NPs were nontoxic to cultured cells in vitro and to mice in vivo. Encapsulation of Dex into the NPs (Dex-NP) did not compromise the bioactivity of the drug. Dex-NPs induced glucocorticoid phosphorylation and showed cytotoxicity similar to the free Dex in leukemic cells. Studies using NPs labeled with fluorescent dyes revealed leukemic cell surface binding and internalization. In vivo biodistribution studies showed NP accumulation in the liver and spleen with subsequent clearance of the particles with time. In a preclinical model of leukemia, Dex-NPs significantly improved the quality of life and survival of mice as compared to the free drug. To our knowledge, this is the first report showing the efficacy of polymeric NPs to deliver Dex to potentially treat childhood leukemia and reveals that low doses of Dex should be sufficient for inducing cell death and improving survival.

α-Catenin overrides Src-dependent activation of β-catenin oncogenic signaling

Loss of α-catenin is one of the characteristics of prostate cancer. The catenins (α and β) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of β-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where β-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, β-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of β-catenin invariably are altered in cancer. Although a wealth of information is available about β-catenin deregulation during oncogenesis, much less is known about how or whether α-catenin regulates β-catenin functions. In this study, we show that α-catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of β-catenin. In α-catenin-null prostate cancer cells, reexpression of α-catenin increased cell-cell adhesion and decreased β-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of β-catenin is a major mechanism for decreased β-catenin interaction with E-cadherin in α-catenin-null cells. α-Catenin attenuated the effect of Src phosphorylation by increasing β-catenin association with E-cadherin. We also show that α-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that α-catenin is a key regulator of β-catenin transcriptional activity and that the status of α-catenin expression in tumor tissues might have prognostic value for Src targeted therapy. [Mol Cancer Ther 2008;7(6):1386–97]

Dysfunction of ouabain-induced cardiac contractility in mice with heart-specific ablation of Na,K-ATPase β1-subunit

Na,K-ATPase is composed of two essential alpha- and beta-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its alpha(1), alpha(3) and beta(1) isoforms are reduced in the failing human heart. The catalytic alpha-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase beta(1)-subunit (Na,K-beta(1)) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-beta(1) gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-beta(1) gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13-14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-beta(1) KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-beta(1) KO in older mice. Further, we show that intact hearts of Na,K-beta(1) KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-beta(1) plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.

Inhibition of epidermal growth factor signaling by the cardiac glycoside ouabain in medulloblastoma.

Epidermal growth factor (EGF) signaling regulates cell growth, proliferation, and differentiation. Upon receptor binding, EGF triggers cascades of downstream signaling, including the MAPK and phosphoinositide‐3‐kinase (PI3K)/Akt signaling pathways. Aberrant expression/activation of EGFR is found in multiple human cancers, including medulloblastoma, the most prevalent pediatric brain cancer, and often has been associated with metastasis, poor prognosis, and resistance to chemotherapy. Na,K‐ATPase is an ion pump well known for its role in intracellular ion homeostasis. Recent studies showed that Na,K‐ATPase also functions as a signaling platform and revealed a role in EGFR, MAPK, and PI3K signaling. While both EGFR and Na,K‐ATPase seem to modulate similar signaling pathways, cardiac glycosides that are steroid‐like inhibitors of Na,K‐ATPase, exhibit antiproliferative and proapoptotic properties in cancer cells. Thus, we sought to better understand the relationship between EGF and cardiac glycoside signaling. Here, we show that in medulloblastoma cells, both EGF and ouabain activate Erk1/2 and PI3K/Akt signaling. Nevertheless, in medulloblastoma cells ouabain did not transactivate EGFR as has been reported in various other cell lines. Indeed, ouabain inhibited EGF‐induced Erk1/2 and Akt activation and, moreover, prevented EGF‐induced formation of actin stress fibers and cell motility, probably by activating a stress signaling response. Na,K‐ATPase has been proposed to act as a signaling scaffold and our studies suggest that in medulloblastoma cells Na,K‐ATPase might act as a check point to integrate EGF‐associated signaling pathways. Thus, Na,K‐ATPase might serve as a valid target to develop novel therapeutic approaches in tumors with aberrant activation of the EGFR signaling cascades.

Regulation of Na,K-ATPase β1-subunit in TGF-β2-mediated epithelial-to-mesenchymal transition in human retinal pigmented epithelial cells

Proliferative vitreo retinopathy (PVR) is associated with extracellular matrix membrane (ECM) formation on the neural retina and disruption of the multilayered retinal architecture leading to distorted vision and blindness. During disease progression in PVR, retinal pigmented epithelial cells (RPE) lose cell-cell adhesion, undergo epithelial-to-mesenchymal transition (EMT), and deposit ECM leading to tissue fibrosis. The EMT process is mediated via exposure to vitreous cytokines and growth factors such as TGF-β2. Previous studies have shown that Na,K-ATPase is required for maintaining a normal polarized epithelial phenotype and that decreased Na,K-ATPase function and subunit levels are associated with TGF-β1-mediated EMT in kidney cells. In contrast to the basolateral localization of Na,K-ATPase in most epithelia, including kidney, Na,K-ATPase is found on the apical membrane in RPE cells. We now show that EMT is also associated with altered Na,K-ATPase expression in RPE cells. TGF-β2 treatment of ARPE-19 cells resulted in a time-dependent decrease in Na,K-ATPase β1 mRNA and protein levels while Na,K-ATPase α1 levels, Na,K-ATPase activity, and intracellular sodium levels remained largely unchanged. In TGF-β2-treated cells reduced Na,K-ATPase β1 mRNA inversely correlated with HIF-1α levels and analysis of the Na,K-ATPase β1 promoter revealed a putative hypoxia response element (HRE). HIF-1α bound to the Na,K-ATPase β1 promoter and inhibiting the activity of HIF-1α blocked the TGF-β2 mediated Na,K-ATPase β1 decrease suggesting that HIF-1α plays a potential role in Na,K-ATPase β1 regulation during EMT in RPE cells. Furthermore, knockdown of Na,K-ATPase β1 in ARPE-19 cells was associated with a change in cell morphology from epithelial to mesenchymal and induction of EMT markers such as α-smooth muscle actin and fibronectin, suggesting that loss of Na,K-ATPase β1 is a potential contributor to TGF-β2-mediated EMT in RPE cells.

Expression of Na,K-ATPase-β1 subunit increases uptake and sensitizes carcinoma cells to oxaliplatin

PurposeThe ovarian carcinoma subline A2780/C10B (C10B) is an oxaliplatin resistant clone derived from the human ovarian carcinoma cell line A2780. The C10B cells are characterized by mesenchymal phenotype, decreased platinum uptake and increased glutathione levels (Hector et al. in Cancer Lett 245:195–204, 2007; Varma et al. in Oncol Rep 14:925–932, 2005). Na,K-ATPase-β subunit (Na,K-β1) functions as a cell–cell adhesion molecule in epithelial cells and is reduced in a variety of carcinoma cells that show mesenchymal phenotype. The purpose of this study is to evaluate the relationship between Na,K-β expression and sensitivity to oxaliplatin.MethodsCell lines used include A2780, C10B, C10B transfected with Na,K-β1 (C10B-Na,K-β) and a canine kidney carcinoma cell line MSV-MDCK also transfected with Na,K-β1 (MSV-MDCK-β subunit). Cytotoxicity studies were performed by sulforhodamine-blue assay. The Na,K-α1 and Na,K-β1 subunit localization and expression were by immunofluorescence microscopy and Western blot analysis. Platinum accumulation measurements were by atomic absorption spectrophotometry.ResultsC10B cells express highly reduced levels of Na,K-β1 subunit. Exogenous expression of Na,K-β1 increased platinum accumulation and sensitized C10B cells to oxaliplatin. The pharmacological inhibitor of Na,K-ATPase ouabain did not alter the oxaliplatin accumulation indicating that Na,K-β1 sensitizes cells in a Na,K-ATPase enzyme activity independent manner. These findings were also confirmed in MSV-MDCK-β subunit cells.ConclusionsThis study for the first time reveals that reduced expression of the Na,K-β1 protein is associated with oxaliplatin resistance in cancer cells and demonstrates a novel role for this protein in sensitizing the cells to oxaliplatin. This study suggests a potentially important role for Na,K-β1 in both prognosis and therapy of oxaliplatin resistant malignancies.

Na,K-ATPase β-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells

Summary Na,K-ATPase is a hetero-oligomer of an &agr;- and a &bgr;-subunit. The &agr;-subunit (Na,K-&agr;) possesses the catalytic function, whereas the &bgr;-subunit (Na,K-&bgr;) has cell-cell adhesion function and is localized to the apical junctional complex in polarized epithelial cells. Earlier, we identified two distinct conserved motifs on the Na,K-&bgr;1 transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-&bgr;1 and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-&agr;1. We now provide evidence that knockdown of Na,K-&bgr;1 prevents lumen formation and induces activation of extracellular regulated kinases 1 and 2 (ERK1/2) mediated by phosphatidylinositol 3-kinase in MDCK cells grown in three-dimensional collagen cultures. These cells sustained cell proliferation in an ERK1/2-dependent manner and did not show contact inhibition at high cell densities, as revealed by parental MDCK cells. This phenotype could be rescued by wild-type Na,K-&bgr;1 or heptad repeat motif mutant of Na,K-&bgr;1, but not by the glycine zipper motif mutant that abrogates Na,K-&bgr;1 cis homo-oligomerization. These studies suggest that Na,K-&bgr;1 cis homo-oligomerization rather than hetero-oligomerization with Na,K-&agr;1 is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.

Gramicidin A Induces Metabolic Dysfunction and Energy Depletion Leading to Cell Death in Renal Cell Carcinoma Cells

Ionophores are lipid-soluble organic molecules that disrupt cellular transmembrane potential by rendering biologic membranes permeable to specific ions. They include mobile-carriers that complex with metal cations and channel-formers that insert into the membrane to form hydrophilic pores. Although mobile-carriers possess anticancer properties, investigations on channel-formers are limited. Here, we used the channel-forming ionophore gramicidin A to study its effects on the growth and survival of renal cell carcinoma (RCC) cells. RCC is a histologically heterogeneous malignancy that is highly resistant to conventional treatments. We found that gramicidin A reduced the in vitro viability of several RCC cell lines at submicromolar concentrations (all IC50 < 1.0 μmol/L). Gramicidin A exhibited similar toxicity in RCC cells regardless of histologic subtype or the expression of either the von Hippel-Lindau tumor suppressor gene or its downstream target, hypoxia-inducible factor-1α. Gramicidin A decreased cell viability equal to or greater than the mobile-carrier monensin depending on the cell line. Mechanistic examination revealed that gramicidin A blocks ATP generation by inhibiting oxidative phosphorylation and glycolysis, leading to cellular energy depletion and nonapoptotic cell death. Finally, gramicidin A effectively reduced the growth of RCC tumor xenografts in vivo. These results show a novel application of gramicidin A as a potential therapeutic agent for RCC therapy. Mol Cancer Ther; 12(11); 2296–307. ©2013 AACR.

Glucocorticoids suppress renal cell carcinoma progression by enhancing Na,K-ATPase beta-1 subunit expression.

Glucocorticoids are commonly used as palliative or chemotherapeutic clinical agents for treatment of a variety of cancers. Although steroid treatment is beneficial, the mechanisms by which steroids improve outcome in cancer patients are not well understood. Na,K-ATPase beta-subunit isoform 1 (NaK-β1) is a cell-cell adhesion molecule, and its expression is down-regulated in cancer cells undergoing epithelial-to mesenchymal-transition (EMT), a key event associated with cancer progression to metastatic disease. In this study, we performed high-throughput screening to identify small molecules that could up-regulate NaK-β1 expression in cancer cells. Compounds related to the glucocorticoids were identified as drug candidates enhancing NaK-β1 expression. Of these compounds, triamcinolone, dexamethasone, and fluorometholone were validated to increase NaK-β1 expression at the cell surface, enhance cell-cell adhesion, attenuate motility and invasiveness and induce mesenchymal to epithelial like transition of renal cell carcinoma (RCC) cells in vitro. Treatment of NaK-β1 knockdown cells with these drug candidates confirmed that these compounds mediate their effects through up-regulating NaK-β1. Furthermore, we demonstrated that these compounds attenuate tumor growth in subcutaneous RCC xenografts and reduce local invasiveness in orthotopically-implanted tumors. Our results strongly indicate that the addition of glucocorticoids in the treatment of RCC may improve outcome for RCC patients by augmenting NaK-β1 cell-cell adhesion function.

Sodium-Calcium Exchanger 1 Regulates Epithelial Cell Migration via Calcium-dependent Extracellular Signal-regulated Kinase Signaling

Background: Sodium-calcium exchanger (NCX1) regulates calcium in renal epithelial cells. Results: Na,K-ATPase β-subunit regulates NCX1 membrane localization and reduced NCX1 expression or its functional inhibition increases cell migration. Conclusion: NCX1 plays a pivotal role in activation of calcium dependent migration via calmodulin/PI3K/ERK. Significance: Identifying regulators of epithelial cell motility is important in establishing novel therapeutic targets in fibrosis and cancer. Na+/Ca2+ exchanger-1 (NCX1) is a major calcium extrusion mechanism in renal epithelial cells enabling the efflux of one Ca2+ ion and the influx of three Na+ ions. The gradient for this exchange activity is provided by Na,K-ATPase, a hetero-oligomer consisting of a catalytic α-subunit and a regulatory β-subunit (Na,K-β) that also functions as a motility and tumor suppressor. We showed earlier that mice with heart-specific ablation (KO) of Na,K-β had a specific reduction in NCX1 protein and were ouabain-insensitive. Here, we demonstrate that Na,K-β associates with NCX1 and regulates its localization to the cell surface. Madin-Darby canine kidney cells with Na,K-β knockdown have reduced NCX1 protein and function accompanied by 2.1-fold increase in free intracellular calcium and a corresponding increase in the rate of cell migration. Increased intracellular calcium up-regulated ERK1/2 via calmodulin-dependent activation of PI3K. Both myosin light chain kinase and Rho-associated kinase acted as mediators of ERK1/2-dependent migration. Restoring NCX1 expression in β-KD cells reduced migration rate and ERK1/2 activation, suggesting that NCX1 functions downstream of Na,K-β in regulating cell migration. In parallel, inhibition of NCX1 by KB-R7943 in Madin-Darby canine kidney cells, LLC-PK1, and human primary renal epithelial cells (HREpiC) increased ERK1/2 activation and cell migration. This increased migration was associated with high myosin light chain phosphorylation by PI3K/ERK-dependent mechanism in HREpiC cells. These data confirm the role of NCX1 activity in regulating renal epithelial cell migration.