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Purpose: The 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-beta subunit (Na,K-beta(1)) 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-beta expression and sensitivity to oxaliplatin.Methods: Cell lines used include A2780, C10B, C10B transfected with Na,K-beta(1) (C10B-Na,K-beta) and a canine kidney carcinoma cell line MSV-MDCK also transfected with Na,K-beta(1) (MSV-MDCK-beta subunit). Cytotoxicity studies were performed by sulforhodamine-blue assay. The Na,K-alpha(1) and Na,K-beta(1) subunit localization and expression were by immunofluorescence microscopy and Western blot analysis. Platinum accumulation measurements were by atomic absorption spectrophotometry.Results: C10B cells express highly reduced levels of Na,K-beta(1) subunit. Exogenous expression of Na,K-beta(1) 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-beta(1) sensitizes cells in a Na,K-ATPase enzyme activity independent manner. These findings were also confirmed in MSV-MDCK-beta subunit cells.Conclusions: This study for the first time reveals that reduced expression of the Na,K-beta(1) 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-beta(1) in both prognosis and therapy of oxaliplatin resistant malignancies. [ABSTRACT FROM AUTHOR]

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive rare disease condition in which audiological deficit is also observed. A 4-year-old male child with PMD underwent an audiological evaluation. The results suggested normal middle ear and outer hair cells functioning, with only peak I of the auditory brainstem response present until 30 dBnHL. Further, the cervical vestibular evoked myogenic potential showed delayed latencies with normal amplitudes. In this case report, we attempt to explain the audio-vestibular test results and correlate them with the pathophysiology. This is the first report on the cervical vestibular myogenic potentials in patients with PMD.

Background: In India, persons with hearing impairment who benefit from hearing aids can acquire a driving license. The law mandates helmet use while driving two-wheelers. Using a hearing aid with a helmet on may be a challenge, but it is paramount to understand the difficulties the hearing aid users face., Methods: A cross-sectional research design was used to study the experience of hearing aid users who ride two-wheelers. A checklist was developed and administered to 15 individuals with hearing impairment who ride two-wheelers. The checklist had 11 questions under three domains; 'Helmet related,' 'Comfort related', and 'Driving/Listening related. The study was carried out in and around Bangalore. The data was collected through the interview method., Results: The subjects reported difficulties in the comfort-related and driving/listening-related domains. Many participants felt the need to modify the helmet design to suit hearing aid users. They also felt a need for a special program for driving mode in the hearing aids., Conclusions: Hearing-impaired two-wheeler riders face problems in localization, fear of hearing aid falling, and sweating using hearing aids while driving. It affects them on two fronts. One, it may pose a risk to others or the hearing impaired themselves on the road. Second, avoiding riding two-wheelers may limit their rights and mobility., Competing Interests: Conflict of interest: None declared

Background and objective Temporal processing abilities help perceive signal changes over time. Efficient temporal processing is necessary for pitch perception, voice identification, and speech perception. It plays a significant role in language development. Internal redundancy of the central auditory nervous plays a role in processing sensory information. There is a need to gain more insights into the maturation of neural hardwiring that supports binaural temporal processing at a young age. The purpose of the present study was to evaluate the difference between monaural and binaural temporal processing in children aged 7-11 years. Methods Temporal processing was assessed using gap detection and temporal modulation transfer function (TMTF) tests. The tests were administered in 40 typically developing children with normal clinical auditory sensitivity. The maximum likelihood procedure (MLP), a MATLAB toolbox, was employed to deliver the stimulus. A multivariate analysis followed by post hoc analysis was performed to analyze the data. Results There was a significant difference between binaural and monoaural stimulation in children aged 7-11 years. However, there was no statistically significant difference between the right and left ears for gap detection threshold (GDT) and TMTF across all test frequencies. Conclusion Based on our findings, binaural stimulation enhances temporal processing in young children., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Yuvaraj et al.)

Introduction:  Vestibular dysfunction is a debilitating disorder frequently encountered in neurological and otological settings. The vestibular system is a complex network between peripheral and central mechanisms. This innate complexity of the vestibular system necessitates objective test procedures for evidence-based diagnostic formulations and intervention. Objective tests aid in the evaluation of both peripheral and central vestibular pathologies. Establishing and availability of comprehensive normative data for these objective tests is crucial for clinicians and researchers alike., Materials and Methods: This is a prospective study involving 120 participants (both males and females) aged between 18 and 55 years. All participants were right-handed individuals and had no significant medical history. On pre-set protocols, cVEMP (cervical vestibular evoked myogenic potential), oVEMP (ocular vestibular evoked myogenic potential), vHIT (video head impulse test), and VNG (videonystagmography) were done., Results: While all participants (n=120) underwent cVEMP, oVEMP, vHIT, saccade, smooth pursuit, and optokinetic tests, only 109 participants consented to the caloric test. Each test's mean, standard deviation, median, quartile, and third quartiles have been recorded. A right-left comparison yielded no significant difference on cVEMP, oVEMP, caloric test, smooth pursuit, and optokinetic test. However, few vHIT and saccade parameters did reveal significant differences., Discussion: This study presents comprehensive normative data for cVEMP, oVEMP, vHIT, caloric test on VNG, and oculomotor tests (smooth pursuit, saccade, optokinetic) on VNG. The test results were in concordance with previously published data. The significant difference between the right and left sides in vHIT may be because of the monocular goggles used for the testing., Conclusion: This study brings out the normative data for various vestibular tests on individuals aged between 18-55 years. This information could aid both clinicians and researchers working in the field of vestibular science., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Narayana Swamy et al.)

Purpose: Susac syndrome (SS) is a rare autoimmune disorder that affects the brain and the retina and causes unilateral or bilateral sensorineural hearing loss. Although vestibular dysfunction is reported in SS, limited information is available underlying the vestibular pathophysiology., Method: The diagnosis of SS was established based on symptoms and diagnostic tools such as magnetic resonance imaging and fundus fluorescein angiography. The audiovestibular evaluation was done on the seventh day of admission (Session 1) into the emergency unit, whereas the second and third evaluations were done at 3-month (Session 2) and 8-month (Session 3) follow-ups after discharge, respectively. The audiovestibular test battery consisted of routine audiological tests, auditory brainstem response, and vestibular evoked myogenic potentials (VEMPs; both cervical and ocular)., Results: We found unilateral sensorineural hearing loss and absent cervical VEMPs(cVEMPs) when testing the left ear during Session 1. In the following sessions, the unilateral hearing loss did not recover; however, cVEMPs were present bilaterally. The ocular VEMPs showed an increase in amplitude during Sessions 2 and 3., Conclusions: SS can selectively disrupt auditory and vestibular structures. It may present with unique findings of audiovestibular tests. A detailed audiovestibular evaluation may be essential in patients with SS., Supplemental Material: https://doi.org/10.23641/asha.21513843.

Demand for fresh water increases day by day. Solar desalination is one of the promising technologies to meet this demand in an economical fashion which uses solar still. For the current study, single-basin single-slope conventional solar still and a modified single-basin single-slope solar still with inbuilt condenser and agitator were designed and fabricated. Both the stills were tested under the same ambient conditions to compare the performance. Through experimental results, it was found that modified still with inbuilt condenser and agitator had 98.69% more productivity than conventional solar still. Modified still productivity was recorded as 4.856 L/m 2 /day and that of conventional still was 2.44 L/m 2 /day. The agitation effect caused by the agitator in the modified still led to an increase in the rate of evaporation. The increase in condensing area for the same evaporation area of the modified still improved the condensation rate. These two synergized effects resulted in an overall performance improvement of the modified still over the conventional still. An energy analysis revealed that modified still is 24.42% more efficient than its counterpart. The energy efficiency of modified and conventional stills was calculated as 4.82% and 2.04% respectively., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Purpose: The National Institute of Health Stroke Scale (NIHSS) is widely used in clinical practice to evaluate stroke-related neurological deficits. The aim of this study was to develop Kannada language version of the NIHSS (Ka-NIHSS) and determine its validity and reliability., Materials and Methods: In the first phase of the study, Ka-NIHSS was adapted based on cultural and linguistic peculiarities. In the next phase, 51 acute stroke patients were prospectively enrolled in the study. The concurrent validity of the Ka-NIHSS was evaluated by comparison with the Glasgow Coma Scale (GCS) and the modified Rankin Scale (mRS). The predictive validity was assessed by comparison with Barthel Index (BI) score and mRS at a 90-day telephonic follow-up. The reliability was evaluated using the kappa statistics for inter-rater agreement between two independent raters and intra-class correlation coefficient (ICC) analysis. The inter-rater agreement of videotaped assessment of items 9 and 10 between four independent raters was evaluated using kappa statistics., Results: Ka-NIHSS scores highly correlated with GCS ( P = -0.74 P < 0.001) and mRS ( P = 0.85, P < 0.001) at baseline. It moderately correlated with mRS ( P = 0.67, P < 0.001) and BI ( P = -0.64, P < 0.001) at 90 days follow up. Inter-rater reliability was high between the two examiners, with kappa values ranging from 0.66 to 0.95. The inter-rater agreements of the video assessment of items 9 and 10 for nine patients among four raters were 0.81 and 1 respectively., Conclusions: Ka-NIHSS is a valid and reliable tool for assessing neurological deficits in Kannada-speaking stroke patients., Competing Interests: There are no conflicts of interest., (Copyright: © 2006 - 2022 Annals of Indian Academy of Neurology.)

Background: Charcot-Marie-Tooth disease type 4F (CMT4F) is an autosomal recessive disorder with symptoms presenting in early adulthood. This clinical case series demonstrates atypical findings in cervical and ocular vestibular evoked myogenic potentials (VEMP) in siblings with CMT4F., Purpose: The aim of this study was to highlight the audiovestibular test findings in CMT4F., Research Design: Case series study sample: 4 siblings, 3 of whom diagnosed with CMT4F., Data Collection and Analysis: Audiological test battery and electrophysiological tests comprising auditory brainstem response (ABR) and VEMP (both cervical and ocular) were performed in our patient population., Results: Older siblings, in whom the hearing loss was present, manifested prolonged peak V latencies in ABR. Three out of four siblings with CMT4F showed prolongation of latencies on cervical and ocular VEMP., Conclusions: In many neurodegenerative conditions, prolongation of ABR peak latencies has often been reported in the literature. There have also been a few reports of prolonged VEMP peak latencies. This article reports prolongation of only VEMP peak latencies (in both cervical and ocular recordings). The youngest sibling had prolongation of VEMP latencies, with ABR peak latencies being normal. The assumption we put forth that CMT4F may affect the vestibular pathway first requires to be tested on a larger sample and by longitudinally studying the individuals with disease condition., Competing Interests: None declared., (American Academy of Audiology. This article is published by Thieme.)

Context: Temporal lobe epilepsy can affect central auditory processing (CAP) skills. Auditory temporal ordering (ATO) is a CAP skill that can be evaluated using duration pattern test (DPT)., Aim: The aim is to evaluate ATO in patients with medial temporal lobe epilepsy (MTLE) with hippocampal sclerosis (MTLE + HS) and without hippocampal sclerosis (MTLE-HS) and in their subgroups., Settings and Design: It was a prospective cross-sectional behavioral observational study conducted in a tertiary neuropsychiatric hospital., Subjects and Methods: The subjects were patients with refractory MTLE (N = 100), comprising 50 "MTLE + HS" patients and 50 "MTLE-HS". Age-range matched normal healthy subjects (n = 50) formed the control group. Both groups were administered duration pattern test (DPT)., Statistical Analysis Used: Analysis of variance (ANOVA) with post hoc analysis, Dunnett's two-sided and Bonferroni, paired sample t-test, Pearson's correlation, and independent t-test., Results: The clinical groups performed significantly poorer than the control group, and however, did not differ significantly between them. The age at onset and the duration of the seizures did not have significant relation with the test measures., Conclusions: Patients with "MTLE + HS" as well as those with "MTLE-HS" and their respective subgroups revealed abnormal ATO indicating CAP dysfunction., Competing Interests: None

Objective: Pilocytic astrocytoma commonly occurs in children and depending on the extension of the lesion may cause varied audiovestibular dysfunctions. However, audiovestibular findings are scarcely reported in the literature. Design: Audiovestibular testing was performed on a single subject on two occasions pre-surgically. Study sample: A 6 year old girl with pilocytic astrocytoma. Results: All audiological tests revealed normal findings except for the cervical vestibular evoked myogenic potential testing (cVEMP). The amplitude of cVEMP was higher in the lesion side indicating a hypersensitive vestibulocollic reflex pathway functioning. Conclusions: This case study reported a unique finding of hypersensitive cVEMP findings in the lesion side in a patient with pilocytic astrocytoma. The pathophysiological basis for this hypersensitivity is attributed to anatomical connections between the cerebellum and the vestibular nuclei through the inferior cerebellar peduncle.

Fibrous proteins found in the natural extracellular matrix (ECM) function as host substrates for migration and growth of endogenous cells during wound healing and tissue repair processes. Although various fibrous scaffolds have been developed to recapitulate the microstructures of the native ECM, facile synthesis of hydrogel microfibers that are mechanically robust and biologically active have been elusive. Described herein is the use of interfacial bioorthogonal polymerization to create hydrogel-based microfibrous scaffolds via tetrazine ligation. Combination of a trifunctional strained trans-cyclooctene monomer and a difunctional s-tetrazine monomer at the oil-water interface led to the formation of microfibers that were stable under cell culture conditions. The bioorthogonal nature of the synthesis allows for direct incorporation of tetrazine-conjugated peptides or proteins with site-selectively, genetically encoded tetrazines. The microfibers provide physical guidance and biochemical signals to promote the attachment, division and migration of fibroblasts. Mechanistic investigations revealed that fiber-guided cell migration was both F-actin and microtubule-dependent, confirming contact guidance by the microfibers. Prolonged culture of fibroblasts in the presence of an isolated microfiber resulted in the formation of a multilayered cell sheet wrapping around the fiber core. A fibrous mesh provided a 3D template to promote cell infiltration and tissue-like growth. Overall, the bioorthogonal approach led to the straightforward synthesis of crosslinked hydrogel microfibers that can potentially be used as instructive materials for tissue repair and regeneration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chemical modification of engineered microenvironments surrounding living cells represents a means for directing cellular behaviors through cell-matrix interactions. Presented here is a temporally controlled method for modulating the properties of biomimetic, synthetic extracellular matrices (ECM) during live cell culture employing the rapid, bioorthogonal tetrazine ligation with trans-cyclooctene (TCO) dienophiles. This approach is diffusion-controlled, cytocompatible, and does not rely on light, catalysts, or other external triggers. Human bone-marrow-derived mesenchymal stem cells (hMSCs) were initially entrapped in a hydrogel prepared using hyaluronic acid carrying sulfhydryl groups (HA-SH) and a hydrophilic polymer bearing both acrylate and tetrazine groups (POM-AT). Inclusion of a matrix metalloprotease (MMP)-degradable peptidic cross-linker enabled hMSC-mediated remodeling of the synthetic environment. The resultant network displayed dangling tetrazine groups for subsequent conjugation with TCO derivatives. Two days later, the stiffness of the matrix was increased by adding chemically modified HA carrying multiple copies of TCO (HA-TCO) to the hMSC growth media surrounding the cell-laden gel construct. In response, cells developed small processes radially around the cell body without a significant alteration of the overall shape. By contrast, modification of the 3D matrix with a TCO-tagged cell-adhesive motif caused the resident cells to undergo significant actin polymerization, changing from a rounded shape to spindle morphology with long cellular processes. After additional 7 days of culture in the growth media, quantitative analysis showed that, at the mRNA level, RGD tagging upregulated cellular expression of MMP1, but downregulated the expression of collagen I/III and tenascin C. RGD tagging, however, was not sufficient to induce the classic osteoblastic, chondrogenic, adipogenic, or fibroblastic/myofibroblastic differentiation. The modular approach allows facile manipulation of synthetic ECM to modulate cell behavior, thus potentially applicable to the engineering of functional tissues or tissue models.

Background: Schwannoma of the hypoglossal nerve is rare. This case report documents an atypical abnormality of the cervical vestibular evoked myogenic potential (cVEMP) in a patient with schwannoma of the hypoglossal nerve. The observed abnormality was attributed to the proximity of the hypoglossal nerve to the spinal accessory nerve in the medullary cistern and base of the skull., Purpose: To report cVEMP abnormality in a patient with hypoglossal nerve schwannoma and provide an anatomical correlation for this abnormality., Research Design: Case report., Study Sample: A 44-yr-old woman., Data Collection: Pure-tone and speech audiometry, tympanometry, acoustic stapedial reflex, auditory brainstem response, and cVEMP testing were performed., Results: The audiological test results were normal except for the absence of cVEMP on the lesion side (right)., Conclusions: A cVEMP abnormality indicating a compromised spinal accessory nerve was observed in a patient with hypoglossal nerve schwannoma. This case report highlights the importance of recording cVEMP in relevant neurological conditions and provides clinical proof for the involvement of the spinal accessory nerve in the vestibulocollic reflex pathway., (American Academy of Audiology)

Toward the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of tert-butyl methacrylate (tBMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of tert-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ∼95 μm by day 28. Cells in these structures were viable, formed cell-cell contacts through E-cadherin (E-CAD), and displayed cortical organization of F-actin. Compared with the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids, and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD are a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs.

Epithelial-to-mesenchymal transition (EMT) is a well-studied biological process that takes place during embryogenesis, carcinogenesis, and tissue fibrosis. During EMT, the polarized epithelial cells with a cuboidal architecture adopt an elongated fibroblast-like morphology. This process is accompanied by the expression of many EMT-specific molecular markers. Although the molecular mechanism leading to EMT has been well-established, the effects of matrix topography and microstructure have not been clearly elucidated. Synthetic scaffolds mimicking the meshlike structure of the basement membrane with an average fiber diameter of 0.5 and 5 μm were produced via the electrospinning of poly(ε-caprolactone) (PCL) and were used to test the significance of fiber diameter on EMT. Cell-adhesive peptide motifs were conjugated to the fiber surface to facilitate cell attachment. Madin-Darby Canine Kidney (MDCK) cells grown on these substrates showed distinct phenotypes. On 0.5 μm substrates, cells grew as compact colonies with an epithelial phenotype. On 5 μm scaffolds, cells were more individually dispersed and appeared more fibroblastic. Upon the addition of hepatocyte growth factor (HGF), an EMT inducer, cells grown on the 0.5 μm scaffold underwent pronounced scattering, as evidenced by the alteration of cell morphology, localization of focal adhesion complex, weakening of cell-cell adhesion, and up-regulation of mesenchymal markers. In contrast, HGF did not induce a pronounced scattering of MDCK cells cultured on the 5.0 μm scaffold. Collectively, our results show that the alteration of the fiber diameter of proteins found in the basement membrane may create enough disturbances in epithelial organization and scattering that might have important implications in disease progression.

In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells., (© 2015. Published by The Company of Biologists Ltd.)

Nanomedicine has advanced to clinical trials for adult cancer therapy. However, the field is still in its infancy for treatment of childhood malignancies such as acute lymphoblastic leukemia (ALL). Nanotherapy offers multiple advantages over conventional therapy. It facilitates targeted delivery and enables controlled release of drugs to reduce treatment-related side effects. Here, we demonstrate that doxorubicin (DOX) encapsulated in polymeric nanoparticles (NPs) modified with targeting ligands against CD19 (CD19-DOX-NPs) can be delivered in a CD19-specific manner to leukemic cells. The CD19-DOX-NPs were internalized via receptor-mediated endocytosis and imparted cytotoxicity in a CD19-dependent manner in CD19-positive ALL cells. Leukemic mice treated with CD19-DOX-NPs survived significantly longer and manifested a higher degree of agility, indicating reduced apparent systemic toxicity during treatment compared to mice treated with free DOX. We suggest that targeted delivery of drugs used in childhood cancer treatment should improve therapeutic efficacy and reduce treatment-related side effects in children.

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.

Gramicidin A (GA) is a channel-forming ionophore that renders biological membranes permeable to specific cations which disrupts cellular ionic homeostasis. It is a well-known antibiotic, however it's potential as a therapeutic agent for cancer has not been widely evaluated. In two recently published studies, we showed that GA treatment is toxic to cell lines and tumor xenografts derived from renal cell carcinoma (RCC), a devastating disease that is highly resistant to conventional therapy. GA was found to possess the qualities of both a cytotoxic drug and a targeted angiogenesis inhibitor, and this combination significantly compromised RCC growth in vitro and in vivo. In this review, we summarize our recent research on GA, discuss the possible mechanisms whereby it exerts its anti-tumor effects, and share our perspectives on the future opportunities and challenges to the use of GA as a new anticancer agent., Competing Interests: Conflicts of Interest: Authors A.K. Rajasekaran and J.M. David have filed a patent.

The Wilms' tumor transcription factor (WT1) was originally classified as a tumor suppressor, but it is now known to also be associated with cancer progression and poor prognosis in several malignancies. WT1 plays an essential role in orchestrating a developmental process known as mesenchymal-to-epithelial transition (MET) during kidney development, but also induces the reverse process, epithelial-to-mesenchymal transition (EMT) during heart development. WT1 is not expressed in the adult kidney, but shows elevated expression in clear cell renal cell carcinoma (ccRCC). However, the role of WT1 in this disease has not been characterized. In this study, we demonstrate that WT1 is upregulated in ccRCC cells that are deficient in the expression of the von Hippel-Lindau tumor suppressor protein (VHL). We found that WT1 transcriptionally activated Snail, a master transcriptional repressor that is known to induce EMT. Although Snail represses E-cadherin and induces mesenchymal characteristics, we found partial maintenance of E-cadherin and associated epithelial characteristics in kidney cells and ccRCC cells that express WT1, since WT1 upregulates E-cadherin expression and competes with Snail repression. These findings support a novel paradigm in which WT1 induces an epithelial-mesenchymal hybrid transition (EMHT), characterized by Snail up-regulation with E-cadherin maintenance, a tumor cell differentiation state in which cancer cells keep both EMT and MET characteristics which may promote tumor cell plasticity and tumor progression.

Ionophores are hydrophobic organic molecules that disrupt cellular transmembrane potential by permeabilizing membranes to specific ions. Gramicidin A is a channel-forming ionophore that forms a hydrophilic membrane pore that permits the rapid passage of monovalent cations. Previously, we found that gramicidin A induces cellular energy stress and cell death in renal cell carcinoma (RCC) cell lines. RCC is a therapy-resistant cancer that is characterized by constitutive activation of the transcription factor hypoxia-inducible factor (HIF). Here, we demonstrate that gramicidin A inhibits HIF in RCC cells. We found that gramicidin A destabilized HIF-1α and HIF-2α proteins in both normoxic and hypoxic conditions, which in turn diminished HIF transcriptional activity and the expression of various hypoxia-response genes. Mechanistic examination revealed that gramicidin A accelerates O(2)-dependent downregulation of HIF by upregulating the expression of the von Hippel-Lindau (VHL) tumor suppressor protein, which targets hydroxylated HIF for proteasomal degradation. Furthermore, gramicidin A reduced the growth of human RCC xenograft tumors without causing significant toxicity in mice. Gramicidin A-treated tumors also displayed physiologic and molecular features consistent with the inhibition of HIF-dependent angiogenesis. Taken together, these results demonstrate a new role for gramicidin A as a potent inhibitor of HIF that reduces tumor growth and angiogenesis in VHL-expressing RCC.

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na(+) and uptake of K(+) across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β 1 (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients' tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2'-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.

Although chemotherapy-based treatment of leukemia has tremendously improved survival rates in children, induction of treatment-related side effects is a major concern in clinical oncology. The development of nanotechnology-based drug delivery techniques to target clinically approved anticancer agents specifically to leukemic cells should diminish toxic side effects. This review aims to address the rational design of nanotherapeutics in treating hematologic malignancies with a focus on acute lymphoblastic leukemia (ALL)--the most prominent form of pediatric cancer.

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., (©2013 AACR.)

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.

Na,K-ATPase is a hetero-oligomer of an α- and a β-subunit. The α-subunit (Na,K-α) possesses the catalytic function, whereas the β-subunit (Na,K-β) 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-β(1) transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-β(1) and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-α(1). We now provide evidence that knockdown of Na,K-β(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-β(1) or heptad repeat motif mutant of Na,K-β(1), but not by the glycine zipper motif mutant that abrogates Na,K-β(1) cis homo-oligomerization. These studies suggest that Na,K-β(1) cis homo-oligomerization rather than hetero-oligomerization with Na,K-α(1) is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.

Strong cell-cell interactions represent a major barrier against cancer cell mobility, and loss of intercellular adhesion by E-cadherin is a fundamental change that occurs during the progression of cancer to invasive disease. However, some aggressive carcinomas retain characteristics of differentiated epithelial cells, including E-cadherin expression. Emerging evidence indicates that proteolysis of E-cadherin generates fragments that promote tumor growth, survival, and motility, suggesting that E-cadherin cleavage converts this tumor suppressor into an oncogenic factor. In this review we discuss the emerging roles of cleaved E-cadherin fragments as modulators of cancer progression, and explore the translational and clinical implications of this research.

Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.

Background: Tissue microarray (TMA) data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups., Methods: We apply weighted correlation network analysis (WGCNA) to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%), moderate (22%) and high (50%) mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-β1, and TGF β receptor II) that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis., Results: We find that the rule-based grouping variable (referred to as WGCNA*) is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data), it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance). We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach., Conclusions: We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes.

High levels of the soluble form of E-cadherin can be found in the serum of cancer patients and are associated with poor prognosis. Despite the possible predictive value of soluble E-cadherin, little is understood concerning its patho-physiological consequences in tumor progression. In this study, we show that soluble E-cadherin facilitates cell survival via functional interaction with cellular E-cadherin. Exposure of cells to a recombinant form of soluble E-cadherin, at a concentration found in cancer patient's serum, prevents apoptosis due to serum/growth factor withdrawal, and inhibits epithelial lumen formation, a process that requires apoptosis. Further, soluble E-cadherin-mediated cell survival involves activation of the epidermal growth factor receptor (EGFR) and EGFR-mediated activation of both phosphoinositide-3 kinase (PI3K)/AKT and ERK1/2 signaling pathways. These results are evidence of a complex functional interplay between EGFR and E-cadherin and also suggest that the presence of soluble E-cadherin in cancer patients' sera might have relevance to cell survival and tumor progression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair, and occurs during pathologic processes of tumor invasiveness, metastasis, and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. Although it is well documented that transforming growth factor (TGF)-beta plays a central role in the induction of EMT, the targets of TGF-beta signaling are poorly defined. We have shown earlier that Na,K-ATPase beta(1)-subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-beta(1)-mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-beta(1), the surface expression of the beta(1)-subunit of Na,K-ATPase is reduced, before well-characterized EMT markers, and is associated with the acquisition of a mesenchymal phenotype. RNAi-mediated knockdown confirmed the specific involvement of the Na,K-ATPase beta(1)-subunit in the loss of the epithelial phenotype and exogenous overexpression of the Na,K-ATPase beta(1)-subunit attenuated TGF-beta(1)-mediated EMT. We further show that both Na,K-ATPase alpha- and beta-subunit levels are highly reduced in renal fibrotic tissues. These findings reveal for the first time that Na,K-ATPase is a target of TGF-beta(1)-mediated EMT and is associated with the progression of EMT in cancer and fibrosis.

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.

Tight junctions are unique organelles in polarized epithelial and endothelial cells that regulate the flow of solutes and ions across the epithelial barrier. The structure and functions of tight junctions are regulated by a wide variety of signaling and molecular mechanisms. Several recent studies in mammals, drosophila, and zebrafish reported a new role for Na,K-ATPase, a well-studied ion transporter, in the modulation of tight junction development, permeability, and polarity. In this review, we have attempted to compile these new reports and suggest a model for a conserved role of Na,K-ATPase in the regulation of tight junction structure and functions.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in advanced and metastatic prostate cancers. The pathologic consequence of elevated PSMA expression in not known. Here, we report that PSMA is localized to a membrane compartment in the vicinity of mitotic spindle poles and associates with the anaphase-promoting complex (APC). PSMA-expressing cells prematurely degrade cyclin B and exit mitosis due to increased APC activity and incomplete inactivation of APC by the spindle assembly checkpoint. Further, expression of PSMA in a karyotypically stable cell line induces aneuploidy. Thus, these findings provide the first evidence that PSMA has a causal role in the induction of aneuploidy and might play an etiologic role in the progression of prostate cancer.

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

The Na,K-ATPase, consisting of two essential subunits (alpha, beta), plays a critical role in the regulation of ion homeostasis in mammalian cells. Recent studies indicate that reduced expression of the beta1 isoform (NaK-beta1) is commonly observed in carcinoma and is associated with events involved in cancer progression. In this study, we present evidence that repletion of NaK-beta1 in Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK), a highly tumorigenic cell line, inhibits anchorage independent growth and suppresses tumor formation in immunocompromised mice. Additionally, using an in vitro cell-cell aggregation assay, we showed that cell aggregates of NaK-beta1 subunit expressing MSV-MDCK cells have reduced extracellular regulated kinase (ERK) 1/2 activity compared with parental MSV-MDCK cells. Finally, using immunohistochemistry and fully quantitative image analysis approaches, we showed that the levels of phosphorylated ERK 1/2 are inversely correlated to the NaK-beta1 levels in the tumors. These findings reveal for the first time that NaK-beta1 has a potential tumor-suppressor function in epithelial cells.

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.

Purpose: Na,K-adenosine triphosphatase, which is composed of a catalytic alpha-subunit and a regulatory beta-subunit, generates an electrochemical gradient across the plasma membrane. Previous studies demonstrated altered Na,K-adenosine triphosphatase subunit expression in renal clear cell carcinoma and an association of subunit levels with the prediction of recurrent bladder cancer. We determined the clinical association of protein expression patterns of the Na,K-adenosine triphosphatase alpha1 and beta1-subunits in renal clear cell carcinoma using tissue microarrays with linked clinicopathological data., Materials and Methods: The UCLA kidney cancer tissue microarray was used to investigate the protein expression of Na,K-adenosine triphosphatase alpha1 and beta1-subunits by immunohistochemistry in 342 patients with renal clear cell carcinoma who were treated with radical nephrectomy. Of these patients clinical outcomes studies were performed in 317. The resultant expression reactivity was correlated with clinicopathological variables., Results: We found that the alpha1-subunit was a significant and independent predictor of disease specific death from renal clear cell carcinoma on multivariate Cox proportional hazards analysis that included established prognostic factors Eastern Cooperative Oncology Group performance status, pT status, metastasis status and tumor grade. Significance was found when examining all patients with clear cell renal cell carcinoma as well as patient substrata with low or high grade tumors and localized or metastatic disease, suggesting that the Na,K-adenosine triphosphatase alpha1-subunit could be used as a new prognosticator for disease specific death from renal clear cell carcinoma., Conclusions: These results suggest that Na,K-adenosine triphosphatase alpha1-subunit expression patterns may be a useful clinical prognosticator for renal clear cell carcinoma. The Na,K-adenosine triphosphatase beta1-subunit was not found to be a useful prognosticator in this setting.

Prostate-specific membrane antigen (PSMA) is an integral membrane glycoprotein expressed in prostatic epithelia and is being evaluated as a therapeutic target in prostate cancer. It undergoes constitutive receptor-mediated endocytosis via clathrin-coated pits, which is enhanced in the presence of monoclonal antibodies directed against it. We describe distinct interactions of PSMA with clathrin and the clathrin adaptor protein-2 (AP-2) complex, two components of clathrin-coated pits. The intracellular N-terminal domain of PSMA interacts with the N-terminal globular domain of clathrin heavy chain. Deletion analysis revealed an important determinant of this interaction residing within the proximal portion of the clathrin heavy chain N-terminal domain (amino acids 1-85) distinct from the clathrin binding sites of other known clathrin-binding proteins. Furthermore, PSMA interacts with the ear domain of alpha-adaptin (an AP-2 subunit), and a glutamic acid residue at position 7 in the cytoplasmic tail of PSMA is essential for this interaction. These data indicate that PSMA exhibits a high affinity, specific association with the clathrin-based endocytic machinery by distinct interactions with both clathrin and AP-2. Thus, although PSMA is a new member of the dual AP and clathrin binding proteins, its alpha-adaptin and clathrin heavy chain binding determinants are distinct from those of other members.

Prostate cancer is the most common non-cutaneous cancer of men in the United States and represents their second-leading cause of cancer-related death. Metastatic disease is largely resistant to conventional chemotherapies, and targeted therapies are urgently needed. Prostate-specific membrane antigen (PSMA) is a prototypical cell-surface marker of prostate cancer. PSMA is an integral, non-shed, type 2 membrane protein with abundant and nearly universal expression in prostate carcinoma, but has limited extra-prostatic expression. In addition, PSMA is expressed in the neovasculature of other solid tumors. These findings have spurred development of PSMA-targeted therapies for cancer, and first-generation products have entered clinical testing. Vaccine approaches have included recombinant protein, nucleic acid and cell-based strategies, and anti-PSMA immune responses have been demonstrated in the absence of significant toxicity. Therapy with drug-conjugated and radiolabeled antibodies has yielded objective clinical responses as measured by reductions in serum prostate-specific antigen and/or imageable tumor volume. However, responses were observed in a minor fraction of patients and at doses near the maximum tolerated dose. Overall, these initial studies have provided measured proof of concept for PSMA-based therapies, and second-generation antibody and vaccine products may hold the key to exploit PSMA for molecularly targeted therapy of prostate and other cancers.

Prostate specific membrane antigen (PSMA) is a transmembrane glycoprotein expressed almost exclusively in prostatic epithelial cells. Expression of PSMA is elevated in prostate cancer, with levels closely correlated with disease grade. Although the highest levels of PSMA expression are associated with high-grade, hormone-refractory and metastatic prostate cancer, the significance of elevated PSMA expression in advanced prostate cancer has yet to be fully elucidated. We provide evidence that prostatic carcinoma cells expressing PSMA exhibit reduced motility and increased attachment when grown on a bone marrow matrix substrate. This phenomenon occurs via activation of focal adhesion kinase and provides the first evidence of a link between PSMA expression and prostate cancer metastasis to the bone.

Na,K-ATPase is a hetero-oligomer of alpha and beta-subunits. The Na,K-ATPase beta-subunit (Na,K-beta) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-beta transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-beta with Na,K-alpha, and the glycine zipper face is involved in the homo-oligomerization of Na,K-beta. Point mutations in the heptad repeat motif reduced Na,K-beta binding to Na,K-alpha, and Na,K-ATPase activity. Na,K-beta TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-beta links ion transport and cell-cell adhesion.

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.

We have shown that repletion of Na,K-ATPase Beta1-subunit (Na,K-Beta) in Moloney Sarcoma virus transformed MDCK (MSV-Na,K-Beta) cells induced lamellipodia and suppressed motility in a PI3-Kinase dependent manner. In this study, we provide evidence that decreased cell motility is due to increased attachment of Na,K-Beta expressing cells to the substratum. Treatment of MSV-Beta-GFP cells with bisindolylmalemide, a general Protein Kinase C (PKC) inhibitor, abolished PI3-Kinase activation and its down stream effects of Rac1 activation, binding of Na,K-Beta to annexin II, and suppression of cell motility and attachment. Thus, these studies unraveled that a PKC is involved upstream of PI3-Kinase in the suppression of Na,K-Beta mediated cell motility in carcinoma cells.

Prostate-specific membrane antigen (PSMA) is a protein up-regulated in the vast majority of prostate cancers. Antibodies to PSMA have proved highly specific for prostate cancer cells, and the therapeutic potential of such antibodies is currently being assessed in clinical trials. We have previously shown that PSMA at the cell surface of polarized epithelial cells is predominantly expressed at the apical plasma membrane and that microtubule depolymerization abolishes apical PSMA targeting. In the current report, we implicate a functional role for a target membrane soluble N-ethylmaleimide-sensitive factor adaptor protein receptor, syntaxin 3, in the microtubule-dependent apical targeting of PSMA. PSMA and syntaxin 3 are similarly localized to the apical plasma membrane of the prostatic epithelium and Madin-Darby canine kidney cells. Introduction of a point mutation into syntaxin 3 abolishes its polarized distribution and causes PSMA to be targeted in a nonpolarized fashion. Additionally, treatment of polarized Madin-Darby canine kidney cells with vinblastine, a microtubule depolymerizing chemotherapeutic agent, causes both syntaxin 3 and PSMA to redistribute in a nonpolarized fashion. However, following treatment with the microtubule stabilizing chemotherapeutic agent Taxotere, both syntaxin 3 and PSMA continue to localize in a polarized manner at the apical plasma membrane. Thus, microtubule depolymerizing and stabilizing chemotherapeutic drugs might exact similar cytotoxic effects but have disparate effects on protein targeting. This phenomenon might have important clinical implication, especially related to antibody-mediated immunotherapy, and could potentially be exploited for therapeutic benefit.