Ion transport regulation by P2Y receptors, protein kinase C and phosphatidylinositol 3-kinase within the semicircular canal duct epithelium

<p>Abstract</p> <p>Background</p> <p>The ionic composition of the luminal fluid in the vestibular labyrinth is maintained within tight limits by the many types of epithelial cells bounding the lumen. Regulatory mechanisms include systemic, paracrine and autocrine hormones along with their associated intracellular signal pathways. The epithelium lining the semicircular canal duct (SCCD) is a tissue that is known to absorb sodium and calcium and to secrete chloride.</p> <p>Findings</p> <p>Transport function was assessed by measurements of short circuit current (<it>I</it>_<it>sc</it>) and gene transcript expression was evaluated by microarray. Neither ATP nor UTP (100 microM) on the apical side of the epithelium had any effect on <it>I</it>_<it>sc</it>. By contrast, basolateral ATP transiently increased <it>I</it>_{<it>sc </it>}and transepithelial resistance dropped significantly after basolateral ATP and UTP. P2Y2 was the sole UTP-sensitive purinergic receptor expressed. <it>I</it>_{<it>sc </it>}was reduced by 42%, 50% and 63% after knockdown of α-ENaC, stimulation of PKC and inhibition of PI3-K, while the latter two increased the transepithelial resistance. PKCdelta, PKCgamma and PI3-K were found to be expressed.</p> <p>Conclusions</p> <p>These observations demonstrate that ion transport by the SCCD is regulated by P2Y2 purinergic receptors on the basolateral membrane that may respond to systemic or local agonists, such as ATP and/or UTP. The sodium absorption from endolymph mediated by ENaC in SCCD is regulated by signal pathways that include the kinases PKC and PI3-K. These three newly-identified regulatory components may prove to be valuable drug targets in the control of pathologic vestibular conditions involving dysfunction of transport homeostasis in the ear, such as Meniere's disease.</p

An evaluation of the combination effect of zoledronate and chemotherapeutic agents in canine osteosarcoma cells

IntroductionOsteosarcoma (OSA) is an aggressive form of bone cancer in both dogs and humans. The treatment options for metastatic (stage III) OSA are currently limited and the prognosis is poor. Zoledronate, a second generation amino-bisphosphonate, is commonly used for palliation of cancer induced bone pain. Zoledronate has also demonstrated anti-cancer properties and possibly enhances the cytotoxicity of doxorubicin in a canine histiocytosis cell line and human prostatic cancer cell line. The goal of this study was to evaluate the combination effect of zoledronate and various chemotherapeutic drugs in canine OSA cells.MethodsCanine OSA cell line (D17), cells from two canine primary OSAs, and MDCK, a canine kidney cell line, were used to evaluate the therapeutic potential of these drugs. Carboplatin, doxorubicin, vinorelbine, toceranib, and isophosphoramide mustard (active metabolite of ifosfamide) were used as chemotherapeutic agents. First, cells were treated with either zoledronate or chemotherapy drug alone for 72 hours. Cell viability was assessed using CellTiter Glo and IC5, IC10, IC20, and IC50 were calculated. Second, cells were treated with a combination of zoledronate and each chemotherapeutic agent at their IC5, IC10, IC20, and IC50 concentrations. After 72 hours, cell viability was assessed by CellTiter Glo.Results and discussionZoledronate, carboplatin, doxorubicin, vinorelbine, and isophosphoramide mustard showed concentration dependent decrease in cell viability. Toceranib showed decreased cell viability only at higher concentrations. When zoledronate was used in combination with chemotherapy drugs, while it showed potential synergistic effects with toceranib, potential antagonistic effects with vinorelbine and isophosphoramide mustard were observed. However, the results differed by cell line and thus, further evaluation is warranted to understand the exact mechanism of action

Early transcriptomic response of mouse adrenal gland and Y-1 cells to dexamethasone

Glucocorticoids have short- and long-term effects on adrenal gland function and development. RNA sequencing (RNA-seq) was performed to identify early transcriptomic responses to the synthetic glucocorticoid, dexamethasone (Dex), in vitro and in vivo. In total, 1711 genes were differentially expressed in the adrenal glands of the 1-h Dex-treated mice. Among them, only 113 were also considered differentially expressed genes (DEGs) in murine adrenocortical Y-1 cells treated with Dex for 1 h. Gene ontology analysis showed that the upregulated DEGs in the adrenal gland of the 1-h Dex-treated mice were highly associated with the development of neuronal cells, suggesting the adrenal medulla had a rapid response to Dex. Interestingly, only 4.3% of Dex-responsive genes in the Y-1 cell line under Dex treatment for 1 h were differentially expressed under Dex treatment for 24 h. The heatmaps revealed that most early responsive DEGs in Y-1 cells during 1 h of treatment exhibited a transient response. The expression of these genes under treatment for 24 h returned to basal levels similar to that during control treatment. In summary, this research compared the rapid transcriptomic effects of Dex stimulation in vivo and in vitro. Notably, adrenocortical Y-1 cells had a transient early response to Dex treatment. Furthermore, the DEGs had a minimal overlap in the 1-h Dex-treated group in vivo and in vitro

Pregnane X Receptor and Cancer: Context-Specificity is Key

Pregnane X receptor (PXR) is an adopted orphan nuclear receptor that is activated by a wide-range of endobiotics and xenobiotics, including chemotherapy drugs. PXR plays a major role in the metabolism and clearance of xenobiotics and endobiotics in liver and intestine via induction of drug-metabolizing enzymes and drug-transporting proteins. However, PXR is expressed in several cancer tissues and the accumulating evidence strongly points to the differential role of PXR in cancer growth and progression as well as in chemotherapy outcome. In cancer cells, besides regulating the gene expression of enzymes and proteins involved in drug metabolism and transport, PXR also regulates other genes involved in proliferation, metastasis, apoptosis, anti-apoptosis, inflammation, and oxidative stress. In this review, we focus on the differential role of PXR in a variety of cancers, including prostate, breast, ovarian, endometrial, and colon. We also discuss the future directions to further understand the differential role of PXR in cancer, and conclude with the need to identify novel selective PXR modulators to target PXR in PXR-expressing cancers

cAMP-stimulated Clˉ secretion is increased by glucocorticoids and inhibited by bumetanide in semicircular canal duct epithelium

Background: The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Clˉ under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids. Results: Short circuit current (I[subscript sc]) from rat SCCD epithelia demonstrated stimulation by forskolin (EC[subscript 50]: 0.8 μM), 8-Br-cAMP (EC[subscript 50]: 180 μM), 8-pCPT-cAMP (100 μM), IBMX (250 μM), and RO-20-1724 (100 μM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated I[subscript sc]. Partial inhibition of stimulated I[subscript sc] individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated Isc was also partially inhibited by CFTR[subscript inh]-172 (5 & 30 μM), flufenamic acid (5 μM) and diphenylamine-2,2′-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/− mice showed a stimulation of I[subscript sc] from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR−/− mice had no responses. Nonetheless, CFTR−/− mice showed no difference from CFTR+/− mice in their ability to balance (rota-rod). Stimulated I[subscript sc] was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 μM), prednisolone (0.3, 1 & 3 μM), hydrocortisone (0.01, 0.1 & 1 μM), and corticosterone (0.1 & 1 μM) and mineralocorticoid aldosterone (1 μM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined. Conclusions: These results are consistent with a model of Clˉ secretion whereby Clˉ is taken up across the basolateral membrane by a Na+-K+-2Clˉ cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Clˉ channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids

Cellular interactions in tumor microenvironment during breast cancer progression: new frontiers and implications for novel therapeutics

The breast cancer tumor microenvironment (TME) is dynamic, with various immune and non-immune cells interacting to regulate tumor progression and anti-tumor immunity. It is now evident that the cells within the TME significantly contribute to breast cancer progression and resistance to various conventional and newly developed anti-tumor therapies. Both immune and non-immune cells in the TME play critical roles in tumor onset, uncontrolled proliferation, metastasis, immune evasion, and resistance to anti-tumor therapies. Consequently, molecular and cellular components of breast TME have emerged as promising therapeutic targets for developing novel treatments. The breast TME primarily comprises cancer cells, stromal cells, vasculature, and infiltrating immune cells. Currently, numerous clinical trials targeting specific TME components of breast cancer are underway. However, the complexity of the TME and its impact on the evasion of anti-tumor immunity necessitate further research to develop novel and improved breast cancer therapies. The multifaceted nature of breast TME cells arises from their phenotypic and functional plasticity, which endows them with both pro and anti-tumor roles during tumor progression. In this review, we discuss current understanding and recent advances in the pro and anti-tumoral functions of TME cells and their implications for developing safe and effective therapies to control breast cancer progress