Shigekuni Hosogi

V-ATPase as an effective therapeutic target for sarcomas

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9-6.0pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highests levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment.

Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell

The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl−) in regulation of lysosomal acidification [intra‐lysosomal pH (pHlys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl− condition elevated pHlys and reduced the intra‐lysosomal Cl− concentration ([Cl−]lys) via reduction of cytosolic Cl− concentration ([Cl−]c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G0/G1 arrest without induction of apoptosis. We also studied effects of direct modification of H+ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V‐type H+‐ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na+/H+ exchanger (NHE)] elevated pHlys and decreased [Cl−]lys associated with inhibition of cell proliferation via induction of G0/G1 arrest similar to the culture under a low Cl− condition. However, unlike low Cl− condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl−]c compared with low Cl− condition. These observations suggest that the lowered [Cl−]c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra‐lysosomal acidification. This is the first study showing that cytosolic Cl− is a key factor of lysosome acidification and autophagy.

Improvement of insulin resistance, blood pressure and interstitial pH in early developmental stage of insulin resistance in OLETF rats by intake of propolis extracts.

Propolis, a resinous mixture collected from plants by the Apis mellifera bee, contains high level nutrient factors including vitamins, polyphenols, and amino acids that would be expected to improve insulin sensitivity. Insulin resistance would secondarily cause elevation of blood pressure and increase the risk of cardiovascular diseases. The purpose of this study is to investigate the effect of propolis extracts on blood glucose levels and blood pressures in an early developmental stage of insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (10 weeks old) were divided into three different groups: normal diet, 0.1% propolis diet, and 0.5% propolis diet. After 8 weeks, blood glucose levels, blood pressures, plasma metabolic factors and hormones, and interstitial fluid pH were measured. Casual blood glucose levels were decreased associated with a reduction of plasma insulin levels in both propolis diet groups compared with normal diet group. Propolis decreased systolic blood pressure with no significant changes in plasma aldosterone levels. We also found that interstitial fluid pH in ascites, liver, and skeletal muscle was higher in rats fed propolis diet than rats fed normal diet. These data suggests that dietary propolis improves insulin sensitivity and blood pressures in the early stage of the process in development of insulin resistance, which may be mediated by suppression of metabolic acidosis.

Impairment of Lysosomal Activity as a Therapeutic Modality Targeting Cancer Stem Cells of Embryonal Rhabdomyosarcoma Cell Line RD

Rhabdomyosarcoma is the most frequent soft tissue sarcoma in children and adolescents, with a high rate of relapse that dramatically affects the clinical outcome. Multiagent chemotherapy, in combination with surgery and/or radiation therapy, is the treatment of choice. However, the relapse rate is disappointingly high and identification of new therapeutic tools is urgently needed. Under this respect, the selective block of key features of cancer stem cells (CSC) appears particularly promising. In this study, we isolated rhabdomyosarcoma CSC with stem-like features (high expression of NANOG and OCT3/4, self-renewal ability, multipotency). Rhabdomyosarcoma CSC showed higher invasive ability and a reduced cytotoxicity to doxorubicin in comparison to native cells, through a mechanism unrelated to the classical multidrug resistance process. This was dependent on a high level of lysosome acidity mediated by a high expression of vacuolar ATPase (V-ATPase). Since it was not associated with other paediatric cancers, like Ewing’s sarcoma and neuroblastoma, V-ATPase higher expression in CSC was rhabdomyosarcoma specific. Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole, or by specific siRNA silencing, significantly enhanced doxorubicin cytoxicity. Unexpectedly, lysosomal targeting also blocked cell growth and reduced the invasive potential of rhabdomyosarcoma CSC, even at very low doses of omeprazole (10 and 50 µM, respectively). Based on these observations, we propose lysosome acidity as a valuable target to enhance chemosensitivity of rhabdomyosarcoma CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease.

Regulation of Epithelial Sodium Transport via Epithelial Na+ Channel

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane.

An Inhibitor of Na + /H + Exchanger (NHE), Ethyl-Isopropyl Amiloride (EIPA), Diminishes Proliferation of MKN28 Human Gastric Cancer Cells by Decreasing the Cytosolic Cl - Concentration via DIDS-Sensitive Pathways

Background/Aims: Tumor cells produce a large amount of acidic metabolites due to their high metabolic condition. However, cytosolic pH (pH_c) of tumor cells is identical to or even slightly higher than that of normal cells. To maintain pH_c at a normal or higher level, tumor cells would have to have higher expression and/or activity of H⁺ transporting systems than normal cells. The purpose of the present study was to identify effects of ethyl-isopropyl amiloride (EIPA, an inhibitor of Na⁺/H⁺ exchanger (NHE)) on proliferation of human gastric cancer MKN28 cells. Methods: Effects of EIPA on proliferation, pH_c, [Cl^-]_c and expression of proteins regulating cell cycle and MAPKs were studied in MKN28 expressing NHE exposed to EIPA for 48 h. Results: EIPA suppressed proliferation of MKN28 cells by causing G₀/G₁ arrest without any significant effects on pH_c, but associated with reduction of [Cl^-]_c. Although EIPA alone had no effects on pH_c, EIPA co-applied with DIDS (an inhibitor of Cl^-/HCO₃^- exchangers; i.e., anion exchanger (AE) and Na+-driven Cl^-/HCO₃^- exchanger (NDCBE)) reduced pH_c, suggesting that DIDS-sensitive Cl^-/HCO₃^- transporters such as AE and/or NDCBE keep pH_c normal by stimulating HCO₃^- uptake coupled with Cl^- release under an NHE-inhibited condition. EIPA-induced lowered [Cl^-]_c up-regulated expression of p21associated with phosphorylation of MAPKs, suppressing proliferation associated with G₀/G₁ arrest. Conclusions: EIPA suppressed proliferation of MKN28 cells through up-regulation of p21 expression via reduction of [Cl^-]_c as a result from DIDS-sensitive Cl^-/HCO₃^- exchanger-mediated compensation for keeping pH_c normal under an NHE-inhibited condition. This is the first study revealing that an NHE inhibitor suppressed the proliferation of cancer cells by reducing [Cl^-]_c but not pH_c.

Photodynamic therapy with acridine orange in musculoskeletal sarcomas

Limb salvage involving wide resection and reconstruction is now well established for managing musculoskeletal sarcomas. However, involvement of major nerves and vessels with a large volume of muscle and skin may result in a useless limb, contributing to depression and a low quality of life. We have been studying alternative treatments for musculoskeletal sarcoma since 1990, and have recently established a regime using photodynamic surgery with cells labelled with acridine orange, photodynamic therapy with cells treated similarly and radiodynamic treatment using the effect of X-rays on such cells. These techniques have been used after marginal or intralesional resection of tumours since 1999 and have enabled maintenance of excellent limb function in patients with sarcomas.

A regulatory role of K+–Cl− cotransporter in the cell cycle progression of breast cancer MDA-MB-231 cells

K(+)-Cl(-) cotransporter (KCC) has been shown to be involved in cell proliferation as well as cell volume regulation. A regulatory role of KCC in cell cycle progression of breast cancer MDA-MB-231 cells was explored by using synchronized MDA-MB-231 cells and dihydro-indenyloxy-alkanoic acid (DIOA), a potent inhibitor of KCC. MDA-MB-231 cells cultured in the presence of DIOA exhibited an increase in cell volume, a decrease in intracellular Cl(-) concentration, and reduction in cell proliferation with the G0/G1 phase arrest, which was accompanied with down-regulation of cyclin D1 and cyclin E2, and up-regulation of p21. Among these molecules, the expression of cyclin E2, a molecule essential for the transition from G1 to S phase, was markedly suppressed by DIOA treatment. DIOA-mediated up- or down-regulation of these molecules occurred at the transcriptional level. These findings suggest that KCC plays an important role in the early phase of cell cycle progression by regulating the expression of cyclin D1, cyclin E2, and p21, the molecules essential for the cell cycle progression.

Quercetin is a useful medicinal compound showing various actions including control of blood pressure, neurite elongation and epithelial ion transport

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.

Low pH of interstitial fluid around hippocampus of the brain in diabetic OLETF rats.

BackgroundWe have reported that pH values of ascites and interstitial fluids around the liver in Otsuka Long-Evans Tokushima Fatty (OLETF) rats are significantly lower than normal pH, 7.40, of mammalian body fluids (Biochem Biophys Res Commun 2013, 432:650), and that this lowered pH of interstitial fluid causes the insulin resistance in diabetic patients by decreasing insulin-binding to its receptors (J Physiol Sci 2013, 63:S199). In the preset study, we tried to measure the interstitial fluid pH in diabetic OLETF rats, since the interstitial fluid pH plays key factors in the brain function from a viewpoint of the binding affinity of neurotransmitters to their receptors.FindingsWe found that the pH value of interstitial fluids around hippocampus, the most important area for memory, in diabetic OLETF rats was lower than that in normal rats by measuring pH with antimony pH electrodes.ConclusionsThe lowered pH of interstitial fluid around hippocampus of the brain in diabetic rats observed in the present study suggests that the function of hippocampus of the brain would be diminished due to low affinity of various types of neurotransmitters, playing key roles in the hippocampus function, to their receptors. Therefore, we indicate that maintenance of the interstitial fluid pH at the normal level would be one of the most important key factors for molecular and cellular therapies in various types of diseases including diabetes mellitus.