7,8-Dihydroxy-3-(4'-hydroxyphenyl)coumarin inhibits invasion and migration of osteosarcoma cells.

Advances in pharmacy and medicine have led to the development of many anti-cancer and molecular targeted agents; however, there are few agents capable of suppressing metastasis. To prevent cancer recurrence, it is essential to develop novel agents for inhibiting metastasis. Coumarin-based compounds have multiple pharmacological activities including anti-cancer effects. We screened a compound library constructed at Kyoto Pharmaceutical University and showed that 7,8-dihydroxy-3-(4'-hydroxyphenyl)coumarin (DHC) inhibited invasion and migration of LM8 mouse osteosarcoma cells and 143B human osteosarcoma cells in a concentration-dependent manner. DHC decreased intracellular actin filament formation by downregulating Rho small GTP-binding proteins such as RHOA, RAC1, and CDC42, which regulate actin reorganization. However, DHC did not downregulate the corresponding mRNA transcripts, whereas it downregulated Rho small GTP-binding proteins in the presence of cycloheximide, suggesting that DHC enhances the degradation of these proteins. DHC treatment inhibited metastasis and prolonged overall survival in a spontaneous metastasis mouse model. These results indicate that DHC has the potential to suppress metastasis of osteosarcoma cells by downregulating Rho small GTP-binding proteins.

Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl-]i Decrease in the Lung Airway Epithelial Cells of Mice.

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl-]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3- cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15-20%), and a decrease in [Cl-]i, which was induced by the Cl- release via anoctamine 1 (ANO1), enhanced the CBA (by 10-15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl-]i in the c-LAECs. In conclusion, the pHi increase and the [Cl-]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

Expression level of BTN3A1 on the surface of CD14+ monocytes is a potential predictor of γδ T cell expansion efficiency.

Human γδ T cells expressing Vγ9Vδ2 T cell receptors exert a robust response to pathogens and malignant cells. These cells are activated by BTN3A1, which is expressed by pathogen-derived phosphoantigens (pAgs) or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Activated Vδ2 (+) T cells exert multiple effector functions; therefore, they are a promising candidate for immunotherapy. However, not all donors have γδ T cells with adequate proliferative activity. Here, we performed ex vivo culture of γδ T cells from 20 healthy donors and explored factors that may affect their expansion efficiency. Consistent with previous studies, we found that amplification of γδ T cells requires CD14+ monocytes to act as accessory cells. We also show here that surface expression of BTN3A1 by monocytes correlates positively with γδ T cell expansion. Moreover, treatment with BTN3A1-Fc increased the expansion efficiency of peripheral blood mononuclear cells (PBMCs) from donors harboring γδ T cells with poor expansion capacity. Taken together, the data suggest that the level of BTN3A1 expressed on the surface of monocytes is a useful biomarker for predicting the degree of expansion of γδ T cells.

The CDK4/6-UCHL5-BRD4 axis confers resistance to BET inhibitors in MLL-rearranged leukemia cells by suppressing BRD4 protein degradation.

Among acute leukemias, mixed-lineage leukemia-rearranged (MLL-r) leukemia is associated with poor prognosis. Bromodomain and extra-terminal inhibitors (BETi) are promising agents for treatment of hematological malignancies; however, the mechanisms underlying sensitivity to BETi and biomarkers to predict sensitivity are yet to be clarified. Here, we established OTX015-resistant MLL-r cell lines (OTX015-R cells) and used them to explore therapeutic targets in BETi-resistant MLL-r leukemia. OTX015-R cells exhibited resistance to various BETi, and levels of bromodomain-containing protein 4 (BRD4) and BRD4-regulated molecules, such as c-MYC and B-cell/CLL lymphoma-2 (BCL-2), were remarkably increased in OTX015-R cells relative to those in the parental cells; however, BRD4 mRNA transcript levels were not elevated. These results suggest that overexpression of BRD4 protein, through suppression of BRD4 degradation, may contribute to BETi-resistance. Notably, expression of ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) was increased in OTX015-R cells. Further, a UCHL5 inhibitor, b-AP15, and UCHL5 knockdown had antitumor effects by degrading BRD4. In addition, sensitivity to OTX015 was partially recovered in OTX015-R cells pretreated with b-AP15. Furthermore, cyclin-dependent kinase 4/6 (CDK4/6) inhibition decreased UCHL5 expression, suppressed OTX015-R cell proliferation, and induced apoptosis. These results indicate that the CDK4/6-UCHL5-BRD4 axis confers resistance to BETi by suppressing BRD4 degradation. We propose that this pathway is a potential novel therapeutic target in BETi-resistant MLL-r leukemia with BRD4 overexpression.

CN470 is a BET/CBP/p300 multi-bromodomain inhibitor and has an anti-tumor activity against MLL-rearranged acute lymphoblastic leukemia.

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEMLuc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

A novel dipeptide type inhibitor of the Wnt/ß-catenin pathway suppresses proliferation of acute myelogenous leukemia cells.

The Wnt/ß-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/ß-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated "31" that was found to be an inhibitor of the Wnt/ß-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/ß-catenin signaling pathway.

Human Vγ9Vδ2 T cells exert anti-tumor activity independently of PD-L1 expression in tumor cells.

Human γδ T cells expressing Vγ9Vδ2 T cell receptors play a crucial role in the innate immune system and have an attracted interest as effector cells in adoptive cellular immunotherapy. However, the efficacy of adoptive cellular immunotherapy for the treatment of tumors requires overcoming the immunosuppressive microenvironment. αß T cell inhibition in the tumor microenvironment is associated with programmed death-ligand 1 (PD-L1) expression level. Vγ9Vδ2 T cells (abbreviated as γδ T cells here) exert potent cytotoxic effects in various cancers; however, γδ T cell activity in relation to the level of PD-L1 expression in cancer cells remains unclear, and the association between the PD-1/PD-L1 axis and γδ T cell cytotoxicity needs to be investigated. In this study, PD-1 blockade did not increase the cytotoxicity of γδ T cells against PD-L1high cancer cells. However, the anti-PD-L1 monoclonal antibody (mAb) enhanced the cytotoxicity of γδ T cells against a subset of cancer cells, whereas PD-L1 knockdown did not increase the cytotoxicity of γδ T cells. We also found that the expression levels of PD-L1 were positively correlated with the changes of γδ T cells cytotoxicity induced by anti-PD-L1 mAb. These observations suggest that anti-PD-L1 mAb treatment adds ADCC activity to the cytotoxicity of γδ T cells itself against PD-L1high cancer cells. The present results suggest that ex vivo expanded γδ T cells have antitumor activity independently of PD-L1 expression and may be promising effector cells for γδ T cell immunotherapy.

Ciliary beating amplitude controlled by intracellular Cl- and a high rate of CO2 production in ciliated human nasal epithelial cells.

The ciliary transport is controlled by two parameters of the ciliary beating, frequency (CBF) and amplitude. In this study, we developed a novel method to measure both CBF and ciliary bend distance (CBD, an index of ciliary beating amplitude) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which are prepared from patients contracting allergic rhinitis and chronic sinusitis. An application of Cl--free NO3- solution or bumetanide (an inhibitor of Na+/K+/2Cl- cotransport), which decreases intracellular Cl- concentration ([Cl-]i), increased CBD, not CBF, at 37 °C; however, it increased both CBD and CBF at 25 °C. Conversely, addition of Cl- channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4-[[4-Oxo-2-thioxo-3-[3-trifluoromethyl]phenyl]-5-thiazolidinylidene]methyl] benzoic acid (CFTR(inh)-172)), which increase [Cl-]i, decreased both CBD and CBF, suggesting that CFTR plays a crucial role for maintaining [Cl-]i in these cells. We speculate that Cl- modulates activities of the molecular motors regulating both CBD and CBF in cHNECs. Moreover, application of the CO2/HCO3--free solution did not change intracellular pH (pHi), and addition of an inhibitor of carbonic anhydrase (acetazolamide) sustained pHi increase induced by the NH4+ pulse, which transiently increased pHi in the absence of acetazolamide. These results indicate that the cHNEC produces a large amount of CO2, which maintains a constant pHi even under the CO2/HCO3--free condition.

Carbocisteine stimulated an increase in ciliary bend angle via a decrease in [Cl-]i in mouse airway cilia.

Carbocisteine (CCis), a mucoactive agent, is widely used to improve respiratory diseases. This study demonstrated that CCis increases ciliary bend angle (CBA) by 30% and ciliary beat frequency (CBF) by 10% in mouse airway ciliary cells. These increases were induced by an elevation in intracellular pH (pHi; the pHi pathway) and a decrease in the intracellular Cl- concentration ([Cl-]i; the Cl- pathway) stimulated by CCis. The Cl- pathway, which is independent of CO2/HCO3-, increased CBA by 20%. This pathway activated Cl- release via activation of Cl- channels, leading to a decrease in [Cl-]i, and was inhibited by Cl- channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid and CFTR(inh)-172). Under the CO2/HCO3--free condition, the CBA increase stimulated by CCis was mimicked by the Cl--free NO3- solution. The pHi pathway, which depends on CO2/HCO3-, increased CBF and CBA by 10%. This pathway activated HCO3- entry via Na+/HCO3- cotransport (NBC), leading to a pHi elevation, and was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. The effects of CCis were not affected by a protein kinase A inhibitor (1 µM PKI-A) or Ca2+-free solution. Thus, CCis decreased [Cl-]i via activation of Cl- channels including CFTR, increasing CBA by 20%, and elevated pHi via NBC activation, increasing CBF and CBA by 10%.

Spontaneous oscillation of the ciliary beat frequency regulated by release of Ca2+ from intracellular stores in mouse nasal epithelia.

Ciliary beating frequency (CBF) was investigated in ciliated nasal epithelial cells (cMNECs) isolated from mice using video microscopy equipped with a high-speed camera. In cMNECs, a spontaneous CBF oscillation was observed. The CBF oscillation was abolished by BAPTA-AM but not by Ca2+-free solution. The addition of thapsigargin, which depletes Ca2+ from internal stores, also abolished CBF oscillation. Moreover, the intracellular Ca2+ concentration [Ca2+]i, spontaneously oscillated even with the Ca2+-free solution. Moreover, 2APB (an inhibitor of the IP3 receptor) abolished CBF oscillation in cMNECs. Overall, these findings suggest that the CBF oscillation in cMNECs is triggered by the release of Ca2+ from the IP3-sensitive internal stores. Moreover, IBMX, an inhibitor of phosphodiesterase, did not affect CBF oscillation in cMNECs, although it slightly increased CBF. These results suggest that CBF oscillations were induced by [Ca2+]i oscillation controlled via the release of Ca2+ from IP3-sensitive stores, rather than via cAMP accumulation. CBF oscillation possibly plays a crucial role in maintaining an efficient mucociliary clearance in the nasal epithelia.

[Ca2+ ]i modulation of cAMP-stimulated ciliary beat frequency via PDE1 in airway ciliary cells of mice.

NEW FINDINGS: What is the central question of this study? The ciliary beat frequency (CBF) of the airway is controlled by [Ca2+ ]i . However, the effects of a reduction in [Ca2+ ]i on CBF are still controversial (an increase, a decrease or no change). What is the main finding and its importance? This study demonstrated that [Ca2+ ]i directly regulates CBF (direct action) and also indirectly regulates CBF via cAMP accumulation controlled by Ca2+ -dependent PDE1 activity (indirect action). The final CBF is determined by the balance of direct and indirect actions. PDE1 plays crucial roles in the regulation of airway CBF. ABSTRACT: [Ca2+ ]i plays crucial roles in the regulation of ciliary beat frequency (CBF) and ciliary bend angle (CBA) of airway cilia. Moreover, Ca2+ -dependent PDE1A existing in the CBF-regulating metabolon of cilia modifies the CBF by regulating the cAMP accumulation. This study demonstrated that the CBF is regulated by a direct and an indirect action of [Ca2+ ]i ; the direct action changes CBF mediated via [Ca2+ ]i , and the indirect action changes CBF mediated via cAMP, the accumulation of which is controlled by PDE1 activity. Upon reducing [Ca2+ ]i to various levels, the direct action decreases CBF and the indirect action increases CBF. The final CBF is determined by the extent of cAMP accumulation, which is determined by the amount of inhibition of PDE1 activity, dependent on a reduction in [Ca2+ ]i ; a slight decrease induced by a nominally Ca2+ -free solution (no cAMP accumulation via PDE1) decreases CBF, and an extreme decrease induced by 50 µm BAPTA-AM increases CBF via cAMP accumulation by inhibiting PDE1 in a similar manner to a PDE1 inhibitor (8MmIBMX). The increase in CBA in response to a reduction in [Ca2+ ]i is smaller than the increase in CBF, because no PDE1A exists in the CBA-regulating metabolon. On the contrary, an increase in [Ca2+ ]i induced by ionomycin, which decreases cAMP accumulation by PDE1A activation, caused a slower procaterol-stimulated increase in CBF than that decreased by a Ca2+ -free solution. A decrease in [Ca2+ ]i stimulates cAMP accumulation, whereas an increase in [Ca2+ ]i inhibits cAMP accumulation in airway ciliary cells. Thus, changes in [Ca2+ ]i modulate CBF and CBA via cAMP accumulation by controlling the activity of PDE1.

Seihai-to (TJ-90)-Induced Activation of Airway Ciliary Beatings of Mice: Ca2+ Modulation of cAMP-Stimulated Ciliary Beatings via PDE1.

Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3',5'-cyclic adenosine monophosphate) accumulation modulated by Ca2+-activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 µg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca2+ concentration ([Ca2+]i) by 10 µM BAPTA-AM (Ca2+-chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca2+/calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 µg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 µg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP]i) and [Ca2+]i in airway ciliary cells of mice. These small increases in [cAMP]i and [Ca2+]i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca2+-signal, or cAMP-signal.

A low [Ca2+]i-induced enhancement of cAMP-activated ciliary beating by PDE1A inhibition in mouse airway cilia.

This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an ß2-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca2+ concentration ([Ca2+]i) caused by switching to an EGTA-containing Ca2+-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca2+-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca2+ chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia.

Knockdown of ezrin causes intrahepatic cholestasis by the dysregulation of bile fluidity in the bile duct epithelium in mice.

UNLABELLED: Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2(kd/kd) ) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal cross-linker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na(+) /H(+) exchanger regulatory factor were disturbed in bile ducts of Vil2(kd/kd) mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl(-) efflux activity. Furthermore, bile flow and biliary HCO3 (-) concentration were also significantly reduced in Vil2(kd/kd) mice. CONCLUSION: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2(kd/kd) mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies.

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 5.9­6.0 pH) 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 highest 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.

POLD1 Is Required for Cell Cycle Progression by Overcoming DNA Damage in Malignant Pleural Mesothelioma.

BACKGROUND/AIM: The prognosis of patients with malignant pleural mesothelioma (MPM) remains poor due to lack of effective therapeutic targets. DNA damage caused by long-time exposure to asbestos fibers has been associated with the development of MPM, with mutations at genes encoding DNA damage repair (DDR)-related molecules frequently expressed in patients with MPM. The present study was designed to identify novel therapeutic targets in MPM using large public databases, such as The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression project (GTEx) focused on DDR pathways. MATERIALS AND METHODS: The correlations between mRNA expression levels of DDR-related genes and overall survival (OS) were analyzed in mesothelioma patients in TCGA mesothelioma (TCGA-MESO) datasets. The anti-tumor effects of small interfering RNAs (siRNA) against DDR-related genes associated with OS were subsequently tested in MPM cell lines. RESULTS: High levels of mRNA encoding DNA polymerase delta 1, catalytic subunit (POLD1) were significantly associated with reduced OS in patients with MPM (p<0.001, Log-rank test). In addition, siRNA targeting POLD1 (siPOLD1) caused cell cycle arrest at the G1/S checkpoint and induced apoptosis involving accumulation of DNA damage in MPM cell lines. CONCLUSION: POLD1 plays essential roles in overcoming DNA damage and cell cycle progression at the G1/S checkpoint in MPM cells. These findings suggest that POLD1 may be a novel therapeutic target in MPM.

Comparing region of interest selection and whole-field analysis for measurement of ciliary beat frequency in high-speed video analysis.

BACKGROUND: Ciliary beat frequency (CBF) is crucial in mucociliary clearance. High-speed video analysis (HSVA) is commonly used to measure CBF but lacks standardization. We compared visual observation and computer-assisted calculation using fast Fourier transformation (FFT) in freshly collected bronchial ciliary epithelial cells and cultured cells. METHODS: Bronchial epithelial cells were obtained from 12 patients who required bronchoscopic examination. Eighty-five videos of ciliary movement of freshly collected and cultured cells were recorded and used to calculate CBF using manual observation, region of interest (ROI) selection, and whole-field analysis. RESULTS: CBF measured by the ROI selection method strongly correlated with that measured using manual observation, especially in freshly collected cells. However, 27.8% of the manual observation method values were doubled in the ROI selection method, probably because a round trip of cilia was calculated as two cycles and needed to be corrected to 1/2 value. Upon increasing the number of ROIs, the results of the ROI selection method came closer to that of WFA. CONCLUSIONS: Computer-assisted calculation using FFT can aid in measuring CBF; however, current methods require visual confirmation. Further automated evaluation techniques are needed to establish more standardized and generalized CBF measurement methods using HSVA.

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.

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.