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Background: Despite emerging research on new treatment strategies, chemotherapy remains one of the most important therapeutic modalities for cancers. Imidazopyridines are important targets in organic chemistry and, given their numerous applications, they are worthy of attention. Objective: The objective of this study was to design and synthesize a novel series of imidazo[1,2-a]pyridine-derived compounds and investigate their antitumor effects and the underlying mechanisms. Methods: Imidazo[1,2-a]pyridine-derived compounds were synthesized with new strategies and conventional methods. The antitumor activities of the new compounds were evaluated by MTT assay. Flow cytometry and immunofluorescence were performed to examine the effects of the most effective antiproliferative compound on cell apoptosis. Western blot analysis was used to assess the expression of apoptotic proteins. Results: Fifty-two new imidazo[1,2-a]pyridine compounds were designed and successfully synthesized. The compound, 1-(imidazo[1,2-a]pyridin-3-yl)-2-(naphthalen-2-yl)ethane-1,2-dione, named La23, showed high potential for suppressing the viability of HeLa cells (IC50 15.32 μM). La23 inhibited cell proliferation by inducing cell apoptosis, and it reduced the mitochondrial membrane potential of HeLa cells. Moreover, treatment with La23 appeared to increase the expression of apoptotic-related protein P53, Bax, cleaved caspase-3, and cytochrome c at a low concentration range. Conclusion: The novel imidazo[1,2-a]pyridine compound, La23, was synthesized and it suppressed cell growth by inducing cell apoptosis via the p53/Bax mitochondrial apoptotic pathway.

Iodide (I−) is crucial to thyroid function, and its regulation in thyrocytes involves ion transporters and reactive oxygen species (ROS). However, the extent of 2Cl−/H+ exchanger (ClC-3) involvement in the iodide (I−) efflux from thyrocytes remains unclear. Therefore, we examined the effects of ClC-3 on I− efflux. ClC-3 expression was found to significantly alter the serum TT3 and TT4 concentrations in mice. We further found that excess I− stimulation affected ClC-3 expression, distribution, and I− efflux in FRTL-5 cells. Immunofluorescence analyses indicated that ClC-3 mainly accumulated in the cell membrane and co-localized with β-tubulins after 24 h of excess I− treatment, and that this process depended on ROS production. Thus, ClC-3 may be involved in I− efflux at the apical pole of thyrocytes via excess I−-induced ROS production and β-tubulin polymerization. Our results reveal novel insights into the role of ClC-3 in I− transport and thyroid function.

T cells play an important role in antitumor immunity. Numbers and function of T cells are controlled by regulating the uptake and utilization of nutrients, and their antitumor activity can be promoted by targeting metabolic pathways. In this review, we highlight the relationship between metabolism and cellular function of T cells. Specifically, we emphasize the metabolic state of tumor-infiltrating T cells and review key pathways that affect the antitumor function of T cells. In the field of tumor immunotherapy, targeting T cell metabolism to enhance the immune response is a new therapeutic strategy for enhancing immunotherapy combined with traditional treatments.

The representative morphological features of pyroptosis are excessive cell swelling and subsequent membrane rupture. However, the mechanism underlying the cell's inherent inability to regulate volume during the progression of pyroptosis is poorly understood. In the current study, we found that both volume-activated chloride currents (Icl, vol) and the regulatory volume decrease (RVD) were markedly decreased in bone marrow-derived macrophages (BMDMs) undergoing pyroptosis induced by lipopolysaccharides (LPS) and nigericin. The inhibition of ICl, vol and RVD by the chloride channel blockers, tamoxifen or DCPIB, led to the emergence of pyroptosis-like phenotypes such as activated-caspase-1, pyroptotic-body-like bubbles, and a fried-egg-like appearance. Moreover, the expression of the volume-activated chloride channel (VRAC) constituent protein Leucine-Rich Repeat-Containing 8A (LRRC8A) was significantly down-regulated in pyroptotic BMDMs treated with LPS and nigericin. The silencing of LRRC8A expression by small interfering RNA (si)-LRRC8A transfection not only reduced ICl, vol and RVD, but also caused BMDMs to show pyroptosis-like manifestations such as activated-caspase-1, membrane bubbles, and have a fried-egg-like appearance. These results reveal a new mechanism for the loss of volume regulation in the process of pyroptotic cell swelling and strongly suggest that a functional deficiency of VRAC/LRRC8A plays a key role in this disorder.

Wound exudate holds great clinical and research potential in wound repair via paracrine signaling. In essence, exudate is modified serum that contains a high concentration of exosomes. The aim of this study was to investigate the role of serum-derived exosomes in scald wound healing of NIH mice skin and to explore the underlying mechanisms. Hence, we constructed a deep second-degree scald model in NIH mice, testing the benefits of exosomes in the scald wound healing. The scratch wound assay, apoptosis assay and MTT assay were conducted to assess the effects of serum-derived exosomes on migration, apoptosis and proliferation of HaCaT cells and fibroblasts. Our results showed that serum-derived exosomes injected subcutaneously entered cells and effectively accelerated wound healing processes in mice. Additionally, serum-derived exosomes optimized functions of cells related to skin injury repair by stimulating fibroblast proliferation, promoting HaCaT cell migration, and suppressing apoptosis of HaCaT cells induced by heat stress. Further study revealed that serum-derived exosomes enhanced phosphorylation of the serine-threonine kinase Akt in scalded skin tissue. These results suggest a potential clinical use of serum-derived exosomes for treating skin scald.

5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a non-specific chloride channel blocker. Peritoneal adhesion is an inevitable complication of abdominal surgery and remains an important clinical problem, leading to chronic pain, intestinal obstruction, and female infertility. The aim of this study is to observe the effects of NPPB on peritoneal adhesions and uncover the underlying mechanism. The formation of postoperative peritoneal adhesions was induced by mechanical injury to the peritoneum of rats. MTT assay and wound-healing assay were used to evaluate proliferation and migration of primary cultured adhesion fibroblasts (AFB) respectively. Whole-cell chloride currents were measured using a fully automated patch-clamp workstation. Cell volume changes were monitored by light microscopy and video imaging. Our results demonstrated that NPPB could significantly prevent the formation of peritoneal adhesion in rats and inhibit the proliferation of AFB in a concentration-dependent manner. NPPB also reduced the migration of AFB cells with an IC50 of 53.09 μM. A 47% hypotonic solution successfully activated the ICl,vol in AFB cells. The current could be blocked by extracellular treatment with NPPB. Moreover, 100 μM NPPB almost completely eliminated the capacity of regulatory volume decrease (RVD) in these cells. These data indicate that NPPB could prevent the formation of postoperative peritoneal adhesions. The possible mechanism may be through the inhibition of the proliferation and migration of AFB cells by modulating ICl,vol and cell volume. These results suggest a potential clinical use of NPPB for preventing the formation of peritoneal adhesions.

Wound exudate holds great clinical and research potential in wound repair via paracrine signaling. In essence, exudate is modified serum that contains a high concentration of exosomes. The aim of this study was to investigate the role of serum-derived exosomes in scald wound healing of NIH mice skin and to explore the underlying mechanisms. Hence, we constructed a deep second-degree scald model in NIH mice, testing the benefits of exosomes in the scald wound healing. The scratch wound assay, apoptosis assay and MTT assay were conducted to assess the effects of serum-derived exosomes on migration, apoptosis and proliferation of HaCaT cells and fibroblasts. Our results showed that serum-derived exosomes injected subcutaneously entered cells and effectively accelerated wound healing processes in mice. Additionally, serum-derived exosomes optimized functions of cells related to skin injury repair by stimulating fibroblast proliferation, promoting HaCaT cell migration, and suppressing apoptosis of HaCaT cells induced by heat stress. Further study revealed that serum-derived exosomes enhanced phosphorylation of the serine-threonine kinase Akt in scalded skin tissue. These results suggest a potential clinical use of serum-derived exosomes for treating skin scald.

FGF signaling pathway is imperative for definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs), which always accompanies an epithelial-to-mesenchymal transition (EMT) process. However, whether there is an association between FGF signaling and the EMT during DE formation in vitro has remained elusive. In the present study, we identify that several FGF family members were significantly activated during the differentiation of hESCs toward DE. Inhibition of FGF signaling by an efficient and selective inhibitor BGJ398 abolishes both the EMT and DE induction by blocking the activation of the zinc-finger transcription factor SNAI1 which is a direct transcriptional repressor of cell adhesion protein CDH1. In addition, cell proliferation is also severely influenced by attenuating the FGF signaling. Collectively, we propose that the FGF signaling promotes the DE formation through mediating the EMT and cell proliferation.

Fibroblasts migrating to peritoneum injuries play an important role in the development of postoperative peritoneal adhesions due to the excessive synthesis and deposition of extracellular matrix (ECM). This effect is mainly induced by the transforming growth factor-β (TGF-β). Studies indicate that elevated TGF-β1 levels and TGF-β1/Smad signaling are both implicated in the formation of peritoneal adhesions. To confirm the effect of TGF-β1/Smad signaling interference in regulating excessive ECM synthesis, a total of four different R-Smad-targeting small interference RNA (siRNA) duplexes (Smad2-500, Smad2-956, Smad3-378, Smad3-1385) were tested in this study using a TGF-β1-stimulated adhesion tissue fibroblasts (ATFs) cell model. The in vitro assessments show that all proposed siRNAs are capable of significantly downregulating the mRNA and protein levels of Smad2 and Smad3 in ATFs. They also inhibit the phosphorylation of both Smads, which confirms their effect in blocking the TGF-β1/Smad signaling pathway. Moreover, the siRNA duplexes can appreciably lower the elevated levels of fibronectin and collagen 3 alpha 1 (COL3A1) in TGF-β1-stimulated ATFs, and the Smad3-378 siRNA can actually restore both molecules (fibronectin and COL3A1) to normal levels. Therefore, the Smad3-378 siRNA is suitable for both adhesion prevention and wound healing. Overall, our results indicate that postoperative adhesion prophylaxis may be achieved by temporarily blocking TGF-β1/Smad signaling transduction.

Serine-52 (Ser52) is the major physiologic site of keratin 18 (K18) phosphorylation. Here, we report that serine-52 phosphorylated K18 (phospho-Ser52 K18) accumulated on centrosomes in a cell cycle-dependent manner. Moreover, we found that phospho-Ser52 K18 was located at the proximal end of the mother centriole. Transfection with the K18 Ser52 → Ala (K18 S52A) mutant prevented centriole localization of phospho-Ser52 K18 and resulted in separation of the mother–daughter centrioles. Inhibition of microtubule polymerization led to the disappearance of aggregated phospho-Ser52 K18 on the centrosome; removal of inhibitors resulted in reaccumulation of phospho-Ser52 K18 in microtubule-organizing centers. Transfection with a K18 S52A mutant inhibited microtubule nucleation. These results reveal a cell cycle-dependent change in centrosome localization of phospho-Ser52 k18 and strongly suggest that the phosphorylation status of Ser52 K18 of mother centrioles plays a critical role in maintaining a tight engagement between mother and daughter centrioles and also contributes to microtubule nucleation.

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl- channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.

Whether mitochondrial remodeling and metabolic reprogramming occur during the differentiation of human embryonic stem cells (hESCs) to definitive endoderm (DE) is unknown. We found that fragmented and punctate mitochondria in undifferentiated hESCs progressively fused into an extensive and branched network upon DE differentiation. Mitochondrial mass and mitochondrial DNA (mtDNA) content were significantly increased with the upregulated expression of mitochondrial biogenesis regulator PGC1-A upon DE differentiation, accompanied by the rise of the amount of ATP (2.5-fold) and its by-product reactive oxygen species (2.0-fold). We observed that in contrast to a shutoff of glycolysis, expressions of oxidative phosphorylation (OXPHOS) genes were increased, indicating that a transition from glycolysis to OXPHOS was tightly coupled to DE differentiation. In the meantime, we discovered that inhibition of TGF-β signaling led to impaired mitochondrial biogenesis and disturbed metabolic switch upon DE differentiation. Our work, for the first time, reports that TGF-β signaling-dependent mitochondrial biogenesis and metabolic reprogramming occur during early endodermal differentiation.

P-glycoprotein (P-gp) is encoded by the multidrug resistance (MDR1) gene and is well studied as a multi-drug resistance transporter. Peritoneal adhesion formation following abdominal surgery remains an important clinical problem. Here, we found that P-gp was highly expressed in human adhesion fibroblasts and promoted peritoneal adhesion formation in a rodent model. Knockdown of P-gp expression by intraperitoneal injection of MDR1-targeted siRNA significantly reduced both the peritoneal adhesion development rate and adhesion grades. Additionally, we found that operative injury up-regulated P-gp expression in peritoneal fibroblasts through the TGF-β1/Smad signaling pathway and histone H3 acetylation. The overexpression of P-gp accelerated migration and proliferation of fibroblasts via volume-activated Cl(-) current and cell volume regulation by enhancing phosphorylation of the chloride channel-3. Therefore, P-gp plays a critical role in postoperative peritoneal adhesion formation and may be a valuable therapeutic target for preventing the formation of peritoneal adhesions.

// Bin Xu 1,4,* , Xiaobao Jin 1,2,* , Ling Min 5,* , Qin Li 1,2 , Lulu Deng 1,6 , Hui Wu 2 , Guixian Lin 2 , Lixin Chen 3 , Haifeng Zhang 3 , Chunmei Li 2 , Liwei Wang 3 , Jiayong Zhu 1,2 , Weizhang Wang 1,2 , Fujiang Chu 1,2 , Juan Shen 1,4 , Hongzhi Li 2 and Jianwen Mao 1,2 1 Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, Guangzhou, China 2 School of Basic Medicine, Guangdong Pharmaceutical University, Guangzhou, China 3 Department of Pharmacology and Department of Physiology, Medical College, Jinan University, Guangzhou, China 4 School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China 5 Cancer Center of Guangzhou Medical University, Guangzhou, China 6 Department of Pharmacology, Guangdong Pharmaceutical University, Guangzhou, China * These authors contributed equally to this work Correspondence: Jianwen Mao, email: // Lixin Chen, email: // Keywords : chloride channel-3, tumor metastasis, membrane ruffling, cell migration Received : September 10, 2014 Accepted : December 10, 2014 Published : December 11, 2014 Abstract The chloride channel-3 (ClC-3) protein is known to be a component of Cl - channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo . High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl − channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cells are insensitive to BCR-ABL tyrosine kinase inhibitor imatinib, the underlying mechanisms remain largely unknown. Here, we showed that imatinib treatment induced significant upregulation of miR-21 and downregulation of PTEN in Ph+ ALL cell line Sup-b15. Transient inhibition of miR-21 resulted in increased apoptosis, PTEN upregulation and AKT dephosphorylation, whereas ectopic overexpression of miR-21 further conferred imatinib resistance. Furthermore, knockdown of PTEN protected the cells from imatinib-induced apoptosis achieved by inhibition of miR-21. Additionally, PI3K inhibitors also notably enhanced the effects of imatinib on Sup-b15 cells and primary Ph+ ALL cells similar to miR-21 inhibitor. Therefore, miR-21 contributes to imatinib resistance in Ph+ ALL cells and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to imatinib therapy.

Isoprenaline, an activator of β-adrenergic receptor, has been found to induce cardiac hypertrophy in vivo and in vitro, but the exact mechanism is still unclear. ClC-3 is a member of the chloride channel family and is highly expressed in mammalian myocardium. In the present study, the role of ClC-3 in isopronaline-induced cardiac hypertrophy was investigated. We found that ClC-3 expression was reduced in isoprenaline-induced hypertrophic H9c2 cells, primary rat neonatal cardiomyocytes and myocardium of C57/BL/6 mice, and this reduction was prevented by the pretreatment of propranolol. Adeno-associated virus 9 (AAV9)-mediated ClC-3 expression in myocardium decreased heart mass index, thinned interventricular septum and left ventricular wall and lowered the mRNA expression of natriuretic peptide type A (ANF) and β-myosin heavy chain (β-MHC). Our results showed that ClC-3 played an important role in β-adrenergic cardiac hypertrophy which could be associated with ANF and β-MHC, and all these findings suggested that ClC-3 may be a novel therapeutic target for the prevention or treatment of myocardiac hypertrophy.

Cisplatin-based concomitant chemoradiotherapy is considered as the standard treatment for locally advanced nasopharyngeal carcinoma patients. However, the curative efficacy of cisplatin-based chemotherapy is limited because of the occurrence of cisplatin resistance. Some researches indicate that activating the volume-sensitive Cl(-) channel might be a new strategy for the reduction of cisplatin resistance. However, little is known about the activation pathway of the Cl(-) channels activated by cisplatin. In this study, the cisplatin-activated chloride current was investigated using the whole cell patch-clamp technique in the poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells), and the activation pathway of the current was also discussed. The results showed that extracellular application of cisplatin activated a Cl(-) current, showing the properties of significant outward rectification, intracellular ATP dependency, and a selectivity sequence of I(-) > Br(-) > Cl(-) > gluconate, and being inhibited by the Cl(-) channel inhibitors tamoxifen and extracellular ATP. These characteristics are similar to those of the volume-sensitive Cl(-) current in CNE-2Z cells, indicating that cisplatin induces the Cl(-) current by activating the volume-sensitive like chloride channel. The cisplatin-activated current was blocked by suramin (a wide-spectrum purinergic antagonist) and RB2 (a relatively selective P2Y antagonist). In addition, the current was depressed by extracellular application of apyrase. The apoptotic volume decrease induced by cisplatin was also attenuated by RB2. P2Y receptors were expressed in CNE-2Z cells. These results suggest that cisplatin can induce a Cl(-) current by activating volume-sensitive like Cl(-) channels through the P2Y purinoceptor pathway.

An efficient copper-catalyzed selective cross coupling of imidazo[1,2-a]pyridines with methyl hetarenes has been reported. This transformation opened a new route to synthesize the C-3 carbonyl imidazo[1,2-a]pyridine derivative, which is a common structural motif in natural products and pharmaceuticals. (18)O-labeling experiments indicated that the oxygen source of products originated from O2.

It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6 and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression.

An efficient copper-catalyzed selective cross coupling of imidazo[1,2-a]pyridines with methyl hetarenes has been reported. This transformation opened a new route to synthesize the C-3 carbonyl imidazo[1,2-a]pyridine derivative, which is a common structural motif in natural products and pharmaceuticals. (18)O-labeling experiments indicated that the oxygen source of products originated from O2.

Stretch-activated chloride currents (I(Cl,SA) ) have been considered to be a component of volume-activated chloride currents (I(Cl,vol) ) for some time. This is due to a similarity in biophysical and pharmacological properties that involve a membrane curvature-induced mechanism and rearrangement of the cytoskeleton induced by cell swelling or membrane stretch. In the present study, we demonstrated that current density, along with the time taken from the activation of currents to the peak, were significantly different between the two currents, in highly metastatic human hepatocellular carcinoma cells. In addition, the activation of I(Cl,vol) or I(Cl,SA), induced maximally by hypotonic solutions or membrane stretch, respectively, did not affect the following activation of the other one. Moreover, neither inhibition of I(Cl,vol) by sh-ClC-3 transfection, nor functional blocking of I(Cl,vol) by intracellular dialysis of anti-ClC-3 antibody had an effect on the activation and properties of I(Cl,SA). Collectively, our results suggest that I(Cl,SA) is different from I(Cl,vol) in activation mechanism and/or in molecular entity responsible for formation of the currents. ClC-3 is involved in the activation of I(Cl,vol), but not of I(Cl,SA).

Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H(2)O(2))-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H(2)O(2) activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H(2)O(2) elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1h and induced apoptosis of most PC12 cells tested in 24h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H(2)O(2)-induced high membrane permeability and cell shrinkage, suppressed H(2)O(2)-activated chloride currents and protected PC12 cells from apoptosis induced by H(2)O(2). The results suggest that chloride channels may contribute to H(2)O(2)-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.

Recent studies suggest that chloride (Cl-) channels regulate tumor cell migration. In this report, we have used antisense oligonucleotides specific for ClC-3, the most likely molecular candidate for the volume-activated Cl- channel, to investigate the role of ClC-3 in the migration of nasopharyngeal carcinoma cells (CNE-2Z) in vitro. We found that suppression of ClC-3 expression inhibited the migration of CNE-2Z cells in a concentration-dependent manner. Whole-cell patch-clamp recordings and image analysis further demonstrated that ClC-3 suppression inhibited the volume-activated Cl- current (I(Cl,vol)) and regulatory volume decrease (RVD) of CNE-2Z cells. The expression of ClC-3 positively correlated with cell migration, I(Cl,vol) and RVD. These results strongly suggest that ClC-3 is a component or regulator of the volume-activated Cl- channel. ClC-3 may regulate CNE-2Z cell migration by modulating cell volume. ClC-3 may be a new target for cancer therapies.

Cell volume regulation is a fundamental activity to maintain cell survival, and aquaporins and chloride channels play important roles in this process. However, the interactions between these channels are far from clear. In this study, the interactions between AQP-3 and ClC-3 were investigated in CNE-1 and CNE-2Z nasopharyngeal carcinoma cells, which are well and poorly differentiated, respectively. The correlation coefficient of AQP-3 and ClC-3 protein phylogenetic trees was 0.319. In CNE-1 cells, there are overlapping distributions of AQP-3 and ClC-3, mainly in the plasma membrane. This was confirmed by the co-immunoprecipitation of AQP-3 and ClC-3, showing that they could be interlinked and form complexes. AQP-3 over-expression had no significant effects on swelling-induced Cl(-) currents (ICl,swell); however, ICl,swell could be inhibited by aquaporin blockers, anti-AQP-3 antibodies and AQP-3-siRNAs. In addition, the AQP-3 expression was decreased by down-regulation of ClC-3 expression, indicating that ClC-3 can modulate the expression of AQP-3 proteins. The effects of aquaporin blockers, anti-AQP-3 antibodies and AQP-3 over-expression on ICl,swell in CNE-2Z cells were consistent with those in CNE-1 cells. In conclusion, AQP-3 and ClC-3 are functionally-related integral membrane channel proteins, and their interactions are involved in cell volume regulation in CNE-1 and CNE-2Z cells. The opening of ClC-3 transports Cl(-) across the cell membrane and then drives the efflux of water through AQP-3 channels and ion channels; AQP-3 may interact with ClC-3 in order to regulate the effluxes of chloride and water.

Microgels are defined as intramolecularly crosslinked macromolecules. They have in common with crosslinked polymers a three-dimensional structure and fixed surfaces but are still soluble in certain solvents like most linear or branched polymers. A general hypothesis by which microgels would be expected to be formed from polyfunctional polymerizable monomers in good solvents, and at complete conversion to polymer but without macrogelation, has been previously proposed and demonstrated for radical addition polymerization (Graham and Hayes, Macromolecular Symposium, Polymer Networks 94, 93 (1995) 293). This paper extends the generality of the practical demonstration of this hypothesis by experimental extension to stepgrowth polymerization. The polyurethane microgels of this study were synthesized using stoichiometric amounts of poly(ethylene glycol) of molecular weights ranging from 3800 to 8000, a trifunctional hydroxylic crosslinking agent, trimethylolpropane, and a diisocyanate, diphenylmethane-4,4′-diisocyanate (MDI). The polymerization was done in various solvents. It was found that in every solvent/monomer system there is a critical monomer concentration below which no macrogelation occurred but microgels were formed. The monomer concentration and the solubility parameter of the solvent were found to be the two most important factors influencing the formation of microgel or macrogel. Auto-steric stabilization, is believed to have played an important role in the formation of small microgel particles and in preventing macrogelation.

This paper describes studies on the formation of microgels from high functionality mixtures of monomers which are able to polymerise by a step-growth process to polyurethanes in the specially selected solvent butan-2-one. Using the reactants poly(ethylene glycol) 6000, 1,2,6-hexanetriol and biscyclohexylmethane-4,4'-diisocyanate in formulations made to comprise exactly one isocyanate group per hydroxyl, it was found that microgels of number average molecular weight, as determined by Get Permeation Chromatography,of less than 20,000 were obtained up to the Critical Gelation Concentration which ranged from w/w reactant to solvent concentrations of from ∼0.24 to 0.74 at PEG/HT ratios of 1 :4 to 4 :4 respectively. These polymerisations surprisingly, went to complete conversion without gelation and produced 95 - 99% yields of microgel product after precipitation into a non-solvent. Intrinsic viscosity determinations on the microgels dissolved in chloroform were determined and used to obtain estimates of the swollen coil densities at infinite dilution from which the critical packing concentration of these swollen coils in the solvent used for the solvent could be estimated. The critical packing concentration was in all cases well below the Critical Gelation Concentration. It was thus clearly demonstrated that the formation ofsoluble microgels is progressing in spite of the system being critically packed. This is a surprising observation and especially so when it is combined with the very low measured weight average molecular weights and high conversions of the products in the critically packed region. The GPC molecular weights decreased at the higher concentrations which is contrary to expectations. The ability to form microgels above critical packing confirms the results obtained and reported previously by us, that a similar phenomenom occurs in the case of the polymerisation of polyfunctional free-radically polymerised monomer systems. These observations will need to be taken into account when formulating mathematical analyses of the gelation process. This class of products are able to be crystallised from organic solvents and are soluble in water. They are potentially useful as components of pharmaceutical formulations for the controlled release of drugs, high molecular weight active materials and living microorganisms.

The functions of chloride channels in preconditioning-induced cell protection remain unclear. In this report, we show that the volume-activated chloride channels play a key role in hydrogen peroxide (H2O2) preconditioning-induced cell protection in pheochromocytoma PC12 cells. The preconditioning with 100 μM H2O2 for 90 min protected the cells from injury induced by long period exposure to 300 μM H2O2. The protective effect was attenuated by pretreatment with the chloride channel blockers, 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB) and tamoxifen. H2O2 preconditioning directly activated a chloride current, which was moderately outward-rectified and sensitive to the chloride channel blockers and hypertonicity-induced cell shrinkage. H2O2 preconditioning functionally up-regulated the activities of volume-activated chloride channels and enhanced the regulatory volume decrease when exposure to extracellular hypotonic challenges. In addition, acute application of H2O2 showed distinctive actions on cell volume and membrane permeability in H2O2 preconditioned cells. In H2O2 preconditioned cells, acute application of 300 μM H2O2 first promptly induced a decrease of cell volume and enhancement of cell membrane permeability, and then, cell volume was maintained at a relatively stable level and the facilitation of membrane permeability was reduced. Conversely, in control cells, 300 μM H2O2 induced a slow but persistent apoptotic volume decrease (AVD) and facilitation of membrane permeability. H2O2 preconditioning also significantly up-regulated the expression of ClC-3 protein, the molecular candidate of the volume-activated chloride channel. These results suggest that H2O2 preconditioning can enhance the expression and functional activities of volume-activated chloride channels, thereby modulate cell volume and cell membrane permeability, which may contribute to neuroprotection against oxidant-induced injury.

It has been demonstrated previously by us that cyclin D1 and ClC-3 play important roles in regulation of the cell cycle in nasopharyngeal carcinoma cells. The action of cyclin D1 on the functional activities and expression of chloride channels were investigated in nasopharyngeal carcinoma CNE-2Z cells in this study. The results indicated that enhanced cyclin D1 expression increased the activation of volume-activated chloride currents and promoted the expression of ClC-3 chloride channel proteins. The fluorescence resonance energy transfer (FRET) experiments demonstrated that the distance between cyclin D1 and ClC-3 was less than 10nm, and there existed interaction between the two proteins. ClC-3 was partially colocalized with cyclin D1 and CDK4/6. Dialyzing CDK4 antibodies into cells via recording pipettes activated a chloride current, but dialysis of CDK6 antibodies inhibited basal and volume-activated Cl(-) currents. The CDK4/6 inhibitor fascaplysin chloride hydrate (highly selective for CDK4/cyclin D1 with IC(50) = 0.35 μM and less selective for CDK6/D1 with IC(50) = 3.4 μM) activated a chloride current in low concentration, but did not show significantly facilitative effects on the current in high concentration. In conclusion, our data suggest that the ClC-3 chloride channel is an important target of cyclin D1. Cyclin D1 may regulate the functional activities of the chloride channel via CDK4 and CDK6, and/or the expression of the chloride channel. Cyclin D1-CDK4 complexes may phosphorylate chloride channels resulting in inhibition or inactivation of the channels, and cyclin D1-CDK6 complexes may facilitate the activation of chloride channels.

Aim The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. Main methods Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. Key findings Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. Significance Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells.

It is accepted that K(+) and Cl(-) flows are coupled tightly in regulatory volume decrease (RVD). However, using self referencing microelectrodes, we proved that K(+) and Cl(-) transport mainly by channels in RVD was uncoupled in nasopharyngeal carcinoma CNE-2Z cells, with the transient K(+) efflux activated earlier and sustained Cl(-) efflux activated later. Hypotonic challenges decreased intracellular pH (pH(i)), and activated a proton pump-dependent H(+) efflux, resulting in a decline of extracellular pH (pH(o)). Modest decreases of pH(o) inhibited the volume-activated K(+) outflow and RVD, but not the Cl(-) outflow, while inhibition of H(+) efflux or increase of pH(o) buffer ability promoted K(+) efflux and RVD. The results suggest that the temporal dynamics of K(+) channel activities is different from that of Cl(-) channels in RVD, due to differential sensitivity of K(+) and Cl(-) channels to pH(o). H(+) efflux may play important roles in cell volume regulation, and may be a therapeutic target for human nasopharyngeal carcinoma.

Chloride channel-3 (ClC-3) is suggested to be a component and/or a regulator of the volume-activated Cl(-) channel in the plasma membrane. However, ClC-3 is predominantly located inside cells and the role of intracellular ClC-3 in tumor growth is unknown. In this study, we found that the subcellular distribution of endogenous ClC-3 varied in a cell cycle-dependent manner in HeLa cells. During interphase, ClC-3 was distributed throughout the cell and it accumulated at various positions in different stages. In early G1, ClC-3 was mainly located in the nucleus. In middle G1, ClC-3 gathered around the nuclear periphery as a ring. In late G1, ClC-3 moved back into the nucleus, where it remained throughout S phase. In G2, ClC-3 was concentrated in the cytoplasm. When cells progressed from G2 to the prophase of mitosis, ClC-3 from the cytoplasm translocated into the nucleus. During metaphase and anaphase, ClC-3 was distributed throughout the cell except for around the chromosomes and was aggregated at the spindle poles and in between two chromosomes, respectively. ClC-3 was then again concentrated in the nucleus upon the progression from telophase to cytokinesis. These results reveal a cell cycle-dependent change of the subcellular distribution of ClC-3 and strongly suggest that ClC-3 has nucleocytoplasmic shuttling dynamics that may play key regulatory roles during different stages of the cell cycle in tumor cells.

Volume-activated chloride channels have been studied by us extensively in human nasopharyngeal carcinoma cells. However, the chloride channels in the counterpart of the carcinoma cells have not been investigated. In this study, volume-activated chloride currents (I(cl,vol)) were characterized in normal fetal human nasopharyngeal epithelial cells using the whole-cell patch-clamp technique. Under isotonic conditions, nasopharyngeal epithelial cells displayed only a weak background current. Exposure to 47% hypotonic solution activated a volume-sensitive current. The reversal potential of the current was close to the calculated equilibrium potential for Cl(-). The peak values of the hypotonicity-activated current at +80 mV ranged from 0.82 to 2.71 nA in 23 cells. Further analysis indicated that the density of the hypotonicity-activated current in most cells (18/23) was smaller than 60 pA/pF. Only five cells presented a current larger than 60 pA/pF. The hypotonicity-activated current was independent of the exogenous ATP. Chloride channel inhibitors ATP, tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibited the current dramatically. The anion permeability of the hypotonicity-activated chloride channels was I(-)Br(-)Cl(-)gluconate. Unexpectedly, in isotonic conditions, ATP (10 mM) activated an inward-rectified current, which had not been observed in the nasopharyngeal carcinoma cells. These results suggest that, under hypotonic challenges, fetal human nasopharyngeal epithelial cells can produce I(cl,vol), which might be involved in cell volume regulation.

We have previously shown that chloride channel activities were cell cycle-dependent and were involved in cell proliferation in nasopharyngeal carcinoma cells. In this study, the expression and roles of volume-activated chloride channels in cell growth were investigated in the poorly-differentiated human nasopharyngeal carcinoma cell (CNE-2Z) and its counterpart, the normal human nasopharyngeal epithelial cell (NP69-SV40T). Consistent with growth ability, the background chloride currents recorded under isotonic condition, the volume-activated chloride currents induced by 47% hypotonic challenges and the hyponinicity-induced regulatory volume decrease (RVD) were much larger in CNE-2Z cells than in NP69-SV40T cells, suggesting the up-regulation of expression of volume-activated chloride channels in cancerous cells. This was proved by the up-regulation of ClC-3 proteins, a candidate of volume-activated chloride channels, in the cancerous cells. Functional inhibition of chloride channel activities by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and tamoxifen, and knock-down of ClC-3 expression by specific ClC-3 siRNA attenuated the background currents, suppressed the activation of volume-activated chloride currents, decreased the hyponinicity-induced RVD and inhibited cell growth in the cancerous and normal cells. However, the sensitivities of the cancerous cells were much higher than that of the normal cells. Our data suggest that volume-activated chloride channels play a more important role in control cell proliferation in the cancerous cells than in the normal cells; the growth of cancerous cells is more dependent on the activities of volume-activated chloride channels than that of the normal cells. ClC-3 protein may be considered as a potential tumor marker and therapeutic target for human nasopharyngeal carcinoma.

ClC-3, a gene encoding a candidate protein for volume-activated chloride (C(-)) channels, may be involved in tumor development. Herein we report a study using an antisense "knock-down" strategy to investigate the mechanism by which ClC-3 affects cell proliferation in nasopharyngeal carcinoma CNE-2Z cells. With immunoblots and MTT assays we demonstrated that the expression of ClC-3 was cell cycle dependent and in a similar concentration-dependent manner, an antisense oligonucleotide specific for ClC-3 inhibited ClC-3 protein expression and cell proliferation. The expression level of ClC-3 correlated with cell proliferation. Moreover, in the cells exposed to a ClC-3 antisense oligonucleotide, the cloning efficiency was inhibited, and cells were arrested in the S phase. The ClC-3 antisense oligonucleotide inhibited the volume-activated C(-) current (I(Cl,vol)) and the regulatory volume decrease (RVD) in a concentration-dependent manner. Additionally, the I(Cl,vol) or RVD was positively correlated with cell proliferation in the treated cells. In conclusion, ClC-3 is involved in cell proliferation and cell cycle progression through a mechanism involving modulation of I(Cl,vol) and RVD. CIC-3 may represent a therapeutic target in human cancer.

The activation of volume-activated chloride Cl(-) channels has been implicated to play important roles in modulating cell cycle and cell migration. The aim of this study was to determine whether volume-activated Cl(-) channels are involved in cell-cycle-dependent regulation of cell migration in HeLa cells. Using techniques including cell-cycle synchronization, transwell migration assays and the patch-clamp technique, we demonstrate in this study that both the expression of volume-activated chloride current (I(Cl,vol)) and the potential of cell migration are cell-cycle-dependent; specifically, these events were high in G(0)/G(1) phase, low in S phase, and medium in G(2)/M phase. Moreover, the mean density of I(Cl,vol) was positively correlated to the rate of cell migration during cell-cycle progression. Additionally, endogenous suppression of I(Cl,vol) by transfecting cells with ClC-3 antisense oligonucleotides arrested cells in S phase and slowed cell migration. Collectively, our results suggest that volume-activated Cl(-) channels contribute to the cell-cycle-dependent regulation of cell migration.

Cell migration is crucial for tumor metastasis. Membrane ion channels may play a major role in tumor cell migration because the cells must undergo changes in shape and volume during migration. In the present study, we used the transwell migration assay, an in vitro model for cell migration, and the patch-clamp technique to investigate the role of the volume-activated Cl(-) current (I(cl,vol)) in the regulation of the migration of nasopharyngeal carcinoma CNE-2Z cells. 5-Nitro-2- (3-phenylpropylamino) benzoic acid (NPPB) inhibited the I(cl,vol) and the migration of CNE-2Z cells with almost identical dose-dependent pattern (IC(50) of 98.1 microM and 97.7 microM for I(cl,vol) and cell migration, respectively). Extracellular adenosine triphosphate (ATP) also showed similar dose-dependent inhibitory effects on the currents and migration (IC(50) of 1.07mM, and 1.11mM for I(cl,vol) and cell migration, respectively). Hypotonic treatments, which activated I(cl,vol), increased cell migration. Exposure to hypertonic solutions, which was shown to suppress I(cl,vol), inhibited cell migration. Replacement of Cl(-) with gluconate, which is relatively chloride channel-impermeable, impaired cell migration, whereas substitution of Cl(-) by I(-) and Br(-), the chloride channel-permeable ions, did not significantly affect cell migration. Analysis of the effects of all the above treatments on I(cl,vol) and cell migration indicated that the inhibition of migration was positively correlated with the blockage of I(cl,vol), with a correlation coefficient (r) of 0.97, suggesting a functional relationship between I(cl,vol) and cell migration. These data suggest that the volume-activated Cl(-) channels are involved in cell migration.