Enhanced Gap Junction Research Articles

Abstract 3125: Upregulation of gap junctions with substituted quinolines in colon cancer cells

Abstract Colon cancer is one of the most common cancers in the United States with a high predisposition to metastasize. Furthermore, 30-40% of patients have metastatic disease at the initial diagnosis. Cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of cell cycle, cell differentiation, and cell signaling. Previous work demonstrated that connexin proteins such as Cx43, Cx32 and Cx26 have a distinct pattern in SW480, SW620, HT29 and Caco-2 human colon cancer cell lines. The findings suggest that the lack of GJIC, in part due to low level of connexin, may contribute to facilitate colon cancer progression. Since the regulation of gap junction is lost in colon cancer cells, the goal of this study is to restore GJIC in colon cancer cells. Recently, substituted quinolines (PQs), gap junction enhancers, have been shown to increase gap junction activity in breast cancer cells. The level of Cx43 is low in SW620 and SW480 human colon cancer cells. Transfected Cx43 cells have a 6-fold increase of gap junction activity compared to control using scrape load/dye transfer assay. Western blot analysis confirmed that a significant level of Cx43 was expressed in transfected Cx43 cells compared to control. This suggests that overexpressing Cx43 can restore GJIC. Interestingly, 200 nM PQ1 causes a 4-fold increase of gap junction activity as well and subsequently causes a decrease in cell proliferation by 15%. However, TPA, GJIC inhibitor, can reverse the gap junction activity of transfected Cx43 or treated PQ1 cells. Further analysis of Cx43, survivin, and cyclin D1 in treated cells was observed. Overall, the results show that substituted quinolines can directly enhance gap junction activity and attenuate cell proliferation in colon cancer cells. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2011-3125

Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50

Both connexin 50 (Cx50) and aquaporin 0 (AQP0) have important roles in lens development and homeostasis, and their mutations are associated with human congenital cataracts. We have previously shown that Cx50 directly interacts with AQP0. Here, we demonstrate the importance of the Cx50 intracellular loop (IL) domain in mediating the interaction with AQP0 in the lens in vivo. AQP0 significantly increased (~20-30%) the intercellular coupling and conductance of Cx50 gap junctions. However, this increase was not observed when the IL domain was replaced with those from other lens connexins. The Cx50-AQP0 interaction had no effect on Cx50 hemichannel function. A fusion protein containing three extracellular loop domains of AQP0 efficiently blocked the cell-to-cell adhesion of AQP0 and attenuated the stimulatory effect of AQP0 on Cx50 gap junction conductance. These data suggest that the specific interaction between Cx50 and AQP0 enhances the coupling of Cx50 gap junctions, but not hemichannels, through the cell adhesion function of AQP0. This result establishes a physiological role of AQP0 in the functional regulation of gap junction channels.

Supercritical CO2-Extracted Tomato Oleoresins Enhance Gap Junction Intercellular Communications and Recover from Mercury Chloride Inhibition in Keratinocytes

A nutritionally relevant phytochemical such as lycopene, found in tomatoes and other fruits, has been proposed to have health-promoting effects by modulating hormonal and immune systems, metabolic pathways, and gap junction intercellular communication (GJIC). This work analyzes lycopene extracts, obtained from tomato and tomato added with grape seeds by using a safe and environmentally friendly extraction process, based on supercritical carbon dioxide technology (S-CO(2)). Analysis of the innovative S-CO(2)-extracted oleoresins showed peculiar chemical composition with high lycopene concentration and the presence of other carotenoids, lipids, and phenol compounds. The oleoresins showed a higher in vitro antioxidant activity compared with pure lycopene and beta-carotene and the remarkable ability to enhance the GJIC and to increase cx43 expression in keratinocytes. The oleoresins, (0.9 microM lycopene), were also able to overcome, completely, the GJIC inhibition induced by 10 nM HgCl(2), mercury(II) chloride, suggesting a possible action mechanism.

Oscillating Pressure Treatment Upregulates Connexin43 Expression in Skeletal Myoblasts and Enhances Therapeutic Efficacy for Myocardial Infarction

Transplantation of autologous skeletal myoblasts (SMBs) is a potential therapeutic approach for myocardial infarction. However, their clinical efficacy and safety is still controversial. Electrical coupling through gap junction between SMBs and host myocardium is essential for synchronized contraction and electrical stability. Here, we investigated the effect of heart beat-simulating environment, oscillating pressure, on the expression of connexin43 in two types of SMBs from rat and mouse. We found that connexin43 is markedly decreased under ischemia-mimicking conditions such as serum starvation and hypoxia (1% O(2)) in rat primary cultured SMBs and mouse C2C12 SMB cell line. Interestingly, the decrease of connexin43 expression under serum starvation was attenuated by oscillating pressure. Oscillating pressure treatment increased the expression of connexin43 twofold through AP-1 stimulation, which was blocked by PD98059, ERK inhibitor. In coculture of cardiomyocytes and C2C12, pressure-treated C2C12 and cardiomyocytes were able to form functional gap junction, which was demonstrated by both calcein-AM dye transfer assay and measurement of simultaneous contraction. In rat myocardial infarction model, transplantation of SMBs pretreated with oscillating pressure resulted in lesser ventricular dilatation and better systolic function than transplantation of untreated SMBs and control group. These results suggested that application of oscillating pressure on SMBs before transplantation may be useful to promote therapeutic efficacy for myocardial infarction by enhancing gap junction formation between transplanted and host cells.

Interaction of BDNF and Connexin43– two downstream targets of CRH-activated gene transcription

Corticotropin-releasing hormone (CRH) is a major central stress mediator, but also a potent neuroprotective effector. In an attempt to unravel the mechanisms by which CRH mediates its neuroprotective actions we identified two downstream targets of CRH-activated gene transcription, the brain-derived neurotrophic factor (BDNF; Bayatti et al., Endocrinology 2005; 146: 1205–1213 and Hanstein et al., Neuroscience 2008; 156 (3):712–21) and the gap junction molecule connexin 43 (Cx43; Hanstein et al., Molecular Endocrinology 2009; in press). Both molecules are known to exert neuroprotective effects and may thus mediate the neuroprotective actions of CRH. By treating neural cells with BDNF and knocking down BDNF expression using BDNF siRNA we could show that BDNF itself upregulates Cx43 expression and enhances gap junction communication. On the contrary, downregulation of Cx43 expression levels by Cx43 siRNA showed no influence on BDNF expression. In summary, our experiments show that CRH may increase gap junction communication by either acting directly on Cx43 gene expression or via an indirect way by stimulation of BDNF expression. Both ways may be involved in the CRH-induced neuroprotection.

Increased Connexin 43 Expression as a Potential Mediator of the Neuroprotective Activity of the Corticotropin-Releasing Hormone

CRH is a major central stress mediator, but also a potent neuroprotective effector. The mechanisms by which CRH mediates its neuroprotective actions are largely unknown. Here, we describe that the gap junction molecule connexin43 (Cx43) mediates neuroprotective effects of CRH toward experimentally induced oxidative stress. An enhanced gap junction communication has been reported to contribute to neuroprotection after neurotoxic insults. We show that CRH treatment up-regulates Cx43 expression and gap junctional communication in a CRH receptor-dependent manner in IMR32 neuroblastoma cells, primary astrocytes, and organotypic hippocampal slice cultures. MAPKs and protein kinase A-cAMP response element binding protein -coupled pathways are involved in the signaling cascade from CRH to enhanced Cx43 function. Inhibition of CRH-promoted gap junction communication by the gap junction inhibitor carbenoxolone could prevent neuroprotective actions of CRH in cell and tissue culture models suggesting that gap junction molecules are involved in the neuroprotective effects of CRH. The extent of oxidative stress-induced protein carbonylation and cell death inversely correlated with Cx43 protein levels as shown by Cx43 small interfering RNA knockdown experiments. Coculture studies of primary neurons and astrocytes revealed that astrocytic Cx43 likely contributes to the neuroprotective effects of CRH. To our knowledge this is the first description of Cx43 as a potential mediator of the neuroprotective actions of CRH.

Pre-Treatment of Mesenchymal Stem Cells With a Combination of Growth Factors Enhances Gap Junction Formation, Cytoprotective Effect on Cardiomyocytes, and Therapeutic Efficacy for Myocardial Infarction

The goal of this study was to investigate the effect of pre-treatment of mesenchymal stem cells (MSCs) with growth factors (GFs) on cardiomyogenic differentiation, cytoprotective action on cardiomyocytes (CMCs), and their therapeutic efficacy in myocardial infarction. Mechanisms of myocardial repair with MSC transplantation have not been fully elucidated, and therapeutic efficacy needs to be enhanced. The MSCs obtained from the bone marrow of Fisher344 rats were treated with fibroblast growth factor-2, insulin-like growth factor-1, and bone morphogenetic protein-2. The expression of cardiac specific markers and the cytoprotective effect of MSCs with its mechanism were evaluated. Efficacy of MSCs transplantation was studied in rat myocardial infarction model. Treatment of MSCs with cocktails of GFs enhanced expression of cardiac transcription factors and survival. Induction of cardiac specific markers by coculture with CMCs and gap junctional communication with CMCs was more active in GF-treated MSCs than untreated MSCs. The GF-treated MSCs reduced apoptosis of neighboring CMCs in a hypoxic condition and enhanced the phosphorylated Akt and phosphorylated c-AMP response element binding protein expression of CMCs, which was markedly reduced by gap junction blockade. In a rat myocardial infarction model, transplantation of GF-treated MSCs resulted in smaller infarct size and better cardiac function than transplantation of untreated MSCs. Additionally, GF treatment enhanced gap junction formation of transplanted MSCs, which did not aggravate arrhythmia. Pre-treatment of MSCs with GFs enhanced cytoprotective effects on neighboring CMCs through gap junction and improved the therapeutic efficacy of MSC transplantation for myocardial repair. "Priming of MSCs with GFs" before transplantation might improve the therapeutic efficacy of cell therapy.

Resveratrol and X rays affect gap junction intercellular communications in human glioblastoma cells

Resveratrol (3,4',5-trihydroxystilbene) is a polyphenol synthesized by a wide variety of plant species in response to injury, UV irradiation and fungal attack. Many studies have revealed a variety of resveratrol intracellular targets whose modulation gives rise to overlapping responses leading to growth arrest and death. Many authors have reported different human cancer cell lines, treated with resveratrol at micromolar concentrations, arrested their proliferative cycle in the G1/S boundary or in the S phase and this cell cycle arrest was followed by apoptotic death. Less is known about the ability of resveratrol to modify the effect of radiation exposure in normal and cancer cells. Considering that controlled exposure to ionizing radiation is one of the most used treatments in cancer patients and that these schedules are not always effective in medical practice, as in the case of glioma patients, the testing of combined treatment protocols (resveratrol and ionizing radiation) could be of interest, opening the door to future studies which would examine the pharmacological activity of resveratrol. In this study we have looked into whether resveratrol is able to modulate cell cycle progression in human glioblastoma cells and to regulate GJs expression in cancer cells. With this aim in mind we have performed a cytofluorimetric multiparameter assay to quantify the presence of GJs in U87 glioma cells treated with resveratrol and/or X rays. We report that resveratrol induces a delay in cell cycle progression and both alone and in combination with X rays is able to enhance gap junction Intercellular Communications.

New antiarrhythmic treatment of atrial fibrillation

Antiarrhythmic pharmaceutical development for the treatment of atrial fibrillation (AF) is moving in several directions. The efficacy of existing drugs, such as carvedilol, for rate control and, possibly, suppression of AF, is more appreciated. Efforts are being made to modify existing agents, such as amiodarone, in an attempt to ameliorate safety and adverse effect concerns. This has resulted in promising data from the deiodinated amiodarone analog, dronedarone, and further work with celivarone and ATI-2042. In an attempt to minimize ventricular proarrhythmia, atrial selective drugs, such as intravenous vernakalant, have demonstrated efficacy in terminating AF in addition to promising data in suppression recurrences when used orally. Several other atrial selective drugs are being developed by multiple manufacturers. Other novel therapeutic mechanisms, such as drugs that enhance GAP junction conduction, are being developed to achieve more effective drug therapy than is offered by existing compounds. Finally, nonantiarrhythmic drugs, such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, high-mobility group coenzyme A enzyme inhibitors and omega-3 fatty acids/fish oil, appear to have a role in suppressing AF in certain patient subtypes. Future studies will clarify the role of these drugs in treating AF.

Impaired cAMP signaling does not account for the attenuated EDHF-mediated dilations in female rat middle cerebral artery

Dilations to endothelium-derived hyperpolarizing factor (EDHF) are significantly attenuated in the middle cerebral artery (MCA) isolated from female compared to male rats. Since gap junctions appear to be involved in the EDHF pathway and cAMP has been shown to enhance gap junction permeability, we tested the hypothesis that elevation of cAMP would enhance EDHF-mediated dilations in female rat MCA. Vascular diameter was measured in perfused MCA segments using videomicroscopy in the presence and absence of IBMX, an inhibitor of cAMP phosphodiesterase. In the presence of l-NAME and indomethacin, dilation to 10 − 4 M ATP was significantly reduced in females (48 ± 12%) compared to males (92 ± 2%). IBMX, an inhibitor of cAMP phosphodiesterase, had no significant effect on ATP-mediated dilations in both males and females. Basal cAMP levels were comparable in male and female MCAs (1.7 pmol/mg protein). Incubation with IBMX (2 × 10 − 4 M) significantly elevated cAMP in both male (12.8 pmol/mg protein) and female (11.2 pmol/mg protein) MCAs. Our results demonstrate that reduced EDHF dilations in female rat MCA cannot be solely attributed to impaired cAMP signaling. Future studies will target other potential sites along the EDHF pathway in order to identify why EDHF dilations are reduced in the female compared to the male rat MCA.

Docosahexaenoic acid (22:6n‐3) enrichment of membrane phospholipids increases gap junction coupling capacity in cultured astrocytes

Although it is agreed that n-3 polyunsaturated fatty acids (PUFAs) are important for brain function, it has yet to be demonstrated how they are involved in precise cellular mechanisms. We investigated the role of enhanced n-3 PUFA in astrocyte membranes on the gap junction capacity of these cells. Astrocytes isolated from newborn rat cortices were grown in medium supplemented with docosahexaenoic acid (DHA), the main n-3 PUFA in cell membranes, or arachidonic acid (AA), the main n-6 PUFA, plus an antioxidant (alpha-tocopherol or N-acetyl-cystein) to prevent peroxidation. The resulting three populations of astrocytes differed markedly in their n-3:n-6 PUFA ratios in phosphatidylethanolamine and phosphatidylcholine, the main phospholipids in membranes. DHA-supplemented cells had a physiological high n-3:n-6 ratio (1.58), unsupplemented cells had a low n-3:n-6 ratio (0.66) and AA-supplemented cells had a very low n-3:n-6 ratio (0.36), with excess n-6 PUFA. DHA-supplemented astrocytes had a greater gap junction capacity than unsupplemented cells or AA-supplemented cells. The enhanced gap junction coupling of DHA-enriched cells was associated with a more functional distribution of connexin 43 at cell interfaces (shown by immunocytochemistry) and more of the main phosphorylated isoform of connexin 43. These findings suggest that the high n-3:n-6 PUFA ratio that occurs naturally in astrocyte membranes is needed for optimal gap junction coupling in these cells.

CAMP elevation does not enhance EDHF‐mediated dilations in female rat middle cerebral artery

Dilations to endothelium-derived hyperpolarizing factor (EDHF) are significantly attenuated in the rat middle cerebral artery (MCA) isolated from females compared to males. We tested two hypotheses; first that gap junctions mediate EDHF dilations in male rat MCA and second that elevation of cAMP, by enhancing gap junction permeability, would augment EDHF dilations in female rat MCA. MCAs were isolated from male and female rats. Vascular diameter was measured in pressurized (85mmHg) and luminally perfused (150μl/min) MCAs using videomicroscopy. cAMP levels were determined using a radioimmunoassay. EDHF dilations were elicited by luminal application of either UTP or ATP in the presence of L-NAME and indomethacin. Carbenoxolone (10−4M), a gap junction inhibitor, caused a significant reduction in dilations to 10-4M UTP (87±8%) compared to vehicle (23±10%). Dilations to 10−4M ATP were significantly reduced in females (48±12%) compared to males (92±2%). IBMX (2x10−4M), a cAMP phosphodiesterase inhibitor, had no effect on EDHF dilations in either males or females. Basal cAMP levels were comparable in male and female MCAs (1.7 pmol/mg protein). Incubation with IBMX significantly elevated cAMP in both male (12.8 pmol/mg protein) and female (11.2 pmol/mg protein) MCAs. Our results demonstrate that first, gap junctions mediate EDHF dilations in male rat MCA and second, reduced EDHF dilations in female rat MCA cannot be solely attributed to impaired cAMP signaling. Supported by AHA SDG 0130250N and NIH HL72954.

Hypoxia disrupts intestinal gap junctions through activation of a PP1-dependent phosphatase and the de-phosphorylation of connexin43 in the pathogenesis of necrotizing enterocolitis

Introduction: Necrotizing enterocolitis (NEC) develops after a period of global hypoxia, and is characterized by a disruption in the intestinal barrier. The mechanisms by which hypoxia leads to barrier dysfunction remain largely unknown. We have shown that intestinal barrier function requires gap junctions comprised of connexin43 (Cx43) that must be phosphorylated to function. We now hypothesize that hypoxia leads to an impairment in intestinal gap junction function in vitro and in vivo, and sought to determine the mechanisms involved. Methods: IEC-6 cells were subjected to hypoxia in a flow regulated, vacuum sealed hypoxia chamber (1% O2, 37°C) for 1-12h. The expression and phosphorylation of Cx43 were assessed by SDS-PAGE and confocal immunofluorescence. To induce NEC, newborn rats were subjected to global hypoxia (10mins 5% O2) and formula gavage, and mucosal scrapings were obtained from the terminal ileum and subjected to SDS-PAGE. To restore Cx43 phosphorylation, IEC-6 cells were pre-treated with a variety of tyrosine phosphatase inhibitors, including pervanadate(10uM,1h) and calyculin A(2nM,30nM). Results: Hypoxic treatment caused a time-dependent decrease in the phosphorylation of Cx43, which correlated with a decrease in gap junction communication between adjacent enterocytes. There were no effects of hypoxia on the expression of total Cx43, or on cell viability. To verify the in vivo significance of these findings, phosphorylation of Cx43 was also significantly decreased in ileal mucosal scrapings from newborn rats subjected to global hypoxia and formula gavage as compared to control, breast fed animals. Strikingly, pre-treatment of IEC-6 cells with the protein phosphatase 1 (PP1) dependent phosphatase inhibitor calyculin A led to the restoration of phosphorylation of Cx43 and enhanced gap junction connectivity, indicating that a hypoxia-induced phosphatase mediated the effect. Conclusion: Hypoxia inhibits gap junction function in enterocytes through the activation of a protein phosphatase 1 dependent phosphatase resulting in the de-phosphorylation of Cx43. These findings suggest that further targeting of this hypoxia-induced phosphatase may provide a novel therapeutic strategy in the management of diseases of intestinal inflammation like NEC.

Epidermal growth factor enhances intestinal gap junction function through post-translational modification of connexin 43 in vitro and in vivo

Introduction: Necrotizing enterocolitis is characterized by impaired mucosal barrier function, in part through alterations in gap junctions comprised of Connexin43 (Cx43) which must be phosphorylated in order to function. Cx43 half-life is tightly regulated by ubiquitination, leading to its degradation over time. Epidermal growth factor (EGF) restores barrier integrity, yet the mechanisms involved remain undefined. We now hypothesize that EGF enhances gap junction function through post-translational modification of Cx43 and sought to determine the mechanisms involved.

Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.

Iodide sensitizes genetically modified non-small cell lung cancer cells to ionizing radiation

While external ionizing radiation has been used for treating non-small cell lung cancer (NSCLC), improved efficacy of this modality would be an important advance. Ectopic expression of the sodium iodide symporter (NIS) and thyroperoxidase (TPO) genes in NSCLC cells facilitated concentration of iodide in NSCLC cells, which markedly induced apoptosis in vitro and in vivo. Pre-incubation of the NIS/TPO-modified NSCLC cells in iodide followed by ionizing radiation generates bystander tumoricidal effects and potently enhances tumor cell killing. This iodide-induced bystander effect is associated with enhanced gap junction intercellular communication (GJIC) activity and increased connexin-43 (Cx43) expression. Thus, iodide may serve as an enhancer to markedly improve the efficacy of radiation therapy in combined therapeutic modalities.

306. A Novel Thymidine Kinase in Enhanced Gap Junction Mediated Suicide Gene Therapy

Top of pageAbstract Patients with malignant gliomas have a poor prognosis due to failure of conventional therapy, and new treatment paradigms are urgently needed. The extensive heterogeneity of glioblastoma cells calls for a treatment strategy with multiple (cellular) targets. Suicide gene therapy for malignant gliomas has showed promising results in experimental animal models, but has failed in clinical trials due to low transfection rate, insufficient expression of the transgene in tumor cells, and low bystander killing effect. Gap junction communication (GJC) - allowing intercellular transport of small water soluble molecules - is generally low in malignant cells, leading to insufficient bystander effect. We have previously shown that treatment of glioma cells with the histone deacetylase inhibitor 4-phenyl butyrate (PB) leads to upregulation of the gap junction protein connexin 43 expression in both its phosphorylated and non-phosphorylated forms 1. This correlates with enhanced intercellular gap junction communication and increased bystander killing effect in a herpes simplex thymidine kinase (HSVtk)/ganciclovir suicide gene therapy in vitro model 2. A novel plant thymidine kinase with high affinity for the nucleoside analogue AZT was employed in a suicide gene therapy in vitro model. We show that the plant-TK/AZT system is superior to the HSV-TK/GCV system (app. 450- vs 55-fold time of activation) in the glioblastoma cell line U-87 MG. In addition, U-87 MG cells recombinantly expressing this thymidine kinase, showed a synergistic sensitivity to an AZT/PB combination therapy. This effect was shown to be partially gap junction mediated. Similar to glioblastoma cells, we show that neural stem cells (NSC) are subject to enhanced gap junction communication by treatment with PB. This was shown by enhanced fluorescent dye transfer between NSCs and glioblastoma cells. Furthermore, the bystander killing of tumor cells was investigated using plant kinase expressing NSCs co-cultured with glioblastoma cells in the presence of an AZT/PB combination compared to AZT alone. The presence of PB also resulted in an upregulation of connexin 43 in NSCs, as was shown for glioma cells. The successful cell killing in vitro using the plant-TK/AZT system prompted us to test this in vivo. An intracranial glioblastoma xenograft model in immuno-suppressed rats was developed. Plant-TK expressing U-87 MG glioma cell xenografts were established. The sensitivity of the tumor cells to AZT was assessed by intracranial infusion by use of an osmotic minipump. Drug concentrations of 40, 80, and 160 mM, with a flow of 5 ml/hour were used. 80 mM AZT was well tolerated by the rats for a 2 week period. The anti-tumor effect was assessed by histological and immunohistochemical techniques after sacrifice of animals and sectioning of the brain. To explore the NSC migration in rat brains carrying glioma xenografts, BrdU labeled NSCs or NSCs recombinantly expressing green fluorescent protein have been injected at some distance from the tumor site and their migrational capacity is being studied.

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates “suicide gene” expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.

Upregulation of connexin43 gap junctions between neointimal smooth muscle cells

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P < 0.05), but subsequently increased four-fold, attaining levels double that of non-injured controls in the 10-day group (P < 0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis.

Oxidized β-carotene inhibits gap junction intercellular communication in the human lung adenocarcinoma cell line A549

In addition to its antioxidant activity, β-carotene (BC) is known to enhance gap junction intercellular communication (GJIC) by up-regulation of connexin 43 (Cx43), an action that may be important in its control of tumor growth. Surprisingly, two clinical trials on supplemental BC suggest that BC may increase lung cancer incidence in smokers. Recently, an animal study indicated that a very high dose of BC (50 mg/kg b.w./day for 5 days) decreases GJIC in rat liver, while a lower dose (5 mg/kg b.w./day) increases GJIC. It is unclear how high-doses of BC inhibit GJIC. In this study, we tested whether oxidized BC (OBC, obtained by heating BC at 60 °C in open air for 1 h) may inhibit GJIC. We incubated a human lung cancer cell line (A549) with OBC or BC at 2–10 μM for 5 days. Cell viability (by Trypan-blue assay), GJIC (by scrape-loading dye transfer) and Cx43 expression (by western blotting and immunocytochemical localization) were measured to investigate the effects of OBC and BC on GJIC and the possible mechanisms. The results show that OBC at concentrations lower than 10 μM did not significantly affect cell viability. However, OBC at 5 μM inhibited GJIC, whereas BC at 5 μM markedly increased GJIC. The loss of GJIC in A549 induced by OBC accompanied the aberrant localization and phosphorylation of connexin43 (Cx43). These changes in the expression of Cx43 induced by OBC were similar to those induced by 12- O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter. Thus, our results suggest that in vivo inhibition of GJIC by a high dose of BC on GJIC is, at least in part, attributable to the effect of OBC.