The Impact of Repeated Steam Sterilization Cycles on the Efficacy of Chairside Adjustment Kits for Polishing Monolithic Multi-Layered Zirconia Dental Restoration Material.

BACKGROUND Before insertion, chairside adjustment kits are heat sterilized for positioning and polishing dental restorations. This study aimed to evaluate the effects of 2 steam sterilization cycles on the efficacy of polishing highly translucent monolithic zirconia (HTMLZ) dental restoration material. MATERIAL AND METHODS 100 HTMLZ disc-shaped specimens were adjusted (grinding, finishing, polishing) with EVE Diacera kit. Two steam sterilization techniques [standard (Gp S), immediate/flash (Gp (F)] of CAK were further subgrouped based on number of sterilization cycles [cycle 1 (control), cycle 5, 10, 15, and 20 (experimental)] (n=10 each). Each subgroup accordingly was evaluated for average surface roughness (Ra) and root mean square roughness (Rq) using a profilometer. Mean and standard deviation of 5 subgroups were statistically analyzed using one-way ANOVA/post hoc Tukey's test. Scanning electron microscopy complemented Ra, Rq measurements. Statistical differences of P≤0.05 were considered significant. RESULTS HTMLZ specimens in both groups showed increased (Ra/Rq) values after repeated sterilization of EVE Diacera kit, with Gp F showing lesser increase than Gp S (20 cycles). Gp F at 10 cycles and Gp S at 15 cycles showed clinically unacceptable roughness threshold (0.25 µm). Differences between subgroups for Ra and Rq values were significant (P≤0.05) with less differences within groups observed in early cycles (1, 10). Results validate the manufacturer's recommendations of using flash sterilization/10 cycles for EVE Diacera kit. CONCLUSIONS Repeated sterilization reduces efficacy of chairside adjustment kit to produce smooth surfaces on HTMLZ. This study recommends flash sterilization to a maximum of 10 times to get the clinically acceptable results of Ra and Rq.

Influence of Chairside Simulated Adjustment (Finishing and Polishing) Protocol and Chlorhexidine Mouthwash Immersion on Color Stability and Translucency of 2 and 3 Preshaded Multilayered Monolithic Zirconia.

BACKGROUND Preshaded monolithic zirconia (MLZ) is reported to have high translucency. This study aimed to assess the effect of chlorhexidine gluconate (ChG) mouthwash on color and translucency parameter (TP) of 2 different preshaded MLZ dental ceramics after clinical adjustment. MATERIAL AND METHODS Two MLZ disk-shaped specimens [NPM (Nacera Pearl Multi-Shade) (n=72) and CZM (Ceramill Zolid FX Multilayer)] (n=72) were simulated for clinical adjustment, finished, and polished using 2 adjustment kits [recommended kit, third-party kit: Diasynt Plus and SUN (n=12 each)] and later immersed in ChG mouthwash (Avohex) for 2 weeks. Difference in color (ΔE) and TP (Y) were calculated using the CIELab formula after measuring the coordinates (Lab) with a colorimeter. Individual changes in color and TP were assessed on the Clinical acceptance (perceptible) threshold (CAT/CPT) and Translucency perception threshold (TPT), respectively. Differences between the 2 ceramics were assessed using one-way ANOVA and post hoc tests, with all differences considered significant at P<0.05. RESULTS NPM and CZM differed in color at baseline despite having the same Vita shade combination. Between the 2 preshaded MLZ ceramics, NPM showed significant changes in color when adjusted with a third-party kit. Chlorhexidine produced changes in color and TP that were designated as clinically perceptible (ΔE=1.0 to 3.3) on the CAT/CPT and TPT scales, irrespective of the adjustment kit used. ChG produced the least or no changes in glazed MLZ specimens. CONCLUSIONS ChG mouthwash, whenever prescribed for preshaded MLZ restoration, should be adjusted prior to final glazing to avoid clinical adjustments that adversely affects color and translucency of the restoration.

Shear Strength of Repaired 3D-Printed and Milled Provisional Materials Using Different Resin Materials with and without Chemical and Mechanical Surface Treatment.

The aim of this study was to assess the shear bond strength of 3D-printed and milled provisional restorations using various resin materials and surface finishes. There were 160 preliminary samples in all, and they were split into two groups: the milled group and the 3D-printed group. Based on the resin used for repair (composite or polymethylmethacrylate (PMMA)) and the type of surface treatment utilized (chemical or mechanical), each group was further divided into subgroups. The specimens were subjected to thermocycling from 5 °C to 55 °C for up to 5000 thermal cycles with a dwell time of 30 s. The mechanical qualities of the repaired material underwent testing for shear bond strength (SBS). To identify the significant differences between the groups and subgroups, a statistical analysis was carried out. Three-way ANOVA was used to analyze the effects of each independent component (the material and the bonding condition), as well as the interaction between the independent factors on shear bond strength. Tukey multiple post-hoc tests were used to compare the mean results for each material under various bonding circumstances. The shear bond strengths of the various groups and subgroups differed significantly (p < 0.05). When compared to the milled group, the 3D-printed group had a much greater mean shear bond strength. When compared to PMMA repair, the composite resin material showed a noticeably greater shear bond strength. In terms of surface treatments, the samples with mechanical and chemical surface treatments had stronger shear bonds than those that had not received any. The results of this study demonstrate the effect of the fabrication method, resin type, and surface treatment on the shear bond strength of restored provisional restorations. Particularly when made using composite material and given surface treatments, 3D-printed provisional restorations showed exceptional mechanical qualities. These results can help dentists choose the best fabrication methods, resin materials, and surface treatments through which to increase the durability and bond strength of temporary prosthesis.

Comparison of the Degree of Staining of Computer-Aided Design-Computer-Aided Manufacture (CAD-CAM) Ceramic Veneers by Green Tea, Coffee, and Coca-Cola Using a Digital Spectrophotometer.

BACKGROUND The majority of dental professionals currently recognize lithium disilicate E-max ceramic veneers as a the most widely used, conservative, and effective cosmetic materials in dentistry. This study aimed to compare the degree of surface changes - roughness (Ra), depth (Rz), and mean color changes (ΔE00) - of computer-aided design-computer-aided manufactured (CAD/CAM) ceramic veneers materials of varying thicknesses caused by staining by green tea, coffee, and Coca-Cola using digital spectrophotometer. MATERIAL AND METHODS This study was conducted at King Khalid University, College of Dentistry. Lithium disilicate glass ceramic (LDGC) material was used to create 60 rectangular slices using the CAD/CAM system. The material thickness and the type of beverage were measured. The specimens were immersed in beverages according to the manufacturer's instructions. Specimen description and tomography were completed with a 3D noncontact surface metrology using interferometry. The "VITA Easy-Shade" spectrophotometer was used to measure ΔE00. It was recorded after 2 weeks for different material thicknesses after immersing samples in green tea, coffee, and Coca-Cola staining materials.f RESULTS Significant changes in ceramic thickness were found in Ra and Rz of 0.07 and 1.00 mm after 14 days of staining. Coca-Cola showed a significant difference in Ra and Rz with 1.00 mm thickness measurement compared to the 0.07 mm group with ≤ of 0.05, which was considered statistically significant. Highest ΔE00 were recorded among samples stained by Coca-Cola, followed by coffee, for both thicknesses. CONCLUSIONS Those findings support previous studies using spectrophotometric analysis of staining of CAD-CAM ceramic veneers that Coca-Cola followed by coffee resulted in the greatest color ΔE00 change.

Accuracy Comparison between Robot-Assisted Dental Implant Placement and Static/Dynamic Computer-Assisted Implant Surgery: A Systematic Review and Meta-Analysis of In Vitro Studies.

Background and Objectives: The present systematic review and meta-analysis undertake a comparison of studies that examine the accuracy of robot-assisted dental implant placement in relation to static computer-assisted implant surgery (SCAIS), dynamic computer-assisted implant surgery (DCAIS), and freehand procedures. This study aims to provide a comprehensive understanding of the precision of robot-assisted dental implant placement and its comparative efficacy in relation to other placement techniques. Methods: The guidelines recommended by Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) were used to organize and compose this review. Four electronic databases (PubMed, Web of Science, Scopus, and Cochrane) were systematically searched for pertinent articles. Articles were selected following the inclusion and exclusion criteria. Qualitative and quantitative analyses of the selected articles were performed. Results: The initial electronic search resulted in 1087 hits. Based on the inclusion and exclusion criteria, five articles were selected for qualitative analysis, out of which three were considered for quantitative analysis. Three parameters were considered for accuracy evaluation (angular, coronal, and apical deviation). The mean angular deviation was -1.22 degrees (95% CI, -1.06--1.39), the mean coronal deviation was -0.15 mm (95% CI, -0.24--0.07), and the mean apical deviation was -0.19 mm (95% CI, -0.27--0.10). Conclusions: The robotic implant system was found to have significantly lower angular deviations and insignificantly lower coronal and apical deviations compared to DCAIS. Within the limitations of this review, it can be concluded that robot-assisted implant placement in resin models permits higher accuracy compared to DCAIS and SCAIS systems. However, due to the limited number of comparative studies with high heterogeneity, the findings of this review should be interpreted with caution. Further research is necessary to confirm the clinical application of robotics in implant surgery.

Physical and Mechanical Properties of 3D-Printed Provisional Crowns and Fixed Dental Prosthesis Resins Compared to CAD/CAM Milled and Conventional Provisional Resins: A Systematic Review and Meta-Analysis.

Newly introduced provisional crowns and fixed dental prostheses (FDP) materials should exhibit good physical and mechanical properties necessary to serve the purpose of their fabrication. The aim of this systematic literature review and meta-analysis is to evaluate the articles comparing the physical and mechanical properties of 3D-printed provisional crown and FDP resin materials with CAD/CAM (Computer-Aided Designing/Computer-Aided Manufacturing) milled and conventional provisional resins. Indexed English literature up to April 2022 was systematically searched for articles using the following electronic databases: MEDLINE-PubMed, Web of Science (core collection), Scopus, and the Cochrane library. This systematic review was structured based on the guidelines given by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The focused PICO/PECO (Participant, Intervention/exposure, Comparison, Outcome) question was: 'Do 3D-printed (P) provisional crowns and FDPs (I) have similar physical and mechanical properties (O) when compared to CAD/CAM milled and other conventionally fabricated ones (C)'. Out of eight hundred and ninety-six titles, which were recognized after a primary search, twenty-five articles were included in the qualitative analysis, and their quality analysis was performed using the modified CONSORT scale. Due to the heterogeneity of the studies, only twelve articles were included for quantitative analysis. Within the limitations of this study, it can be concluded that 3D-printed provisional crown and FDP resin materials have superior mechanical properties but inferior physical properties compared to CAD/CAM milled and other conventionally fabricated ones. Three-dimensionally printed provisional crowns and FDP materials can be used as an alternative to conventional and CAD/CAM milled long-term provisional materials.

Clinical and Radiological Outcomes for Guided Implant Placement in Sites Preserved with Bioactive Glass Bone Graft after Tooth Extraction: A Controlled Clinical Trial.

The goal of the study was to evaluate marginal bone loss (MBL) after 1-year implant placement using a guided implant surgical (GIS) protocol in grafted sockets compared to non-grafted sites. We followed a parallel study design with patients divided into two groups: grafted group (Test group, n = 10) and non-grafted group (Control, n = 10). A bioactive glass bone graft was used for grafting. A single edentulous site with a minimum bone height ≥11 mm and bone width ≥6 mm confirmed by cone-beam computerized tomography (CBCT) was chosen for implant placement. Tapered hybrid implants that were sandblasted and acid-etched (HSA) were placed using the GIS protocol and immediately loaded with a provisional prosthesis. MBL and implant survival rates (ISR) were assessed based on standardized radiographs and clinical exams. Patients were followed up for 1-year post-loading. MBL after one year, in the control group, was −0.31 ± 0.11 mm (mesial) and −0.28 ± 0.09 mm (distal); and in the test group was −0.35 ± 0.11 mm (mesial) and −0.33 ± 0.13 mm (distal), with no statistical significance (p > 0.05). ISR was 100% in both groups after one year. ISR was similar between groups and the marginal bone changes were comparable one year after functional loading, without statistical significance, suggesting that bioactive glass permitted adequate bone formation. The GIS protocol avoided raising flaps and provided a better position to place implants, preserving the marginal bone around implants.

A Comparison in Patient Comfort Using Conventional Syringe and Needleless Jet Anesthesia Technique in Periodontal Surgery-A Split-Mouth Randomized Clinical Trial.

Background and Objectives: Periodontal surgery requires local anesthetic coverage to alleviate patient discomfort. Needles and injections can engender feelings of fear and anxiety in individuals. This study aimed to assess the level of comfort and anxiety in patients during the administration of local anesthesia using needleless jet anesthesia (JA) when compared to a conventional syringe (CS) in periodontal surgery. Method and Materials: 60 sites were designated for injection in a split-mouth design in 30 subjects who required periodontal surgery. Local anesthesia was administered in two appointments scheduled one week apart using either a JA system or a CS. The Visual Analogue Scale (VAS), Verbal Rating Scale (VRS), and Beck's anxiety inventory were used to report the pain and anxiety levels while injecting local anesthesia. Statistical analysis of the results was performed using the Shapiro-Wilks test and Paired t-test. Results: Patients reported greater comfort with JA. The VAS and VRS values were statistically significant-(p = 0.003) and (p = 0.001), respectively. Patients showed fear and were nervous about receiving a local anesthetic using a CS. A few subjects experienced lingering pain with the CS, whereas greater comfort and no lingering soreness were reported post-operatively at the site of JA administration. Conclusions: This study provides the first comprehensive assessment of using JA for periodontal surgical procedures. Lower pain scores were consistently observed with the use of jet injectors. Patients were at ease and reported lesser anxiety and greater comfort with jet injectors, making it ideally suited for providing local anesthesia in periodontal surgery.

Antimicrobial Efficacy of Different Pulp-Capping Materials against Enterococcus faecalis: An In vitro Study.

AIM: The present study aims to assess the antimicrobial action of three different pulp-capping agents against Enterococcus faecalis. MATERIALS AND METHODS: Three pulp-capping agents were chosen for this study: Calcicur, mineral trioxide aggregate (MTA)-Angelus, and Dycal. The zone of inhibition produced by these three pulp-capping agents was measured at 24 h and 72 h to assess their antimicrobial efficacy against E. faecalis. The agar diffusion method was used to examine the antimicrobial effect of pulp-capping agents. Mueller-Hinton agar plates were used to inoculate the microorganisms. Analysis of variance (ANOVA) and Tukey's post hoc tests were done to compare the different groups. P < 0.05 was considered as statistically significant. RESULTS: At 24 h, the highest zone of inhibition was found in MTA-Angelus (3.32 ± 0.11 mm), followed by Dycal (2.02 ± 0.46 mm) and Calcicur (1.84 ± 0.92 mm). After 72 h, MTA-Angelus demonstrated a zone of inhibition of 4.60 ± 0.22 mm, followed by Dycal (3.48 ± 0.74 mm) and Calcicur (2.90 ± 0.18 mm). ANOVA test showed a highly statistical significance. A statistically significant difference (P < 0.001) was shown between MTA-Angelus and Dycal. Calcicur did not show any significant difference. CONCLUSION: This trial found that the freshly mixed MTA-Angelus has a significantly superior antimicrobial effect against E. faecalis than Dycal and Calcicur.

Assessment of Marginal Integrity and Color Stability of Provisional Restoration Fabricated from Different Autopolymerizing Acrylic Resins - A Comparative Study.

AIM: The present study is aimed to assess the marginal integrity and color stability of provisional restorations fabricated from different autopolymerizing acrylic resins. MATERIALS AND METHODS: Totally, 60 provisional crowns were fabricated. A mandibular first molar artificial typodont was mounted on a base of dental stone. The mandibular first molar which was mounted was prepared for full cast crown, using the tooth preparation standard principles with shoulder finish line of 1 mm and taper 6°. There were 20 samples in each acrylic resin group: Group I: Polyvinyl-ethyl methacrylate resin, Group II: Autopolymerizing bis-acrylic material, and Group III: Polymethyl methacrylate (PMMA) autopolymerizing resin. Crowns were verified for marginal adaptation using stereomicroscope at a ×40. The color stability was measured using spectrophotometer poststaining period. RESULTS: Autopolymerizing bis-acrylic material group showed minimum mean vertical marginal discrepancy (128.68 ± 18.036 µm) followed by PMMA autopolymerizing resin group (147.49 ± 20.128 µm) and polyvinyl-ethyl methacrylate resin group (172.89 ± 22.118 µm). Analysis of variance demonstrated a statistically significant difference between different autopolymerizing acrylic resins. The color change values did not show any significant difference between the groups on numerous comparisons between different autopolymerizing acrylic resin groups. A statistically significant difference was seen between Groups I and II, Groups I and III, and Groups II and III (P < 0.05). CONCLUSION: This study concluded that the autopolymerizing bis-acrylic material demonstrated significantly improved marginal integrity when compared to PMMA autopolymerizing resin and polyvinyl-ethyl methacrylate resin.

Examining the Effect of Gravity on Different Irrigation Systems: An In vitro Study.

BACKGROUND: Periapical extrusion is frequently observed during endodontic therapy. It can lead to acute injury of periapical tissues, resulting in interappointment pain or swelling. The effect is pronounced in teeth with immature apex, which are more susceptible to the extrusion of irrigant. The aim of this study was to evaluate the effect of gravity on the apical extrusion of irrigating solution with different irrigation protocols in single-rooted premolars. METHODOLOGY: A total number of 80 permanent single-rooted teeth (premolars) with same working length (WL) were divided into two main groups: Group A: Penetration depth of irrigation syringe to 2 mm from the WL and Group B: Penetration depth of irrigation syringe to 4 mm from the WL. Each group was subdivided into four subgroups. (n = 10). The extruded debris and irrigants were weighed, and the data were statistically analyzed by the analysis of variance and the Tukey test. RESULTS: Irrespective of the irrigation technique used, the amount of irrigant extruded from the apex showed a statistically significant difference related to the effect of gravity (P < 0.05). There was no statistically significant difference observed between irrigation methods (P > 0.05). CONCLUSION: The degree of apical extrusion of irrigant was dependent on the type of irrigation technique and gravity. Greater caution should be taken during irrigation to prevent postoperative pain.

Prevention of pancreatic cancer in a hamster model by cAMP decrease.

Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). We have previously shown that these cancers overexpressed stress neurotransmitters and cyclic adenosine monophosphate (cAMP) while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) was suppressed. Using a hamster model, the current study has tested the hypothesis that cAMP decrease by GABA supplementation in the drinking water prevents the development of pancreatitis-associated PDAC. Our data reveal strong preventive effects of GABA supplementation on the development of PDAC and pancreatic intraductal neoplasia (PanIN). ELISA assays and immunohistochemistry revealed significant decreases in the levels of cAMP and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers and phosphorylated signaling proteins, which stimulate cell proliferation, and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP decrease by GABA supplementation inhibits multiple cancer stimulating pathways in cancer stem cells, differentiated cancer cells and the immune system, identifying this approach as promising novel tool for the prevention of PDAC in individuals with a history of smoking and alcoholism.

Nicotine induces self-renewal of pancreatic cancer stem cells via neurotransmitter-driven activation of sonic hedgehog signalling.

A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects.

Differential modulation of nicotine-induced gemcitabine resistance by GABA receptor agonists in pancreatic cancer cell xenografts and in vitro.

BACKGROUND: Pancreatic cancer is frequently resistant to cancer therapeutics. Smoking and alcoholism are risk factors and pancreatic cancer patients often undergo nicotine replacement therapy (NRT) and treatment for alcohol dependence. Based on our report that low dose nicotine within the range of NRT causes gemcitabine resistance in pancreatic cancer, our current study has tested the hypothesis that GABA or the selective GABA-B-R agonist baclofen used to treat alcohol dependence reverse nicotine-induced gemcitabine resistance in pancreatic cancer. METHODS: Using mouse xenografts from the gemcitabine--sensitive pancreatic cancer cell line BXPC-3, we tested the effects of GABA and baclofen on nicotine-induced gemcitabine resistance. The levels of cAMP, p-SRC, p-ERK, p-AKT, p-CREB and cleaved caspase-3 in xenograft tissues were determined by ELISA assays. Expression of the two GABA-B receptors, metalloproteinase-2 and 9 and EGR-1 in xenograft tissues was monitored by Western blotting. Mechanistic studies were conducted in vitro, using cell lines BXPC-3 and PANC-1 and included analyses of cAMP production by ELISA assay and Western blots to determine protein expression of GABA-B receptors, metalloproteinase-2 and 9 and EGR-1. RESULTS: Our data show that GABA was as effective as gemcitabine and significantly reversed gemcitabine resistance induced by low dose nicotine in xenografts whereas baclofen did not. These effects of GABA were accompanied by decreases in cAMP, p-CREB, p-AKT, p-Src, p-ERK metalloproteinases-9 and -2 and EGR-1 and increases in cleaved caspase-3 in xenografts whereas baclofen had the opposite effects. In vitro exposure of cells to single doses or seven days of nicotine induced the protein expression of MMP-2, MMP-9 and EGR-1 and these responses were blocked by GABA. Baclofen downregulated the protein expression of GABA-B-Rs in xenograft tissues and in cells exposed to baclofen for seven days in vitro. This response was accompanied by inversed baclofen effects from inhibition of cAMP formation after single dose exposures to stimulation of cAMP formation in cells pretreated for seven days. CONCLUSIONS: These findings suggest GABA as a promising single agent for the therapy of pancreatic cancer and to overcome nicotine-induced gemcitabine resistance whereas treatment with baclofen may increase gemcitabine resistance.

Hydraulic fracturing: paving the way for a sustainable future?

With the introduction of hydraulic fracturing technology, the United States has become the largest natural gas producer in the world with a substantial portion of the production coming from shale plays. In this review, we examined current hydraulic fracturing literature including associated wastewater management on quantity and quality of groundwater. We conclude that proper documentation/reporting systems for wastewater discharge and spills need to be enforced at the federal, state, and industrial level. Furthermore, Underground Injection Control (UIC) requirements under SDWA should be extended to hydraulic fracturing operations regardless if diesel fuel is used as a fracturing fluid or not. One of the biggest barriers that hinder the advancement of our knowledge on the hydraulic fracturing process is the lack of transparency of chemicals used in the practice. Federal laws mandating hydraulic companies to disclose fracturing fluid composition and concentration not only to federal and state regulatory agencies but also to health care professionals would encourage this practice. The full disclosure of fracturing chemicals will allow future research to fill knowledge gaps for a better understanding of the impacts of hydraulic fracturing on human health and the environment.

Gamma-amino butyric acid (GABA) prevents the induction of nicotinic receptor-regulated signaling by chronic ethanol in pancreatic cancer cells and normal duct epithelia.

Pancreatic cancer has a high mortality rate and alcoholism is a risk factor independent of smoking. We have shown that nicotinic acetylcholine receptors (nAChR) regulate pancreatic ductal epithelia and pancreatic ductal adenocarcinoma (PDAC) cells in an autocrine fashion by stimulating their production of the stress neurotransmitters noradrenaline and adrenaline that signal through ß-adrenergic receptors (ß-AR). Our current study has investigated the modulation of this autocrine regulatory loop by chronic ethanol and explored the potential prevention of these effects by γ-amino butyric acid (GABA). Using MTT assays, cell migration assays, Western blotting, immunoassays, and gene knockdown of individual nAChRs in two PDAC cell lines and in immortalized human pancreatic duct epithelial cells, our data show that treatment for seven days with ethanol induced the protein expression and sensitivity of nAChRs α3, α5, and α7 resulting in increased production of noradrenaline and adrenaline, which drive proliferation and migration via cyclic AMP (cAMP)-dependent signaling downstream of ß-ARs. Treatment with GABA prevented all of these responses to chronic ethanol, reducing cell proliferation and migration below base levels in untreated cells. Our findings suggest that alcoholism induces multiple cAMP-dependent PDAC stimulating signaling pathways by upregulating the protein expression and sensitivity of nAChRs that regulate stress neurotransmitter production. Moreover, our data identify GABA as a promising agent for the prevention of PDAC in individuals at risk due to chronic alcohol consumption.

Celecoxib and GABA cooperatively prevent the progression of pancreatic cancer in vitro and in xenograft models of stress-free and stress-exposed mice.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is associated with high levels of psychological distress. We have shown that beta-adrenergic receptors (ß-ARs), which are activated by stress neurotransmitters, regulate PDAC cells via cyclic AMP (cAMP)-dependent signaling in vitro, that social stress promotes PDAC progression in mouse xenografts and that γ-aminobutyric acid (GABA) inhibits these responses in vitro and in vivo. The targeted inhibition of stress-induced regulatory pathways may abolish the potentially negative impact of psychological stress on clinical outcomes. Our current data show that chronic exposure of PDAC cell lines Panc-1 (activating point mutations in K-ras) and BXPC-3 (no mutations in K-ras) in vitro to the stress neurotransmitter epinephrine at the concentration (15 nM) previously measured in the serum of mice exposed to social stress significantly increased proliferation and migration. These responses were inhibited in a concentration-dependent manner by celecoxib. The effects of celecoxib alone and in combination with γ-aminobutyric acid (GABA) on the progression of subcutaneous mouse xenografts from the cell line (BXPC-3) most responsive to epinephrine were then investigated in the presence and absence of social stress. Cancer-stimulating factors (VEGF & prostaglandin E(2) [PGE(2)]) and levels of cAMP were measured by immunoassays in blood and xenograft tissue. Phosphorylation of the signaling proteins ERK, CREB, Src, and AKT was assessed by ELISA assays and Western blotting. Expression of COX-2, 5-lipoxygenase, and p-5-LOX were determined by semi-quantitative Western blotting. Celecoxib alone significantly inhibited xenograft progression and decreased systemic and tumor VEGF, PGE2, and cAMP as well as phosphorylated signaling proteins in stress-exposed and stress-free mice. These responses were significantly enhanced by co-treatment with GABA. The celecoxib-induced downregulation of COX-2 protein and p-5-LOX was also significantly enhanced by GABA under both experimental conditions. Our findings identify the targeted inhibition of stress-induced pathways as a promising area for more effective cancer intervention in pancreatic cancer.

Effects of chronic nicotine on the autocrine regulation of pancreatic cancer cells and pancreatic duct epithelial cells by stimulatory and inhibitory neurotransmitters.

Pancreatic ductal adenocarcinoma (PDAC) has a mortality rate near 100%. Smoking is a documented risk factor. However, the mechanisms of smoking-associated pancreatic carcinogenesis are poorly understood. We have shown that binding of nicotine to nicotinic acetylcholine receptors (nAChRs) expressing subunits α7, α3 and α5 in PDAC and pancreatic duct epithelial cells in vitro triggered the production of the neurotransmitters noradrenaline and adrenaline by these cells. In turn, this autocrine catecholamine loop significantly stimulated cell proliferation via cyclic adenosine 3',5'-monophosphate-dependent signaling downstream of beta-adrenergic receptors. However, the observed responses only represent acute cellular reactions to single doses of nicotine whereas nicotine exposure in smokers is chronic. Using the PDAC cell lines BxPC-3 and Panc-1 and immortalized pancreatic duct epithelial cell line HPDE6-C7, our current experiments reveal a significant sensitization of the nAChR-driven autocrine catecholamine regulatory loop in cells pre-exposed to nicotine for 7 days. The resulting increase in catecholamine production was associated with significant inductions in the phosphorylation of signaling proteins ERK, CREB, Src and AKT, upregulated protein expression of nAChR subunits α3, α4, α5 and α7 and increased responsiveness to nicotine in 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and cell migration assays. All three cell lines produced the inhibitory neurotransmitter γ-aminobutyric acid, an activity inhibited by gene knockdown of the α4ß2nAChR and suppressed by chronic nicotine via receptor desensitization. All of the observed adverse effects of chronic nicotine were reversed by treatment of the cells with γ-aminobutyric acid, suggesting the potential usefulness of this agent for the improvement of PDAC intervention strategies in smokers.

Intercepting neoplastic progression in lung malignancies via the beta adrenergic (ß-AR) pathway: implications for anti-cancer drug targets.

The understanding of signaling cascades involved in the induction, promotion, and progression of cancer, although advanced in recent years, is still incomplete. Tracing the imbalance of the impaired, physiologically-essential cellular signaling that drives the neoplastic process is a complex issue. This review discusses the role of the regulator of the fight or flight response, the beta-adrenergic signaling cascade, as a mediator of cancer growth and progression in in vitro and in vivo cancer models. We review a series of experiments from our own laboratory and those of others examining the contribution of this signaling network to lung and other human malignancies and thereby identifying potential targets for chemotherapeutic interventions. The stimulation of the ß-adrenergic receptor by lifestyle and environmental factors, as well as a preexisting risk for neoplasm, activates downstream effector molecules (adenylyl cyclase/cAMP/PKA/CREB) concomitant to the transactivation of related pathways (EGFR) that lead to pro-oncogenic signaling; this ß-adrenergic pathway thereby encourages cancer growth by evasion of apoptosis, invasion, angiogenesis, and metastasis. GABAergic signaling acts as an antagonist to the ß-adrenergic cascade by intercepting adenylyl cyclase activation, and thereby neutralizing the pro-oncogenic effects of ß-adrenergic stimulation. The regulation of cancer cell growth by neurobiological signals expands the possibilities for pharmacological interventions in cancer therapy.

Cooperative regulation of non-small cell lung carcinoma by nicotinic and beta-adrenergic receptors: a novel target for intervention.

Lung cancer is the leading cause of cancer death; 80-85% of lung cancer cases are non-small cell lung cancer (NSCLC). Smoking is a documented risk factor for the development of this cancer. Although nicotine does not have the ability to initiate carcinogenic events, recent studies have implicated nicotine in growth stimulation of NSCLC. Using three NSCLC cell lines (NCI-H322, NCI-H441 and NCI-H1299), we identified the cooperation of nicotinic acetylcholine receptors (nAChRs) and ß-adrenergic receptors (ß-ARs) as principal regulators of these effects. Proliferation was measured by thymidine incorporation and MTT assays, and Western blots were used to monitor the upregulation of the nAChRs and activation of signaling molecules. Noradrenaline and GABA were measured by immunoassays. Nicotine-treated NSCLC cells showed significant induction of the α7nAChR and α4nAChR, along with significant inductions of p-CREB and p-ERK1/2 accompanied by increases in the stress neurotransmitter noradrenaline, which in turn led to the observed increase in DNA synthesis and cell proliferation. Effects on cell proliferation and signaling proteins were reversed by the α7nAChR antagonist α-BTX or the ß-blocker propranolol. Nicotine treatment also down-regulated expression of the GABA synthesizing enzyme GAD 65 and the level of endogenous GABA, while treatment of NSCLC cells with GABA inhibited cell proliferation. Interestingly, GABA acts by reducing ß-adrenergic activated cAMP signaling. Our findings suggest that nicotine-induced activation of this autocrine noradrenaline-initiated signaling cascade and concomitant deficiency in inhibitory GABA, similar to modulation of these neurotransmitters in the nicotine-addicted brain, may contribute to the development of NSCLC in smokers. Our data suggest that exposure to nicotine either by tobacco smoke or nicotine supplements facilitates growth and progression of NSCLC and that pharmacological intervention by ß blocker may lower the risk for NSCLC development among smokers and could be used to enhance the clinical outcome of standard cancer therapy.