Drosophila melanogaster is an attractive model of familial Parkinson's disease, as flies with loss-of-function mutations of the parkin gene exhibit many pathologies observed in PD patients. Progressive motor deficits found in homozygous parkin mutants seem to result from mitochondrial pathology that causes indirect flight muscle and dopaminergic neuronal degeneration [1,2]. We have found that heterozygous parkin mutants have decreased lifespan, generally progressive motor dysfunction and olfactory deficits compared to control flies, suggesting that mutation of this gene produces a dominant phenotype. Tobacco smokers are dose-dependently less likely to develop PD [3,4]; subsequent in vitro and in vivo studies show that nicotine is protective in models of sporadic PD [6]. Literature addressing the potential protection by nicotine in Parkin loss-of-function models spans limited concentrations and selected time points in the organism's lifespan. We have found that parkin heterozygotes have late-onset climbing and flying deficits as well as decreased viability and olfactory deficits that precede motor defects. While chronic nicotine exposure decreases lifespan and climbing and flying abilities in control flies, it can improve viability and flying capability as well as rescue climbing and olfactory deficits in parkin heterozygotes. Dopaminergic neurons are spared in the parkin heterozygote, perhaps because this phenotype is less severe than in the homozygous parkin mutants. Nicotine pretreatment may be protective in sporadic PD patients and models; however, timely diagnosis remains to be an obstacle. Our results suggest that nicotine also may be protective in familial PD patients, who can be easily identified before motor symptoms occur.

We describe a patient who developed upper respiratory tract symptoms following long-term treatment of Wilson's disease with penicillamine. These symptoms were attributed to areas of pharyngeal thickening and were treated with a laser. Histological examination of the lesions showed proliferations of abnormal elastic fibres similar to those previously described at other sites, especially the skin, in patients receiving penicillamine. This drug impairs the maturation and reduces the stability of elastic fibres and although elastic tissue throughout the body is affected, we are aware of no previous reports of penicillamine- induced changes presenting with upper respiratory tract symptoms.

Strong epidemiological evidence and studies in models of Parkinson’s disease (PD) suggest that nicotine may be therapeutically beneficial in PD patients. However, a number of clinical trials utilizing nicotine in PD patients have had mixed results, indicating that either nicotine is not beneficial in PD patients, or an important aspect of nicotine therapy was absent. We hypothesized that nicotine must be administered early in the adult fly life in order to have beneficial effects. We show that continuous early nicotine administration improves both climbing and flight deficiencies present in homozygous park25 mutant PD model Drosophila melanogaster. Using a new climbing assay, we identify several climbing deficiencies in this PD model that are improved or rescued by continuous nicotine treatment. Amongst these benefits, it appears that nicotine improves the ability of the park25 flies to descend the climbing vial by being able to climb down more. In support of our hypothesis, we show that in order for nicotine benefits on climbing and flight to happen, nicotine administration must occur in a discrete time frame following adult fly eclosure: within one day for climbing or five days for flight. This therapeutic window of nicotine administration in this PD model fly may help to explain the lack of efficacy of nicotine in human clinical trials.

This study aimed to investigate pharmacokinetics of fluoxetine in horses and validate a method for liquid chromatography mass spectrometry analysis of serum levels. Fluoxetine pharmacokinetics were determined using 10 healthy, adult horses.Fluoxetine pharmacokinetics following a single oral dose (0.25 mg/kg) were determined using blood samples collected prior to and at several time points over 7 days following administration. Serum concentrations of fluoxetine and its bioactive metabolite norfluoxetine were measured using liquid chromatography coupled to an accurate mass/high-resolution mass spectrometer. Pharmacokinetic parameters were estimated using a noncompartmental model. Time to maximum serum concentration and serum half-life of fluoxetine was 1.5 and 15.6 h, respectively. Steady-state serum concentrations were evaluated using five horses each receiving fluoxetine (0.25 mg/ kg, PO, q24hrs) for 8 weeks and were found to be 62.9 ± 25.5 ng/ml on average. Norfluoxetine was not detected in any sample.

Introduction: Lung cancer is the leading cause of cancer-related death in the United States, with nearly 85% of cases histologically classified as non-small cell lung cancer (NSCLC). A recent epidemiological study provides evidence of an increased risk ratio for NSCLC in association with adherence to a Western dietary pattern, which has a higher dietary fat content, compared to vegetarian or Tex-Mex dietary patterns. Our laboratory and others have measured increased tumor burden in murine models of NSCLC when feeding diets are high in fat compared to a standard rodent cereal diet. In our laboratory, increased tumor burden is associated with increased tumor multiplicity, but no change in proliferative markers (Ki-67 and pS6) were observed. Dietary fat increases both tumorigenesis in mouse models of pancreatic cancer and circulating levels of the gastrointestinal hormone cholecystokinin (CCK). Inhibition of CCK receptors antagonizes progression of precursor lesions in murine models of pancreatic cancer. Although CCK receptor expression has not been detected in normal human pulmonary tissue, the CCK-B receptor has been identified immunohistologically in human lung tumors. Further, the CCK-A receptor is expressed in normal murine pulmonary epithelium. Hypothesis: At concentrations approximating circulating levels in mice fed a high fat diet, CCK reduces apoptosis of NSCLC cells in vitro. Materials and Methods: The murine NSCLC cell line IO33 was grown in serum-replete RPMI 1640 or serum-deprived RPM 1640 with 0 to 1000 pM exogenous CCK-8 peptide added. Expression of CCK-A and CCK-B receptors were measured in IO33 and human NSCLC cell lines by qPCR. Apoptosis was assessed using a Caspase-3/7 peptide cleavage assay. Results: Expression of CCK-A and CCK-B receptors are reported in IO33 cells and 11 human lung cancer cell lines. Apoptosis of IO33 cells was significantly decreased by greater than 30% at CCK concentrations greater than 100pM (P < 0.03), which is the circulating concentration of CCK in mice fed a high fat diet. Conclusions: CCK receptors are expressed in human and murine NSCLC cells and circulating CCK may reduce NSCLC cell apoptosis. Citation Format: Alexander C. Miller, Shelby J. Subry, Manijeh M. Assar, Krista Pearman, Jeffrey William Norris. Evaluation of cholecystokinin receptor expression and function in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1931.

Lung cancer is the leading cause of cancer-related mortality worldwide, and 85% of lung cancer cases are associated with tobacco use. Within this group, activating mutations in K-ras have been identified in ∼25% of lung adenocarcinomas. Using a mouse model of k-ras-driven lung tumorigenesis, we previously demonstrated that deletion of the IGF-1 gene or reduction of systemic IGF-1 levels using the antidiabetic drug metformin markedly reduced tumor burden. Since preclinical and clinical studies suggest that diet composition is the best predictor of IGF-1 levels, we hypothesized that diets high in fat or carbohydrate would promote lung tumorigenesis by increasing systemic IGF-1 levels. To assess the effect of diet on systemic IGF-1 levels, 9 week old C57Bl/6J and A/J mice were fed standard cereal, high-carbohydrate, or high-fat (HFD) diets for 12 weeks. At the conclusion of the study plasma, liver, and lung samples were collected. Compared to the cereal-fed control mice, IGF-1 and insulin levels were increased in both strains of mice only with HFD. Average body weight only increased for the C57Bl/6J group that was fed HFD. We investigated the effect of HFD on lung tumorigenesis using two mouse models of lung cancer. In the first, C57Bl/6LA2 mice, which are genetically modified with a K-ras mutation present in human smokers, were fed either cereal diet or HFD for 10 weeks following weaning. Lung tumor burden in the mice fed HFD was increased 2.7-fold compared to littermates fed cereal diet. In the second model, the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) was given by IP injection to A/J mice beginning at 6 weeks of age. This carcinogen causes lung tumor formation by inducing K-ras mutations. Following once weekly injections of NNK for 3 weeks, the mice were randomized to cereal diet or HFD. 10 weeks later, mice fed HFD were found to have a 60% increase in lung tumor burden. In both studies, there was no relationship between the final body weight of the mice and tumor burden. These studies show that HFD promotes lung tumor growth resulting from a mutation commonly observed in smokers. Therefore, dietary modification may slow the progression of tumorigenesis resulting from smoking-related genetic changes through IGF-1. Chemopreventative drugs, like metformin, may also have greater efficacy in a HFD model due to the increased insulin and IGF-1 associated with this model. Finally, understanding the molecular mechanisms by which HFD promotes tumor growth may help to identify new targets for cancer prevention. Citation Format: Jeffrey Norris, Krista Pearman, Regan Memmott, Kristin Lastwika, Joell Gills, Phillip Dennis. A high-fat diet promotes tumorigenesis in two mouse models of K-ras-driven lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4330.

Strong epidemiological evidence and studies in models of Parkinson’s disease (PD) suggest that nicotine may be therapeutically beneficial in PD patients. However, a number of clinical trials utilizing nicotine in PD patients have had mixed results, indicating that either nicotine is not beneficial in PD patients, or an important aspect of nicotine therapy was absent. Here we show that continuous early nicotine administration improves both climbing and flight deficiencies present in homozygous park25 mutant PD model Drosophila melanogaster. Using a new climbing assay, we identify several climbing deficiencies in this PD model that are improved or rescued by nicotine treatment. Amongst these benefits, it appears that nicotine improves the ability of the park25 flies to descend the climbing vial by being able to climb down more. Importantly, we show that in order for nicotine benefits on climbing and flight to happen, nicotine administration must occur in a discrete time frame following adult fly eclosure: within one day for climbing or five days for flight. This therapeutic window of nicotine administration may help to explain its lack of efficacy in human clinical trials, suggesting a need to test earlier nicotine therapy in PD patients.

Lung cancer is the leading cause of cancer-related mortality worldwide, and 85% of lung cancer cases are associated with tobacco use. Activating mutations in K-ras have been identified in ~25% of tobacco-associated lung adenocarcinomas. Using mouse models of K-ras-driven lung tumorigenesis, we previously demonstrated that deletion of the IGF-1 gene or reduction of systemic IGF-1 levels using the antidiabetic drug metformin markedly reduced tumor burden. Preclinical and clinical studies suggest that diet composition is the best predictor of IGF-1 levels. Therefore, we hypothesized that diets high in fat or carbohydrate would promote lung tumorigenesis by increasing systemic IGF-1 levels. To assess the effect of diet on systemic IGF-1 levels, 9 week old C57Bl/6J and A/J mice were fed standard cereal, high-carbohydrate, or high-fat (HFD) diets for 12 weeks. Compared to cereal-fed control mice, plasma IGF-1 and insulin levels were increased in both strains of mice fed a HFD, but not in mice fed a diet high in carbohydrate. This was not due to obesity, as only the C57Bl/6J mice fed a HFD had an increase in body weight. We then investigated the effect of HFD on lung tumorigenesis using two mouse models. In the first, C57Bl/6LA2 mice, which harbor a mutation in K-ras, were fed either cereal diet or HFD for 10 weeks following weaning. Lung tumor burden in the mice fed HFD was increased 2.7-fold compared to littermates fed cereal diet. In the second model, the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) was given by intraperitoneal injection to A/J mice beginning at 6 weeks of age. This carcinogen causes lung tumor development by inducing K-ras mutations. After 3 weekly injections of NNK, the mice were randomized to cereal diet or HFD for ten weeks. Mice fed HFD had a 60% increase in lung tumor burden. In both models, there was no relationship between the weight of the mice and lung tumor burden. Immunohistochemical analysis of the proliferation marker Ki-67 showed no significant difference in expression between tumors from mice fed a HFD or a cereal diet. Therefore, we evaluated tumor-infiltrating lymphocytes (TIL) from mice on both diets. Interestingly, both immunohistochemical analysis and flow cytometry demonstrated a 50% reduction in the number of TIL in mice fed HFD compared to mice fed a cereal diet. Additionally, HFD was associated with a 2-fold increase in PD-1+ CD4+ TIL, and the PD-1 staining intensity in these lymphocytes was significantly greater than in mice fed a cereal diet. These results may suggest that a high fat diet increases lung tumorigenesis by increasing systemic IGF-1 levels and by creating an immune-permissive tumor microenvironment. Citation Format: Regan M. Memmott, Krista Pearman, Joell Gills, Tony Tullot, Valerie Wong, Benjamin Singer, Kristin Lastwika, Franco D'Alessio, Phillip Dennis, Jeffrey William Norris. Increased NSCLC tumorigenesis in mice fed a high fat diet is associated with increased plasma IGF-1 levels and PD-1 expression in CD4+ tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2017-237