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Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II–induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy. Cancer Res; 73(4); 1328–39. ©2012 AACR.

PDF file - 490K, Figure S1: Cell line authentication data Figure S2: Full screen western blots for ChAT, VAChT, AChE and CHT1 Figure S3: Immunohistochemistry of ChAT and VAChT in normal human lung tissues Figure S4: Nicotine-induced ACh production in human BAC requires nAChRs, VAChT, and CHT1 Figure S5: VAChT and ChAT are expressed on human BACs Figure S6: Transfection of VAChT siRNA and Sigma-R siRNA suppress the expression of these proteins in human BACs Figure S7: Effect of VAChT siRNA on the apoptotic activity of vesamicol in nicotine-treated BACs Figure S8: Overexpression of pcDNA3-HA-Akt-CA in A549 and H358 human BAC cells

Until recently, NADPH oxidase (NOX) enzymes were thought to be a property of multicellularity, where the reactive oxygen species (ROS) produced by NOX acts in signaling processes or in attacking invading microbes through oxidative damage. We demonstrate here that the unicellular yeast and opportunistic fungal pathogen Candida albicans is capable of a ROS burst using a member of the NOX enzyme family, which we identify as Fre8. C. albicans can exist in either a unicellular yeast-like budding form or as filamentous multicellular hyphae or pseudohyphae, and the ROS burst of Fre8 begins as cells transition to the hyphal state. Fre8 is induced during hyphal morphogenesis and specifically produces ROS at the growing tip of the polarized cell. The superoxide dismutase Sod5 is co-induced with Fre8 and our findings are consistent with a model in which extracellular Sod5 acts as partner for Fre8, converting Fre8-derived superoxide to the diffusible H2O2 molecule. Mutants of fre8Δ/Δ exhibit a morphogenesis defect in vitro and are specifically impaired in development or maintenance of elongated hyphae, a defect that is rescued by exogenous sources of H2O2. A fre8Δ/Δ deficiency in hyphal development was similarly observed in vivo during C. albicans invasion of the kidney in a mouse model for disseminated candidiasis. Moreover C. albicans fre8Δ/Δ mutants showed defects in a rat catheter model for biofilms. Together these studies demonstrate that like multicellular organisms, C. albicans expresses NOX to produce ROS and this ROS helps drive fungal morphogenesis in the animal host., Author summary We demonstrate here that the opportunistic human fungal pathogen Candida albicans uses a NADPH oxidase enzyme (NOX) and reactive oxygen species (ROS) to control morphogenesis in an animal host. C. albicans was not previously known to express NOX enzymes as these were thought to be a property of multicellular organisms, not unicellular yeasts. We describe here the identification of C. albicans Fre8 as the first NOX enzyme that can produce extracellular ROS in a unicellular yeast. C. albicans can exist as either a unicellular yeast or as multicellular elongated hyphae, and Fre8 is specially expressed during transition to the hyphal state where it works to produce ROS at the growing tip of the polarized cell. C. albicans cells lacking Fre8 exhibit a deficiency in elongated hyphae during fungal invasion of the kidney in a mouse model for systemic candidiasis. Moreover, Fre8 is required for fungal survival in a rodent model for catheter biofilms. These findings implicate a role for fungal derived ROS in controlling morphogenesis of this important fungal pathogen for public health.

The dietary compound capsaicin is responsible for the "hot and spicy" taste of chili peppers and pepper extracts. It is a valuable pharmacological agent with several therapeutic applications in controlling pain and inflammation. Emerging studies show that it displays potent anti-tumor activity in several human cancers. On a more basic research level, capsaicin has been used as a ligand to activate several types of ion-channel receptors. The pharmacological activity of capsaicin-like compounds is dependent on several factors like the dose, the route of administration and most importantly on its concentration at target tissues. The present review describes the current knowledge involving the metabolism and bioavailability of capsaicinoids in rodents and humans. Novel drug delivery strategies used to improve the bioavailability and therapeutic index of capsaicin are discussed in detail. The generation of novel capsaicin-mimetics and improved drug delivery methods will foster the hope of innovative applications of capsaicin in human disease.

NADPH oxidase (NOX) enzymes, which produce reactive oxygen species (ROS), were until recently thought to occur only in multicellular organisms. ROS have important physiological effects such as acting as signaling molecules in biological processes or as defense mechanisms against invading microbes through oxidative damage. In this work, we demonstrate that the unicellular fungus Candida albicans is capable of producing an extracellular ROS burst using a member of the NOX enzyme family identified as Fre8. C. albicans is an opportunistic human fungal pathogen which undergoes morphologic transition between yeast, pseudohyphal, and hyphal forms. During morphogenesis, FRE8 is induced to produce ROS specifically at the growing tip of the polarized cell. The extracellular superoxide dismutase, Sod5, is also induced during morphogenesis and our findings are consistent with a model in which Sod5 acts as a partner for Fre8, converting Fre8-derived superoxide to the diffusible hydrogen peroxide molecule. Fre8∆/∆ mutants exhibit a morphogenesis defect in vitro and are specifically impaired in the development or maintenance of elongated hyphae in high density cultures. A fre8∆/∆ defect in hyphal formation was similarly observed in a disseminated candidiasis model. Moreover, in a rat catheter model, C. albicans fre8∆/∆ mutants showed deficiencies in biofilm formation. Together these studies demonstrate that like multicellular organisms, the unicellular C. albicans expresses NOX to produce ROS and this signal helps drive fungal morphogenesis in the animal host.

Clinical studies show that the development of lung adenocarcinomas (LACs) is correlated with smoking habits. Nicotine, the addictive component of cigarettes, accelerates human LAC proliferation thr...

Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II–induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy. Cancer Res; 73(4); 1328–39. ©2012 AACR.