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Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnoses have lower response rates and shorter median survival compared with patients who stop smoking. To provide insight into the biologic basis for these clinical observations, we tested whether two tobacco components, nicotine or the tobacco-specific carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), could activate the Akt pathway and increase lung cancer cell proliferation and survival. Nicotine or NNK, rapidly and potently, activated Akt in non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) cells. Nicotinic activation of Akt increased phosphorylation of multiple downstream substrates of Akt in a time-dependent manner, including GSK-3, FKHR, tuberin, mTOR and S6K1. Since nicotine or NNK bind to cell surface nicotinic acetylcholine receptors (nAchR), we used RT-PCR to assess expression of nine alpha and three beta nAchR subunits in five NSCLC cell lines and two types of primary lung epithelial cells. NSCLC cells express multiple nAchR subunits in a cell line-specific manner. Agonists of alpha3/alpha4 or alpha7 subunits activated Akt in a time-dependent manner, suggesting that tobacco components utilize these subunits to activate Akt. Cellular outcomes after nicotine or NNK administration were also assessed. Nicotine or NNK increased proliferation of NSCLC cells in an Akt-dependent manner that was closely linked with changes in cyclin D1 expression. Despite similar induction of proliferation, only nicotine decreased apoptosis caused by serum deprivation and/or chemotherapy. Protection conferred by nicotine was NFkappaB-dependent. Collectively, these results identify tobacco component-induced, Akt-dependent proliferation and NFkappaB-dependent survival as cellular processes that could underlie the detrimental effects of smoking in cancer patients.

Human melanoma cells frequently express CC chemokine receptor (CCR)10, a receptor whose ligand (CCL27) is constitutively produced by keratinocytes. Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors. In vitro, exposure of tumor cells to CCL27 led to rapid activation of Akt, resistance to cell death induced by melanoma antigen-specific cytotoxic T cells, and phosphatidylinositol-3-kinase (PI3K)–dependent protection from apoptosis induced by Fas cross-linking. In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells. We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression.

We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H 2 O 2 ), induces apoptosis of human airway epithelial (HAE) cells. Under oxidant exposure, a lung sphingomyelinase (SMase) is activated and displays continued ceramide generation and proapoptotic signaling, thus leading to the pathological apoptosis that causes lung injury. In a search for a specific SMase that is modulated by oxidative stress, we recently cloned nSMase2 from monkey lung tissue and HAE cells. Here, we show that this nSMase2 is up-regulated by an oxidant (H 2 O 2 ) and is inhibited by an antioxidant (glutathione (GSH)). Moreover, nSMase2 subcellular localization is governed by oxidant exposure, which leads to its preferential trafficking to the plasma membrane, where it generates ceramide and induces apoptosis. On the other hand, exposure to GSH results in nSMase2 trafficking to the nucleus, where it neither generates ceramide nor induces apoptosis. KeywordsNeutral sphingomyelinase 2; Apoptosis; Hydrogen peroxide; Glutathione; Ceramide; Lung Reactive oxidants such as hydrogen peroxide (H 2 O 2 ) cause lung injury and contribute to pulmonary diseases mainly through targeting human airway epithelial (HAE) cells. Yet, the cellular and molecular mechanisms that link these reactive oxidants to the development of lung injury and disease are not fully understood. We have shown that HAE cells exposed to reactive oxygen species (ROS) are stimulated to generate excessive ceramide, which functions as a potent inducer of apoptosis in these cells [1][2][3][4][5][6]. Although antioxidant defenses are constitutively expressed inmammalian cells [7], additional responses are mounted when the amount of environmental oxidants exceeds a threshold level, thereby becoming a threat to overall tissue integrity. Apoptosis is one such cellular adaptive response [8][9][10][11].The pro-apoptotic effects of ceramide are mediated by a variety of mechanisms [12][13][14][15]. Ceramide is synthesized primarily through a de novo pathway involving serine palmitoyl-☆ Abbreviations: BSA, bovine serum albumin; BSO, L-buthionine-SRsulfoximine; DAPI, 4′,6-diamidino-2-phenylindole; GSH, glutathione; H 2 O 2 , hydrogen peroxide; HAE, human airway epithelial; nSMase, neutral sphingomyelinase; ROS, reactive oxygen species; siRNA, small interfering RNA. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptCoA transferase and ceramide synthase or from membrane sphingomyelin breakdown by sphingomyelinases (SMases). Sphingomyelin (N-acylsphingosin-1-phosphocholine) is a phospholipid preferentially concentrated in the plasma membrane of mammalian cells [16]. Sphingomyelin catabolism occurs via the action of sphingomyelin-specific forms of phospholipase C termed sphingomyelinases (SMases), which hydrolyze the phosphodiester bond of sphingomyelin, yielding ceramide and phosphorylcholine. Ceramide then serves as a second messenger, leading to apoptotic DNA degradation. The main forms of SMases are distinguished by ...

ABSTRACT

Achondroplasia (ACH), the most common form of human dwarfism, is caused by an activating autosomal dominant mutation in the fibroblast growth factor receptor-3 gene. Genetic overexpression of C-type natriuretic peptide (CNP), a positive regulator of endochondral bone growth, prevents dwarfism in mouse models of ACH. However, administration of exogenous CNP is compromised by its rapid clearance in vivo through receptor-mediated and proteolytic pathways. Using in vitro approaches, we developed modified variants of human CNP, resistant to proteolytic degradation by neutral endopeptidase, that retain the ability to stimulate signaling downstream of the CNP receptor, natriuretic peptide receptor B. The variants tested in vivo demonstrated significantly longer serum half-lives than native CNP. Subcutaneous administration of one of these CNP variants (BMN 111) resulted in correction of the dwarfism phenotype in a mouse model of ACH and overgrowth of the axial and appendicular skeletons in wild-type mice without observable changes in trabecular and cortical bone architecture. Moreover, significant growth plate widening that translated into accelerated bone growth, at hemodynamically tolerable doses, was observed in juvenile cynomolgus monkeys that had received daily subcutaneous administrations of BMN 111. BMN 111 was well tolerated and represents a promising new approach for treatment of patients with ACH.

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