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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.

Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histologic subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome short hairpin RNA (shRNA) screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC., (©2012 AACR.)

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, exerts a wide range of activities in cellular redox control, antioxidant function, cell viability, and proliferation. Recently, the selenocysteine (Sec)-containing mammalian TrxR has emerged as a new target for anticancer drug development because TrxR and Trx are overexpressed in many aggressive tumors and the tumor cells seem to be more dependent on Trx system than normal cells. Here we have investigated the inhibition of mammalian TrxR by flavonoids which have been presumed to be cancer chemoprevention agents because of their antioxidant activities. Myricetin and quercetin were found to have strong inhibitory effects on mammalian TrxRs with IC50 values of 0.62 and 0.97 micromol/L, respectively. The inhibition was shown to be concentration, NADPH, and time dependent and involved an attack on the reduced COOH-terminal -Cys-Sec-Gly active site of TrxR. Oxygen-derived superoxide anions enhanced the inhibitory effect whereas anaerobic conditions attenuated inhibition. Spectral analysis suggested that the flavonols might perform their inhibitory effects via semiquinone radicals. Additionally, the flavonols had the potential to inhibit the growth of A549 cells with the same potency as inhibition of TrxR. TrxR activity in the cell lysates was reduced on treatment with myricetin >50 micromol/L, which coincided with the oxidization of Trx. The cell cycle was arrested in S phase by quercetin and an accumulation of cells in sub-G1 was observed in response to myricetin. Thus, the anticancer activity of quercetin and myricetin may be due to inhibition of TrxR, consequently inducing cell death.

The antitumor efficiency of secondary lymphoid organ chemokine (SLC), a CC chemokine that chemoattracts both dendritic cells (DCs) and T lymphocytes,was evaluated in SV40 large T-antigen transgenic mice that develop bilateral multifocal pulmonary adenocarcinomas. Injection of recombinant SLC in the axillary lymph node region led to a marked reduction in tumor burden with extensive lymphocytic and DC infiltration of the tumors and enhanced survival. SLC injection led to significant increases in CD4 and CD8 lymphocytes as well as DC at the tumor sites, lymph nodes, and spleen. The cellular infiltrates were accompanied by the enhanced elaboration of Type 1 cytokines and the antiangiogenic chemokines IFN-gamma inducible protein 10, and monokine induced by IFN-gamma (MIG). In contrast, lymph node and tumor site production of the immunosuppressive cytokine transforming growth factor beta was decreased in response to SLC treatment. In vitro, after stimulation with irradiated autologous tumor, splenocytes from SLC-treated mice secreted significantly more IFN-gamma and granulocyte macrophage colony-stimulating factor, but reduced levels of interleukin 10. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for additional evaluation of SLC in regulation of tumor immunity and its use in lung cancer immunotherapy.