Capsaicin, the pungent ingredient of chili peppers, displays potent anti-neoplastic activity in a wide array of human cancer cells. The present manuscript examines the signaling pathways underlying the apoptotic activity of capsaicin in human small cell lung cancer (SCLC) in vitro and in vivo. Studies in neuronal cells show that capsaicin exerts its biological activity via the transient receptor potential vanilloid (TRPV) superfamily of cation-channel receptors. The TRPV family is comprised of six members (TRPV1-6). Capsaicin is an agonist of the TRPV1 receptor. We observed that capsaicin-induced apoptosis in human SCLC cells was mediated via the TRPV receptor family; however it was independent of TRPV1. Surprisingly, the apoptotic activity of capsaicin required the TRPV6 receptor. Depletion of TRPV6 receptor by siRNA methodology abolished the apoptotic activity of capsaicin in SCLC cells. Immunostaining and ELISA showed that TRPV6 receptor was robustly expressed on human SCLC tissues (from patients) and SCLC cell lines but almost absent in normal lung tissues. This correlates with our results that capsaicin induced very little apoptosis in normal lung epithelial cells. The proapoptotic activity of was mediated by the intracellular calcium and calpain pathway. The treatment of human SCLC cells with capsaicin induced increased the activity of calpain 1 and 2 by three-fold relative to untreated SCLC cells. Such calpain activation, in response to capsaicin, was downstream of the TRPV6 receptor. Taken together, our data provide insights into the mechanism underlying the apoptotic activity of capsaicin in human SCLCs.

BackgroundSmall cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vitro. However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo.Methodology/Principal FindingsBrdU assays and PCNA ELISAs showed that capsaicin displays robust anti-proliferative activity in four human SCLC cell lines. Furthermore, capsaicin potently suppressed the growth of H69 human SCLC tumors in vivo as ascertained by CAM assays and nude mice models. The second part of our study attempted to provide insight into molecular mechanisms underlying the anti-proliferative activity of capsaicin. We found that the anti-proliferative activity of capsaicin is correlated with a decrease in the expression of E2F-responsive proliferative genes like cyclin E, thymidylate synthase, cdc25A and cdc6, both at mRNA and protein levels. The transcription factor E2F4 mediated the anti-proliferative activity of capsaicin. Ablation of E2F4 levels by siRNA methodology suppressed capsaicin-induced G1 arrest. ChIP assays demonstrated that capsaicin caused the recruitment of E2F4 and p130 on E2F-responsive proliferative promoters, thereby inhibiting cell proliferation.Conclusions/SignificanceOur findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs.

Background: Nicotine promotes the proliferation of human squamous cell lung cancer (SCC-L) via the α7-nicotinic receptor (nAChR).Results: Nicotine increases α7-nAChR expression via transcriptional mechanisms involving Sp1 and GATA proteins.Conclusion: Nicotine-induced up-regulation of α7-nAChR accelerates the growth of human SCC-L.Significance: SCC-L patients exposed to nicotine display fast growing lung tumors and worse clinical outcomes.

The neurotransmitter acetylcholine (ACh) acts as an autocrine growth factor for human lung cancer. Several lines of evidence show that lung cancer cells express all of the proteins required for the uptake of choline (choline transporter 1, choline transporter-like proteins) synthesis of ACh (choline acetyltransferase, carnitine acetyltransferase), transport of ACh (vesicular acetylcholine transport, OCTs, OCTNs) and degradation of ACh (acetylcholinesterase, butyrylcholinesterase). The released ACh binds back to nicotinic (nAChRs) and muscarinic receptors on lung cancer cells to accelerate their proliferation, migration and invasion. Out of all components of the cholinergic pathway, the nAChR-signaling has been studied the most intensely. The reason for this trend is due to genome-wide data studies showing that nicotinic receptor subtypes are involved in lung cancer risk, the relationship between cigarette smoke and lung cancer risk as well as the rising popularity of electronic cigarettes considered by many as a "safe" alternative to smoking. There are a small number of review articles which review the contribution of the other cholinergic proteins in the pathophysiology of lung cancer. The primary objective of this review article is to discuss the function of the acetylcholine-signaling proteins in the progression of lung cancer. The investigation of the role of cholinergic network in lung cancer will pave the way to novel molecular targets and drugs in this lethal malignancy.

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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 a7-, a3b2-, and b3-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. Ó2012 AACR.

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