Cody A. Stover

Inhibition of Cholinergic Signaling Causes Apoptosis in Human Bronchioalveolar Carcinoma

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.

Abstract 1678: Bioavailability and anti-tumor activity of capsaicin in human small cell lung cancer

The nutritional compound capsaicin has been shown to display anti-neoplastic activity in breast, prostate and colon tumors xenografted in nude mice. Based on these data from other research laboratories, we wanted to determine the bioavailability of capsaicin in nude mice in vivo. We found that capsaicin was rapidly metabolized primarily in the liver. The bioavailability of intact capsaicin was highest in the lung. Therefore, we hypothesized that capsaicin should suppress the growth of lung tumors. We found that capsaicin induced robust apoptosis in human SCLC cell lines. Capsaicin decreased the growth rates of human SCLC tumors in two in vivo models, namely the CAM model and the nude mouse model. HPLC studies showed intact capsaicin in the tumors excised from nude mice. The heat-sensation activity of capsaicin is mediated by the transient receptor potential vanilloid (TRPV) family of proteins. Capsaicin functions as an agonist of the TRPV1 receptor. The apoptotic activity of capsaicin was found to be mediated by TRPV6 and not TRPV1. Preliminary data shows that capsaicin increases the expression of the TRPV6 receptor, causing apoptosis in human SCLCs. Citation Format: John D. Hurley, William D. Rollyson, Cody A. Stover, Kathleen C. Brown, Haley E. Perry, Cathryn D. Stevenson, Clayton M. Crabtree, Aaron M. Dom, Jamie K. Lau, Theodore R. Witte, W E. Hardman, Piyali Dasgupta. Bioavailability and anti-tumor activity of capsaicin in human small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1678. doi:10.1158/1538-7445.AM2015-1678

Abstract 3480: Combinatorial apoptotic activity of capsaicin and camptothecin in human small cell lung cancer

Small cell lung cancer (SCLC) accounts for about 13% of all lung cancer cases and is the most aggressive form of lung cancer. Cisplatin and cisplatin-based combination therapies are the cornerstone of SCLC treatment. Platinum refractoriness is defined as disease which has persisted or progressed (grown) while receiving cisplatin chemotherapy. Patients with platinum resistant or refractory disease have very limited options, as the only standard chemotherapy with an FDA-approved drug, topotecan, has an objective response rate of approximately 3% and little or no survival benefit. Topotecan is a derivative of camptothecin. The objective of our study was to examine whether capsaicin could sensitize human SCLC cells to the apoptotic effects of camptothecin. We observed that the combination of capsaicin and camptothecin displays significantly higher apoptotic activity in human SCLC cell lines as compared to either agent alone. Chou-Talalay analysis showed that the interactions between capsaicin and camptothecin were synergistic. We also measured the effect of capsaicin-camptothecin combination on several Bcl-2 family proteins, namely Bcl-2, Bax, Bak, Bcl-XL, Mcl-1, etc. The results of our studies may be of importance in therapy of platinum-refractory SCLCs. Citation Format: Haley E. Perry, Kathleen C. Brown, Cathryn D. Stevenson, William D. Rollyson, Cody A. Stover, Piyali Dasgupta. Combinatorial apoptotic activity of capsaicin and camptothecin in human small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3480. doi:10.1158/1538-7445.AM2015-3480

Abstract 1032: The acetylcholine signaling pathway: A novel molecular target for lung cancers

Cigarette smoking is a major risk factor for all types of lung cancers. Nicotine, the addictive component of cigarettes, accelerates the growth and angiogenesis of human lung cancers. The biological activity of nicotine is mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand of nAChRs is acetylcholine (ACh). We show that both human SCLCs and NSCLCs contain all proteins of the acetylcholine signaling pathway, namely nAChRs, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), choline transporter (ChT1) and acetylcholinesterase (AChE). ACh functions as an autocrine growth factor for human lung cancer cells. Lung adenocarcinoma (LAC), squamous cell carcinoma (SCC-L) and invasive mucinous adenocarcinoma (IMA) express a diverse array of nAChRs. In addition, normal human lung cells also express nAChRs and other ACh signaling proteins. Nicotine amplifies the ACh signaling loop in human lung cancer cells. It increases the levels of alpha7-nAChR subunit in human SCC-Ls. The alpha7-nAChR is responsible for the proliferative and pro-angiogenic activity of nicotine in lung cancer. The level of alpha7-nAChR was analyzed in human SCC-L samples isolated from patients. It was found that the level of alpha7-nAChR in SCC-L patients (who are heavy smokers) was much higher than that of moderate smoker suffering from SCC-Ls. Nicotine was also found to elevate the levels of ChAT and VAChT in human lung cancers. The acetylcholine signaling pathway may be a useful molecular target for the diagnosis and therapy of human lung cancers in smokers. Our results are also relevant to lung cancer patients who are exposed to nicotine via secondhand smoke, nicotine patches, gums or electronic cigarettes. Citation Format: Kathleen C. Brown, Jamie K. Lau, Haley E. Perry, Brent A. Thornhill, Cathryn D. Stevenson, William D. Rollyson, Cody A. Stover, Dennie V. Jones, Joseph F. Pulliam, Piyali Dasgupta. The acetylcholine signaling pathway: A novel molecular target for lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1032. doi:10.1158/1538-7445.AM2015-1032

Abstract 5229: Disruption of the acetylcholine signaling pathway suppresses the growth and angiogenesis of human lung cancers

Cigarette smoking bears a strong association with the development of lung cancer. Nicotine is the addictive component of cigarettes. Several convergent studies show that nicotine facilitates the growth and angiogenesis of human lung cancers. The biological activity of nicotine is mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand of nAChRs is acetylcholine (ACh). Data from our laboratory and other research groups show that lung cancers express all of the genes for synthesis, transport and degradation of ACh. These include nAChRs, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), choline transporter (ChT1) and acetylcholinesterase (AChE). Nicotine upregulates the ACh-signaling loop in human lung cancer cells. Therefore, we conjectured that disruption of ACh-signaling pathway should suppress the growth of human cancers. We show that alpha7-nAChR antagonists robustly suppress angiogenesis in human lung and retinal microvascular endothelial cells. The anti-angiogenic activity of alpha7-nAChR antagonists was also observed in chicken chorioallantoic membrane (CAM) and nude mouse models. Similarly, vesamicol, a small molecule antagonist of VAChT, decreases nicotine-induced tumor growth in human NSCLCs. Our studies suggest that the acetylcholine signaling pathway may be have potential applications in the therapy of human lung cancers. Our results are also relevant to lung cancer patients who are exposed to nicotine via secondhand smoke, nicotine patches, gums or electronic cigarettes. Citation Format: Piyali Dasgupta, Kathleen C. Brown, Jamie K. Lau, Aaron M. Dom, Brent A. Thornhill, Clayton M. Crabtree, Theodore R. Witte, W E. Hardman, Cody A. Stover, A B. Carpenter, Yi C. Chen. Disruption of the acetylcholine signaling pathway suppresses the growth and angiogenesis of human lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5229. doi:10.1158/1538-7445.AM2015-5229