Haley E. Perry

Nicotine Induces the Up-regulation of the α7-Nicotinic Receptor (α7-nAChR) in Human Squamous Cell Lung Cancer Cells via the Sp1/GATA Protein Pathway

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. Nicotine, the addictive component of cigarettes, promotes lung cancer proliferation via the α7-nicotinic acetylcholine receptor (α7-nAChR) subtype. The present manuscript explores the effect of nicotine exposure on α7-nAChR levels in squamous cell carcinoma of the lung (SCC-L) in vitro and in vivo. Nicotine (at concentrations present in the plasma of average smokers) increased α7-nAChR levels in human SCC-L cell lines. Nicotine-induced up-regulation of α7-nAChR was confirmed in vivo by chicken chorioallantoic membrane models. We also observed that the levels of α7-nAChR in human SCC-L tumors (isolated from patients who are active smokers) correlated with their smoking history. Nicotine increased the levels of α7-nAChR mRNA and α7-nAChR transcription in human SCC-L cell lines and SCC-L tumors. Nicotine-induced up-regulation of α7-nAChR required GATA4 and GATA6. ChIP assays showed that nicotine induced the binding of GATA4 or GATA6 to Sp1 on the α7-nAChR promoter, thereby inducing its transcription and increasing its levels in human SCC-L. Our data are clinically relevant because SCC-L patients smoked for decades before being diagnosed with cancer. It may be envisaged that continuous exposure to nicotine (in such SCC-L patients) causes up-regulation of α7-nAChRs, which facilitates tumor growth and progression. Our results will also be relevant to many SCC-L patients exposed to nicotine via second-hand smoke, electronic cigarettes, and patches or gums to quit smoking.

The flavonoid nobiletin inhibits tumor growth and angiogenesis of ovarian cancers via the Akt pathway

Despite its importance, the death rate of ovarian cancer has remained unchanged over the past five decades, demanding an improvement in prevention and treatment of this malignancy. With no known carcinogens, targeted prevention is currently unavailable, and efforts in early detection of this malignancy by screening biomarkers have failed. The inhibition of angiogenesis, also known as angioprevention, is a promising strategy to limit the growth of solid tumors, including ovarian cancers. Nobiletin, a polymethoxy flavonoid compound isolated from the tiansheng plant, has been shown to inhibit the growth of multiple types of human cancers. However, there are no reports involving the effect on nobiletin on human ovarian cancer. The present report shows that nobiletin potently decreases the viability of ovarian cancer cells in vitro. However, nobiletin does not affect the viability of normal ovarian epithelial cells at <40 μM. The antitumor activity of nobiletin was also observed in athymic mouse models and in chicken chorioallantoic membrane (CAM) models. The anti-neoplastic activity of nobiletin was due to its ability to inhibit angiogenesis. We also studied the molecular mechanisms by which nobiletin suppresses angiogenesis. We observed that nobiletin inhibits secretion of the key angiogenesis mediators, Akt, HIF-1α, NF-κB and vascular epithelial growth factor (VEGF) by ovarian cancer cells. Transient transfection experiments showed that nobiletin inhibits production of HIF-1α by downregulation of Akt. Such decreased levels of HIF-1α were responsible for nobiletin-induced suppression of VEGF. Our data suggest that nobiletin may be a promising anti-angiogenic agent relevant for therapy of ovarian cancers.

Oligonucleotide delivery by nucleofection does not rescue the reduced proliferation phenotype of gene-edited cells.

Gene editing using single-stranded oligonucleotides (ODNs) can be used to reverse or create a single base mutation in mammalian cells. This approach could be used to treat genetic diseases caused, at least in part, by a nucleotide substitution. The technique could also be used as a tool to establish single base polymorphisms at multiple sites and thus aid in creating cell lines that can be used to define the basis for drug resistance in human cells. A troubling outcome of the gene-editing reaction is the effect on normal growth of cells that have undergone nucleotide exchange. In this work, we attempt to overcome this reduced proliferation phenotype by changing the method by which the ODN is introduced into the target cell. Using a series of assays that measure gene editing, DNA damage response, and cell viability, we report that chemically modified ODNs, the most active form of ODN for gene editing, can be used successfully if introduced into the cell by the method of nucleofection. Unlike electroporation, which has been used as the standard mode of ODN delivery, one new result is that nucleofection does not induce a dramatic loss of viability within the first 24 hours after the start of gene editing. In addition, and importantly, ODNs introduced to the cell by nucleofection do not activate the DNA damage response pathway as dramatically as ODNs introduced by electroporation. These 2 novel findings are encouraging for the application of gene editing in other systems. However, reduced proliferation phenotype is still observed when the population of corrected cells is monitored out to 8 days, and thus, delivery by nucleofection does not solve the proliferation problem encountered by cells bearing an edited gene.

Capsaicin synergizes with camptothecin to induce increased apoptosis in human small cell lung cancers via the calpain pathway

Graphical abstract Figure. No Caption available. ABSTRACT Small cell lung cancer (SCLC) is characterized by excellent initial response to chemotherapy and radiation therapy with a majority of the patients showing tumor shrinkage and even remission. However, the challenge with SCLC therapy is that patients inevitably relapse and subsequently do not respond to the first line treatment. Recent clinical studies have investigated the possibility of camptothecin‐based combination therapy as first line treatment for SCLC patients. Conventionally, camptothecin is used for recurrent SCLC and has poor survival outcomes. Therefore, drugs which can improve the therapeutic index of camptothecin should be valuable for SCLC therapy. Extensive evidence shows that nutritional compounds like capsaicin (the spicy compound of chili peppers) can improve the anti‐cancer activity of chemotherapeutic drugs in both cell lines and animal models. Statistical analysis shows that capsaicin synergizes with camptothecin to enhance apoptosis of human SCLC cells. The synergistic activity of camptothecin and capsaicin is observed in both classical and variant SCLC cell lines and, in vivo, in human SCLC tumors xenotransplanted on chicken chorioallantoic membrane (CAM) models. The synergistic activity of capsaicin and camptothecin are mediated by elevation of intracellular calcium and the calpain pathway. Our data foster hope for novel nutrition based combination therapies in SCLC.

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