Piyali Dasgupta

Nicotine induces cell proliferation by β-arrestin–mediated activation of Src and Rb–Raf-1 pathways

Recent studies have shown that nicotine, a component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. While nicotine is not carcinogenic by itself, it has been shown to induce cell proliferation and angiogenesis. Here we find that mitogenic effects of nicotine in non-small cell lung cancers (NSCLCs) are analogous to those of growth factors and involve activation of Src, induction of Rb-Raf-1 interaction, and phosphorylation of Rb. Analysis of human NSCLC tumors show enhanced levels of Rb-Raf-1 complexes compared with adjacent normal tissue. The mitogenic effects of nicotine were mediated via the alpha7-nAChR subunit and resulted in enhanced recruitment of E2F1 and Raf-1 on proliferative promoters in NSCLC cell lines and human lung tumors. Nicotine stimulation of NSCLC cells caused dissociation of Rb from these promoters. Proliferative signaling via nicotinic acetylcholine receptors (nAChRs) required the scaffolding protein beta-arrestin; ablation of beta-arrestin or disruption of the Rb-Raf-1 interaction blocked nicotine-induced proliferation of NSCLCs. Additionally, suppression of beta-arrestin also blocked activation of Src, suppressed levels of phosphorylated ERK, and abrogated Rb-Raf-1 binding in response to nicotine. It appears that nicotine induces cell proliferation by beta-arrestin-mediated activation of the Src and Rb-Raf-1 pathways.

Nicotine induces cell proliferation, invasion and epithelial-mesenchymal transition in a variety of human cancer cell lines

Cigarette smoking is strongly correlated with the onset of nonsmall cell lung cancer (NSCLC). Nicotine, an active component of cigarettes, has been found to induce proliferation of lung cancer cell lines. In addition, nicotine can induce angiogenesis and confer resistance to apoptosis. All these events are mediated through the nicotinic acetylcholine receptors (nAChRs) on lung cancer cells. In this study, we demonstrate that nicotine can promote anchorage‐independent growth in NSCLCs. In addition, nicotine also induces morphological changes characteristic of a migratory, invasive phenotype in NSCLCs on collagen gel. These morphological changes were similar to those induced by the promigratory growth factor VEGF. The proinvasive effects of nicotine were mediated by α7‐nAChRs on NSCLCs. RT‐PCR analysis showed that the α7‐nAChRs were also expressed on human breast cancer and pancreatic cancer cell lines. Nicotine was found to promote proliferation and invasion in human breast cancer. The proinvasive effects of nicotine were mediated via a nAChR, Src and calcium‐dependent signaling pathway in breast cancer cells. In a similar fashion, nicotine could also induce proliferation and invasion of Aspc1 pancreatic cancer cells. Most importantly, nicotine could induce changes in gene expression consistent with epithelial to mesenchymal transition (EMT), characterized by reduction of epithelial markers like E‐cadherin expression, ZO‐1 staining and concomitant increase in levels of mesenchymal proteins like vimentin and fibronectin in human breast and lung cancer cells. Therefore, it is probable that the ability of nicotine to induce invasion and EMT may contribute to the progression of breast and lung cancers.

Nicotinic acetylcholine receptors in cancer: multiple roles in proliferation and inhibition of apoptosis.

Nicotinic acetylcholine receptors (nAChRs) constitute a heterogeneous family of ion channels that mediate fast synaptic transmission in neurons. They have also been found on non-neuronal cells such as bronchial epithelium and keratinocytes, underscoring the idea that they have functions well beyond neurotransmission. Components of cigarette smoke, including nicotine and NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], are agonists of nAChRs. Given the association of tobacco use with several diseases, the non-neuronal nAChR signaling pathway has considerable implications for cancer and cardiovascular disease. Recent studies have shown that alpha7 is the main nAChR subunit that mediates the proliferative effects of nicotine in cancer cells. As a result, alpha7 nAChR might be a valuable molecular target for therapy of cancers such as lung cancer and mesothelioma. Future studies involving the design of nAChR antagonists with improved selectivity might identify novel strategies for the treatment of tobacco-related cancers. Here we review the cellular roles of non-neuronal nAChRs, including regulation of cell proliferation, angiogenesis, apoptosis, migration, invasion and secretion.

Nicotine-mediated cell proliferation and angiogenesis: new twists to an old story.

Tobacco smoking is one of the major etiologic factors associated with cancer. While there are many carcinogenic compounds present in tobacco smoke, its main addictive component, nicotine, is not carcinogenic by itself. The addictive properties of nicotine are achieved through the nicotinic acetylcholine receptors (nAChRs) that are widely distributed in the brain and neuromuscular junctions; at the same time, they were found to be expressed in a variety of non-neuronal tissues in the body including those of the lung. Recent studies show that these non-neuronal nAChRs can induce cell proliferation and angiogenesis. Analysis of the molecular mechanisms underlying nicotine-mediated cell proliferation showed the involvement of Src kinase and the scaffolding protein β-arrestin-1. Further, nAChRs were found to activate the basic components of the cell cycle machinery similar to growth factor receptors. This involved increased binding of Raf-1 kinase to the Rb protein, activation of cyclins D and E as well as induction of proliferative promoters. This article describes pathway involved in nicotine-induced cell proliferation and angiogenesis and the potential steps that are amenable for developing novel anti-cancer therapies.

Disruption of the Rb-Raf-1 Interaction Inhibits Tumor Growth and Angiogenesis

ABSTRACT The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G1 phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.

Prohibitin Facilitates Cellular Senescence by Recruiting Specific Corepressors To Inhibit E2F Target Genes

ABSTRACT Prohibitin is a growth regulatory gene that has pleiotropic functions in the nucleus, mitochondria, and cytoplasmic compartments. Earlier studies had proposed a role for prohibitin in modulating cellular senescence, but the underlying mechanisms remain unknown. Here we show that senescence induced by DNA-damaging agents causes the localization of prohibitin to specific heterochromatic foci. Prohibitin could bind to heterochromatin protein 1 (HP1) family proteins and colocalized with HP1γ in senescence-associated heterochromatic foci. Further, HP1γ could synergize with prohibitin to repress E2F1-mediated transcriptional activity. The depletion of prohibitin by small interfering RNA or antisense techniques led to a reduction in the senescent phenotype, correlating with a reduced expression of senescence-associated β-galactosidase and fewer numbers of senescence-associated heterochromatic foci. Chromatin immunoprecipitation assays showed that prohibitin is needed for the recruitment of HP1γ to E2F1-regulated proliferative promoters, leading to their repression. The ablation of prohibitin prevented the recruitment of HPIγ, but not Suv39H, to the promoters upon senescence. Prohibitin-mediated recruitment of HP1γ occurred in only senescent cells, not in quiescent cells; thus, there is a dichotomy in the recruitment of different corepressors by prohibitin, depending on the type of growth arrest. These studies show that prohibitin plays a vital role in inducing cellular senescence.

Angiogenic activity of nicotinic acetylcholine receptors : Implications in tobacco-related vascular diseases

Cigarette smoking bears a strong etiological association with many neovascularization-related diseases like cancer, cardiovascular disease and macular degeneration. Although cigarette smoke is a complex mixture of many compounds, nicotine is the major active and addictive component of tobacco. Recent studies have shown that nicotine can enhance angiogenesis and arteriogenesis in several experimental systems and animal models. The pro-angiogenic activity of nicotine is mediated by nicotinic acetylcholine receptors, which have been found to be expressed on several types of cells in the vasculature like endothelial cells, smooth muscle cells and immune cells. The present review summarizes the pro-angiogenic activity of nicotine in neoplastic and non-neoplastic disease. The present article focuses on the role of nAChRs, particularly alpha7-nAChR in mediating the pro-angiogenic effects of nicotine. The expression patterns of nAChRs on various components of the vasculature are discussed. The complex signaling pathways underlying the angiogenic effect of nAChRs are described. The review also takes a look at the therapeutic potential of nAChR agonists and antagonists in angiogenesis-related diseases. More basic research as well as patient-oriented clinical studies is needed to firmly establish the clinical potential of nAChR ligands in angiogenesis-based therapies. Also the side effects of targeting nAChRs remain to be established in patients. The development of selective nAChR agonists and antagonists with improved specificity may represent novel therapeutic regimens in the treatment of angiogenesis-related diseases.

ARRB1-Mediated Regulation of E2F Target Genes in Nicotine-Induced Growth of Lung Tumors

BACKGROUND Nicotine induces the proliferation of non-small cell lung cancer (NSCLC) cells via nicotinic acetylcholine receptors and the arrestin, β1 (ARRB1) protein. However, whether ARRB1 translocates to the nucleus upon nicotinic acetylcholine receptor activation and how it regulates growth of human NSCLCs are not known. METHODS We investigated nuclear localization of ARRB1 in human NSCLC cell lines (A549 and H1650), normal lung cell lines (NHBE and SAEC), and lung cancer tissue microarray. A549 cells were transfected with ARRB1-specific short hairpin RNA (A549-sh) to knockdown ARRB1 expression, or with empty vector (A549-EV), to examine the role of ARRB1 in the mitogenic and antiapoptotic effects of nicotine, binding of ARRB1 to E2F transcription factors, and the role of ARRB1 in nicotine-induced expression of E2F-regulated survival and proliferative genes cell division cycle 6 homolog (CDC6), thymidylate synthetase (TYMS), and baculoviral IAP repeat-containing 5 (BIRC5). Real-time polymerase chain reaction was performed for quantitative analysis of mRNA expression. Chromatin immunoprecipitation assays were performed on A549 cells and fresh-frozen human NSCLC tumors (n = 8) to examine the binding of ARRB1, E1A binding protein (EP300), and acetylated histone 3 (Ac-H3) on the E2F-regulated genes. All statistical tests were two-sided. RESULTS Nicotine induced the nuclear translocation of ARRB1 in NSCLC and normal lung cells, and lung tumor tissues from smokers showed an increased nuclear localization. The mitogenic and antiapoptotic effects of nicotine were reduced in A549-sh cells. Nuclear ARRB1 bound to E2F transcription factors in normal lung cells, NSCLC cells, and tumors. Nicotine treatment induced a statistically significant increased expression of E2F-regulated genes in A549-EV but not in A549-sh cells; the maximum difference being observed in BIRC5 (A549-EV vs A549-sh, mean fold-increase in mRNA level upon nicotine treatment = 20.7-fold, 95% confidence interval = 19.2- to 22.2-fold, vs mean = 0.8-fold, 95% confidence interval= 0.78- to 0.82-fold, P < .001). Furthermore, nicotine induced the binding of ARRB1, EP300, and Ac-H3 on E2F-regulated genes. CONCLUSION Nicotine induced the nuclear translocation of ARRB1 and showed increased expression of proliferative and survival genes, thereby contributing to the growth and progression of NSCLCs.

Capsaicin Displays Anti-Proliferative Activity against Human Small Cell Lung Cancer in Cell Culture and Nude Mice Models via the E2F Pathway

Background Small 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 Findings BrdU 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/Significance Our findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs.

A Small Molecule Disruptor of Rb/Raf-1 Interaction Inhibits Cell Proliferation, Angiogenesis, and Growth of Human Tumor Xenografts in Nude Mice

Although it is well established that cyclin-dependent kinases phosphorylate and inactivate Rb, the Raf-1 kinase physically interacts with Rb and initiates the phosphorylation cascade early in the cell cycle. We have identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251), that potently and selectively disrupts the Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E, and Rb/HDAC binding. The selective inhibition of Rb/Raf-1 binding suppressed the ability of Rb to recruit Raf-1 to proliferative promoters and inhibited E2F1-dependent transcriptional activity. RRD-251 inhibited anchorage-dependent and anchorage-independent growth of human cancer cells and knockdown of Rb with short hairpin RNA or forced expression of E2F1 rescued cells from RRD-251-mediated growth arrest. P.o. treatment of mice resulted in significant tumor growth suppression only in tumors with functional Rb, and this was accompanied by inhibition of angiogenesis, inhibition of proliferation, decreased phosphorylated Rb levels, and inhibition of Rb/Raf-1 but not Rb/E2F1 binding in vivo. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents.