Shaker A. Mousa, PhD, MBA, FACC, FACB, Professor of Pharmacology and Executive Vice President, Chairman of the Pharmaceutical Research Institute at Albany, Albany College of Pharmacy and Health Sciences, 1 Discovery Drive (Room 238), Rensselaer, NY 12144, USA, Tel.: 518-694-7397, Fax: 518-694-7567, e-mail: firstname.lastname@example.org
Abstract: Nicotine, one of the thousands of chemicals in cigarette smoke has a highly debated effect on cell proliferation and tissue healing. Recent studies documented its pro-angiogenesis effects by stimulating endothelial cell α7-non-neronal nicotinic acetyl choline receptors (α7 N-nACHR). It is well known that individuals who smoke or have diabetes experience impaired wound healing although for different reasons. This review evaluates several current studies relating to nicotine’s ability to mediate cellular activation, migration and angiogenesis in attempts to correlate these data with nicotine’s ability to repair wounds in ischaemic tissue. While its beneficial effects are still under investigation, important findings regarding nicotine’s acceleration of atherosclerosis, tumor angiogenesis, cell proliferation e and resistance to apoptosis put its systemic use into question. Based on the good and bad sides of nicotine, it is recommended to restrict its utility to local applications.
Nicotine is an organic compound, an alkaloid found naturally throughout the tobacco plant, with a high concentration in the leaves. It constitutes 0.3–5% of the plant by dry weight, with biosynthesis taking place in the roots, and accumulates in the leaves. In lower concentrations, the substance is a stimulant and is one of the main factors leading to the pleasure and habit-forming qualities of tobacco smoking (1). In addition to the tobacco plant, nicotine is found in lower quantities in other members of the Solanaceae (nightshade) family, which includes tomato, potato, eggplant and green pepper (2). Nicotine alkaloids are also found in the leaves of the coca plant (3).
Nicotine is called 3-(1-methyl-2-pyrrolidinyl) pyridine. The molecule possesses an asymmetric carbon and so exists in two enantiomeric compounds. Nicotine can be provided in the form of chewing gum, transdermal patch, nasal spray, inhaler and sublingual tablets/lozenges to replace tobacco as the source of nicotine. However, the body’s ability to degrade and remove nicotine rapidly has been a major obstacle to realizing the full benefits of this approach because of the difficulty in maintaining blood levels that are sufficient to curb craving while remaining below levels that cause nausea and toxicity (4).
Neuronal nicotinic receptors in non-neuronal cells
Nicotinic acetylcholine receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors (5). All nicotinic receptors are formed as pentamers of subunits (6). Genes encoding a total of 17 subunits (α1–10, β1–4, δ, ε and γ) have been identified (7). All subunits are of mammalian origin with the exception of α8 (avian). Each subunit possesses 4 TM domains. All subunits possess two tandem cysteine residues near the site involved in acetylcholine binding. Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1εδ, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle. Other nicotinic receptors are assembled as combinations of α(2–6) and β(2–4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4, α2β4) are sufficient to form a functional receptor in vitro, but more complex isoforms may exist in vivo (8,9). α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors (e.g. α4α5αβ2, α6β2β3) when co-expressed with two other subunits. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. avian α7α8) (10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. This combination appears to be largely confined to cochlear and vestibular hair cells (11).
The nicotinic acetylcholine receptors (nAChR) are prototypic ionotropic receptors that mediate fast synaptic transmission. However, also non-excitable cells and particularly the tegumental cells that line external and internal body surfaces, express acetylcholine receptors of neuronal type sensitive to nicotine. Bronchial epithelial cells (12), endothelial cells of blood vessels (13) and skin keratinocytes (14) express neuronal nicotinic receptors composed of α3, α5, β2 and β4 subunits, similar to those expressed in sympathetic ganglia, and neuronal nicotinic receptors composed of α7 subunits (15). Neuronal nicotinic receptors in tegumental cells are involved in modulating cell shape and motility and therefore in maintaining the integrity of the surfaces lined by those cells. Neuronal nicotinic receptors in non-neuronal tissues may modulate other functions including cell proliferation and differentiation. The presence of neuronal nicotinic receptors sensitive to nicotine in tissues known to be involved in tobacco toxicity, like bronchi and blood vessels, raises the possibility that they mediate some of the toxic effects of smoking (15).
The α3 subunit seems to be a key component of the various nAChR subtypes expressed by autonomic neurons (16). Studies in heterologous systems indicate that homomeric and heteromeric complexes of α7 subunits can be formed (17). In addition, inclusion of α5 in hetero-oligomeric nAChRs comprising other α and β subunits yields receptors with lower agonist affinity and larger conductance (18). The expression of all but the smallest conductance class of nAChRs recorded in controls was suppressed in neurons contacting heart, despite increased levels of expression of α5, α7 and β4 (19).
The metabolic pathway of nicotine
The alkaloid nicotine metabolic pathway is mediated by a CYP 450 isoform, namely CYP2A6. Nicotine is oxidized to cotinine, which is catalysed by CYP2A6 enzyme (20, 21). Recent research indicates that chemical compounds that block the activity of this enzyme can stabilize nicotine levels and enhance the effectiveness and reliability of nicotine replacement therapy. The structure of the CYP2A6 enzyme was studied using a technique of x-ray crystallography. A three dimensional model of the enzyme enables researchers to identify and evaluate nicotine inhibitors.
Dual functions of nicotine
Nicotine, which is currently being used to help tobacco smokers quit and is being considered as a potential treatment for a number of other disorders such as ulcerative colitis (22), Alzheimer’s disease (23), Parkinson’s disease (24), has been found to harbour previously undiscovered side affects. Recent findings, however, suggest that there is a risk in using nicotine and its products.
Role of nicotine in healing mechanism
While cigarette smoke has been shown to decrease wound healing (25–30), recent studies have documented the effect of topical nicotine on improving dermal wounds. Wound healing is a complex process which involves different cells and cellular messengers that respond to injury. After injury, platelets aggregate and stop blood loss (31). However, the formation of a clot serves another function. The clot mass releases growth factors such as platelet derived growth factor (PDGF) and even provides a surface on which other cells can move across to access the entire wounded area. Macrophages are one of the cells which are attracted to the area by PDGF and are key during the inflammatory phase of healing (32). Activated macrophages help clean the wound of foreign bodies and release more cellular mediators such as vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) in addition to more PDGF (33), both of which trigger and continue the next phase of healing, granulation. During this stage, the high amounts of PDGF from both clot masses and macrophages summon fibroblasts into the wound. This cell type is responsible for producing a new extracellular matrix to replace the damaged or missing basement layer in the wound (34–36). At the same time, new capillaries are being formed by the process of angiogenesis. This occurs when endothelial progenitor cells (EPC) are stimulated by the high amounts of VEGF to form new blood vessels. This part of the healing process is essential as adequate blood floor provides oxygen, nutrients and cells needed and sustain any newly formed tissue. Keratinocytes located around the wound multiply and migrate over the extracellular matrix before differentiating into the epidermal layers that will become the skin (34). While tissue remodeling still needs to occur, this takes place during the first few days after injury is a key in starting and promoting wound healing (31,32). Wang et al., 2004, discovered that in vitro stimulation of human EPC with nicotine causes an increase in the EPC number, proliferation, migration and adhesion and an increase in their ability to form new vasculature (37). All of these effects demonstrate a potential for nicotine to aid in the wound healing process, but not impair it. Previous research proposes that this relationship is caused by stimulation of nAChR on endothelial cells (38) even though current studies are still not able to prove the exact mechanism by which nicotine elicits its effects. A variety of nicotine levels were examined versus a control media for up to 48 h. It was concluded that 10−8m nicotine caused maximum stimulatory effect on the EPC, especially between the 18 and 32 h marks, whereas 10−4m concentrations of nicotine caused a significant decrease in the number of EPC, cell proliferation, migration, adhesion and ability to form new vasculature (37).
A 2007 study by Morimoto et al., (39) looking at nicotine’s effects on wound healing, demonstrates that 10 μl of 10−4m nicotine resulted in smaller wound areas, longer neoepithelium and a larger area of newly formed capillaries compared with phosphate buffered saline (PBS). As Galiano et al. 2004 looked at cultured EPC (40) and this study was run on animal wounds, the effects of 10−4m nicotine on healing may not be mediated by just EPC. Morimoto et al. also used multiple dosing intervals and ultimately a longer duration of exposure which helps to differentiate further the results of the two studies (39).
The 2007 study evaluated wounds treated with bFGF alone and in combination with 10−4m nicotine. These dosages were administered topically every day for 7 days and the results were analysed on day 8. In this study and others, data showed that either nicotine or bFGF resulted in comparable effects on wound healing (37–39,41). In the study by Arredondo et al. 2003, 24 h exposure to 10−6m of nicotine was found to cause changes in the dermal fibroblasts expression of cell cycle regulators (35), apoptosis regulators and dermal matrix proteins, which can all ultimately alter skin function and healing (42). Nicotine of 10−6m was chosen because it is like the levels found in the plasma of smokers.
The influence of nicotine on bone healing remains controversial. Zheng et al., 2008 (43) stated that nicotine exposure increased microvessel density, whereas inhibited blood flow and bone formation. In this study, the expression of bone morphogenetic protein-2 in osteoblasts was also decreased. Frequent appearance of cartilage islands suggested ischaemia and low oxygen tension in the distraction regenerate. They concluded that nicotine compromises bone regeneration by causing ischaemia and directly by inhibitory effect on osteoblastic cells. They concluded that nicotine exposure enhances angiogenesis, but cannot compensate for the adverse effect of vasoconstriction.
Role of nicotine in impairments of healing mechanism
Diabetic ulcers are an example of how impaired healing can be detrimental to a person’s health. Diabetic patients are thought to experience a number of impaired cellular functions, such as reduced levels of growth factor (40), defective macrophages and even altered interactions among endothelial, fibroblast and keratinocyte cells (44), which ultimately impair their ability to mediate the phases of healing and generate new tissue and blood vessels. In this case, it would seem that simply replacing such growth factors or stimulating other key cells involved in the process would solve some of these issues. Current studies report that administration of exogenous PDGF, VEGF and even bFGF can improve diabetic wound healing (31,40,45).
Smokers are another group of patients who are known to experience poor wound healing (25,26). The thousands of chemicals of nicotine elicit a plethora of effects on the healing process that range from vasoconstriction, which can decrease blood flow to the site of injury, to impair the activation or migration of key cells, like macrophages and fibroblasts, during recovery (25,26,46,47). Studies reveal that exposure to either first or second hand cigarette smoke results in decreased fibroblast migration and increased cell survival, impaired wound healing and scarring (29,30). Wong et al. 2004 (30), determined that mice exposed to side stream second hand smoke experienced delays in healing 7 days after wounding .
An older but numerously cited study, Jacobi et al. 2002, (48) evaluates nicotine’s endothelial stimulating ability to enhance wound healing in diabetic mice. While the study found that diabetic mice experience impaired healing compared to non-diabetic mice when both were treated with PBS alone, it also found that diabetic mice experienced significant increases in wound closure with treatment of either 10−8m nicotine, 10−9m nicotine, or 25 ug/kg bFGF. The diabetic mice treated with nicotine 10−8m also showed increased wound vascularity compared with diabetic mice treated with just PBS (48). Jacobi et al. 2002 could not rule out nicotine’s potential effects on dermal keratinocytes. The study claims that nicotine may have aided in the healing process (48). The review article of Misery 2004, discussed the effects of smoking on dermal cells and its subsequent effect on wound healing. The review summarizes that while nicotine enhances keratinocyte adhesion and differentiation through nAChR modulation, it also causes apoptosis and inhibits cell migration (42). However, nicotine dosages have a bimodal effect and may act using several nAChR subtypes or other cellular means of stimulation (17,21). This makes it possible that low versus high concentrations or acute versus chronic stimulation with nicotinic agonists can result in varying affects on different cells, such as in this case, keratinocytes (14).
One interesting result of the Jacobi et al. 2002 study (48) is that on day 14, there was no apparent difference in wound closure among non-diabetic mice treated with either PBS or nicotine. This is strange when compared with the results of the Morimoto et al. 2007 study (39), where non-diabetic mice experienced improved wound healing with use of topical nicotine. Perhaps this is because the mice in the older study were treated with lower concentrations of nicotine (10−8m and 10−9m) than the 10−4m nicotine used in the 2007 study. From these results, it is observed that non-diabetic wounds require larger doses of nicotinic agonists to produce healing effects. Perhaps as diabetic patients have lower concentrations of the needed growth factors to promote healing (40), their cells become more sensitive and thus would be more likely to respond to stimulation. This could explain how smaller concentrations elicit response in diabetic animals while larger concentrations of nicotinic agonists are needed for similar effects in non-diabetic animals (39,48). These results warrant further investigation into the effects nicotine has on the wounds of diabetic versus non-diabetic animals. If non-diabetic mice could receive wound healing benefit from nicotine or nicotinic agonists then it is possible that one day patients with normal wound healing might also receive benefit from such treatments.
Nicotine and angiogenesis
As nicotine’s effect in wound healing is at least mediated in part through the modulation of angiogenesis (49,50), ischaemic or infarct tissues may benefit from nicotine’s ability to stimulate new blood vessel formation (51). In this regard, Li et al. 2005 study evaluated nicotine effects in a rat model of myocardial infarction (MI) (51). In that study, rats received 9 ug/kg of nicotine intramuscularly (IM) on the left flank daily for 21 days. Then their left ventricles were removed and examined for changes capillary growth versus control rats. It was determined that capillary density increased in the rats given nicotine and the capillary growth was localized to the area of infarcted tissue (51). Future studies that explore the salvage of ischaemic tissue should take into account the time to treatment post infarction, duration of treatment and dose of nicotinic agonist. It would also be important to investigate improvements in the heart’s function and hence clinical significance could be determined (51).
Angiogenesis under hypoxic conditions was also examined in the Heeschen et al. 2001 (38). The study used the hind limbs of mice whose femoral artery was briefly occluded as a model for ischaemic tissue. Mice received either 0.03 ug/kg nicotine locally into the hind limb or systemically with 100 ug/ml nicotine orally to mimic plasma levels seen in ‘moderate smokers’. The study found that these doses, administered by either of these routes, result in the greatest increase in capillary density (38) compared to lower and higher concentrations of nicotine.
Another study, Heeschen et al. 2006, uses the same dosages (41), routes and hypoxic model to demonstrate again that nicotine augments the angiogenesis that takes place during ischaemia. While both studies look at local and systemic application, this updated study compares the routes head-to-head. Systemic administration was found to produce nearly a 75% increase in capillary density whereas local administration produced only a 46% increase (41). These findings indicate that an oral, systemic route for nicotine, versus an IM route, might generate greater results, putting the results of the Li et al. 2005 study into new light, as it only investigated IM injections to the left flank (51). In this regard, the kinetics of effect of dermal application of nicotine on systemic levels need to be examined.
In fact a 2002 study by Conklin et al. (52) describes how both nicotine and cotinine, at doses similar to those seen in habitual smokers, significantly increase endothelial cell expression of VEGF, which may stimulate angiogenesis and thereby worsen atherosclerosis and increase tumor growth.
One of the objectives of the Heeschen et al. 2001 study was whether nicotine enhanced the vascularization associated with increases in tumor and atherosclerotic plaque growth. The researchers found that by day 16, growth of tumor cells stimulated by nicotine exceeded the growth of tumor cells in non-nicotine treated mice. Also, the researchers found that stimulated mice had significantly increased vasculature and capillary density of their tumor tissue. The study further revealed that atherosclerotic plaque areas in nicotine treated mice were significantly greater than those in control mice (38).
Nicotine mediated angiogenesis was found to stimulate angiogenesis and promote tumor growth in another study published in 2006 by Mousa and Mousa (53). The study found that 1 μg of nicotine significantly increased the growth weight of breast, colon and lung cancer tumor cells implanted in a chorioallantoic membrane model after only 1 week (53) of exposure. These findings indicate again that patients using nicotine or other nicotinic agonists systemically may accelerate any underlying angiogenic diseases states subsequent to any possible tissue repair. This means that future therapies aiming to use such agonists will need to either develop ways to administer the drug locally or weigh a patient’s risk before treatment. Mousa and Mousa (53) demonstrated total blocked of nicotine-mediated angiogenesis by nicotinic receptor antagonists or integrin αvβ3 antagonists highlighting molecular and cellular mechanisms of nicotine-mediated angiogenesis (Fig. 1).
The changes in cellular behaviours of human umbilical vein endothelial cells (HUVECs) treated with nicotine were studied by Park et al., 2008 (54). They examined changes in cell count and morphology and assayed cellular migration with Boyden chamber and microcapillary tube formation in a Matrigel matrix following treatment with various concentrations of nicotine. Compared with the control, nicotine stimulated cell proliferation, migration and tube formation at concentrations similar to those found in smokers. Although there were no specific morphological changes in HUVECs treated with nicotine at the concentration similar to that in smokers, at high concentration (10−4m), (54) morphological changes such as cytoplasmic vacuolization and irregular cell shape were observed, which were assumed to be the result of direct cytotoxicity of nicotine. In HUVECs, nicotine enhanced cellular proliferation, migration and angiogenesis in vitro and thus caused a functional change, but not a morphological change at a concentration similar to that in habitual smokers.
The mechanism of action for nicotine on HUVECs was explained by Li et al., 2006. They investigated the α7 nAChR in human umbilical vein endothelial cells. The cellular function was examined using MTT, fluorescence confocal microscopy and angiogenesis assay in vitro. The authors investigated the capillary density in the rat model of MI immunohistochemistry (51). They showed that α7 nAChR agonist’s choline increased the expression of α7 nAChR mRNA and protein, the intracellular Ca2+ concentration, proliferation and tube formation of ECs. Reverse effects were observed by using α7 nAChR antagonist alpha-BTX. Furthermore, in the rat model of MI, α7 nAChR agonist enhanced the capillary density in ischaemic tissues, whereas antagonist mecamylamine and alpha-BTX inhibited the effect. Li et al., 2006 suggest that α7 nAChR is involved in the regulation of cellular function in ECs and capillary formation in MI, which are the important steps of angiogenesis. Therefore, α7 nAChR on ECs may be a new endothelium target for revascularization in therapeutic angiogenesis of ischaemic heart disease (51).
Ng et al. at 2007 studied the endothelial nAChR and its participation in atherogenesis and tumorigenesis (55) by promoting neovascularization. The mechanisms of nAChR-mediated angiogenesis and their relationship to angiogenic factors, e.g. VEGF and bFGF, are unknown (55). Nicotine induced dose-dependent human microvascular endothelial cell (HMVEC) migration, a key angiogenesis event, to an extent which was equivalent in magnitude to bFGF (10 ng/ml) but less than for that VEGF (10 ng/ml). nAChR antagonism abolished nicotine-induced HMVEC migration and abolished migration induced by bFGF and attenuated migration induced by VEGF. Transcriptional profiling identified gene expression programmes, which were concordantly regulated by all the three pro-angiogenesis factor (nicotine, VEGF and bFGF), a notable feature of which includes co-repression of thioredoxin-interacting protein (TXNIP), endogenous inhibitor of the redox regulator thioredoxin. Furthermore, TXNIP repression by all the three pro-angiogensis factors induced thioredoxin activity. Silencing thioredoxin by small interference RNA abrogated all angiogenic-induced migration whereas silencing TXNIP strongly induced HMVEC migration. Interestingly, nAChR antagonism abrogates growth factor (VEGF and bFGF)-mediated induction of thioredoxin activity. Authors concluded that nicotine promotes angiogenesis via stimulation of nAChR-dependent endothelial cell migration. Furthermore, growth factor-induced HMVEC migration, a key angiogenesis event, requires nAChR activation, an effect mediated in part by nAChR-dependent regulation of thioredoxin activity (55).
Sugimoto et al., 2007 hypothesized that nicotine may stimulate postnatal vasculogenesis (56) on EPCs. The proliferation and migration activities of human EPCs cultured from peripheral blood mononuclear cells of non-smoking healthy volunteers were not affected by nicotine. The effect of nicotine on EPC survival was significantly enhanced under serum starvation. Nicotine (100 ng/ml) was administered orally for 7 days before and 4 weeks after injection of cultured EPCs (1 × 10(5)/mouse) into the tail veins of 8-week-old athymic nude mice with ischaemic hind limbs. Laser Doppler imaging analysis indicated that blood perfusion in the ischaemic hind limb was significantly enhanced in EPCs plus nicotine compared with EPCs alone. These findings suggest nicotine improves blood flow following EPC transplantation in patients with ischaemic diseases.
Nicotine and apoptosis
Although nicotine itself is usually not referred to as a carcinogen, there is ongoing debate whether nicotine functions as a tumor enhancer. By binding to nAChR, nicotine modulates essential biological processes like angiogenesis, apoptosis and cell-mediated immunity. Apoptosis plays critical roles in a wide variety of physiological processes during foetal development and in adult tissue and is also a fundamental aspect of the biology of malignant diseases. Dasgupta et al., 2006 show that nicotine inhibits apoptosis (57) induced by the drugs gemcitabine, cisplatin and taxol, which are used to treat non-small cell lung cancer. The anti-apoptotic effects of nicotine were mediated by dihydro beta-erythroidine-sensitive alpha3-containing nAChR and required the Akt pathway. They stated that nicotine stimulation caused an increased recruitment of E2F1 and concomitant dissociation of retinoblastoma tumor suppressor protein (Rb) from survivin promoter in A549 cells. These studies suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutic drugs and that survivin and XIAP play a key role in the anti-apoptotic activity of nicotine (57).
Nicotine and carcinogenesis
For a long time, the tumorigenic potential of smoking was attributed to compounds other than nicotine. However, more recently, data have accumulated which suggest that nicotine may add to the cancer risk by stimulating cellular growth via non-neuronal acetylcholine receptors, by suppressing apoptosis and by inducing angiogenesis in tumors. Kleinsasser et al., 2006 (58) found nicotine induced dose-dependent DNA damage in all cell types of the upper aero-digestive tract at low cytotoxic concentrations that allowed viabilities well above 80%. The lowest nicotine concentrations eliciting a significant increase in DNA migration were 1 mm for tonsillar cells and 0.25 mm for all other cell types. They concluded that Nicotine induces genotoxic effects in human target cells of carcinogenesis in the upper aero-digestive tract at relevant concentrations. Thus, nicotine may contribute directly to tumor initiation resulting from smoking (58).
Ye et al., 2005 used the human colon adenocarcinoma cell line, SW1116, and human umbilical vascular endothelial cells (HUVECs) to elucidate the possible mechanisms of nicotine in tumorgenesis in vitro (59). They showed that cigarette smoke extract enhanced cell proliferation and the expression of 5-lipoxygenase (5-LOX), VEGF, MMP 2 and 9 in SW1116 cells. Inhibition of 5-LOX decreased cell proliferation and expressions of VEGF, MMP-2 and MMP-9 induced by cigarette smoke extract. In addition, cigarette smoke extract indirectly stimulated HUVEC proliferation, a biological activity closely related to angiogenesis during tumor growth. This was again blocked by the 5-LOX inhibitor (59). The same conclusion was reached by Natori et al., 2003 that nicotine promotes tumor growth, at least in part, by stimulating tumor-associated neovascularization (60). Meanwhile, Shin et al., 2005 show that nicotine stimulates the progression of tumor growth, through a cyclooxygenase-2-dependent pathway in animal xenograft models and cell culture systems. On the basis of these findings, nicotine seems to be a potent mitogenic agent in modulating tumor cell proliferation and selective cyclooxygenase-2 inhibitors are promising antitumor agents for gastric cancer in smokers (61).
Another study on colon cancer and its relation to nicotine carried out by Wong et al., 2007 demonstrated that oral nicotine administration (50 or 200 microg/ml) for 25 days stimulated growth of human colon cancer xenograft in nude mice (62). It also increased vascularization in the tumors and elevated cotinine and adrenaline plasma levels. Beta-Adrenoceptors, cyclooxygenase-2 (COX-2), prostaglandin E (2) (PGE (2)), and VEGF in tumor tissues were also increased by nicotine. Intraperitonial injection of beta (1)-selective antagonist (atenolol, 5 or 10 mg/kg) or beta (2)-selective antagonist (ICI 118,551, 5, or 10 mg/kg) blocked the nicotine-stimulated tumor growth dose dependently, in which beta (2)-selective antagonist produced a more prominent effect. Beta-Adrenoceptors blockade also abrogated the stimulatory action of nicotine on microvessel densities as well as cell expression of COX-2, PGE (2), and VEGF, in which beta (2)-selective antagonist produced a significant effect. These findings provide a direct evidence that nicotine can enhance colon tumor growth mediated partly by stimulation of beta-adrenoceptors, preferentially the beta (2)-adrenoceptors. Activation of beta-adrenoceptors and the subsequent stimulation of COX-2, PGE (2) and VEGF expression are perhaps important mechanisms in the tumorigenic action of nicotine on colon tumor growth. These data suggest that beta-adrenoceptors play a modulatory role in the development of colon cancer and partly elucidate the carcinogenic action of cigarette smoke (62).
Zhu et al., 2003 conducted their experiment on the second hand smoke (SHS) which is believed to cause lung cancer (63). In this study, Lewis lung cancer cells were injected subcutaneously into mice, which were then exposed to SHS or clean room air and administered vehicle, cerivastatin, or mecamylamine. SHS significantly increased tumor size, weight, capillary density, VEGF and MCP-1 levels and circulating EPC. Cerivastatin (an inhibitor of HMG-coA reductase) or mecamylamine (an inhibitor of nAChR) suppressed the effect of SHS to increase tumor size and capillary density. Cerivastatin reduced MCP-1 levels, whereas mecamylamine reduced VEGF levels and EPC. Authors revealed that SHS promotes tumor angiogenesis and growth. These effects of SHS are associated with increases in plasma VEGF and MCP-1 levels and EPC mediated in part by isoprenylation and nAChR. Zhang et al., 2007 identified novel mechanisms by which nicotine promotes tumor angiogenesis and metastasis (64) and provide further evidences that HIF-1alpha is a potential anticancer target in nicotine-associated lung cancer. Authors used in this study Human non-small lung cancer cell lines A549 and H157 after their stimulation with nicotine. In the previous study, there was loss of HIF-1alpha function using specific small interfering RNA. Zhang et al. found increase in HIF-1alpha and VEGF expression in non-small cell lung cancer cells treated with nicotine. They stated that pharmacologically blocking nicotinic acetylcholine receptor-mediated signaling cascades, including the Ca2+/calmodulin, c-Src, protein kinase C, PI3-kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2, and the mammalian target of rapamycin pathways, significantly attenuated nicotine-induced up-regulation of HIF-1alpha protein. Functionally, nicotine stimulated in vitro tumor angiogenesis by promoting tumor cell migration and invasion (64). These proangiogenic and invasive effects were abrogated by treatment with small interfering RNA specific for HIF-1alpha.
The role of specific nAChR subtypes in tobacco-related carcinogenesis was also established by Arredondo et al., at 2006. They opened a novel avenue for oral cancer chemoprevention (65). Nicotine contributes to tumorigenesis in oral cancer also through stimulation of nicotinic acetylcholine (nAChRs) in target cells (68). nAChRs can be stimulated by the nicotine-derived nitrosamines 4-(methylnitrosamino)-1-(3–pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) that can induce oral cavity tumors in laboratory animals. Authors in this study used Het-1A cells that were found to express α3, α5, α7, α9, α2 and α4 nAChR subunits. NNK and NNN increased proliferative potential of Het-1A cells and produced an anti-apoptotic effect, which was alleviated by antagonists. α-Bungarotoxin was most effective against NNK and mecamylamine against NNN. Treatment of Het-1A cells with either NNK or NNN led to ability to produce tumors in nude mice. Authors studied transcription of the genes encoding the cell cycle, apoptosis and signal transduction regulators at both the mRNA and protein levels. The Het-1A cells stimulated with nitrosamines showed multifold increases of the mRNA transcripts encoding PCNA and Bcl-2, and up-regulated expression of the transcription factors GATA-3, NF-kB, and STAT-1 (65).
A couple of studies by Dasgupta et al., 2006,2008 show the mitogenic and proliferation effects of nicotine in non-small cell lung cancers (66,67). They find that nicotine activates Src, induction of Rb-Raf-1 interaction and phosphorylation of Rb. Analysis of human non-small lung tumors shows enhanced levels of Rb-Raf-1 complexes compared with adjacent normal tissue. The mitogenic effects of nicotine were mediated via the α7 nAChR subunit and resulted in enhanced recruitment of E2F1 and Raf-1 on proliferative promoters in non-small lung cancer cell lines and human lung tumors (69). Nicotine stimulation of non small lung cancer cells caused dissociation of Rb from these promoters. Proliferative signalling via nAChRs required the scaffolding protein beta-arrestin; ablation of beta-arrestin or disruption of the Rb-Raf-1 interaction blocked nicotine-induced proliferation of non-small cell lung cancer. 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. Nicotine induces cell proliferation by beta-arrestin-mediated activation of the Src and Rb-Raf-1 pathways (67). In the recent study of Dasgupta et al. 2008, and Jarzynka et al., 2006 (67,68) they found morphological changes in the cells similar to those induced by the pro-migratory growth factor VEGF. They suggested that pro-invasive effects of nicotine were mediated by α7 nAChRs on non-small cell lung cancer. The pro-invasive effects of nicotine were mediated via a nAChR, Src and calcium-dependent signalling 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.
Dermatological applications of nicotine and analogues
Nicotine is highly lipophillic and can pass through dermal tissue as well as the blood-brain barrier. Transdermal nicotine patches (TNPs) are established systems of drug delivery that, in previous clinical trials, have demonstrated efficacy in helping adults to curtail cigarette smoking (69). These products were first made available commercially in the United States in 1992 on a prescription-only basis. Four brands of TNP deliver up to 22 mg of nicotine in a 24-h period; however, 27–74% of the total nicotine may remain in a TNP after use. As much as 83 mg of residual nicotine may remain in a used TNP, the equivalent to nicotine content of 4–7 cigarettes.
In 1996, TNPs were made available to the public without prescription to encourage cigarette smoking cessation. An estimated 16 million Americans will spend over one billion dollars on such over-the-counter nicotine replacement systems annually in an attempt to quit smoking (69,70). As the availability of these products increases, it is anticipated that physicians and poison centres will be contacted with increasing frequency concerning inadvertent exposures to them among children.
Therapeutic use of TNPs in adults has been associated with a variety of adverse effects including rashes, allergic skin reactions, nausea and vomiting, sleep disturbances, headaches and chest pain (70).
As it is well known that nicotine can stimulate many intracellular processes by binding to nAChR and increasing intracellular Calcium (71), it may be possible that using acute doses of nicotine to stimulate dermal cells and allowing enough time between doses for the cells to migrate to their needed site of action could result in improved wound healing. Thus, there could be a difference between acute therapeutic use and chronic pathological use. The researchers of future studies should look at a range of concentrations as we have seen that different patient types and even different cell types can be affected differently by different concentrations of nicotine.
The use of novel drug delivery systems of nicotine that limit its systemic absorption or selective nicotine analogues that work specifically at the α7 N-nACHR along with limited systemic absorption might provide an optimal topical dermatological formulation for wound healing and tissue repair. Molecules like nicotine that stimulate vascular growth may even hold potential in rescuing oxygen deprived tissue. However, until more research is carried out, the systemic consequences of nicotine, like atherosclerotic plaque and tumor growth, are currently a limiting factor to the dermal applications of nicotine or analogues.