摘要
肺癌是目前世界范围内最常见的恶性肿瘤之，发病率和死亡率逐年上升。非小细胞肺癌（non-small cell lung cancer, NSCLC）占肺癌患者的80%以上。尽管外科手术与放疗化疗的联合应用日臻完善，对肺癌的疗效不断提高，但其5年生存率仍低于20%。因此迫切需要发展新的治疗方法。目前研究表明，肺癌细胞能够合成和分泌乙酰胆碱（Acetylcholine,ACh）作为种生长因子，构建自分泌促增殖网络，参与并促进肺癌的发展进程。毒蕈碱型胆碱能受体（muscarinic cholinergic receptor, M受体）的M3亚型与烟碱型胆碱能受体（nicotinic cholinergic receptor, N受体）的7亚型的选择性拮抗剂，均能抑制肺癌细胞中过度激活的胆碱能信号，从而抑制肺癌细胞增殖，为肺癌的预防和治疗提供了新的药物研发策略。R2HBJJ是由抗胆碱药盐酸戊乙奎醚衍生得到的季铵盐，本研究的目的是考查R2HBJJ的抗胆碱特性，评价其对非小细胞肺癌细胞增殖的影响，并探讨可能的作用机制以及用于NSCLC治疗的潜在可能性。结果如下：
1.放射性配体受体结合实验表明，R2HBJJ能竞争性抑制[3H]-NMS与M受体结合，抑制
作用随药物浓度增加而增强，对M1-5受体的竞争抑制平衡解离常数Ki值依次为7.86±3.39×10~(-11)、1.33±0.52×10~(-9)、6.79±3.71×10~(-11)、1.06±0.12×10~(-10)和2.64±0.98×10~(-10)M（n=3, F=121.2, P <0.01），R2HBJJ对M3和M1受体显示出较高的亲和力。R2HBJJ抑制卡巴胆碱所诱发豚鼠支气管平滑肌收缩的IC50为7.58±1.05×10~(-9)M（n=5）。
2.采用全细胞膜片钳记录ACh诱发的大鼠背根神经节（Dorsal root ganglion, DRG）神经元电流，R2HBJJ能浓度依赖性阻断该电流，1μM、10μM、20μM R2HBJJ对电流的阻断率分别为29.31±5.76%、91.35±1.03%、99.07±0.14%（n=3或5）。利用爪蟾卵母细胞分别表达大鼠7和9/10受体，使用双电极电压钳技术记录ACh诱发的电流。100μM R2HBJJ能完全阻断100μM ACh诱发的7受体电流（n=3），200μM R2HBJJ对200μM ACh诱发的9/10受体电流的阻断率为76.74±6.17%（n=4）。提示R2HBJJ具有阻断DRG神经元N受体以及在爪蟾卵母细胞上表达的大鼠7受体和9/10受体亚型的作用。
3.用磺酰罗丹明B(sulforhodamine B, SRB)法测定了R2HBJJ作用后细胞活力变化。R2HBJJ在体外能浓度依赖性地抑制NSCLC细胞H1299、H460和H157生长，作用72h后半数生长抑制浓度GI50依次为8.5±0.15、8.8±0.42、28.5±0.75μM（n=6），但对A549细胞生长无明显抑制作用。R2HBJJ在10-40μM明显抑制肺癌细胞生长的浓度范围内，不抑制正常人支气管上皮细胞BEP2D生长。对其它17种不同组织类型的肿瘤细胞研究显示，不同的细胞对R2HBJJ具有不同的敏感性。
4. ACh能浓度依赖性拮抗R2HBJJ对H1299细胞增殖的抑制作用。以正常细胞为对照，分别伴随给予0、0.3、1、3、10、30、100μM ACh，细胞活力显著增加，72h后R2HBJJ10μM组细胞活力分别为57.08±1.62%、65.61±4.52%、56.64±7.01%、74.92±5.42%、82.92±6.08%、88.76±0.86%和91.41±3.99%（n=6）；R2HBJJ20μM组细胞活力分别为19.67±2.15%、33.37±1.77%、24.53±1.76%、29.88±5.18%、35.72±4.26%、42.37±2.12%和58.17±4.38%（n=6）。
5.在H1299、H157、H460、A549和BEP2D细胞上应用RT-PCR方法检测了胆碱能受体（Acetylcholine receptors,AChRs）和胆碱乙酰转移酶（Cholineacetyltransferase, ChAT）的表达。ChAT在这几种细胞中均有表达；M1-5、7、9、10、β2和β4受体的表达有明显差异，但并没有发现与对R2HBJJ敏感性特异相关的受体亚型表达。
6.以H1299细胞为模型，对比R2HBJJ和不同亚型选择性AChRs拮抗剂对其增殖的抑制作用。M3受体选择性拮抗剂达非那新的抑制作用与R2HBJJ相似，但弱于R2HBJJ。7受体选择性拮抗剂-Bungarotoxin(-bgt)和N受体非选择性拮抗剂美加明，在10μM内对H1299生长抑制作用均强于R2HBJJ。M1受体选择性拮抗剂哌仑西平、M2/M4受体选择性拮抗剂AF-DX116、M受体非选择性拮抗剂阿托品、4/β2和4/β4受体选择性拮抗剂DhβE抑制作用弱。提示R2HBJJ抑制H1299细胞生长的作用主要由M3受体和7受体介导，其它受体亚型可能同时参与该过程。
7.用流式细胞术（Flow cytometry, FCM）考察了R2HBJJ对细胞凋亡和细胞周期的影响。用20μM R2HBJJ处理H1299细胞24、48、72h，细胞凋亡没有明显变化，但随着作用时间的延长，细胞周期G0/G1期比值由42.54±2.11%上升到76.21±2.75%，而S期则由46.78±1.40%下降到18.37±2.45%（n=3, P＜0.01），表明R2HBJJ能时间依赖性诱导H1299细胞产生G0/G1期阻滞。此外，R2HBJJ还能浓度依赖性诱导H1299和H157细胞产生G0/G1期阻滞。
8.使用Western Blot方法，分析了调控细胞周期G1检查点信号网络的关键蛋白及其上游信号分子表达水平的变化。R2HBJJ可时间及浓度依赖性下调cyclin D1、CDK4、CDK6表达，抑制Rb磷酸化，而对cyclin E和CDK2的表达影响小，这表明R2HBJJ诱导的细胞周期阻滞发生在G0/G1的早期。进步的实验表明R2HBJJ可时间依赖性下调~(Thr308)p-Akt、c-Myc和~(Ser9)p-GSK3β，提示R2HBJJ能在不同机制介导下使得PI3K/Akt通路激活受到抑制，从而导致Akt和它下游的c-Myc和GSK3β的磷酸化水平下调，使得细胞周期蛋白的表达和稳定性发生改变，最终导致细胞周期进程阻滞于G0/G1期。此外，R2HBJJ还能经由JNK通路抑制~(Tyr705)p-Stat3核转位，从而抑制参与G1期向S期转化的下游信号分子转录。
本研究表明新型抗胆碱药R2HBJJ对五种M受体亚型有不同的亲和力，但主要选择拮抗M3和M1受体。R2HBJJ能阻断ACh诱发的DRG神经元电流以及在爪蟾卵母细胞上表达的7受体和9/10受体电流。R2HBJJ通过拮抗M受体和N受体而抑制NSCLC细胞增殖，ACh能浓度依赖性阻断R2HBJJ的抑制作用。R2HBJJ主要经由PI3K/Akt通路下调细胞周期G1检查点调控蛋白~(Ser807/811)p-Rb、cyclin D1、CDK4和CDK6的表达，诱导细胞发生G0/G1期阻滞。此外，R2HBJJ还能经由JNK通路抑制Tyr705p-Stat3核转位，从而抑制参与G1期向S期转化的下游信号分子转录。R2HBJJ诱导NSCLC细胞发生G0/G1期阻滞是其抑制细胞增殖的主要机制，但其体内抗肿瘤作用仍需进步研究。本研究为非神经元胆碱能系统参与NSCLC细胞增殖的调控提供了新的佐证，并为新型抗胆碱药R2HBJJ用于NSCLC治疗的潜在可能性提供了实验依据。
Lung cancer is now among the leading cause of cancer death worldwide and themortality is still increasing. Non-small cell lung cancer (NSCLC) represents approximately80%of all lung cancers, which survival remains poor with5-year survival rates still below20%. Although current therapies, such as surgery, chemotherapy and radiotherapy areincreasingly sophisticated but do not necessarily improve overall survival. Therefore, there isurgent need for further understanding of the biology of NSCLC and development of noveltherapeutic approaches for NSCLC treatment. The recent discovery that lung cancer cellssynthesize and secrete acetylcholine (ACh) that acts as a growth factor and contributes to theprogression phase of cancer development. Activation of mAChRs and nAChRs with ACh andits analogs builds a part of an autocrine-proliferative network, which may provide multiplepotential targets to inhibit lung cancer growth. The M3and7selectivity antagonistsinterrupt the upregulated cholinergic signaling in lung cancer and inhibit cell proliferation,which provide opportunities to develop new precautions and therapeutic approaches. R2HBJJis one of quaternary ammonium salt analogs of Penehyclidine hydrochloride. In order tofurther understand the role of cholinergic antagonists in lung cancer and to explore thepotential therapeutic utility as antitumor agents, we investigated the pharmacologycharacteristics of R2HBJJ then evaluated the effect on several NSCLC cells and explored thepossible mechanism. The results of the exprements are listed as following:
1. The competition of R2HBJJ with five mAChRs was assayed individually. Thebinding of [3H]-NMS to mAChRs was displaced by R2HBJJ with increasing concentration.The competitive inhibition equilibrium dissociation constants (Ki) to M1-M5receptorsubtypes of R2HBJJ were7.86±3.39×10~(-11),1.33±0.52×10~(-9),6.79±3.71×10~(-11),1.06±0.12×10~(-10)and2.64±0.98×10~(-10)M (n=3, F=121.2, P <0.01). Thus R2HBJJ showed higheraffinity to the M3and M1receptor. R2HBJJ suppressed guinea-pig tracheal contractioninduced by carbachol in a concentration dependent manner and the IC50was7.58±1.05×10~(-9)M (n=5).
2. The currents in rat dorsal root ganglion neuron induced by ACh were recordedthrough whole-cell recording technique. R2HBJJ blocked the currents in aconcentration-dependent manner. The percentage of blocking were29.31±5.76%,91.35± 1.03%,99.07±0.14%to1,10,20μM R2HBJJ, respectively (n=3or5). Currents of rat7and9/10receptors expressed in Xenopus oocyte elicited by ACh were obtained bytwo-electrode voltage clamp.100μM R2HBJJ completely blocked the currents of7receptors induced by100μM ACh (n=3), while the current blocking rate of200μM R2HBJJto9/10receptor was76.74±6.17%induced by200μM ACh (n=4). This suggests thatR2HBJJ can block not only the N receptors in the DRG but also the7and9/10subtypereceptors expressed in Xenopus oocyte.
3. Cell viability was determined by sulforhodamine B assay. R2HBJJ caused aconcentration-dependent inhibition of the growth in NSCLC H1299, H460and H157cell invitro. Respectively GI50was8.5±0.15,8.8±0.42and28.5±0.75μM (n=6) after treated byR2HBJJ72h. R2HBJJ did not exhibit antiproliferative activity against A549, and had lessobvious effect on growth of BEP2D during range of10-40μM. Evaluation on the growth ofseventeen other type cancer cell indicated that different cells have various sensitivity toR2HBJJ.
4. ACh abolished the inhibitory effect of R2HBJJ in H1299by a concentrationdependent manner. Pre-adminstration of0,0.3,1,3,10,30,100μM ACh was performedbefore R2HBJJ and then the cells were incubated for72h. The cell viability were57.08±1.62%,65.61±4.52%,56.64±7.01%,74.92±5.42%,82.92±6.08%,88.76±0.86%and91.41±3.99%in the group of10μM R2HBJJ compared with the normal cells(n=6). Whilethe cell viability were19.67±2.15%,33.37±1.77%,24.53±1.76%,29.88±5.18%,35.72±4.26%,42.37±2.12%and58.17±4.38%in the group of20μM R2HBJJ(n=6).
5. To determine whether NSCLC cell lines express cholineacetyltransferase (ChAT)and functional acetylcholine receptors (AChRs), the mRNAs were examined by RT-PCR incell lines of H1299, H157, H460, A549and BEP2D. ChAT was present in all of the cells, butthe level of M1-5,7,9,10, β2and β4receptors expression was obviously different.Furthmore, we did not find the specific expression of subtype receptors which directly relatedto the sensitivity to R2HBJJ.
6. The inhibition effects induced by R2HBJJ were compared with different subtypereceptor antagonist in H1299. The selective M3mAChR antagonist darifenacin also showedsimilar significant inhibition of H1299cell growth but weaker than R2HBJJ. Inhibitioneffects of the selective7mAChR antagonist-Bungarotoxin(-bgt) and the non selectiveantagonist mecamylamine(＜10μM) are stronger than R2HBJJ. Whereas pirenzepine (M1selective antagonist), AF-DX116(M2/M4selective antagonist), atropine (non selectiveantagonist) and DhβE (4/β2,4/β4selective antagonist) had slight effects on cell proliferation. This suggests the role of M3and7subtype receptor is crucial to the inhibitioneffect of R2HBJJ and the other receptors may be involved simultaneously.
7. Flow cytometric analysis demonstrated inhibitory effects of R2HBJJ on H1299cellcycle progression. There was no evidence of R2HBJJ-induced apoptosis because thepopulation of cell in the sub-G1was not showing obvious increase. However, the cell cycleprogression was dramatically blocked in G0-G1. The percentage of G0-G1phase increasedfrom42.54±2.11%to76.21±2.75%, and S phase decreased from46.78±1.40%to18.37±2.45%in the presence of20μM R2HBJJ for72h suggested that R2HBJJ induced G0-G1arrest in a time-dependent manner (n=3, P＜0.01). In addition, R2HBJJ also arrested H1299and H157cell cycle in G0-G1phase in a concentration dependent manner.
8. Western Blot was used to analyze the change of expression level of the majorproteins that regulate the G1check point and its upstream signaling molecular. The levels ofcyclin D1, CDK4and CDK6proteins and phosphorylation of Rb were signicantlydownregulated by R2HBJJ in a concentration-and time-dependent manner, while the levels ofcyclin E which is associated with CDK2, were remained basically unaltered during the sameperiod. These reveal that block was occurred in early G0-G1phase. Further experimentsdemonstrated that R2HBJJ could significantly downregulate the level of~(Thr308)p-Akt, c-Mycand~(Ser9)p-GSK3β in a time dependent manner. Activation of PI3K/Akt pathway wasantagonized by R2HBJJ through distinct mechanisms. The expression, activity,localization and stability of cell cycle regulatory proteins were consistent with decrease ofphosphorylation of Akt and its downstream effector c-Myc and GSK3β, which resulted in cellcycle G0/G1arrest at last. R2HBJJ also reduced the nuclear translocation of~(Tyr705)p-Stat3through JNK pathway, which leaded to decrease of transcription that regulates transition fromG1to S phase of cell cycle.
In conclusion, R2HBJJ showed a graded affinity profile to M1-5and acted as a M3/M1selective antagonist. R2HBJJ block not only the currents of nAChRs in rat dorsal root ganglionneuron induced by ACh but also that of rat7and9/10receptors expressed in Xenopusoocyte. R2HBJJ inhibit the proliferation of NSCLC cell through mAChRs and nAChRs, whichcan be abolished by ACh in a concentration dependent manner. PI3K/Akt pathway plays a keyrole in the downregulation of~(Ser807/811)p-Rb, cyclin D1, CDK4and CDK6induced by R2HBJJ,which resulted in cell cycle G0/G1arrest at last. R2HBJJ reduce the nuclear translocation of~(Tyr705)p-Stat3through JNK pathway also contribute to the G0/G1arrest, which lead to thedecrease of transcription that regulate transition from G1to S phase of cell cycle. These resultsreveal that the G0/G1arrest is one of the primary mechanisms of R2HBJJ-mediated inhibition effects on the proliferation of NSCLC cells. Although antitumor activity of R2HBJJ in vivoneed to be fully explored. Nonetheless, the present study provides new evidences that thenon-neuronal cholinergic system involved in proliferation of NSCLC cell and highlights thepotential of R2HBJJ as effective therapeutic agent that act through a novel mechanism ofaction in the treatment of NSCLC.
引文
[1] Jemal A, Bray F, Center MM, et al. Global cancer statistics [J]. CA: a cancer journal forclinicians,2011,61(2):69-90.
[2] Ginsburgh PJ, Voles, E.E., Rosenzweig, K. Non-small cell lung cancer. DeVita VT,Hellman, S., Rosenberg, S.A.,(ed). Cancer: principles and practice of oncology. Philadelphia:Lippincott, Williams and Wilkins,2005:925–983.
[3] Klapproth H, Reinheimer T, Metzen J, et al. Non-neuronal acetylcholine, a signallingmolecule synthezised by surface cells of rat and man [J]. Naunyn-Schmiedeberg's archives ofpharmacology,1997,355(4):515-523.
[4] Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: The non-neuronal cholinergicsystem in humans [J]. British journal of pharmacology,2008,154(8):1558-1571.
[5] Paleari L, Grozio A, Cesario A, et al. The cholinergic system and cancer [J]. Seminars incancer biology,2008,18(3):211-217.
[6] Shah N, Khurana S, Cheng K, et al. Muscarinic receptors and ligands in cancer [J].American journal of physiology Cell physiology,2009,296(2):C221-232.
[7] Egleton RD, Brown KC, Dasgupta P. Nicotinic acetylcholine receptors in cancer:Multiple roles in proliferation and inhibition of apoptosis [J]. Trends in pharmacologicalsciences,2008,29(3):151-158.
[8] Song P, Sekhon HS, Jia Y, et al. Acetylcholine is synthesized by and acts as an autocrinegrowth factor for small cell lung carcinoma [J]. Cancer research,2003,63(1):214-221.
[9] Song P, Sekhon HS, Fu XW, et al. Activated cholinergic signaling provides a target insquamous cell lung carcinoma [J]. Cancer research,2008,68(12):4693-4700.
[10]Song P, Sekhon HS, Proskocil B, et al. Synthesis of acetylcholine by lung cancer [J]. Lifesciences,2003,72(18-19):2159-2168.
[11] Paleari L, Cesario A, Fini M, et al. Alpha7-nicotinic receptor antagonists at the beginningof a clinical era for NSCLC and Mesothelioma?[J]. Drug discovery today,2009,14(17-18):822-836.
[12]Thunnissen FB. Acetylcholine receptor pathway and lung cancer [J]. Journal of thoraciconcology,2009,4(8):943-946.
[13]Gotti C, Fornasari D, Clementi F. Human neuronal nicotinic receptors [J]. Progress inneurobiology,1997,53(2):199-237.
[14]Schuller HM, Orloff M. Tobacco-specific carcinogenic nitrosamines. Ligands fornicotinic acetylcholine receptors in human lung cancer cells [J]. Biochemical pharmacology,1998,55(9):1377-1384.
[15]Arredondo J, Chernyavsky AI, Grando SA. Nicotinic receptors mediate tumorigenicaction of tobacco-derived nitrosamines on immortalized oral epithelial cells [J]. Cancerbiology&therapy,2006,5(5):511-517.
[16]Schuller HM. Is cancer triggered by altered signalling of nicotinic acetylcholine receptors?[J]. Nature reviews Cancer,2009,9(3):195-205.
[17]Abnet CC. Carcinogenic food contaminants [J]. Cancer investigation,2007,25(3):189-196.
[18]Jakszyn P, Gonzalez CA. Nitrosamine and related food intake and gastric andoesophageal cancer risk: A systematic review of the epidemiological evidence [J]. Worldjournal of gastroenterology,2006,12(27):4296-4303.
[19]Schuller HM. Nitrosamines as nicotinic receptor ligands [J]. Life sciences,2007,80(24-25):2274-2280.
[20]Liu P, Vikis HG, Wang D, et al. Familial aggregation of common sequence variants on15q24-25.1in lung cancer [J]. Journal of the National Cancer Institute,2008,100(18):1326-1330.
[21]Hung RJ, McKay JD, Gaborieau V, et al. A susceptibility locus for lung cancer maps tonicotinic acetylcholine receptor subunit genes on15q25[J]. Nature,2008,452(7187):633-637.
[22]Spitz MR, Amos CI, Dong Q, et al. The CHRNA5-A3region on chromosome15q24-25.1is a risk factor both for nicotine dependence and for lung cancer [J]. Journal ofthe National Cancer Institute,2008,100(21):1552-1556.
[23]Lam DC, Girard L, Ramirez R, et al. Expression of nicotinic acetylcholine receptorsubunit genes in non-small-cell lung cancer reveals differences between smokers andnonsmokers [J]. Cancer research,2007,67(10):4638-4647.
[24]Song P, Sekhon HS, Lu A, et al. M3muscarinic receptor antagonists inhibit small celllung carcinoma growth and mitogen-activated protein kinase phosphorylation induced byacetylcholine secretion [J]. Cancer research,2007,67(8):3936-3944.
[25]Carlisle DL, Liu X, Hopkins TM, et al. Nicotine activates cell-signaling pathwaysthrough muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lungcancer cells [J]. Pulmonary pharmacology&therapeutics,2007,20(6):629-641.
[26]Zheng Y, Ritzenthaler JD, Roman J, et al. Nicotine stimulates human lung cancer cellgrowth by inducing fibronectin expression [J]. American journal of respiratory cell andmolecular biology,2007,37(6):681-690.
[27]Martinez-Moreno P, Nieto-Ceron S, Torres-Lanzas J, et al. Cholinesterase activity ofhuman lung tumours varies according to their histological classification [J]. Carcinogenesis,2006,27(3):429-436.
[28]Paleari L, Catassi A, Ciarlo M, et al. Role of alpha7-nicotinic acetylcholine receptor inhuman non-small cell lung cancer proliferation [J]. Cell proliferation,2008,41(6):936-959.
[29]Paleari L, Sessa F, Catassi A, et al. Inhibition of non-neuronal alpha7-nicotinic receptorreduces tumorigenicity in A549NSCLC xenografts [J]. International journal of cancer,2009,125(1):199-211.
[30]Davis R, Rizwani W, Banerjee S, et al. Nicotine promotes tumor growth and metastasisin mouse models of lung cancer [J]. PloS ONE,2009,4(10):e7524.
[31]Dasgupta P, Kinkade R, Joshi B, et al. Nicotine inhibits apoptosis induced bychemotherapeutic drugs by up-regulating XIAP and survivin [J]. Proceedings of the NationalAcademy of Sciences of the United States of America,2006,103(16):6332-6337.
[32]Grozio A, Paleari L, Catassi A, et al. Natural agents targeting thealpha7-nicotinic-receptor in NSCLC: A promising prospective in anti-cancer drugdevelopment [J]. International journal of cancer,2008,122(8):1911-1915.
[33]Mai H, May WS, Gao F, et al. A functional role for nicotine in Bcl2phosphorylation andsuppression of apoptosis [J]. The Journal of biological chemistry,2003,278(3):1886-1891.
[34]West KA, Brognard J, Clark AS, et al. Rapid Akt activation by nicotine and a tobaccocarcinogen modulates the phenotype of normal human airway epithelial cells [J]. The Journalof clinical investigation,2003,111(1):81-90.
[35]Paleari L, Negri E, Catassi A, et al. Inhibition of nonneuronal alpha7-nicotinic receptorfor lung cancer treatment [J]. American journal of respiratory and critical care medicine,2009,179(12):1141-1150.
[36]吴红晶，楼滨城，黄岱坤，等.盐酸戊乙奎醚治疗有机磷中毒临床研究[J].北京大学学报(医学版),2001,33(1):80-82.
[37]李占强，朱丽.盐酸戊乙奎醚用于临床全麻患者术前给药效果观察[J].中华临床杂志,2002,2(12):65-66.
[38]Han XY, Liu H, Liu CH, et al. Synthesis of the optical isomers of a new anticholinergicdrug, penehyclidine hydrochloride (8018)[J]. Bioorganic&medicinal chemistry letters,2005,15(8):1979-1982.
[39]Niu WZ, Zhao DL, Liu CG. The effects of a new cholinolytic--8018--and its opticalisomers on the central muscarinic and nicotinic receptors [J]. Archives internationales depharmacodynamie et de therapie,1990,Mar-Apr(304):64-74.
[40]倪友洪，蒲春霞，雍萍.盐酸戊乙奎醚的应用[P].中国专利：1475214,2004.02.18.
[41]Xiao H, Liao Z, Meng X, et al. Effects of the selective muscarinic receptor antagonistpenehyclidine hydrochloride on the respiratory tract [J]. Die Pharmazie,2009,64(5):337-341.
[42]Xiao HT, Liao Z, Meng XM, et al. Characterization of the effect of penehyclidinehydrochloride on muscarinic receptor subtypes mediating the contraction of guinea-pigisolated gastrointestinal smooth muscle [J]. The Journal of pharmacy and pharmacology,2009,61(7):949-952.
[43]Shen W, Gan J, Xu S, et al. Penehyclidine hydrochloride attenuates LPS-induced acutelung injury involvement of NF-kappaB pathway [J]. Pharmacological research,2009,60(4):296-302.
[44]Genzen JR, Van Cleve W, McGehee DS. Dorsal root ganglion neurons express multiplenicotinic acetylcholine receptor subtypes [J]. Journal of neurophysiology,2001,86(4):1773-1782.
[45]Haberberger RV, Bernardini N, Kress M, et al. Nicotinic acetylcholine receptor subtypesin nociceptive dorsal root ganglion neurons of the adult rat [J]. Autonomic neuroscience:basic&clinical,2004,113(1-2):32-42.
[46]Fucile S, Sucapane A, Eusebi F. Ca2+permeability of nicotinic acetylcholine receptorsfrom rat dorsal root ganglion neurones [J]. The Journal of physiology,2005,565(Pt1):219-228.
[47]Elgoyhen AB, Vetter DE, Katz E, et al. alpha10: A determinant of nicotinic cholinergicreceptor function in mammalian vestibular and cochlear mechanosensory hair cells [J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(6):3501-3506.
[48]Sgard F, Charpantier E, Bertrand S, et al. A novel human nicotinic receptor subunit,alpha10, that confers functionality to the alpha9-subunit [J]. Molecular pharmacology,2002,61(1):150-159.
[49]New DC, Wong YH. Molecular mechanisms mediating the G protein-coupled receptorregulation of cell cycle progression [J]. Journal of molecular signaling,2007,2(2).
[50]Baudino TA, Cleveland JL. The max network gone mad [J]. Molecular and cellularbiology,2001,21(3):691-702.
[51]Yang K, Guo Y, Stacey WC, et al. Glycogen synthase kinase3has a limited role in cellcycle regulation of cyclin D1levels [J]. BMC cell biology,2006,7(33).
[52]Kim ES, Lee JJ, Wistuba, II. Cotargeting cyclin D1starts a new chapter in lung cancerprevention and therapy [J]. Cancer Prev Res (Phila),2011,4(6):779-782.
[53]Schwindinger WF, Robishaw JD. Heterotrimeric G-protein betagamma-dimers in growthand differentiation [J]. Oncogene,2001,20(13):1653-1660.
[54]Dasgupta P, Rastogi S, Pillai S, et al. Nicotine induces cell proliferation bybeta-arrestin-mediated activation of Src and Rb-Raf-1pathways [J]. The Journal of clinicalinvestigation,2006,116(8):2208-2217.
[55]Liang J, Slingerland JM. Multiple roles of the PI3K/PKB (Akt) pathway in cell cycleprogression [J]. Cell Cycle,2003,2(4):339-345.
[56]Azam F, Mehta S, Harris AL. Mechanisms of resistance to antiangiogenesis therapy [J].European Journal of Cancer,2010,46(8):1323-1332.
[57]Ebos JM, Lee CR, Kerbel RS. Tumor and host-mediated pathways of resistance anddisease progression in response to antiangiogenic therapy [J]. Clinical cancer research,2009,15(16):5020-5025.
[58]Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy [J]. Naturereviews Cancer,2008,8(8):592-603.
[59]Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation [J]. Cell,2011,144(5):646-674.
[1] Klapproth H, Reinheimer T, Metzen J, et al. Non-neuronal acetylcholine, a signalling molecule synthezised by surfacecells of rat and man [J]. Naunyn-Schmiedeberg's archives of pharmacology,1997,355(4):515-523.
[2] Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: The non-neuronal cholinergic system in humans [J]. Britishjournal of pharmacology,2008,154(8):1558-1571.
[3] Paleari L, Grozio A, Cesario A, et al. The cholinergic system and cancer [J]. Seminars in cancer biology,2008,18(3):211-217.
[4] Shah N, Khurana S, Cheng K, et al. Muscarinic receptors and ligands in cancer [J]. American journal of physiologyCell physiology,2009,296(2):C221-232.
[5] Egleton RD, Brown KC, Dasgupta P. Nicotinic acetylcholine receptors in cancer: Multiple roles in proliferation andinhibition of apoptosis [J]. Trends in pharmacological sciences,2008,29(3):151-158.
[6] Song P, Sekhon HS, Jia Y, et al. Acetylcholine is synthesized by and acts as an autocrine growth factor for small celllung carcinoma [J]. Cancer research,2003,63(1):214-221.
[7] Lang K, Bastian P. Neurotransmitter effects on tumor cells and leukocytes [J]. Progress in experimental tumor research,2007,39:99-121.
[8] Schuller HM. Neurotransmitter receptor-mediated signaling pathways as modulators of carcinogenesis [J]. Progress inexperimental tumor research,2007,39:45-63.
[9] Schuller HM. Neurotransmission and cancer: Implications for prevention and therapy [J]. Anti-cancer drugs,2008,19(7):655-671.
[10] Song P, Sekhon HS, Fu XW, et al. Activated cholinergic signaling provides a target in squamous cell lung carcinoma[J]. Cancer research,2008,68(12):4693-4700.
[11] Martinez-Moreno P, Nieto-Ceron S, Torres-Lanzas J, et al. Cholinesterase activity of human lung tumours variesaccording to their histological classification [J]. Carcinogenesis,2006,27(3):429-436.
[12] Thunnissen FB. Acetylcholine receptor pathway and lung cancer [J]. Journal of thoracic oncology,2009,4(8):943-946.
[13] Wess J. Molecular biology of muscarinic acetylcholine receptors [J]. Critical reviews in neurobiology,1996,10(1):69-99.
[14] Caulfield MP, Birdsall NJ. International union of pharmacology. XVII. Classification of muscarinic acetylcholinereceptors [J]. Pharmacological reviews,1998,50(2):279-290.
[15] Guizzetti M CP, Peters J, Costa LG. Acetylcholine as a mitogen: Muscarinic receptor-mediated proliferation of ratastrocytes and human astrocytoma cells.[J]. Europe journal of pharmacol,1996,297:265-273.
[16] Espanol A, Eijan AM, Mazzoni E, et al. Nitric oxide synthase, arginase and cyclooxygenase are involved in muscarinicreceptor activation in different murine mammary adenocarcinoma cell lines [J]. International journal of molecular medicine,2002,9(6):651-657.
[17] Cheng K, Samimi R, Xie G, et al. Acetylcholine release by human colon cancer cells mediates autocrine stimulation ofcell proliferation [J]. American journal of physiology Gastrointestinal and liver physiology,2008,295(3):G591-597.
[18] Oppitz M, Mobus V, Brock S, et al. Muscarinic receptors in cell lines from ovarian carcinoma: Negative correlationwith survival of patients [J]. Gynecologic oncology,2002,85(1):159-164.
[19] Song P, Sekhon HS, Lu A, et al. M3muscarinic receptor antagonists inhibit small cell lung carcinoma growth andmitogen-activated protein kinase phosphorylation induced by acetylcholine secretion [J]. Cancer research,2007,67(8):3936-3944.
[20] Luthin GR, Wang P, Zhou H, et al. Role of m1receptor-g protein coupling in cell proliferation in the prostate [J]. Lifesciences,1997,60(13-14):963-968.
[21] Sailer M, Oppitz M, Drews U. Induction of cellular contractions in the human melanoma cell line SK-mel28aftermuscarinic cholinergic stimulation [J]. Anatomy and embryology,2000,201(1):27-37.
[22] Boss A, Oppitz M, Lippert G, et al. Muscarinic cholinergic receptors in the human melanoma cell line SK-Mel28:Modulation of chemotaxis [J]. Clinical and experimental dermatology,2005,30(5):557-564.
[23] Bowers JW, Schlauder SM, Calder KB, et al. Acetylcholine receptor expression in Merkel cell carcinoma [J]. TheAmerican Journal of dermatopathology,2008,30(4):340-343.
[24] Kodaira M, Kajimura M, Takeuchi K, et al. Functional muscarinic m3receptor expressed in gastric cancer cellsstimulates tyrosine phosphorylation and MAP kinase [J]. Journal of gastroenterology,1999,34(2):163-171.
[25] Albuquerque EX, Pereira EF, Alkondon M, et al. Mammalian nicotinic acetylcholine receptors: From structure tofunction [J]. Physiological reviews,2009,89(1):73-120.
[26] Buckingham SD, Jones AK, Brown LA, et al. Nicotinic acetylcholine receptor signalling: Roles in alzheimer's diseaseand amyloid neuroprotection [J]. Pharmacological reviews,2009,61(1):39-61.
[27] Paleari L, Sessa F, Catassi A, et al. Inhibition of non-neuronal alpha7-nicotinic receptor reduces tumorigenicity inA549NSCLC xenografts [J]. International journal of cancer,2009,125(1):199-211.
[28] Paleari L, Negri E, Catassi A, et al. Inhibition of nonneuronal alpha7-nicotinic receptor for lung cancer treatment [J].American journal of respiratory and critical care medicine,2009,179(12):1141-1150.
[29] Dasgupta P, Rizwani W, Pillai S, et al. Nicotine induces cell proliferation, invasion and epithelial-mesenchymaltransition in a variety of human cancer cell lines [J]. International journal of cancer,2009,124(1):36-45.
[30] Dasgupta P, Rastogi S, Pillai S, et al. Nicotine induces cell proliferation by beta-arrestin-mediated activation of Src andRb-Raf-1pathways [J]. The Journal of clinical investigation,2006,116(8):2208-2217.
[31] Trombino S, Cesario A, Margaritora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation ofhuman mesothelioma cells: Role of mitogen-activated protein kinase pathway [J]. Cancer research,2004,64(1):135-145.
[32] Wong HP, Yu L, Lam EK, et al. Nicotine promotes cell proliferation via alpha7-nicotinic acetylcholine receptor andcatecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29cells [J]. Toxicology andapplied pharmacology,2007,221(3):261-267.
[33] Lee CH, Huang CS, Chen CS, et al. Overexpression and activation of the alpha9-nicotinic receptor duringtumorigenesis in human breast epithelial cells [J]. Journal of the National Cancer Institute,2010,102(17):1322-1335.
[34] Chen CS, Lee CH, Hsieh CD, et al. Nicotine-induced human breast cancer cell proliferation attenuated by garcinolthrough down-regulation of the nicotinic receptor and cyclin D3proteins [J]. Breast cancer research and treatment,2011,125(1):73-87.
[35] Linnoila RI. From nicotine to breast cancer, implications of cholinergic receptor pathway [J]. Journal of the NationalCancer Institute,2010,102(17):1298-1299.
[36] Calleja-Macias IE, Kalantari M, Bernard HU. Cholinergic signaling through nicotinic acetylcholine receptorsstimulates the proliferation of cervical cancer cells: An explanation for the molecular role of tobacco smoking in cervicalcarcinogenesis?[J]. International journal of cancer,2009,124(5):1090-1096.
[37] Chen RJ, Ho YS, Guo HR, et al. Rapid activation of Stat3and ERK1/2by nicotine modulates cell proliferation inhuman bladder cancer cells [J]. Toxicological sciences,2008,104(2):283-293.
[38] Chen RJ, Ho YS, Guo HR, et al. Long-term nicotine exposure-induced chemoresistance is mediated by activation ofStat3and downregulation of ERK1/2via nAChR and beta-adrenoceptors in human bladder cancer cells [J]. Toxicologicalsciences,2010,115(1):118-130.
[39] Guizzetti M, Costa LG. Possible role of protein kinase C zeta in muscarinic receptor-induced proliferation ofastrocytoma cells [J]. Biochemical pharmacology,2000,60(10):1457-1466.
[40] Jimenez E, Montiel M. Activation of MAP kinase by muscarinic cholinergic receptors induces cell proliferation andprotein synthesis in human breast cancer cells [J]. Journal of cellular physiology,2005,204(2):678-686.
[41] Raufman JP, Samimi R, Shah N, et al. Genetic ablation of M3muscarinic receptors attenuates murine colon epithelialcell proliferation and neoplasia [J]. Cancer research,2008,68(10):3573-3578.
[42] Xie G, Cheng K, Shant J, et al. Acetylcholine-induced activation of M3muscarinic receptors stimulates robust matrixmetalloproteinase gene expression in human colon cancer cells [J]. American journal of physiology Gastrointestinal and liverphysiology,2009,296(4):G755-763.
[43] Carlisle DL, Liu X, Hopkins TM, et al. Nicotine activates cell-signaling pathways through muscle-type and neuronalnicotinic acetylcholine receptors in non-small cell lung cancer cells [J]. Pulmonary pharmacology&therapeutics,2007,20(6):629-641.
[44] Zheng Y, Ritzenthaler JD, Roman J, et al. Nicotine stimulates human lung cancer cell growth by inducing fibronectinexpression [J]. American journal of respiratory cell and molecular biology,2007,37(6):681-690.
[45] Paleari L, Catassi A, Ciarlo M, et al. Role of alpha7-nicotinic acetylcholine receptor in human non-small cell lungcancer proliferation [J]. Cell proliferation,2008,41(6):936-959.
[46] Davis R, Rizwani W, Banerjee S, et al. Nicotine promotes tumor growth and metastasis in mouse models of lungcancer [J]. PLoS ONE,2009,4(10):e7524.
[47] Dasgupta P, Chellappan SP. Nicotine-mediated cell proliferation and angiogenesis: New twists to an old story [J]. CellCycle,2006,5(20):2324-2328.
[48] Schuller HM. Is cancer triggered by altered signalling of nicotinic acetylcholine receptors?[J]. Nature reviews Cancer,2009,9(3):195-205.
[49] Shant J, Cheng K, Marasa BS, et al. Akt-dependent NF-kappaB activation is required for bile acids to rescue coloncancer cells from stress-induced apoptosis [J]. Experimental cell research,2009,315(3):432-450.
[50] Dasgupta P, Kinkade R, Joshi B, et al. Nicotine inhibits apoptosis induced by chemotherapeutic drugs by up-regulatingXIAP and survivin [J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(16):6332-6337.
[51] Dan HC, Sun M, Kaneko S, et al. Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein(XIAP)[J]. The Journal of biological chemistry,2004,279(7):5405-5412.
[52] Grozio A, Paleari L, Catassi A, et al. Natural agents targeting the alpha7-nicotinic-receptor in NSCLC: A promisingprospective in anti-cancer drug development [J]. International journal of cancer,2008,122(8):1911-1915.
[53] Mai H, May WS, Gao F, et al. A functional role for nicotine in Bcl2phosphorylation and suppression of apoptosis [J].The Journal of biological chemistry,2003,278(3):1886-1891.
[54] West KA, Brognard J, Clark AS, et al. Rapid Akt activation by nicotine and a tobacco carcinogen modulates thephenotype of normal human airway epithelial cells [J]. The Journal of clinical investigation,2003,111(1):81-90.
[55] Jin Z, Gao F, Flagg T, et al. Tobacco-specific nitrosamine4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotesfunctional cooperation of Bcl2and c-Myc through phosphorylation in regulating cell survival and proliferation [J]. TheJournal of biological chemistry,2004,279(38):40209-40219.
[56] Xu L, Deng X. Tobacco-specific nitrosamine4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylationof mu-and m-calpain in association with increased secretion, cell migration, and invasion [J]. The Journal of biologicalchemistry,2004,279(51):53683-53690.
[57] Nishioka T, Guo J, Yamamoto D, et al. Nicotine, through upregulating pro-survival signaling, cooperates with NNK topromote transformation [J]. Journal of cellular biochemistry,2010,109(1):152-161.
[58] Brognard J, Clark AS, Ni Y, et al. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells andpromotes cellular survival and resistance to chemotherapy and radiation [J]. Cancer research,2001,61(10):3986-3997.
[59] Tsurutani J, Castillo SS, Brognard J, et al. Tobacco components stimulate Akt-dependent proliferation andNFkappaB-dependent survival in lung cancer cells [J]. Carcinogenesis,2005,26(7):1182-1195.
[60] Xin M, Deng X. Nicotine inactivation of the proapoptotic function of Bax through phosphorylation [J]. The Journal ofbiological chemistry,2005,280(11):10781-10789.
[61] Zhao J, Xin M, Wang T, et al. Nicotine enhances the antiapoptotic function of Mcl-1through phosphorylation [J].Molecular cancer research,2009,7(12):1954-1961.
[62] Espanol AJ, de la Torre E, Fiszman GL, et al. Role of non-neuronal cholinergic system in breast cancer progression [J].Life sciences,2007,80(24-25):2281-2285.
[63] Villablanca AC. Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro [J]. J ApplPhysiol,1998,84(6):2089-2098.
[64] Heeschen C, Weis M, Aicher A, et al. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholinereceptors [J]. The Journal of clinical investigation,2002,110(4):527-536.
[65] Heeschen C, Jang JJ, Weis M, et al. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis[J]. Nature medicine,2001,7(7):833-839.
[66] Carty CS, Soloway PD, Kayastha S, et al. Nicotine and cotinine stimulate secretion of basic fibroblast growth factorand affect expression of matrix metalloproteinases in cultured human smooth muscle cells [J]. Journal of vascular surgery,1996,24(6):927-934; discussion934-925.
[67] Lee WO, Wright SM. Production of endothelin by cultured human endothelial cells following exposure to nicotine orcaffeine [J]. Metabolism: clinical and experimental,1999,48(7):845-848.
[68] Cucina A, Sapienza P, Corvino V, et al. Nicotine-induced smooth muscle cell proliferation is mediated through bFGFand TGF-beta1[J]. Surgery,2000,127(3):316-322.
[69] Zhang Q, Tang X, Zhang ZF, et al. Nicotine induces hypoxia-inducible factor-1alpha expression in human lung cancercells via nicotinic acetylcholine receptor-mediated signaling pathways [J]. Clinical cancer research,2007,13(16):4686-4694.
[70] Shin VY, Wu WK, Chu KM, et al. Nicotine induces cyclooxygenase-2and vascular endothelial growth factorreceptor-2in association with tumor-associated invasion and angiogenesis in gastric cancer [J]. Molecular cancer research:MCR,2005,3(11):607-615.
[71] Martinez-Garcia E, Irigoyen M, Gonzalez-Moreno O, et al. Repetitive nicotine exposure leads to a more malignant andmetastasis-prone phenotype of SCLC: A molecular insight into the importance of quitting smoking during treatment [J].Toxicological sciences,2010,116(2):467-476.
[72] Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells[J]. Cell,2008,133(4):704-715.
[73] Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: Acquisition of malignant and stemcell traits [J]. Nature reviews Cancer,2009,9(4):265-273.
[74] Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition [J]. The Journal of clinical investigation,2009,119(6):1420-1428.
[75] Oppitz M, Busch C, Garbe C, et al. Distribution of muscarinic receptor subtype M3in melanomas and their metastases[J]. Journal of cutaneous pathology,2008,35(9):809-815.
[76] Chernyavsky AI, Arredondo J, Wess J, et al. Novel signaling pathways mediating reciprocal control of keratinocytemigration and wound epithelialization through M3and M4muscarinic receptors [J]. The Journal of cell biology,2004,166(2):261-272.
[77] Gautam A, Li ZR, Bepler G. RRM1-induced metastasis suppression through PTEN-regulated pathways [J]. Oncogene,2003,22(14):2135-2142.
[78] Garces YI, Yang P, Parkinson J, et al. The relationship between cigarette smoking and quality of life after lung cancerdiagnosis [J]. Chest,2004,126(6):1733-1741.
[79] Murin S, Inciardi J. Cigarette smoking and the risk of pulmonary metastasis from breast cancer [J]. Chest,2001,119(6):1635-1640.
[80] Murin S, Pinkerton KE, Hubbard NE, et al. The effect of cigarette smoke exposure on pulmonary metastatic disease ina murine model of metastatic breast cancer [J]. Chest,2004,125(4):1467-1471.
[81] Chipitsyna G, Gong Q, Anandanadesan R, et al. Induction of osteopontin expression by nicotine and cigarette smoke inthe pancreas and pancreatic ductal adenocarcinoma cells [J]. International journal of cancer,2009,125(2):276-285.
[82] Lazar M, Sullivan J, Chipitsyna G, et al. Involvement of osteopontin in the matrix-degrading and proangiogenicchanges mediated by nicotine in pancreatic cancer cells [J]. Journal of gastrointestinal surgery,2010,14(10):1566-1577.
[83] Shin VY, Jin HC, Ng EK, et al. Activation of5-lipoxygenase is required for nicotine mediated epithelial-mesenchymaltransition and tumor cell growth [J]. Cancer letters,2010,292(2):237-245.
[84] Nguyen VT, Ndoye A, Grando SA. Novel human alpha9acetylcholine receptor regulating keratinocyte adhesion istargeted by pemphigus vulgaris autoimmunity [J]. The American journal of pathology,2000,157(4):1377-1391.
[85] Elgoyhen AB, Johnson DS, Boulter J, et al. Alpha9: An acetylcholine receptor with novel pharmacological propertiesexpressed in rat cochlear hair cells [J]. Cell,1994,79(4):705-715.
[86] Rothlin CV, Katz E, Verbitsky M, et al. The alpha9nicotinic acetylcholine receptor shares pharmacological propertieswith type A gamma-aminobutyric acid, glycine, and type3serotonin receptors [J]. Molecular pharmacology,1999,55(2):248-254.
[87] Plazas PV, Katz E, Gomez-Casati ME, et al. Stoichiometry of the alpha9alpha10nicotinic cholinergic receptor [J]. TheJournal of neuroscience,2005,25(47):10905-10912.
[88] Chernyavsky AI, Arredondo J, Vetter DE, et al. Central role of alpha9acetylcholine receptor in coordinatingkeratinocyte adhesion and motility at the initiation of epithelialization [J]. Experimental cell research,2007,313(16):3542-3555.