【作者】 华南；

【导师】 郑建全；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2012， 博士

【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^-10M（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^-9M（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^Thr308p-Akt、c-Myc和^Ser9p-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^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。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^Ser807/811p-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。更多还原

【Abstract】 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^-10and2.64±0.98×10^-10M （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^-9M （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^Thr308p-Akt, c-Mycand^Ser9p-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^Tyr705p-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^Ser807/811p-Rb, cyclin D1, CDK4and CDK6induced by R2HBJJ,which resulted in cell cycle G0/G1arrest at last. R2HBJJ reduce the nuclear translocation of^Tyr705p-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.更多还原