| 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 ofThr308p-Akt, c-MycandSer9p-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 ofTyr705p-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 ofSer807/811p-Rb, cyclin D1, CDK4and CDK6induced by R2HBJJ,which resulted in cell cycle G0/G1arrest at last. R2HBJJ reduce the nuclear translocation ofTyr705p-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.
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