br Previous studies have shown that blockade

Previous studies have shown that blockade of α7-nAChR with spe-cific antagonists or siRNA prevents the stimulatory effects of nicotine and NNK on proliferation, migration, EMT or angiogenesis in several cancer cell lines [13,29,31,33]. Nicotine and NNK are α7-nAChR ago-nists, and the affinity of nitrosamine for the receptor is 1,300 times greater than that of nicotine [34]. Since we did not find significant differences in the in vitro tumorigenic effects between nicotine and NNK, we can conclude that both agents produce all the above oncogenic effects mainly through α7-nAChRs expressed in A549 or SK-MES-1 cells.
The present in vitro findings are corroborated in our in vivo study, where we found that nicotine promotes tumor growth of A549 
xenografts transplanted into nude mice (Figs. 6A-6C). This nicotine effect on tumor growth agrees with previous data obtained in xeno-grafts corresponding to different tumor cell lines, including lung, breast, Meropenem or hepatic carcinoma cell lines [35–38]. Moreover, the histological analysis of A549 xenografts in our in vivo study reveals the increased expression of the angiogenesis (VEGF) and proliferative (Ki67) markers in response to nicotine (Fig. 6D), a finding already re-ported in xenografts derived from adenocarcinoma or SCLC cell lines [39–41]. Here, we report, for the first time, that dupα7 overexpression in A549 xenografts completely suppresses tumor growth and the in-creased expression of tumor progression markers in vivo. Although we still do not know the mechanism by which dupα7 interferes with the α7-nAChR function in NSCLC cells, we have already reported that du-plicated subunits assembled with ancestral subunits to form hetero-meric dupα7/α7-nAChRs in the neuroendocrine GH4C1 cell line [20].
Fig. 6. Overexpression of dupα7 in a nude mouse A549 xenograft model suppressed the nicotine-mediated tumorigenic effect in vivo. Wild-type cells (A549) or dupα7 overexpressing cells (A549dupα7) were innoculated into the left flanks of the mice, which would then receive nicotine or not in the drinking water. There were four mouse subgroups (5–6 mice/subgroup) in the study. (A) Tumor volume growth (mean ± SEM) in the four subgroups over the 37 days of the study; tumor volume was expressed in mm3. #p < 0.05 and ##p < 0.01 after multiple comparative analysis of the indicated mice subgroups. (B) Photos of four representative mice from each subgroup as well as their corresponding tumors excised immediately after the sacrifice of the animal. (C) Final tumor volumes (mean ± SEM) determined in the four subgroups after the completion of the study. *p < 0.05, significantly different from the wild-type A549 xenograft subgroup that did not receive nicotine; ns, no significant differences between the two A549dupα7 xenograft subgroups (receiving nicotine or not). #p < 0.05 and ##p < 0.01, significant differences between the indicated subgroups. (D) Microscopic images of expression levels for proliferation (Ki67) and angiogenesis (VEGF) markers, staining (brown) by IHC, in a re-presentative xenograft tumor from each subgroup.
As expected from the structural characteristics of dupα7, these het-eromeric receptors exhibit worse migration from endoplasmic re-ticulum to the cell membrane, as well as less sensitivity to agonists than homomeric α7-nAChRs and thus, reduced functionality [18,20].
It should be noted that SCLC cells express high levels of α7-nAChRs, which have been identified as central regulators of nicotine- or NNK-induced proliferation and migration by these cells through stimulation of the release of autocrine growth factors serotonin, mammalian bom-besin and, possibly, other neuropeptides [see Ref. [42] and references therein]. Given that the strength of association with smoking is stronger for SCLC than for lung adenocarcinoma [43], one of the two major histological types of NSCLC, it is feasible that the tumor suppressor effect of dupα7 on α7-nAChR activity that we have just revealed in A549 cells may also be applicable to SCLC tumors. Additional experi-ments with SCLC cell lines will be necessary to contrast the above proposal.