br Overexpression of dup in NSCLC cells
3.4. Overexpression of dupα7 in NSCLC Ceruletide prevents the epithelial-mesenchymal transition (EMT) elicited by nicotine or NNK in vitro
EMT is the trans-diﬀerentiation of stationary epithelial cells into motile mesenchymal cells; it facilitates the motility of individual cells and the acquisition of an invasive phenotype. Changes in expression of epithelial (β-catenin) or mesenchymal (vimentin and fibronectin) markers in response to nicotine or NNK caused by overexpression of dupα7 were evaluated by Western blot in A549dupα7 or SK-MES-1dupα7
cells and in the corresponding wild-type cells, using β-actin as the loading control protein. Fig. 5A shows representative blots of the ex-pression of the above markers induced by 48 h of incubation of each cell variant with nicotine (1 μM) or NNK (100 nM). Fig. 5B shows pooled immunoblot results, expressed as the marker/β-actin ratio for each cell variant, taking the ratio in non-stimulated (control) cells as 1; bars show the mean values ± SEM obtained in diﬀerent cell cultures. Re-sults reveal reduced expression of β-catenin accompanied by increased
expression of vimentin and fibronectin in A549 or SK-MES-1 upon ni-cotine or NNK incubation compared with their respective non-stimu-lated cell variants (Control). In contrast, in the case of A549dupα7 or SK-MES-1dupα7 cells, changes in the expression of the above markers in-duced by nicotine or NNK were either abolished (β-catenin) or the opposite (decreased expression of vimentin or fibronectin) of those observed in the corresponding wild-type cell variant subjected to the same treatment.
3.5. Overexpression of dupα7 suppressed the nicotine-mediated tumorigenic eﬀect in vivo
Next, we proceeded to evaluate whether the in vitro findings were reproduced in vivo in a nude mouse xenograft model. In this model, wild-type A549 cells or A549dupα7 cells were inoculated into the left flanks of the mice, which would then receive or not receive nicotine (1 μM) in their drinking water. The volumes of the xenograft tumors generated in the four subgroups were measured over the 37 days of the study. Tumor volumes (mean ± SEM) in the two wild-type A549 xe-nograft subgroups (receiving nicotine or not) reflected a progressive and significantly higher growth rate than that observed in the two subgroups of A549dupα7 xenografts subjected to the same conditions (Fig. 6A). In fact, volumes in the two latter subgroups remained quite small and stable throughout the study. Furthermore, the final tumor volumes (mean ± SEM) determined in the four subgroups after the completion of the study revealed that nicotine produced significant diﬀerences between the two wild-type subgroups but not between the two subgroups with dupα7 overexpression (Fig. 6C). Fig. 6B shows the
Fig. 3. Overexpression of dupα7 in NSCLC cell lines suppresses the in vitro pro-migratory eﬀects of nicotine or NNK. Cell migration in A549 cells (top) or SK-MES-1 cells (bottom) was determined by the transwell migration assay. (A) Representative confocal images (40X) of the migration rate in wild-type cells (A549 or SK-MES- 1) or in cells overexpressing dupα7 (A549dupα7 or SK-MES-1dupα7) after 24 h stimulation with nicotine (1 μM) or NNK (100 nM). Blank: unstimulated cells. (B) Bar charts representing the pooled results of cell migration, expressed as a percentage of the spontaneous migration by the same unstimulated cell variant (considered
100%), in response to nicotine or NNK. Values show mean ± SEM from five independent cell cultures; 2-3 microscopic fields were randomly selected for migration analysis in each cell culture. ***p < 0.001 compared to its corresponding unstimulated cell variant. ##p < 0.01 and ###p < 0.001 after comparing the indicated groups.
photos corresponding to four representative mice tested for each sub-group as well as their corresponding tumors excised immediately after their sacrifice. The IHC analysis reveals a clear increase in the expres-sion of the cellular markers for proliferation (Ki67) and angiogenesis (VEGF) in the A549 xenograft tumors of mice receiving nicotine com-pared to those that did not (Fig. 6D). The eﬀect of nicotine on the ex-pression of the two markers was suppressed in the A549dupα7 xeno-grafts. Collectively, our latest findings reveal that dupα7 overexpression in lung adenocarcinoma cells suppresses tumor growth and progression in vivo, just as it does in vitro.
The present study shows the first experimental evidence that dupα7, a human-specific duplicated form of the α7-nAChR subunit, interferes with the tobacco-activated tumorigenic activity mediated by α7-nAChRs in human NSCLC cell lines (A549 or SK-MES-1). Specifically, we show that dupα7: (1) significantly inhibits in vitro cell migration, proliferation and EMT induced by nicotine or NNK in both cells lines; and (2) strongly restrains nicotine-induced tumor growth and increased expression of tumor markers (Ki67 or VEGF) in A549 xenografts transplanted into nude mice.