br were going to be used
were going to be used throughout our study. The results show that, in comparison to wild-type cells, the overexpression of dupα7-Myc in stably-transfected KN93 (Figs. 1A and B) did not modify the endogenous expression of α7 mRNA or protein in either of the two NSCLC cell lines tested (Figs. 1A and C).
Fig. 1. The endogenous expression of α7 in human NSCLC cell lines remains unchanged after stable overexpression of dupα7. The figure reflects the expression of native α7, at the mRNA or protein level, in parallel with the corresponding expression of dupα7 mRNA (native plus foreign) or dupα7-Myc protein (for-eign) in the four cellular variants that were going to be used
throughout present study. These variants correspond to cells stably-transfected with dupα7-Myc (A549dupα7 or SK-MES-1dupα7)
or to non-transfected wild-type cells (A549 or SK-MES-1). (A) Normalized expression of dupα7 or α7 messengers determined by qPCR in the above cell variants. Data show mean ± SEM of three independent cell cultures. *p < 0.05 and ***p < 0.001 com-pared to non-transfected cells. (B) Confocal images showing the successful overexpression of the dupα7-Myc subunit in A549dupα7 or SK-MES-1dupα7 cells compared to expression in wild-type cells; the subunit was detected by cell incubation with the anti-Myc primary antibody followed by the appropriate Alexa Fluor 488 secondary antibody. (C) Immunoblot evaluating the α7-nAChR subunit expression in the above four cellular variants using β-actin as a loading control; the blot is representative of two independent experiments. Specific bands corresponding to α7 protein expres-sion were detected with the anti-α7 primary antibody and the appropriate secondary (HRP)-conjugated antibody.
3.2. Stable overexpression of dupa7 in NSCLC cells prevents in vitro cell migration induced by nicotine or NNK
To get an insight into the functional eﬀect of dupα7 on the onco-genic activity induced by nicotine or NNK in NSCLC cells, we analyzed the eﬀects of both tobacco smoke constituents on migration in non-
transfected (wild-type) cells or in cells with stable dupα7 over-expression (A549dupα7 or SK-MES-1dupα7). The wound-healing assay
performed on wild-type cells showed significantly increased migration after 24 h of stimulation with nicotine (1 μM), NNK (100 nM) or 10% FBS (Fig. 2) compared to the corresponding unstimulated cells (Blank). The overexpression of dupα7 in the transfected cells significantly re-duced the pro-migratory eﬀect of nicotine or NNK, but not that due to FBS. This last observation ruled out the possibility that dupα7 over-expression alters the migratory capacity of these cells. Moreover, the pro-migratory response to any of the three stimuli tested in the cells
overexpressing the empty-vector (A549pcDNA3.1 or SK-MES-1pcDNA3.1) was indistinguishable from that found in the corresponding wild-type cell variant (Fig. 2B). The transwell migratory assay performed with the same cell variants and treatments described above yielded similar re-
sults: the increase in cell migration promoted by nicotine or NNK (24 h) in wild-type cells was abolished in A549dupα7 or SK-MES-1dupα7 cells (Fig. 3).
3.3. Nicotine and NNK lose their ability to stimulate in vitro cell proliferation in NSCLC cells overexpressing dupα7
Changes in cell proliferation elicited by nicotine or NNK due to dupα7 overexpression were assessed by an EdU assay performed on the above NSCLC cell lines. Fig. 4A shows typical confocal images of non-proliferating cells (blue nuclear staining with DAPI) or EdU positive proliferating cells (magenta color staining) obtained in each of the four
Fig. 2. Overexpression of dupα7 in NSCLC cell lines inhibits in vitro cell migration induced by nicotine and NNK. Migration rate in A549 cells (top) or SK-MES-1 cells (bottom) was determined by the wound-healing assay. (A) Representative microscope images (10X) of the wound captured at 0 and 24 h after scratching in wild-type cells (A549 and SK-MES-1) or in dupα7 overexpressing cells (A549dupα7 and SK-MES-1dupα7) after 24 h stimulation with NNK (100 nM). Blank: cells not stimulated.
(B) Pooled results of the migration rate induced by nicotine, NNK or FBS in wild-type cells or in cells that overexpress dupα7 or the empty vector (A549pcDNA3.1 and SK-MES-1pcDNA3.1). The migration rate, expressed as percentage of wound healing (scratch closure), was calculated according to the next equation: % of wound healing = [(a-b/a)x100], where (a) is the distance between both edges of the wound at 0 h and (b) at 24 h. Data show mean ± SEM for the cell migration induced by
cell variants used here (A549, SK-MES-1, A549dupα7 or SK-MES-1dupα7) in the diﬀerent conditions tested. Fig. 4B represents means ± SEM of the number of proliferating cells, expressed as a percentage of the total number of cells found in the microscope field, in response to each sti-mulus. A significant increase in proliferation was found in non-trans-fected cells of both cell types after 24 h of incubation with nicotine (1 μM), NNK (100 nM) or 10% FBS. Once again, the proliferative eﬀect induced by nicotine or NNK was completely lost in A549dupα7 or SK-MES-1dupα7 cells, whereas that of FBS (10%) persisted in cells over-expressing dupα7.