Abstract 36P
Background
Nicotine, one of the core components of tobacco smoke, has been correlated with cancer progression and tumorigenesis in breast cancer. However, the exact mechanism involved remains under heavy debate as different groups have yielded different possible pathways with α7 and α9 being rooted as the potential key mediators of nicotine. Lack of genomic data, use of extreme high concentrations of nicotine and lack of explanation of such further aggravates matters. In this study, we aim to elucidate the role of α9 nAChR/ CHRNA9 as the pivotal receptor for mediating nicotine-induced effects in breast cancer and explore the possible effects of clinically relevant concentrations of nicotine in transitory ER stress and EMT induced by TGF-β1 in breast cancer.
Methods
Real-time PCR was used to evaluate possible expression of CHRNA9 in breast cancer cell-lines MCF-7 and MDA-MB-231. ER stress response and subsequent dose-evaluation of nicotine was then conducted by treating MCF-7 cells with 5 μg/ml tunicamycin for 24 hours. Experimental groups were co-treated with nicotine concentrations of 50 nM, 250 nM and 500 nM respectively. Viability of these MCF-7 cells were done using standard WST-1 assay. For induction of EMT, MCF-7 cells were treated with 10 ng/ml TGF-β1 for 3 days. Experimental groups consisted of co-treatment with low-dose nicotine (TGF-β1 + nicotine), low-dose nicotine and CHRNA9 antagonist methyllycaconitine (TGF-β1+nicotine+MLCN), low dose nicotine only and 10μM nicotine. Subsequent expression of different EMT and ER stress markers were evaluated using western blotting.
Results
Real-time PCR analysis revealed moderate expression of CHRNA9 in untreated MCF-7 and MDA-MB-231 cells with a 3-4 fold increase after TGF-β1 treatment (p<0.001). Action of nicotine is dose dependent as 500nM of nicotine alleviates both tunicamycin-induced ER stress (p< 0.05) and basal ER stress in MCF-7 cells (p<0.05) while 10μM increases ER stress and rapidly induces EMT in MCF-7 cells (p<0.001). No significsnt effects are observed during EMT for low-dose nicotine groups. However, CHRNA9 levels increase steadily with increasing mesenchymal-phenotype markers.
Conclusions
In conclusion, our data suggests that the action of nicotine is entirely mediated via CHRNA9 and subsequent pathway is entirely dose-dependent.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Department of Bioengineering, George Mason University.
Funding
Department of Bioengineering, George Mason University.
Disclosure
All authors have declared no conflicts of interest.