Sulforaphane Inhibits the Acquisition of Tobacco Smoke-Induced Lung Cancer Stem Cell-Like Properties via the IL-6/ΔNp63α/Notch Axis

Background: Tobacco smoke (TS) critically contributes to the development of lung cancer; however, the underlying molecular mechanisms remain unclear. The induction of cancer stem cells (CSCs) by TS represents an early event in tumor initiation. The lung cancer-related gene Delta Np63 alpha is highly expressed in epithelial tissues and drives tumor formation and cancer stem cell properties. This study investigated the role of Delta Np63 alpha in the long-term acquisition of TS-induced lung CSC-like properties. Methods: The expression levels of Delta Np63 alpha, lung CSC markers, and interleukin (IL)-6 in lung carcinoma specimens were determined by western blotting and enzyme linked immunosorbent assays. Human bronchial epithelial (HBE) cells were chronically exposed to 2 % cigarette smoke extract for 55 passages, following which colony formation capacity, expression of proteins associated with malignant transformation, lung CSC markers, and tumor incidence were investigated. The effects of Delta Np63 alpha on long-term TS exposure-induced lung CSC-like properties and Notch activation were analyzed using tumorsphere formation ability, immunofluorescence assays, luciferase reporter assays, and western blotting. The roles of IL-6 on chronic TS exposure-induced lung CSC-like properties and Delta Np63 alpha expression were also examined. Moreover, the effects of sulforaphane (SFN) on TS-transformed lung CSC-like properties, IL-6 and Delta Np63 alpha expression, and Notch signaling were investigated in vitro and in vivo. Results: Higher levels of Delta Np63 alpha were observed in the lung cancer tissues of smokers than in those of non-smokers, whereas Delta Np63 alpha was positively correlated with CD133 and Oct4 expression in lung cancer tissues. Data from the in vivo and in vitro experiments demonstrated that long-term TS exposure-transformed HBE (THBE) cells acquired lung CSC-like properties. Furthermore, Delta Np63 alpha transcriptionally activated the Notch signaling pathway to promote the acquisition of CSC-like properties by the THBE cells. TS upregulated IL-6, which increased Delta Np63 alpha expression in THBE sphere-forming cells. Finally, SFN inhibited the TS-induced CSC-like properties of THBE cells via the IL-6/Delta Np63 alpha Notch axis. Conclusion: Our data suggest that the IL-6/Delta Np63 alpha/Notch axis plays an important role in the long-term TS exposure-induced acquisition of lung CSC-like properties and SFN intervention.