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Sulfur mustard (a type of vesicant) can directly damage lung bronchial epithelium via aerosol inhalation, and prevalent cell death is an early event that obstructs the respiratory tract. JNK/c-Jun is a stress response pathway, but its role in cell death of the injured cells is not clear. Here, we report that JNK/c-Jun was activated in immortalized human bronchial epithelial (HBE) cells exposed to a lethal dose (20 μM) of nitrogen mustard (NM, a sulfur mustard analog). c-Jun silencing using small-interfering RNA (siRNA) rendered the cells resistant to NM-mediated cell death by blocking poly(ADP-ribose) polymerase 1 (PARP1) cleavage and DNA fragmentation. In addition, the transduction of upstream extrinsic (Fasl-Fas-caspase-8) and intrinsic (loss of Bcl-2 and mitochondrial membrane potential, ΔΨm) apoptosis pathways, as well as phosphorylated (p)-H2AX (Ser139), an epigenetic marker contributing to DNA fragmentation and PARP1 activity, was partially suppressed. To mimic the detachment of cells by NM, HBE cells were trypsinized and seeded on culture plates that were pre-coated with poly-HEMA to prevent cell adhesion. The JNK/c-Jun pathway was found to be activated in the detached cells. In conclusion, our results indicate that JNK/c-Jun pathway activation is necessary for NM-caused HBE cell death and further suggest that c-Jun silencing may be a potential approach to protect HBE cells from vesicant damage.

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long‐term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3β activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis‐related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti‐apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin‐1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction., Highlights ► APPL2 protein levels are elevated in 40% cases of glioblastoma multiforme.► Overexpression of APPL2 exhibits cytoprotective effects in glioma cells.► APPL2 depletion reduces survival and transformation abilities of glioma cells.► Silencing of APPL2 promotes expression of proapoptotic genes HRK and UNC5B.