Abstract
BackgroundChronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC) while nasopharyngeal reflux is known to be one of the major aetiological factors of CRS. Bile acid (BA), the component of gastric duodenal contents, has been recognised as a carcinogen. BA-induced apoptosis was suggested to be involved in human malignancies. Cells have the potential and tendency to survive apoptosis. However, cells that evade apoptosis upon erroneous DNA repair may carry chromosome rearrangements. Apoptotic nuclease, caspase-activated deoxyribonuclease (CAD) has been implicated in mediating translocation in leukaemia. We hypothesised that BA-induced apoptosis may cause chromosome breaks mediated by CAD leading to chromosome rearrangement in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is one of the most common deletion sites in NPC.MethodsWe tested the ability of BA at neutral and acidic pH in inducing phosphatidylserine (PS) externalisation, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) disruption, and caspase 3/7 activity in normal nasopharyngeal epithelial (NP69) and NPC (TWO4) cells. Inverse-PCR (IPCR) was employed to detect AF9 gene cleavages. To investigate the role of CAD in mediating these cleavages, caspase inhibition was performed. IPCR bands representing AF9 cleaved fragments were sequenced.ResultsBA-treated cells showed higher levels of PS externalisation, ROS production, MMP loss and caspase 3/7 activity than untreated control cells. The effect of BA in the induction of these intracellular events was enhanced by acid. BA at neutral and acidic pH also induced significant cleavage of the AF9 gene. These BA-induced gene cleavages were inhibited by Z-DEVD-FMK, a caspase-3 inhibitor. Intriguingly, a few chromosome breaks were identified within the AF9 region that was previously reported to participate in reciprocal translocation between the mixed lineage leukaemia (MLL) and AF9 genes in an acute lymphoblastic leukaemia (ALL) patient.ConclusionsThese findings suggest a role for BA-induced apoptosis in mediating chromosome rearrangements in NPC. In addition, CAD may be a key player in chromosome cleavages mediated by BA-induced apoptosis. Persistent exposure of sinonasal tract to gastric duodenal refluxate may increase genomic instability in surviving cells.
Highlights
Chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC) while nasopharyngeal reflux is known to be one of the major aetiological factors of CRS
Representative fluorescence microscopic images showing the Bile acid (BA)-treated NP69 and TWO4 cells were shown in Figs. 4b and 5b. These findings suggest that ai bi aii bii BA-induced apoptosis results in chromosome breaks in the AF9 gene To test if BA-induced apoptosis leads to cleavage in the AF9 gene, BA-treated NP69 and TWO4 cells were subjected to genomic DNA extraction and subsequently IPCR
The effect of BA was amplified by acidic pH, suggesting that there is a synergistic effect between acid and BA in the induction of these events
Summary
Chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC) while nasopharyngeal reflux is known to be one of the major aetiological factors of CRS. We hypothesised that BA-induced apoptosis may cause chromosome breaks mediated by CAD leading to chromosome rearrangement in NPC. A retrospective cohort study involving 231,490 Taiwan Chinese individuals revealed that individuals with chronic rhinosinusitis (CRS) have a 3.55-fold higher risk of developing NPC as compared with those without CRS [12]. Similar findings had been found in a case-control study which comprised 2242 NPC patients and 6726 controls; the odd ratio of prior CRS for subjects with NPC is 3.83 (95% confidence interval [CI], 3.23–4.53) [11] The investigators attributed these findings to the chronic inflammation which may predispose the nasopharyngeal mucosa to transformation by carcinogens [7,8,9, 11, 12]. The causative factors and the underlying mechanisms which contribute to the chronic inflammation of nose or ear leading to NPC remain enigmatic
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