Pepsin Promotes Laryngopharyngeal Carcinoma Through Regulation Of Signaling Pathways That Related To Proliferation And Cell Cycle

Background:The purpose of this study was to investigate whether laryngopharyngeal reflux(LPR)can play a role in the pathogenesis of hypopharyngeal cancer;therefore,we assessed the effect of pepsin on hypopharyngeal squamous cell cancer(HPSCC)patient tissue and cell lines and investigated its effect on signal transduction pathway.Methods:(1)70 HPSCCs and paired tumor adjacent tissues were analyzed for presentation of pepsin using immunohistochemistry.(2)Then,we exposed human hypopharyngeal squamous cancer cells(Fadu cell line)to pepsin at neutral p H,and the effect of pepsin was assessed by cell proliferation bioassay and flow cytometry assays.(3)Lastly,we centered our study on the human signal pathway of pepsin caused Fadu cell proliferation by real-time polymerase chain reaction(PCR)array technology.Results:(1)The presentation of pepsin was markedly higher in cancer tissues(39/70,55.7%))than in tumor-adjacent tissues(21/70,30.0%))(P<0.01)by IHC.A significant correlation was found between high pepsin staining and nodal stage of HPSCC(P<0.05),but not with alcohol consumption,tobacco exposure,T stage or G stage.(2)In vitro study shows us that: 1)Fadu cells exposed to pepsin(0.2mg/ml,0.4mg/ml)at p H 7 for 30 mins demonstrated a significant increase in cell numbers,extending exposure time(1hour,2 hours or 4 hours)did not show more effect on cell numbers.Cell cycle analysis of propidium iodide-stained Fa Du cells treated with pepsin revealed a statistically significant increase in the percentage of cells in S phase and decrease in the percentage of cells in G1 phase following pepsin exposure with a clear dose response.An increase in the cells number was not observed in cells exposed to pepsin that had been irreversibly inactivated.2)Fadu cells treated with Pepsin-Cy3 for 0.5 h,washed and incubated for 24 hours or 36 hours.After be treated with Lyso-tracker red,an indicator of acid environment,subsequent microscopy showed that co-localization of the red tracker with pepsin was detected.Up to 36 hours after treatment,pepsin still mainly located in the lysosome.3)The Human Signal Transduction Pathway PCR Array analysis revealed that 6 of the 84 examined genes that changed at least a 1.5-fold between control and pepsin-treated group.Our study confirmed that pepsin treated group had >1.5-Fold TNF-α(tumor necrosis factor),and BCL2A1(Bcl-2-related protein A1)expression level than control group with significant difference(P< 0.05).Pepsin treatment of Fadu cell did down–regulate the TNFSF10(Tumor necrosis factor superfamily member 10,TRAIL)and HES5(hes family b HLH transcription factor 5)expression for-1.93 fold and-2.83 fold respectively compared to control group with significant difference(P<0.05).The CCND2(Cyclin D2)expression down-regulated for-1.88 fold and the FABP1(Fatty Acid Binding Protein 1)expression up-regulated for1.57 fold compared to control group but without significant difference(P=0.36,P=0.42respectively).These genes related to NF-κB,TRAIL and Notch signaling.4)In consistent with the PCR array results,NF-κB p65,p21 and c-Myc validation were performed by immunofluorescent staining.And,further semi-quantitative analysis of the staining of these three proteins expression confirm that they were higher expressed in pepsin-treated Fadu cell than control(P< 0.05).Western blot of p65 and IκB results show that the phosphorylation levels of these two proteins in pepsin treated Fadu cells were significantly increased compared with control(P< 0.05,respectively).Conclusion:Collectively,according to the findings of the present study,we suggest that pepsin reflux induces a dose-dependent promotion of proliferation in hypopharyngeal cancer cell that is highly related to its enzymatic activity.Although the molecular and cellular mechanisms linking pepsin exposure to promotion of hypopharyngeal cancer is not fully understood,NF-κB and TRAIL pathway,the two major mediators of cell proliferation,differentiation and apoptosis,likely play crucial roles in the cancer development.There is increasing evidence LPR caused chronic inflammation is related to HPSCC,and to develop targeted pharmacologic inhibitors of pepsin activity and early prevention of LPR is essential.