Influence of acid and bile acid on ERK activity, PPAR_Y expression and cell proliferation in normal human esophageal epithelial cells

AIM： To observe the effects of acid and bile acid exposure on cell proliferation and the expression of extracellular signal-regulated protein kinase （ERK） and peroxisome proliferator-activated receptor γ （PPARγ） in normal human esophageal epithelial cells in vitro. METHODS： In vitro cultured normal human esophageal epithelial cells were exposed to acidic media （pH 4.0-6.5）, media containing different bile acid （250 μmol/ L）, media containing acid and bile acid, respectively. Cell proliferation was assessed using MTT and flow cytometry. The expressions of phosphorylated ERK1/2 and PPAR~ protein were determined by the immunoblotting technique. RESULTS： Acid-exposed （3 min） esophageal cells exhibited a significant increase in proliferation ratio, S phase of the cell cycle （P〈0.05） and the level of phosphorylated ERK1/2 protein. When the acid-exposure period exceeded 6 min, we observed a decrease in proliferation ratio and S phase of the cell cycle, with an increased apoptosis ratio （P〈0.05）. Bile acid exposure （3-12 min） also produced an increase in proliferation ratio, S phase of the cell cycle （P〈0.05） and phosphorylated ERKx/2 expression. On the contrary, deoxycholic acid （DCA） exposure （〉 20 rain） decreased proliferation ratio. Compared with bile acid exposure （pH 7.4）, bile acid exposure （pH 6.5, 4） significantly decreased proliferation ratio （P〈0.05）. There was no expression of PPARy in normal human esophageal epithelial cells.CONCLUSION： The rapid stimuli of acid or bile acid increase proliferation in normal human esophageal epithelial cells by activating the ERK pathway.

Cellular proteomic profiling of esophageal epithelial cells cultured under physioxia or normoxia reveals high correlation of radiation response

Objective To investigate the radiation response and proteomic profiling of esophageal epithelial cells cultured under physioxia and normoxia.Methods The human immortalized normal esophageal epithelial cell line SHEE cells were cultured under normoxia(21%)and physioxia(4%),respectively.A clonogenic assay was performed to evaluate the radiation response of SHEE cells.Cellular proteomic profiling of SHEE cells maintained under physioxia and normoxia was conducted to determine the differentially expressed proteins.Then,the identified differentially expressed proteins were validated by Western blot.Results SHEE cells exposed to normoxia showed an increased radiation response compared to physioxia(irradiation dose≥10Gy,P<0.05).Over 1200 non-redundant proteins were identified in the collected samples.Protein expression was compared between physioxia and normoxia,42 proteins were downregulated and 45 proteins upregulated,in which oxidative phosphorylation was the most significantly enriched pathway.When cells were cultured under normoxia conditions,the induction of antioxidant genes appeared to contribute to form a phenotype adapted to the environment with high oxygen-content.Further analysis validated NRF2,BIP,VCP,SOD1,and YAP1 were the key regulators of this phenotype.Conclusions Compared with physioxia,normoxic cell culture condition can enhance the radiation response.This study could stimulate in vivo microenvironment,and provide a basis for radiation-induced normal tissue damage.

Bile salts inhibit growth and induce apoptosis of culture human normal esophageal mucosal epithelial cells

AIM： To investigate the effect of six bile salts： glycocholate （GC）, glycochenodeoxycholate （GCDC）, glycodeoxycholate （GDC）, taurocholate （TC）, taurochenodeoxycholate （TCDC）, taurodeoxycholate （TDC）, and their mixture on cultured human normal esophageal rnucosal epithelial cells. METHODS： Human normal esophageal mucosal epithelial cells were cultured with serum-free keratinocyte medium. 3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide assay was applied to the detection of cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay. Sub-G1 DNA fragmentations and early apoptotic cells were assayed by flow cytometry （FCI） with propidium iodide （PI） staining and annexin V-FITC conjugated with PI staining. Apoptotic DNA ladders on agarose gel electrophoresis were observed. RESULTS： Except for GC, GCDC, GDC, TC, TCDC, TDC and their mixture could initiate growth inhibition of esophageal mucosal epithelial cells in a dose- and time-dependent manner. TUNEL and FCM assays demonstrated that the bile salts at 500 μmol/L and their mixture at 1 500 μmol/L induced apoptosis except for GC. The percentage of sub-G1 detected by FCM with PI staining was 83.5% in cells treated with 500 μmol/L TC for 2 h, and 19.8%, 20.4%, 25.6%, 13.5%, and 75.8% in cells treated with 500 μmol/L GCDC, TCDC, GDC, TDC, and 1 500 μmol/L mixture for 24 h, respectively, which were higher than that of the control （1.5%）. The percentage was 1.4% in cells with 500 μmol/L GC for 24 h. DNA ladders on agarose gel electrophoresis were seen in cells treated with 500 μmol/L TC for 2 h and i 500 μmnol/L mixture for 24 h. CONCLUSION： All GCDC, GDC, TC, TCDC, TDC and their mixture can inhibit growth and induce apoptosis of cultured human normal esophageal mucosal epithelial cells, but GC is well tolerated by the cells.