Bile acid reflux contributes to development of esophageal adenocarcinoma via activation of phosphatidylinositol-specific phospholipase Cgamma2 and NADPH oxidase NOX5-S

Abstract

Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA.

Figure 1. Effect of U73122 on TDCA-induced NOX5-S expression

(A) A typical example of Western blot analysis and summarized data showed PI-PLC inhibitor U73122 significantly decreased TDCA-induced NOX5-S protein expression. (B) U73122 significantly reduced TDCA-induced H₂O₂ production. (C) U73122 significantly reduced TDCA-induced thymidine incorporation. The data suggest that TDCA-induced increase in NOX5-S expression, H₂O₂ and cell proliferation may depend on activation of PI-PLC. (D) A typical example of Western blot analysis of three experiments showed that PI-PLC β1, β3, β4, γ1, and γ2, but not β2 or δ1, were detectable in human EAcells FLO. For TDCA treatment, FLO cells were pretreated with U73122 (10⁻⁶ M) or culture medium (control) for 1 h, and then treated with TDCA (10⁻¹¹ M) or TDCA+U73122 for 24 h. The culture medium was collected for measurement of H₂O₂ and cells for measurement of mRNA expression. N=3, ANOVA, * P<0.02, compared with control, ** P<0.02, compared with TDCA alone group; *** P<0.001, compared with control group; ▲ P<0.001, compared with control, ▲ ▲ P<0.001, compared with TDCA alone group.

Figure 2. Role of PI-PLC γ2 in TDCA-induced NOX5-S expression

(A) A typical example of Western blot analysis and summarized data showed that transfection with PI-PLC γ2 siRNA significantly decreased PI-PLC γ2 protein expression in FLO EA cells, indicating that PI-PLC γ2 siRNA effectively knocked down PI-PLC γ2 protein expression (n=3). (B) A typical example of Western blot analysis and summarized data show that knockdown of PI-PLC γ2 protein significantly decreased TDCA-induced NOX5-S expression. (C) Knockdown of PI-PLC γ2 protein significantly decreased TDCA-induced H₂O₂ production. (D) Knockdown of PI-PLC γ2 protein significantly decreased TDCA-induced thymidine incorporation both in FLO and OE33 cells. The data suggest that PI-PLC γ2 protein may contribute to TDCA-induced NOX5-S expression, H₂O₂ production and cell proliferation in EA cells. Transfection of siRNA was carried out with Lipofectamine 2000. Per well, 75 pmol of siRNA duplex of PI-PLC γ2 or control siRNA formulated into liposomes were applied. After a 4-h transfection, the transfection medium was replaced with regular medium. 24 h after transfection, cells were treated with low dose of TDCA for 24 h, and then culture medium and cells were collected for measurements. N=3, ANOVA, * P<0.02, compared with control group and control siRNA group; ** P<0.02, compared with control siRNA group and PI-PLC γ2 siRNA group; *** P<0.001, compared with control siRNA+ TDCA group, ▲ P<0.001, compared with control siRNA+ TDCA group.

Figure 3. Effect of PD98059 on TDCA-induced NOX5-S expression

(A) MEK1/2 kinase inhibitor PD98059 significantly decreased TDCA-induced NOX5-S expression measured by real time PCR. (B) PD98059 significantly decreased TDCA-induced H₂O₂ production. (C) PD98059 significantly decreased TDCA-induced thymidine incorporation. The data suggest that TDCA-induced increase in NOX5-S expression, H₂O₂ production and cell proliferation may depend on activation of ERK MAP kinases. N=3, ANOVA, * P<0.02, compared with control group, ** P<0.001, compared with TDCA alone group, ▲ P<0.01, compared with control group, ▲ ▲ P<0.01, compared with TDCA alone group.

Figure 4. Role of ERK-2 MAP kinase in TDCA-induced NOX5-S expression

(A) A typical example of Western blot analysis and summarized data show that transfection with ERK-2 MAP kinase siRNA significantly decreased ERK-2 protein expression in FLO EA cells, indicating that ERK-2 siRNA effectively knocks down ERK-2 protein expression. (B) A typical example of Western blot analysis and summarized data show that knockdown of ERK-2 MAP kinase protein significantly decreased TDCA-induced NOX5-S expression. (C) Knockdown of ERK-2 MAP kinase protein significantly decreased TDCA-induced H₂O₂ production. (D) Knockdown of ERK-2 MAP kinase protein significantly decreased TDCA-induced thymidine incorporation both in FLO and OE33 cells. The data suggest that ERK-2 MAP kinase may mediate TDCA-induced NOX5-S expression, H₂O₂ production and cell proliferation in EA cells. Transfection of siRNA was carried out with Lipofectamine 2000. Per well, 75 pmol of siRNA duplex of ERK-2 MAP kinase siRNA or control siRNA formulated into liposomes were applied. After a 4-h transfection, the transfection medium was replaced with regular medium. 24 h after transfection, cells were treated with low dose of TDCA for 24 h, and then culture medium and cells were collected for measurements. N=3, ANOVA, * P<0.02, compared with control siRNA group, ** P<0.05, compared with control siRNA group; ▲ P<0.05, compared with control siRNA+TDCA group.

Figure 5. Role of ERK-1 MAP kinase in TDCA-induced NOX5-S expression

(A) A typical example of Western blot analysis and summarized data showed that transfection with ERK-1 MAP kinase siRNA significantly decreased ERK-1 protein expression in FLO EA cells, indicating that ERK-1 siRNA effectively knocks down ERK-1 protein expression. (B) A typical example of Western blot analysis and summarized data show that knockdown of ERK-1 MAP kinase protein did not significantly affect TDCA-induced NOX5-S expression. The data suggest that TDCA-induced NOX5-S expression may not be mediated by activation of ERK-1 in FLO EA cells. Transfection of siRNA was carried out with Lipofectamine 2000. Per well, 75 pmol of siRNA duplex of ERK-1 or control siRNA formulated into liposomes were applied. After a 4-h transfection, the transfection medium was replaced with regular medium. 24 h after transfection, cells were treated with low dose of TDCA for 24 h, and then culture medium and cells were collected for measurements. N=3, ANOVA, * P<0.01, compared with control group or control siRNA group, ** P<0.01, compared with control siRNA group or ERK-1 siRNA group There no statistically significant difference between control siRNA+ TDCA group and ERK-1 siRNA+ TDCA group.

Figure 6. ERK-2 phosphorylation in FLO EA cells

(A) A typical example of Western blot analysis and summarized data show that TDCA significantly increased ERK-2 MAP kinase phosphorylation, an increase which was significantly reduced by U73122. The data suggest that TDCA-induced ERK-2 activation may depend on activation of PI-PLC. (B) A typical example of Western blot analysis and summarized data show that TDCA-induced ERK-2 phosphorylation was significantly reduced by knockdown of PI-PLC γ2 protein, suggesting that TDCA-induced ERK-2 activation may depend on activation of PI-PLC γ2 in FLO EA cells. Transfection of siRNA was carried out with Lipofectamine 2000. Per well, 75 pmol of siRNA duplex of ERK-2 or control siRNA formulated into liposomes were applied. After a 4-h transfection, the transfection medium was replaced with regular medium. 24 h after transfection, cells were treated with TDCA (10⁻⁹M) for 10 min, and then cells were collected for measurements. N=3, ANOVA, * P<0.001, compared with control group; ▲ P<0.001, compared with TDCA treatment group; ** P<0.05, compared with control siRNA group or PI-PLC γ2 siRNA group; ▲ ▲ P<0.05, compared with control siRNA+TDCA group.