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. 2013;20(33):4142-4152.
doi: 10.2174/09298673113209990249.

Indometacin ameliorates high glucose-induced proliferation and invasion via modulation of e-cadherin in pancreatic cancer cells

Liang Han #  1 , Bo Peng #  2 , Qingyong Ma  1 , Jiguang Ma  3 , Juntao Li  1 , Wei Li  1 , Wanxing Duan  1 , Chao Chen  1 , Jiangbo Liu  1 , Qinhong Xu  1 , Kyle Laporte  4 , Zehui Li  4 , Erxi Wu  4
Affiliations

Indometacin ameliorates high glucose-induced proliferation and invasion via modulation of e-cadherin in pancreatic cancer cells

Liang Han et al. Curr Med Chem. 2013.

Abstract

Indometacin, an inhibitor of cyclooxygenase-2 (COX-2), has been shown to exert anticancer effects in a variety of cancers. However, the effect and mechanism of indometacin on high glucose (HG)-induced proliferation and invasion of pancreatic cancer (PC) cells remain unclear. Multiple lines of evidence suggest that a large portion of pancreatic cancer (PC) patients suffer from either diabetes or HG which contributing PC progression. In this study, we report that indometacin down-regulated HG-induced proliferation and invasion via up-regulating E-cadherin but not COX-2 in PC cells. Additionally, the E-cadherin transcriptional repressors, Snail and Slug, were also involved in the process. Furthermore, the proliferation and invasion of PC cells, incubated in HG medium and treated with indometacin were significantly increased when E-cadherin was knocked down (Si-E-cad). Moreover, the protein levels of MMP-2, MMP-9, and VEGF were increased in PC cells transfected with Si-E-cad. Finally, the activation of the PI3K/AKT/GSK-3β signaling pathway was demonstrated to be involved in indometacin reversing HG-induced cell proliferation and invasion in PC cells. In conclusion, these results suggest that indometacin plays a key role in down-regulating HG-induced proliferation and invasion in PC cells. Our findings indicate that indometacin could be used as a novel therapeutic strategy to treat PC patients who simultaneously suffer from diabetes or HG.

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Figures

Fig. (1)
Fig. (1). HG increased the proliferation rate and invasion of PC cells
(A) Panc-1 cells were seeded in a 96-well plate for 12 h and then treated with LG (5.5 mM) and HG (25 mM) for 24, 48 or 72 h, and equivalent concentrations of mannitol were used as osmotic control. Proliferation rate was measured by MTT assay. (B) The proliferation rate of BXPC-3 cells was detected with the MTT assay. (C) The effects of LG and HG on PC cells invasion (mannitol as osmotic control). (D) The number of migrated cells was quantified by counting the number of cells from 10 random fields at ×200 magnification. * p < 0.05 compared to normal controls. Results are represented by mean ± SEM (n = 3; values without a common letter are significantly different, p < 0.05). Column: mean; bar: SD.
Fig. (2)
Fig. (2). Indometacin down-regulated the effect of HG-induced proliferation and invasion in PC cells
(A, B) Panc-1 and BXPC-3 cells were seeded in a 96-well plate with HG for 12 h and treated with gradually increasing concentrations of indometacin (0 as a control and 50, 100 or 150 mg/L) for 24 h, 48 h, 72 h. Proliferation rate was measured with the MTT assay. (C) Panc-1 and BXPC-3 cells were incubated with HG for 48 h. The invasive ability of cells was detected in DMEM containing 1% FBS. Cells were seeded in the matrigel-coated transwell upper chambers. (D) The number of migrated cells was quantified by counting the number of cells from 10 random fields at ×200 magnification. * p < 0.05 compared to control (Indometacin 0 mg/L). The data represent the results of three independent experiments. Column: mean; bar: SD.
Fig. (3)
Fig. (3). Involvement of E-cadherin but not COX-2 in the event of indometacin reversing HG-induced effect in PC cells
(A, B) The mRNA expression of E-cadherin and COX-2 was estimated by real-time RT-PCR in four groups. The expression of each target gene was quantified using β-actin as a normalization control. (C) The protein expression of E-cadherin and COX-2 was evaluated by Western blotting. The blots were then re-probed with β-actin as a loading control. (D, E) The relative protein expression of E-cadherin and COX-2 in different group. * p < 0.05 compared to controls. The data represent the results of three independent experiments. Column: mean; bar: SD.
Fig. (4)
Fig. (4). The effect of knockdown E-cadherin on proliferation in PC cells
(A) The knockdown of E-cadherin by siRNA for 48 h was confirmed by real-time RT-PCR. (B) The knockdown of E-cadherin by siRNA for 48 h was confirmed by Western blotting. (C, D) Panc-1 and BXPC-3 cells were seeded in a 96-well plate with HG for 12 h and treated with a series of gradually increasing concentrations of indometacin (0 as a control and 50, 100 or 150 mg/L) for 48 h. Proliferation rate was shown by MTT assay. * p < 0.05 compared with Si-control. The data represent the results of three independent experiments. Column: mean; bar: SD.
Fig. (5)
Fig. (5). The effect of knockdown E-cadherin on invasion in PC cells
(A) Panc-1 and BXPC-3 cells were incubated with HG for 48 h. The invasive ability by DMEM containing 1% FBS seeded in matrigel-coated transwell upper chambers. (B) The number of migrated cells was quantified by counting the number of cells from 10 random fields at ×200 magnification. (C) The expression of MMP-2, MMP-9, and VEGF at protein level was evaluated by Western blotting in two groups. The blots were then re-probed with β-actin as a loading control. (D, E) The relative protein expression of MMP-2, MMP-9, and VEGF in two groups. * p < 0.05 compared with Si-control. The data represent the results of three independent experiments. Column: mean; bar: SD.
Fig. (6)
Fig. (6). The effect of indometacin on E-cadherin transcriptional repressors expression in PC cells
(A, B, C, D) The expression of Snail, Slug, Twist and ZEB1 at mRNA level was estimated with real-time RT-PCR. The expression of each target gene was quantified using β-actin as a normalization control. (E, F) Panc-1 and BXPC-3 cells were incubated with HG for 12 h and then treated with a series of gradually increasing concentrations of indometacin (0 as a control and 50, 100 or 150 mg/L) for 48 h. The expression of Snail and Slug at protein level was evaluated by Western blotting. The blots were then re-probed with β-actin as a loading control. * p < 0.05 compared to controls. The data represent the results of three independent experiments. Column: mean; bar: SD.
Fig. (7)
Fig. (7). The activation of the PI3K/AKT/GSK-3β signaling pathway is critical for indometacin reversing HG-induced cell proliferation and invasion in PC cells
(A) The expression of p-AKT, AKT, p-GSK-3β, GSK-3β and β-actin at protein level was evaluated by Western blotting in four groups. The blots were then re-probed with β-actin as a loading control. (B) Panc-1 cells were incubated with HG for 48 h. The invasive ability by DMEM containing 1% FBS seeded in matrigel-coated Transwell upper chambers. The cells were treated with an AKT pathway inhibitor (LY294002) and/or Si-E-cad. (C) Panc-1 cells were seeded in a 96-well plate with HG for 12 h and then treated with AKT pathways inhibitor (LY294002) and/or Si-E-cad for 48 h. Proliferation rate was determined with the MTT assay. (D) The number of migrated cells was quantified by counting the number of cells from 10 random fields at ×200 magnification. Control: [Si-E-cad (−) and LY294002 (−)]. * p < 0.05 compared to controls. The data represent the results of three independent experiments. Column: mean; bar: SD.
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