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. 2013 Feb;51(1):61-8.
doi: 10.3347/kjp.2013.51.1.61. Epub 2013 Feb 18.

Entamoeba histolytica induces cell death of HT29 colonic epithelial cells via NOX1-derived ROS

Kyeong Ah Kim  1 , Ju Young Kim, Young Ah Lee, Arim Min, Young Yil Bahk, Myeong Heon Shin
Affiliations

Entamoeba histolytica induces cell death of HT29 colonic epithelial cells via NOX1-derived ROS

Kyeong Ah Kim et al. Korean J Parasitol. 2013 Feb.

Abstract

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.

Keywords: Entamoeba histolytica; HT29 colon epithelial cell; NADPH oxidase 1 (NOX1); host cell death; reactive oxygen species (ROS).

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Figures

Fig. 1
Fig. 1
Co-incubation with Entamoeba histolytica induces cell death in HT29 colon cells. (A) Percentage of dead cells in HT29 cells incubated with live trophozoites of E. histolytica. Data are presented as means±SEM from 5 independent experiments. Significant differences from controls are as follows. *P<0.05. (B) Addition of D-galactose inhibits DNA fragmentation in HT29 cells induced by E. histolytica. The figure is representative of 3 separate experiments showing similar results.
Fig. 2
Fig. 2
NOX-derived ROS, but not caspases are involved in cell death of HT29 cells induced by E. histolytica. (A) Effect of pan-caspase inhibitor z-VAD-fmk (100 µM) on PI influx in HT29 cells induced by E. histolytica. Data are presented as means±SEM from 4 independent experiments. (B) Effect of z-VAD-fmk or NADPH oxidase inhibitor (DPI) on E. histolytica-induced DNA fragmentation in HT29 cells. The figure is representative of 3 separate experiments showing similar results.
Fig. 3
Fig. 3
Incubation with live trophozoites of E. histolytica induces ROS generation in HT29 cells. (A) Incubation with E. histolytica induces ROS generation in HT29 cells. Data are presented as means±SEM from 3 independent experiments. Significant differences from controls are as follows; *P<0.05. (B) Visualization of intracellular ROS accumulation in HT29 cells adhered to live trophozoites of E. histolytica. The production of intracellular ROS in HT29 cells was observed by inverted fluorescence microscopy (×200).
Fig. 4
Fig. 4
Transfection with Rac1 siRNA inhibits E. histolytica-induced cell death in HT29 cells. (A) Analysis of Rac1 protein levels following Rac1 siRNA in HT29 cells. Blots are representative of 3 independent experiments. (B) Effect of Rac1 siRNA on PI influx in HT-29 cells induced by E. histolytica. Data are means±SD of 3 experiments done in triplicate. Significant differences from controls are as follows; *P<0.05.
Fig. 5
Fig. 5
Transfection with NOX1 siRNA inhibits E. histolytica-induced ROS generation and cell death in HT29 cells. (A) Analysis of rac1 gene or protein levels following NOX1 siRNA in HT29 cells. Blots are representative of 3 independent experiments. (B) Effect of NOX1 siRNA on E. histolytica-induced DNA fragmentation in HT29 cells. The figure is representative of 3 separate experiments showing similar results.
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