α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells

Abstract

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku–DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku–DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin–proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.

Astaxanthin Inhibits Oxidative Stress-Induced Ku Protein Degradation and Apoptosis in Gastric Epithelial Cells

“…Ku–DNA binding activity was determined using EMSA, followed by the method described previously [ 15 ] using a 32 P-labeled double-stranded oligonucleotide 5′-GGGCCAAGAATCTTAGCAGTTTCGGG-3′. Quantification of the Ku complexes was performed by densitometry.…”

“…The cells (5.0 × 10 4 /mL/well) were cultured with or without treatment. Cell viability and DNA fragmentation were determined as previously described [ 15 ]. The number of viable untreated cells was set to 100%.…”

“…Immunofluorescence staining for γ-H2AX and 4,6-diamidino-2-phenylindole (DAPI) was determined as previously described [ 15 ] using a confocal laser-scanning microscope (Zeiss LSM 880, Carl Zeiss Inc., Thornwood, NY, USA). Nuclear γ-H2AX fluorescence was determined using the ZEN Blue 3.1 software (Carl Zeiss Inc., Thornwood, NY, USA).…”

“…These studies suggested that oxidative stress degrades DNA repair proteins via caspase-dependent or AIF-dependent pathways. Recently, we demonstrated that excessive amounts of ROS induce caspase-3 cleavage and truncation of AIF with a loss of Ku proteins, leading to apoptosis in H elicobacter pylori (H. pylori) -infected cells [ 15 ]. Therefore, the mechanism of degradation of DNA repair Ku proteins should be investigated to prevent persistent DNA damage-induced genomic instability and carcinogenesis.…”

“…Ser-139-phosphorylated γ-H2AX is an established DSB marker, and γ-H2AX foci formation is used as an indicator of DNA damage [ 18 ]. We previously demonstrated that H. pylori -induced oxidative stress led to γ-H2AX foci formation, a decrease in Ku proteins, and an increase in ROS levels in gastric epithelial cells [ 15 ]. Therefore, antioxidants may inhibit ROS-induced Ku protein loss and oxidative stress-mediated apoptosis.…”