Study on the regulation of gastric cancer cell apoptosis by LACTB through mitochondrial autophagy pathway

This study aimed to determine whether β-lactamase-like protein (Lactamase-β, LACTB) influences apoptosis in gastric cancer cells by modulating mitochondrial autophagy through the PTEN-induced putative kinase 1 (PINK1) or Parkin pathway. Firstly, the expression level of LACTB in gastric cancer tissues was detected by immunohistochemistry, and the survival data of patients were used to explore the relationship between LACTB expression level and patient prognosis. Secondly, LACTB overexpression (+ LACTB) and knockdown (sh-LACTB) AGS gastric cancer cell lines were constructed; flow cytometry and other experiments were used to detect the effect of LACTB on AGS cell apoptosis; Western Blot was used to detect the expression of PINK1/Parkin mitochondrial autophagy pathway-related proteins and lysosome-related proteins in + LACTB and sh-LACTB gastric cancer cells; kits and electron microscopy were used to detect changes in the number of reactive oxygen species (ROS) and autophagosomes. Finally, Western blot was used to detect the expression of apoptotic proteins Bcl-2 associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2) in + LACTB and sh-LACTB gastric cancer cells treated with mitochondrial autophagy inhibitor 3-methyladenine (3-MA). Immunohistochemistry analysis revealed that LACTB expression in gastric cancer tissues was higher than in adjacent non-cancerous tissues, for patients with tumor diameters exceeding 4.5 cm, high LACTB expression was associated with a poor prognosis (P < 0.05). LACTB overexpression reduced apoptosis in gastric cancer cells. It downregulated the pro-apoptotic protein Bax, while LACTB knockdown promoted apoptosis, upregulated Bax, the expression of pro-apoptotic protein Bax, and downregulated the expression of anti-apoptotic protein Bcl-2. In LACTB overexpressing cell lines, protein sequestosome 1 (P62) protein levels were elevated, lysosomal-associated membrane protein 2 (LAMP2) expression was decreased, Reactive oxygen species (ROS) levels remained significantly stable, and autophagosome counts were reduced. Conversely, LACTB knockdown cells, PINK1, Parkin, protein light chain 3II/I (LC3II/I), LAMP2, cathepsin B (CTSB), continuous traumatic stress disorder (CTSD), and other related proteins, downregulated P62 expression, increased ROS accumulation, and higher number of autophagosomes. In LV-LACTB and sh-LACTB gastric cancer cells treated with the mitochondrial autophagy inhibitor 3-methyladenine (3-MA), apoptotic protein Bax is downregulated, and anti-apoptotic protein Bcl-2 is upregulated. In summary, the LACTB protein may regulate the apoptosis in gastric cancer cells by modulating mitochondrial autophagy through the PINK1/Parkin pathway.