Nie Wei, Hu Lihua, Yan Zhiqiang, Wang Qian, He Shui, Gao Xiaoqiang, Yang Fang
Center of Clinical Laboratories, The Affiliated Hospital of Guizhou Medical University, Guiyang, China.
The People's Hospital of Liuzhi-Special-District, Liupanshui, China.
Sci Rep. 2025 Jul 2;15(1):23273. doi: 10.1038/s41598-025-06047-0.
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.
本研究旨在确定β-内酰胺酶样蛋白（Lactamase-β，LACTB）是否通过磷酸酶和张力蛋白同源物（PTEN）诱导的假定激酶1（PINK1）或帕金蛋白途径调节线粒体自噬，从而影响胃癌细胞的凋亡。首先，采用免疫组织化学法检测胃癌组织中LACTB的表达水平，并利用患者的生存数据探讨LACTB表达水平与患者预后的关系。其次，构建LACTB过表达（+LACTB）和敲低（sh-LACTB）的AGS胃癌细胞系；采用流式细胞术等实验检测LACTB对AGS细胞凋亡的影响；利用蛋白质免疫印迹法检测+LACTB和sh-LACTB胃癌细胞中PINK1/帕金蛋白线粒体自噬途径相关蛋白和溶酶体相关蛋白的表达；使用试剂盒和电子显微镜检测活性氧（ROS）数量和自噬体的变化。最后，采用蛋白质免疫印迹法检测用线粒体自噬抑制剂3-甲基腺嘌呤（3-MA）处理的+LACTB和sh-LACTB胃癌细胞中凋亡蛋白Bcl-2相关X蛋白（Bax）和B细胞淋巴瘤-2（Bcl-2）的表达。免疫组织化学分析显示，胃癌组织中LACTB的表达高于相邻非癌组织，对于肿瘤直径超过4.5 cm的患者，LACTB高表达与预后不良相关（P<0.05）。LACTB过表达减少了胃癌细胞的凋亡。它下调了促凋亡蛋白Bax，而LACTB敲低则促进了凋亡，上调了促凋亡蛋白Bax的表达，并下调了抗凋亡蛋白Bcl-2的表达。在LACTB过表达细胞系中，泛素结合蛋白1（P62）蛋白水平升高，溶酶体相关膜蛋白2（LAMP2）表达降低，活性氧（ROS）水平保持显著稳定，自噬体数量减少。相反，LACTB敲低细胞中，PINK1、帕金蛋白、微管相关蛋白轻链3II/I（LC3II/I）、LAMP2、组织蛋白酶B（CTSB）、组织蛋白酶D（CTSD）等相关蛋白，下调了P62表达，增加了ROS积累，自噬体数量增多。在用线粒体自噬抑制剂3-甲基腺嘌呤（3-MA）处理的LV-LACTB和sh-LACTB胃癌细胞中，凋亡蛋白Bax下调，抗凋亡蛋白Bcl-2上调。综上所述，LACTB蛋白可能通过PINK1/帕金蛋白途径调节线粒体自噬，从而调控胃癌细胞的凋亡。
