Abstract：
BACKGROUND: Allicin regulates macrophage autophagy and senescence, and inhibits hepatoma cell growth. This study investigated the mechanism by which allicin inhibits the growth of hepatoma cells.
METHODS: Hepa1-6 mouse hepatoma cells were subcutaneously injected into C57BL/6 J mice to construct a tumor transplantation model. Macrophages were cultured with the supernatant of hepatoma cells to construct a cell model. The levels of mRNA and proteins and the level of Sestrin2 ubiquitination were measured by RTqPCR, immunofluorescence and Western blotting. The levels of autophagy-related factors and the activity of senescence-associated β-galactosidase were determined by kits, and protein stability was detected by cycloheximide (CHX) tracking.
RESULTS: Data analysis of clinical samples revealed that RBX1 was highly expressed in tumor tissues, while Sestrin2 was expressed at low levels in tumor tissues. Allicin can promote the expression of the autophagy-related proteins LC3 and Beclin-1 in tumor macrophages and inhibit the expression of the aging-related proteins p16 and p21, thus promoting autophagy in macrophages and inhibiting cell senescence. Moreover, allicin can inhibit the expression of RBX1, thereby reducing the ubiquitination of Sestrin2, enhancing the stability of Sestrin2, activating autophagy in tumor macrophages and inhibiting senescence. In addition, allicin treatment inhibited the proliferation and migration of hepatoma carcinoma cells cocultured with macrophages and significantly improved the development of liver cancer in mice.
CONCLUSIONS: Allicin can affect the autophagy of macrophages and restrain the growth of hepatoma cells by regulating the ubiquitination of Sestrin2.
METHODS: Hepa1-6 mouse hepatoma cells were subcutaneously injected into C57BL/6 J mice to construct a tumor transplantation model. Macrophages were cultured with the supernatant of hepatoma cells to construct a cell model. The levels of mRNA and proteins and the level of Sestrin2 ubiquitination were measured by RTqPCR, immunofluorescence and Western blotting. The levels of autophagy-related factors and the activity of senescence-associated β-galactosidase were determined by kits, and protein stability was detected by cycloheximide (CHX) tracking.
RESULTS: Data analysis of clinical samples revealed that RBX1 was highly expressed in tumor tissues, while Sestrin2 was expressed at low levels in tumor tissues. Allicin can promote the expression of the autophagy-related proteins LC3 and Beclin-1 in tumor macrophages and inhibit the expression of the aging-related proteins p16 and p21, thus promoting autophagy in macrophages and inhibiting cell senescence. Moreover, allicin can inhibit the expression of RBX1, thereby reducing the ubiquitination of Sestrin2, enhancing the stability of Sestrin2, activating autophagy in tumor macrophages and inhibiting senescence. In addition, allicin treatment inhibited the proliferation and migration of hepatoma carcinoma cells cocultured with macrophages and significantly improved the development of liver cancer in mice.
CONCLUSIONS: Allicin can affect the autophagy of macrophages and restrain the growth of hepatoma cells by regulating the ubiquitination of Sestrin2.
摘要：
背景：大蒜素调节巨噬细胞自噬和衰老，抑制肝癌细胞生长.本研究探讨了大蒜素抑制肝癌细胞生长的机制。
方法：将Hepa1-6小鼠肝癌细胞皮下注射到C57BL/6J小鼠体内，构建肿瘤移植模型。用肝癌细胞的上清液培养巨噬细胞以构建细胞模型。通过RTqPCR检测mRNA和蛋白水平以及Sestrin2泛素化水平，免疫荧光和蛋白质印迹。试剂盒测定自噬相关因子水平和衰老相关β-半乳糖苷酶活性,通过环己酰亚胺(CHX)示踪检测蛋白质稳定性。
结果：临床样本数据分析显示RBX1在肿瘤组织中高表达，而Sestrin2在肿瘤组织中低水平表达。大蒜素可促进肿瘤巨噬细胞自噬相关蛋白LC3和Beclin-1的表达，抑制衰老相关蛋白p16和p21的表达，从而促进巨噬细胞自噬，抑制细胞衰老。此外，大蒜素可以抑制RBX1的表达,从而减少Sestrin2的泛素化,增强Sestrin2的稳定性,激活肿瘤巨噬细胞的自噬,抑制衰老。此外,大蒜素处理抑制了与巨噬细胞共培养的肝癌细胞的增殖和迁移，并显着改善了小鼠肝癌的发展。
结论：大蒜素通过调节Sestrin2的泛素化作用，影响巨噬细胞自噬，抑制肝癌细胞的生长。
方法：将Hepa1-6小鼠肝癌细胞皮下注射到C57BL/6J小鼠体内，构建肿瘤移植模型。用肝癌细胞的上清液培养巨噬细胞以构建细胞模型。通过RTqPCR检测mRNA和蛋白水平以及Sestrin2泛素化水平，免疫荧光和蛋白质印迹。试剂盒测定自噬相关因子水平和衰老相关β-半乳糖苷酶活性,通过环己酰亚胺(CHX)示踪检测蛋白质稳定性。
结果：临床样本数据分析显示RBX1在肿瘤组织中高表达，而Sestrin2在肿瘤组织中低水平表达。大蒜素可促进肿瘤巨噬细胞自噬相关蛋白LC3和Beclin-1的表达，抑制衰老相关蛋白p16和p21的表达，从而促进巨噬细胞自噬，抑制细胞衰老。此外，大蒜素可以抑制RBX1的表达,从而减少Sestrin2的泛素化,增强Sestrin2的稳定性,激活肿瘤巨噬细胞的自噬,抑制衰老。此外,大蒜素处理抑制了与巨噬细胞共培养的肝癌细胞的增殖和迁移，并显着改善了小鼠肝癌的发展。
结论：大蒜素通过调节Sestrin2的泛素化作用，影响巨噬细胞自噬，抑制肝癌细胞的生长。
