Hu Yi, Lin Xing, Wang Ping, Xue Yi-Xue, Li Zhen, Liu Li-Bo, Yu Bo, Feng Tian-Da, Liu Yun-Hui
Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang, P. R. China.
J Cell Physiol. 2015 Aug;230(8):1713-28. doi: 10.1002/jcp.24798.
CRM197 is a naturally nontoxic diphtheria toxin mutant that binds and inhibits heparin-binding epidermal growth factor-like growth factor. CRM197 serves as carrier protein for vaccine and other therapeutic agents. CRM197 also inhibits the growth, migration, invasion, and induces apoptosis in various tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we aimed to investigate the role and mechanism of CRM197 combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion, and apoptosis of glioblastoma cells. U87 and U251 human glioblastoma cells were treated with CRM197 (10 µg/ml) and shRNA interfering technology was employed to silence VCAM-1 expression. Cell viability, migration, invasiveness, and apoptosis were assessed with CCK8, Transwell and Annexin V-PE/7-AAD staining. Activation of cleaved caspase-3, 8, and 9, activity of matrix metalloproteinase-2/9 (MMP-2/9), and expression of phosphorylated Akt (p-Akt) were also checked. Results showed that CRM197 and shRNA-VCAM-1 not only significantly inhibited the cell proliferation, migration, invasion, but also promoted the apoptosis of U87 and U251 cells. Combined treatment of both displayed enhanced inhibitory effects on the malignant biological behavior of glioma cells. The activation of cleaved caspase-3, 8, 9 was promoted, activity of MMP-2 and MMP-9 and expression of p-Akt were inhibited significantly by the treatment of CRM197 and shRNA-VCAM-1 alone or in combination, indicating that the combination of CRM197 with shRNA-VCAM-1 additively inhibited the malignant behavior of human glioblastoma cells via activating caspase-3, 8, 9 as well as inhibiting MMP-2, MMP-9, and Akt pathway.
CRM197是一种天然无毒的白喉毒素突变体，它能结合并抑制肝素结合表皮生长因子样生长因子。CRM197可作为疫苗及其他治疗药物的载体蛋白。CRM197还能抑制多种肿瘤的生长、迁移、侵袭并诱导其凋亡。血管细胞黏附分子-1（VCAM-1）是一种与胶质瘤恶性程度相关的重要细胞表面黏附分子。在本研究中，我们旨在探讨CRM197联合VCAM-1的短发夹RNA干扰（shRNA-VCAM-1）对胶质母细胞瘤细胞迁移、侵袭和凋亡的作用及机制。用CRM197（10µg/ml）处理U87和U251人胶质母细胞瘤细胞，并采用shRNA干扰技术沉默VCAM-1表达。用CCK8、Transwell和膜联蛋白V-PE/7-AAD染色评估细胞活力、迁移、侵袭能力和凋亡情况。同时检测裂解的半胱天冬酶-3、8和9的激活情况、基质金属蛋白酶-2/9（MMP-2/9）的活性以及磷酸化Akt（p-Akt）的表达。结果显示，CRM197和shRNA-VCAM-1不仅显著抑制U87和U251细胞的增殖、迁移和侵袭，还促进其凋亡。两者联合处理对胶质瘤细胞的恶性生物学行为表现出更强的抑制作用。单独或联合使用CRM197和shRNA-VCAM-1处理均能显著促进裂解的半胱天冬酶-3、8、9的激活，抑制MMP-2和MMP-9的活性以及p-Akt的表达，表明CRM197与shRNA-VCAM-1联合通过激活半胱天冬酶-3、8、9以及抑制MMP-2、MMP-9和Akt途径，对人胶质母细胞瘤细胞的恶性行为产生叠加抑制作用。
