Tang Yi, Li Ming, Wang Ya-Lan, Threadgill Michael D, Xiao Ming, Mou Chun-Feng, Song Guang-Lin, Kuang Jing, Yang Xi, Yang Li, Gao Xing-Jie, Wang Ya-Ping, Meng Yun-Peng
Department of Pathology, Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing, China.
Department of Pharmacy and Pharmacology, University of Bath Bath, UK.
Am J Cancer Res. 2015 Jan 15;5(2):498-513. eCollection 2015.
Autophagy plays a protective role in colorectal carcinoma. Arginine ADP-ribosyltransferase 1 (ART1) is an important mono-ADP-ribose transferase, which has been shown to play a role in biological processes such as proliferation and invasion of cancer cells. Interestingly, the role of ART1 in the regulation of autophagy is still not clear. We examined effects of overexpression or knockdown of ART1 by lentiviral transfection on starvation-induced autophagy of colon carcinoma CT26 cell lines in vivo and in vitro. The formation of autophagosome was detected by electron microscopy, acridine orange staining and expression of LC3 B. The molecular contributions of ART1 in regulation of autophagy were detected by western blotting or by co-immunoprecipitation. Additionally, inhibitors were used to study further the signaling pathway of ART1 in the regulation of autophagy. CCK8 assay, plate cloning assay, soft agar assay, examination of subcutaneous transplanted carcinoma in BALB/c mice, flow cytometry and Hoechst33342 staining were used to assess survival and apoptotic ability when starvation-induced autophagy modulated by ART1 was inhibited by 3-MA. Overexpression of ART1 promoted starvation-induced autophagy, which related to increases in the expression of Rac1, NF-κB, PARP-1, LKB1 and p-AMPK and a decrease in the expression of p-P70S6K. Correspondingly, knockdown of ART1 caused the opposite effects. ART1 also interacted with integrin α7. Additionally, changes of protein expressions were further validated following inhibition of Rac1 and PARP-1 in the starvation-induced ART1-GFP CT26 cells. Inhibition of ART1-stimulated starvation-induced autophagy restrained the growth and promoted apoptosis. ART1 is thus relevant in starvation-induced autophagy in colorectal carcinoma and may play essential roles in therapeutic anticancer strategies.
自噬在结直肠癌中发挥着保护作用。精氨酸ADP核糖基转移酶1(ART1)是一种重要的单ADP核糖转移酶,已被证明在癌细胞增殖和侵袭等生物学过程中发挥作用。有趣的是,ART1在自噬调节中的作用仍不清楚。我们通过慢病毒转染过表达或敲低ART1,研究其对体内外结肠癌CT26细胞系饥饿诱导自噬的影响。通过电子显微镜、吖啶橙染色和LC3 B的表达检测自噬体的形成。通过蛋白质印迹法或免疫共沉淀检测ART1在自噬调节中的分子作用。此外,使用抑制剂进一步研究ART1在自噬调节中的信号通路。当3-MA抑制ART1调节的饥饿诱导自噬时,采用CCK8法、平板克隆法、软琼脂法、BALB/c小鼠皮下移植癌检测、流式细胞术和Hoechst33342染色评估细胞存活和凋亡能力。ART1的过表达促进了饥饿诱导的自噬,这与Rac1、NF-κB、PARP-1、LKB1和p-AMPK表达的增加以及p-P70S6K表达的降低有关。相应地,敲低ART1则产生相反的效果。ART1还与整合素α7相互作用。此外,在饥饿诱导的ART1-GFP CT26细胞中抑制Rac1和PARP-1后,进一步验证了蛋白质表达的变化。抑制ART1刺激的饥饿诱导自噬会抑制生长并促进凋亡。因此,ART1与结直肠癌中饥饿诱导的自噬相关,可能在抗癌治疗策略中发挥重要作用。