Zhennan Wang1, Yuhan Zheng1, Haili Huang2, Huijuan He2, Qingming Jia1. 1. Department of Oncology, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China. 2. Clinical Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China.
Abstract
OBJECTIVE: To investigate the effect of Golgi phosphoprotein 3 (Golph3) on paclitaxel- induced apoptosis and autophagy in HeLa cells. METHODS: HeLa cells were transfected with a lentiviral vector expressing Golph3 or a small interfering RNA (siRNA) targeting Golph3 for up-regulation or down-regulation of Golph3 which was verified by Western blotting. The autophagic bodies in the cells were observed using transmission electron microscopy. The expression of autophagy markers p62 and LC3 were detected using Western blotting, and the cell apoptosis was examined by PI/Anexin V-FITC double staining and flow cytometry. The effects of blocking autophagy was evaluated by treatment of the cells with the autophagy inhibitor 3-MA. RESULTS: Transmission electron microscopy showed that the lentivirus-mediated overexpression of Golph3 significantly increased the number of autophagic bodies and interference of Golph3 expression significantly decreased autophagic bodies in HeLa cells. Western blotting showed that Golph3 overexpression caused an increased expression of LC3 and decreased the accumulation of p62 in the cells, and interference of Golph3 resulted in the reverse changes. The cell apoptosis induced by paclitaxel was significantly decreased in Golph3-overexpressing HeLa cells and increased in the cells with Golph3 knockdown (P>0.01). Treatment with 3-MA alone did not obviously affect HeLa cell apoptosis, but in cells with Golph3 knockdown, 3-MA significantly enhanced paclitaxel-induced apoptosis (P>0.01). CONCLUSIONS: Up-regulation of Golph3 promotes autophagy and inhibits paclitaxel-induced apoptosis, whereas suppression of Golph3 inhibits autophagy and enhances paclitaxel- induced apoptosis in HeLa cells.
OBJECTIVE: To investigate the effect of Golgi phosphoprotein 3 (Golph3) on paclitaxel- induced apoptosis and autophagy in HeLa cells. METHODS:HeLa cells were transfected with a lentiviral vector expressing Golph3 or a small interfering RNA (siRNA) targeting Golph3 for up-regulation or down-regulation of Golph3 which was verified by Western blotting. The autophagic bodies in the cells were observed using transmission electron microscopy. The expression of autophagy markers p62 and LC3 were detected using Western blotting, and the cell apoptosis was examined by PI/Anexin V-FITC double staining and flow cytometry. The effects of blocking autophagy was evaluated by treatment of the cells with the autophagy inhibitor 3-MA. RESULTS: Transmission electron microscopy showed that the lentivirus-mediated overexpression of Golph3 significantly increased the number of autophagic bodies and interference of Golph3 expression significantly decreased autophagic bodies in HeLa cells. Western blotting showed that Golph3 overexpression caused an increased expression of LC3 and decreased the accumulation of p62 in the cells, and interference of Golph3 resulted in the reverse changes. The cell apoptosis induced by paclitaxel was significantly decreased in Golph3-overexpressing HeLa cells and increased in the cells with Golph3 knockdown (P>0.01). Treatment with 3-MA alone did not obviously affect HeLa cell apoptosis, but in cells with Golph3 knockdown, 3-MA significantly enhanced paclitaxel-induced apoptosis (P>0.01). CONCLUSIONS: Up-regulation of Golph3 promotes autophagy and inhibits paclitaxel-induced apoptosis, whereas suppression of Golph3 inhibits autophagy and enhances paclitaxel- induced apoptosis in HeLa cells.
Authors: Holly C Dippold; Michelle M Ng; Suzette E Farber-Katz; Sun-Kyung Lee; Monica L Kerr; Marshall C Peterman; Ronald Sim; Patricia A Wiharto; Kenneth A Galbraith; Swetha Madhavarapu; Greg J Fuchs; Timo Meerloo; Marilyn G Farquhar; Huilin Zhou; Seth J Field Journal: Cell Date: 2009-10-16 Impact factor: 41.582
Authors: Christopher S Wood; Karl R Schmitz; Nicholas J Bessman; Thanuja Gangi Setty; Kathryn M Ferguson; Christopher G Burd Journal: J Cell Biol Date: 2009-12-21 Impact factor: 10.539
Authors: Kenneth L Scott; Omar Kabbarah; Mei-Chih Liang; Elena Ivanova; Valsamo Anagnostou; Joyce Wu; Sabin Dhakal; Min Wu; Shujuan Chen; Tamar Feinberg; Joseph Huang; Abdel Saci; Hans R Widlund; David E Fisher; Yonghong Xiao; David L Rimm; Alexei Protopopov; Kwok-Kin Wong; Lynda Chin Journal: Nature Date: 2009-06-25 Impact factor: 49.962