Int J Med Sci 2013; 10(6):707-718. doi:10.7150/ijms.5726 This issue Cite
1. Institute of virology and AIDS research, The first hospital of Jilin University, 519 Dongminzhu Street, Changchun 130021, P. R. China;
2. Acupuncture department, The affiliated hospital to Changchun University of Chinese Medicine, 1478 Gongnong Road, Changchun 130021, P. R. China;
3. Department of Thyroid Surgery, The First Hospital of Jilin University, 71 Qianjin Street, Changchun 130021, P. R. China;
4. Department of Biotechnology, College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, P. R. China.
Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis.
Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot.
Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence.
Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC.
Keywords: murine fibrosarcoma L929 cells, pseudolaric acid B (PAB), PKC, autophagy, cell cycle arrest, senescence.