Pseudolaric acid B induced autophagy, but not apoptosis, in MRC5 human fibroblast cells

Autophagy and apoptosis are closely associated. In previous studies, pseudolaric acid B (PAB), a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae), was demonstrated to induce apoptosis in various cell lines. However, in L929 murine fibrosarcoma and SW579 human thyroid squamous cell carcinoma cells, only autophagy was induced. In the present study, another cell line, MRC5 human lung fibroblast cells, was identified in which PAB only induced autophagy. The relationship between apoptosis and autophagy subsequent to PAB treatment in MRC5 cells was explored. When autophagy was inhibited by 3-methyladenine (3MA), apoptosis was induced in the PAB-treated MRC5 cells. To study the mechanism for the promotion of apoptosis by 3MA in the PAB-treated cells, the expression of members from the apoptotic signal pathways was assessed. As Bcl-2, Bcl-2 associated X and pro-caspase-9 expression following PAB treatment was not affected by 3MA treatment, it was determined that apoptosis was induced independent of the mitochondrial pathway of apoptosis. As Fas and pro-caspase-8 expression following PAB treatment were not altered by 3MA, it was further determined that the death receptor pathway was not induced. However, the phosphorylation of c-Jun-N-terminal kinase and the expression of pro-caspase-3 were upregulated, and the phosphorylation of extracellular signal-regulated kinase downregulated, by the combination of PAB and 3MA treatment compared with PAB alone. It was also observed that 3MA did not affect the microtubule aggregation ability of PAB. Therefore, inhibiting autophagy in MRC5 cells did not affect the role of PAB in microtubule aggregation, while apoptosis was induced. This may present a strategy to enhance the anti-tumor effects of PAB.