Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways

Biol Pharm Bull. 2011;34(1):47-53. doi: 10.1248/bpb.34.47.

Abstract

Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(•-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(•-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.

MeSH terms

  • Antineoplastic Agents / pharmacology
  • Autophagy / drug effects*
  • Cell Line, Tumor
  • Fibrosarcoma / drug therapy*
  • Humans
  • JNK Mitogen-Activated Protein Kinases / metabolism*
  • Reactive Oxygen Species
  • Silybin
  • Silymarin / pharmacology*
  • Tumor Suppressor Protein p53 / metabolism*
  • p38 Mitogen-Activated Protein Kinases / metabolism*

Substances

  • Antineoplastic Agents
  • Reactive Oxygen Species
  • Silymarin
  • Tumor Suppressor Protein p53
  • Silybin
  • JNK Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases