Sulforaphane-N-Acetyl-Cysteine Induces Autophagy Through Activation of ERK1/2 in U87MG and U373MG Cells

Cell Physiol Biochem. 2018;51(2):528-542. doi: 10.1159/000495274. Epub 2018 Nov 20.

Abstract

Background/aims: Sulforaphane-N-acetyl-cysteine (SFN-NAC) is a sulforaphane (SFN) metabolite with a longer half-life and better blood-brain barrier permeability than those of SFN. Previous studies have found that SFN-NAC can act via ERK to destroy microtubules and inhibit cell growth in lung cancer cells. However, the underlying mechanisms are unclear, and it is unknown whether SFN-NAC can inhibit the growth of glioma. Here, we have demonstrated for the first time that SFN-NAC activates autophagy-mediated downregulation of α-tubulin expression via the ERK pathway.

Methods: U87MG and U373MG cells, two widely used glioma cell lines, were utilized in this study. Apoptosis assay, western blot analysis, co-immunoprecipitation, immunostaining, and electron microscopy were used to analyze the effect of SFN-NAC on α-tubulin and its interaction with microtube-associated protein 1 light-chain 3 (LC3).

Results: SFN-NAC induced cell-cycle arrest in the G2/M phase and dose-dependently induced intracellular ERK activation, autophagy, and α-tubulin downregulation. These SFN-NAC-induced effects were reversed by inhibiting the ERK pathway with its inhibitor PD98059. U87MG and U373MG cells were transfected with LC3 small interfering RNA, and the subsequent inhibition of autophagy reversed the downregulation of α-tubulin by SFN-NAC. Furthermore, co-immunoprecipitation experiments and confocal microscopy confirmed that SFN-NAC promotes the binding of LC3 with α-tubulin in the cytoplasm. Cell viability experiments demonstrate that SFN-NAC inhibits the growth of U87MG and U373MG cell colonies.

Conclusion: These findings suggest that SFN-NAC is a novel potential anti-glioma agent.

Keywords: Apoptosis; Autophagy; Glioma; Sulforaphane; α-tubulin.

MeSH terms

  • Acetylcysteine / analogs & derivatives*
  • Acetylcysteine / metabolism
  • Acetylcysteine / pharmacology*
  • Anticarcinogenic Agents / pharmacology*
  • Apoptosis / drug effects
  • Autophagy / drug effects*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Databases, Factual
  • Flavonoids / pharmacology
  • G2 Phase Cell Cycle Checkpoints / drug effects
  • Humans
  • Isothiocyanates / metabolism
  • Isothiocyanates / pharmacology*
  • MAP Kinase Signaling System / drug effects
  • Microtubule-Associated Proteins / antagonists & inhibitors
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Mitogen-Activated Protein Kinase 3 / metabolism*
  • Proportional Hazards Models
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Sulfoxides
  • Tubulin / genetics
  • Tubulin / metabolism

Substances

  • Anticarcinogenic Agents
  • Flavonoids
  • Isothiocyanates
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • RNA, Small Interfering
  • Sulfoxides
  • TUBA1B protein, human
  • Tubulin
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • sulforaphane
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
  • Acetylcysteine