Withaferin A triggers G2/M arrest and intrinsic apoptosis in glioblastoma cells via ATF4-ATF3-CHOP axis

Cell Prolif. 2020 Jan;53(1):e12706. doi: 10.1111/cpr.12706. Epub 2019 Oct 23.

Abstract

Objective: Withaferin A (WA) is a bioactive compound with a remarkable anti-cancer effect derived from Withania somnifera, commonly known as ashwagandha. However, the anti-cancer mechanisms of WA in glioblastoma multiforme (GBM) are still unclear.

Materials and methods: Cell viability assays and xenografted nude mice were used to evaluate the effects of WA, along with flow cytometry to detect apoptosis and cell cycle of GBM. RNA-seq analysis, Western blotting, immunofluorescence staining, qRT-PCR and siRNA gene silencing were carried out to determine the signalling pathways affected by WA.

Results: Withaferin A significantly inhibited the growth of GBM in vitro and in vivo and triggered the intrinsic apoptosis of GBM cells by up-regulating expression of Bim and Bad. WA arrested GBM cells at the G2/M phase of the cell cycle through dephosphorylating Thr161 of CDK1 by activating p53-independent p21 up-regulation. Knockdown of p21 restored cell cycle progression and cell viability by down-regulating the expression of Bad rather than Bim. We demonstrated that endoplasmic reticulum (ER) stress induced by WA through the ATF4-ATF3-CHOP axis, initiated apoptosis and G2/M arrest in GBM cells.

Conclusion: We revealed a novel pathway that elucidated WA activation of apoptosis and G2/M arrest in GBM cells through the ATF4-ATF3-CHOP axis. This discovery is important for optimization of WA-based regimens for prevention and/or treatment of GBM.

Keywords: apoptosis; cell cycle; endoplasmic reticulum; glioblastoma; unfolded protein response.

MeSH terms

  • Activating Transcription Factor 3 / metabolism*
  • Activating Transcription Factor 4 / metabolism*
  • Animals
  • Cell Line, Tumor
  • G2 Phase Cell Cycle Checkpoints / drug effects*
  • Glioblastoma / drug therapy*
  • Glioblastoma / metabolism
  • Glioblastoma / pathology
  • Humans
  • M Phase Cell Cycle Checkpoints / drug effects*
  • Mice
  • Mice, Nude
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • Signal Transduction / drug effects*
  • Transcription Factor CHOP / metabolism*
  • Withanolides / pharmacology*
  • Xenograft Model Antitumor Assays

Substances

  • ATF3 protein, human
  • ATF4 protein, human
  • Activating Transcription Factor 3
  • DDIT3 protein, human
  • Neoplasm Proteins
  • Withanolides
  • Activating Transcription Factor 4
  • Transcription Factor CHOP
  • withaferin A