Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models

PLoS One. 2013 Oct 18;8(10):e78958. doi: 10.1371/journal.pone.0078958. eCollection 2013.

Abstract

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.

MeSH terms

  • Animals
  • Antibiotics, Antineoplastic / pharmacology
  • Antineoplastic Agents / pharmacology
  • Apoptosis / drug effects*
  • Autophagy / drug effects*
  • Cell Line, Tumor
  • Cell Survival
  • Down-Regulation / drug effects
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism
  • Genistein / pharmacology*
  • Humans
  • Mice, Nude
  • Microtubule-Associated Proteins / genetics*
  • Mitochondria / drug effects
  • Neuroblastoma / pathology*
  • Neuroblastoma / therapy
  • Plasmids / genetics
  • Protein Kinase Inhibitors / pharmacology*
  • RNA Interference
  • RNA, Small Interfering
  • Sirolimus / pharmacology
  • Transfection
  • Up-Regulation / drug effects

Substances

  • Antibiotics, Antineoplastic
  • Antineoplastic Agents
  • Microtubule-Associated Proteins
  • Protein Kinase Inhibitors
  • RNA, Small Interfering
  • light chain 3, human
  • Genistein
  • ErbB Receptors
  • Sirolimus

Grant support

The authors have no support or funding to report.