Synergistic Apoptotic Effects of Bortezomib and Methylstat on Multiple Myeloma Cells

Arch Med Res. 2020 Apr;51(3):187-193. doi: 10.1016/j.arcmed.2020.01.012. Epub 2020 Feb 26.


Background: In this study, we aimed to determine synergistic apoptotic and cytotoxic effects of methylstat and bortezomib on U266 and ARH77 multiple myeloma (MM) cells.

Methods: Cytotoxic effects of the drugs were demonstrated by MTT cell proliferation assay while apoptotic effects were examined by loss of mitochondrial membrane potential (MMP) by JC-1 MMP detection kit, changes in caspase-3 enzyme activity and Annexin-V apoptosis assay by flow cytometry. Expression levels of apoptotic and antiapoptotic genes were examined by qRT-PCR.

Results: Our results showed that combination of methylstat and bortezomib have synergistic antiproliferative effect on MM cells as compared to either agent alone. These results were also confirmed by showing synergistic apoptotic effects determined by increased loss of mitochondrial membrane potential and increased caspase-3 enzyme activity and relocation of phosphotidyleserine on the cell membrane by Annexin-V/PI double staining. Combination of bortezomib with methylstat arrested cells at the S phase of the cell cycle. Methylstat treatment caused upregulation of FASLG, NGFR, TNF, TNFRS10B and TNFRS1B apoptotic genes and downregulation of AKT1, AVEN, BAG1 BCL2L2 and RELA antiapoptotic genes in a dose and time dependent manner.

Conclusion: In conclusion, our data suggested that bortezomib in combination with methylstat decreased cell proliferation and induced apoptosis significantly in U266 and ARH77 cells. When supported with in vivo analyses, methylstat might be considered as a potential new agent for the treatment of MM.

Keywords: Apoptosis; Bortezomib; Methylstat; Multiple myeloma.

MeSH terms

  • Annexin A5 / metabolism
  • Antineoplastic Agents / pharmacology*
  • Antineoplastic Agents / therapeutic use
  • Apoptosis / drug effects*
  • Bortezomib / pharmacology*
  • Bortezomib / therapeutic use
  • Caspase 3 / metabolism
  • Cell Cycle / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects*
  • Down-Regulation / drug effects
  • Drug Combinations
  • Humans
  • Membrane Potential, Mitochondrial / drug effects
  • Multiple Myeloma / drug therapy*
  • Multiple Myeloma / genetics
  • Multiple Myeloma / metabolism
  • Naphthalenes / pharmacology*
  • Naphthalenes / therapeutic use


  • Annexin A5
  • Antineoplastic Agents
  • Drug Combinations
  • Naphthalenes
  • methylstat
  • Bortezomib
  • Caspase 3