ROS-mediated activation and mitochondrial translocation of CaMKII contributes to Drp1-dependent mitochondrial fission and apoptosis in triple-negative breast cancer cells by isorhamnetin and chloroquine

J Exp Clin Cancer Res. 2019 May 28;38(1):225. doi: 10.1186/s13046-019-1201-4.


Background: Triple-negative breast cancer (TNBC) is often aggressive and associated with a poor prognosis. Due to the lack of available targeted therapies and to problems of resistance with conventional chemotherapeutic agents, finding new treatments for TNBC remains a challenge and a better therapeutic strategy is urgently required.

Methods: TNBC cells and xenograft mice were treated with a combination of chloroquine (CQ) and isorhamnetin (IH). Mitochondrial fission, apoptosis, and related signaling pathways were determined by flow cytometry, immunofluorescence, and related molecular biological techniques.

Results: The inhibition of autophagy/mitophagy by CQ selectively enhances IH-induced mitochondrial fission and apoptosis in TNBC cells but not in estrogen-dependent breast cancer cells. These events were accompanied by mitochondrial translocation of Bax and the release of cytochrome c. Mechanistically, these effects were associated with oxidative stress-mediated phosphorylation of CaMKII (Thr286) and Drp1 (S616), and subsequent mitochondrial translocation of CaMKII and Drp1. The interruption of the CaMKII pathway by genetic approaches (e.g. CaMKII mutant or siRNA) attenuated combination-mediated mitochondrial fission and apoptosis. The combination of CQ/IH was a marked inhibitor tumor growth, inducing apoptosis in the TNBC xenograft mouse model in association with the activation of CaMKII and Drp1 (S616).

Conclusions: Our study highlights the critical role of ROS-mediating CaMKII/Drp1 signaling in the regulation of mitochondrial fission and apoptosis induced by combination of CQ/IH. These findings also suggest that IH could potentially be further developed as a novel chemotherapeutic agent. Furthermore, a combination of IH with classic autophagy/mitophagy inhibitor could represent a novel therapeutic strategy for the treatment of TNBC.

Keywords: Apoptosis; Autophagy; CaMKII; Chloroquine; Drp1; Isorhamnetin; Triple-negative breast cancer.

MeSH terms

  • Animals
  • Antineoplastic Combined Chemotherapy Protocols / administration & dosage*
  • Antineoplastic Combined Chemotherapy Protocols / pharmacology
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / genetics
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism*
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Survival / drug effects
  • Chloroquine / administration & dosage*
  • Chloroquine / pharmacology
  • Dynamins
  • Female
  • GTP Phosphohydrolases / metabolism*
  • Humans
  • Mice
  • Microtubule-Associated Proteins / metabolism*
  • Mitochondrial Dynamics / drug effects
  • Mitochondrial Proteins / metabolism*
  • Quercetin / administration & dosage
  • Quercetin / analogs & derivatives*
  • Quercetin / pharmacology
  • Reactive Oxygen Species / metabolism*
  • Triple Negative Breast Neoplasms / drug therapy*
  • Triple Negative Breast Neoplasms / genetics
  • Triple Negative Breast Neoplasms / metabolism
  • Xenograft Model Antitumor Assays


  • Microtubule-Associated Proteins
  • Mitochondrial Proteins
  • Reactive Oxygen Species
  • 3-methylquercetin
  • Chloroquine
  • Quercetin
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • GTP Phosphohydrolases
  • DNM1L protein, human
  • Dynamins