Bioactive compound 1,8-Cineole selectively induces G2/M arrest in A431 cells through the upregulation of the p53 signaling pathway and molecular docking studies

Phytomedicine. 2018 Jul 15:46:57-68. doi: 10.1016/j.phymed.2018.04.007. Epub 2018 Apr 5.


Background: Callistemon citrinus has been traditionally known for its medicinal property. Recently, our research group identified 1,8-Cineole, as one of the predominant compound present in the hexane extract (HE-C), whose leaves have potent anticancer activity.

Hypothesis/purpose: The present study was designed to isolate 1,8-Cineole from Callistemon citrinus plant and to determine their role in anticancer effects in in vitro using skin carcinoma cells. Moreover, the molecular mechanism of apoptosis and molecular docking studies were also investigated.

Study design/methods: In vitro cytotoxicity test was performed with HE-C fractionates 1F, 2F, and 3F against A431 and HaCaT cell lines. MTT and AB assay demonstrated that 1F was toxic to cancer cells with no adverse effect to non-malignant cells and it was subjected to 1H NMR, 13C NMR spectroscopy and further characterized by FTIR and GC-MS analysis. On the basis of spectroscopic data, the metabolite was confirmed as 1,8-Cineole.

Results: Based on the cytotoxicity results, the well-characterized metabolite 1,8-Cineole was investigated upon to understand the mechanism that caused cancer cell death. In this process, the changes in mitochondrial membrane potential (ΔΨm) were confirmed by Rh-123/DAPI staining; the ultra structure was observed by TEM and quantified by flow cytometric analysis. These results proved that the compound effectively induced the apoptosis and G2/M phase arrest in A431 cells by increasing the expression of p53 and that it was monitored by FACS. Further, the expression of apoptotic proteins, such as Bax/Bcl-2, Cyt-c, caspase-9, and caspase-3 was confirmed by western blot. The molecular docking simulations predicted the hydrophobic interaction between 1,8-cineole with Bcl-2 and PARP1 receptor.

Conclusions: 1,8-Cineole is a potential candidate for skin carcinoma, which is possible by regulating the p53 apoptotic signaling pathway.

Keywords: 1,8-Cineole; Apoptosis; Caspase; Cell cycle arrest; Mitochondria; p53.

MeSH terms

  • Apoptosis / drug effects*
  • Caspase 3 / metabolism
  • Caspase 9 / metabolism
  • Cell Cycle Checkpoints / drug effects*
  • Cell Death
  • Cell Line, Tumor
  • Cyclohexanols / pharmacology*
  • Cytochromes c / metabolism
  • Eucalyptol
  • Humans
  • Membrane Potential, Mitochondrial / drug effects
  • Mitochondria / drug effects
  • Molecular Docking Simulation
  • Monoterpenes / pharmacology*
  • Myrtaceae / chemistry
  • Poly (ADP-Ribose) Polymerase-1 / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Signal Transduction / drug effects*
  • Tumor Suppressor Protein p53 / metabolism*
  • Up-Regulation


  • BCL2 protein, human
  • Cyclohexanols
  • Monoterpenes
  • Proto-Oncogene Proteins c-bcl-2
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Cytochromes c
  • PARP1 protein, human
  • Poly (ADP-Ribose) Polymerase-1
  • CASP3 protein, human
  • CASP9 protein, human
  • Caspase 3
  • Caspase 9
  • Eucalyptol