Mechanism of apoptosis induced by quinoxalone from the myxobacterium Stigmatella eracta WXNXJ-B in B16 mouse melanoma cell line
- Published
- Accepted
- Subject Areas
- Biotechnology, Microbiology, Pharmacology
- Keywords
- Apoptosis, Stigmatella eracta WXNXJ-B, Quinoxalone, Mitochondrial membrane potential, Bax, Caspase-3
- Copyright
- © 2017 Wang et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2017. Mechanism of apoptosis induced by quinoxalone from the myxobacterium Stigmatella eracta WXNXJ-B in B16 mouse melanoma cell line. PeerJ Preprints 5:e2703v1 https://doi.org/10.7287/peerj.preprints.2703v1
Abstract
The biological activities of quinoxalone, a novel small molecular substance isolated from the broth of the myxobacterium Stigmatella eracta WXNXJ-B, was investigated. This study was designed to determine the anti-proliferative, apoptotic property of quinoxalone, using B16 mouse melanoma cells as a model system. The results showed that quinoxalone has antitumor activity and can significantly inhibit the proliferation of B16 cells. The extent and the timing of apoptosis were strongly dependent on the dose. After treating with quinoxalone and staining with Hoechst 33342, B16 cells showed typical apoptotic morphological features such as chromatin condensation by fluorescent microscopy. DNA isolated from B16 cells cultured with quinoxalone showed a typical DNA ladder of apoptosis in agarose gel electrophoresis. Further investigation results showed that the apoptotic machinery of B16 induced by quinoxalone was associated with drop in mitochondrial membrane potential from 5.35% to 23.7%, up-regulation of Bax and down-regulation of Bcl-2 in a dose-dependent manner. And a significant increased activation of caspase-3. Our finding suggests that quinoxalone could suppress the growth of B16 cells and reduces cell survival via disturbing mitochondrial membrane potential and inducing apoptosis of tumor cells.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
The influence of quinoxalone on B16 cells observed by inverted microscope
B16 cells were plated onto glass cover slips in 6-well plates and treated with quinoxalone for 48 h. Then, cells were washed twice with PBS, fixed with 1% glutaraldehyde, stained with Hoechst 33342 for 15 min at room temperature. Nuclear morphology was examined by fluorescence microscope.
Fluorescence micrographs of B16 cells
B16 cells were treated without (A) and with quinoxalone (B: 5 μg/mL, C: 10 μg/mL) for 48 h. White arrow were the normal cells in A. White arrow were the apoptosis cells in B and C .
Effect of quinoxalone on DNA of B16 cells
B16 cells were treated with different dose of quinoxalone for 48h. Isolated DNA was analysed in agarose gel electrophoresis as described in Material and Methods. 200 bp DNA ladder marker (novoprotein, China) was used as marker (M) of DNA fragment size.
Effect of quinoxalone on mitochondrial transmembrane potential in B16 cells
After treatment without (A,control) and with 2.5 µg/mL (B) , 5 µg/mL(C), 10 µg/mL (D) quinoxalone for 48 h, the cells were double-stained with Rhodamine-123 and PI for 30 min, respectively. The percentages of PI negative and low-staining (Rh123-PI-) group represent the apoptotic cell group.
Effect of quinoxalone on the expression of Bax, Bcl-2 and P53 protein
Cells were treated without (control) and with 5, 10 µg/mL quinoxalone for 48 h. After washing with 75% ethanol, the cells were respectively incubated with anti-Bcl-2 antibody, anti-Bax antibody and anti-53 antibody. Then, the cells were incubated with FITC-conjugated secondary goat anti-mouse IgG. Bax (A), Bcl-2 (B) and p53 (C) levels were checked by flow cytometry.