Background: BNS‐22 is a chemically synthesized derivative of the plant natural product GUT‐70 isolated from the stem bark of Calophyllum brasiliense. BNS‐22 possesses antiproliferative activity on human cancer cells, but its mechanism of action is unknown. We recently developed a proteome‐based profiling system to predict molecular targets and mechanisms of action of bioactive small molecules. Here we report on the identification of a molecular target for BNS‐22 by using the proteomic profiling approach.
Materials and Methods: HeLa cells were treated with BNS‐22 and 19 well‐characterized small molecules including signal transduction inhibitors and anticancer drugs for 18 h, and the cell lysates were subjected to 2‐dimensional fluorescence differential gel electrophoresis (2D‐DIGE). Then, we extracted 314 spots detected in all analysis gels, and performed cluster analysis based on the expression levels of those spots.
Results: Proteomic profiling analysis indicated that BNS‐22 was classified into the same cluster as ICRF‐193, a DNA topoisomerase II (TOP2) catalytic inhibitor. This profiling analysis also distinguished clearly the action between ICRF‐193 and VP‐16 (etoposide), a TOP2 poison. These results suggested that BNS‐22 acted as a TOP2 catalytic inhibitor and not a TOP2 poison in cells. BNS‐22 inhibited the DNA decatenation activity of human TOP2 in vitro, with an IC50 value of 2.4 M. BNS‐22, like ICRF‐193, induced the abnormal mitotic figure characterized by the unusually‐distorted spindle and the failure of chromosome alignment and segregation, as assessed by immunofluorescence staining with anti‐ ‐tubulin antibody, anti‐ ‐tubulin antibody, and DAPI. Flow cytometric analysis showed that BNS‐22‐treated cells were accumulated at G2/M phase after 24 h in a dose‐dependent manner. In the time course experiment, peaks corresponding to 8C were observed after 36 h in cells treated with 10 M BNS‐22, indicating that BNS‐22 as well as ICRF‐193 induced polyploidy. BNS‐22, unlike VP‐16, did not cause DNA double‐strand breaks, as measured by the phosphorylation of histone H2AX on Ser 139 (‐H2AX), and furthermore antagonized VP‐16‐mediated DNA damage.
Conclusion: These results strongly suggest that BNS‐22 targets TOP2 and acts as its catalytic inhibitor in cells.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C157.
- Copyright © 2009, American Association for Cancer Research