Benastatin A, isolated from Streptomyces bacteria, is reported to inhibit mammalian
glutathione transferases (
GSTs). Since GST inhibitors such as
ethacrynic acid are suggested to induce apoptosis in some cell lines, the effect of
benastatin A on the survival of mouse colon 26
adenocarcinoma cells was compared with that of
ethacrynic acid. When cells in stationary phase were treated with
benastatin A, viable cells were found to be dose-dependently decreased after 3 days. In the case of
ethacrynic acid, this became apparent within 24 h. Electrophoretic analysis revealed DNA fragmentation, indicating that cell loss was due to apoptosis in both cases. The dominant GST in colon 26 cells was identified as the class Pi-form (
GST-II), and the activities in
crude extracts as well as purified
GST-II were almost completely inhibited by 50 microM
ethacrynic acid. Immunoblot and northern blot analyses revealed increased
GST-II protein and
mRNA levels in cells treated with
ethacrynic acid.
Benastatin A did not significantly affect the activity in the
crude extract even at 20 microM, a 10-fold higher concentration than that which almost completely inhibited the activity of purified
GST-II. However, GST activity and
GST-II protein were decreased in colon 26 cells treated with
benastatin A for 5 days, no significant activity being detected in the range of 16 - 20 microM. In addition,
beta-actin and bax mRNAs were also decreased in a dose-dependent manner. Furthermore, flow cytometric analysis of colon 26 cells revealed that
benastatin A blocked the cell cycle at the G1/G0 phase. Thus,
benastatin A also induces apoptosis of colon 26 cells, but this is unlikely to be due to inhibition of GST activity.