Related Experiment Videos
Induction of apoptosis and cell cycle arrest in mouse colon 26 cells by benastatin A
I Kakizaki1, K Ookawa, T Ishikawa
1Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
Abstract:
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.
Insights
Benastatin A induces apoptosis in colon 26 cells, leading to decreased cell survival and DNA fragmentation. This effect is dose-dependent but unlikely caused by glutathione transferase (GST) inhibition.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Mammalian glutathione transferases (GSTs) are enzymes involved in cellular detoxification.
- GST inhibitors, like ethacrynic acid, can induce apoptosis in cancer cells.
- Benastatin A, a bacterial compound, is known to inhibit mammalian GSTs.
Purpose of the Study:
- To investigate the effect of Benastatin A on the survival of mouse colon 26 adenocarcinoma cells.
- To compare the apoptotic effects of Benastatin A with ethacrynic acid.
- To elucidate the mechanism of Benastatin A-induced apoptosis, particularly its relationship with GST inhibition.
Main Methods:
- Cell viability assays on colon 26 cells treated with Benastatin A and ethacrynic acid.
- Electrophoretic analysis for DNA fragmentation.
- Identification and activity assays of GST-II in colon 26 cells.
- Immunoblot and northern blot analyses for GST-II expression.
- Analysis of beta-actin and bax mRNA levels.
- Flow cytometry for cell cycle analysis.
Main Results:
- Benastatin A and ethacrynic acid both dose-dependently decreased colon 26 cell viability and induced DNA fragmentation, indicating apoptosis.
- Ethacrynic acid rapidly inhibited GST-II activity and increased its expression, while Benastatin A showed delayed effects on GST activity and protein levels.
- Benastatin A decreased beta-actin and bax mRNA levels and caused G1/G0 cell cycle arrest.
- Benastatin A's apoptotic effect was observed at concentrations that did not significantly inhibit GST activity in crude extracts.
Conclusions:
- Benastatin A induces apoptosis in mouse colon 26 adenocarcinoma cells.
- The mechanism of apoptosis induction by Benastatin A is likely independent of direct glutathione transferase (GST) inhibition.
- Benastatin A may exert its effects through modulation of cell cycle progression and gene expression (beta-actin, bax).