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Caspase-3 activation during apoptosis caused by glutathione-doxorubicin conjugate
T Asakura1, T Sawai, Y Hashidume
1Department of Biochemistry (I), Jikei University School of Medicine, Tokyo, Japan.
Abstract:
Glutathione-doxorubicin (GSH-DXR) effectively induced apoptosis in rat hepatoma cells (AH66) at a lower concentration than DXR. After 24 h of drug treatment, DNA fragmentation of the cells was observed at the concentration of 1.0 microM DXR or 0.01 microM GSH-DXR. Increase in caspase-3 activity and DNA fragmentation were observed within 12 h and 15 h after treatment with either drug. Intracellular caspase-3 activity was increased in a dose-dependent manner after treatment with DXR or GSH-DXR, and caspase-3 activity correlated well with the ability to induce DNA fragmentation. When the cells were treated with either DXR or GSH-DXR for only 6 h, apoptotic DNA degradation and caspase-3 activation occurred 24 h after treatment. DNA fragmentation caused by these drugs was prevented completely by simultaneous treatment with the caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), at 10 microM. By contrast, DNA fragmentation was not prevented by the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO), at the same concentration as DEVD-CHO, and caspase-1 was not activated at all by the treatment of AH66 cells with both DXR and GSH-DXR. These results demonstrate that DXR and GSH-DXR induce apoptotic DNA fragmentation via caspase-3 activation, but not via caspase-1 activation, and that GSH-DXR enhances the activation of caspase-3 approximately 100-fold more than DXR. Moreover, the findings suggested that an upstream apoptotic signal that can activate caspase-3 is induced within 6 h by treating AH66 cells with the drug.
Insights
Glutathione-doxorubicin (GSH-DXR) induces apoptosis in rat hepatoma cells more effectively than doxorubicin (DXR). GSH-DXR activates caspase-3, a key enzyme in programmed cell death, at lower concentrations and faster rates than DXR.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Doxorubicin (DXR) is a widely used chemotherapy drug, but its efficacy can be limited by resistance and toxicity.
- Investigating novel drug formulations, such as glutathione-doxorubicin (GSH-DXR), may enhance therapeutic outcomes.
- Understanding the molecular mechanisms of apoptosis induction is crucial for developing more effective cancer treatments.
Purpose of the Study:
- To compare the efficacy of GSH-DXR and DXR in inducing apoptosis in rat hepatoma cells (AH66).
- To elucidate the role of caspase-3 and caspase-1 activation in the apoptotic pathways triggered by these drugs.
- To determine the dose- and time-dependency of apoptosis induction by GSH-DXR and DXR.
Main Methods:
- Treatment of AH66 cells with varying concentrations of DXR and GSH-DXR.
- Assessment of DNA fragmentation and caspase-3/caspase-1 activity at different time points post-treatment.
- Inhibition studies using specific caspase-3 (DEVD-CHO) and caspase-1 (YVAD-CHO) inhibitors.
Main Results:
- GSH-DXR induced apoptosis at significantly lower concentrations (0.01 microM) compared to DXR (1.0 microM).
- Both drugs increased caspase-3 activity and DNA fragmentation in a dose- and time-dependent manner.
- GSH-DXR demonstrated approximately 100-fold greater enhancement of caspase-3 activation than DXR.
- Apoptosis induction was dependent on caspase-3 activation, as it was blocked by DEVD-CHO but not by YVAD-CHO, and caspase-1 was not activated.
Conclusions:
- GSH-DXR is a more potent inducer of apoptosis in AH66 cells than DXR.
- The apoptotic effect of both drugs is mediated through caspase-3 activation, independent of caspase-1.
- GSH-DXR represents a promising chemotherapeutic agent with enhanced efficacy and potentially reduced side effects.