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Failure to activate caspase 3 in phorbol ester-resistant leukemia cells is associated with resistance to apoptotic
Yun-Jung Choi1, Jong-Wook Park, Ju-Hyung Woo
1Department of Immunology, School of Medicine, Keimyung University, 194 DongSan-Dong Jung-Gu, 700-712, Taegu, South Korea.
Abstract:
The protein kinase C (PKC)-specific inhibitor, Ro-31-8220, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. In the present study, we determined the molecular pathways that lead to apoptosis after treatment of cells with the PKC-specific inhibitor RO-31-8220. For this, we used the U937 human leukemia cell line and a phorbolmyristate acetate (PMA)-resistant derivative cell line, R-U937. Ro-31-8220 treatment of U937 cells leads to apoptosis, which is accompanied by activation of caspase 3 (as measured by decreased levels of the 32kDa inactive form and increased proteolytic cleavage of phospholipase C (PLC)-gamma1). The broad-range caspase inhibitor z-VAD-fmk inhibits this induction of apoptosis, supporting a direct link between caspase activation and Ro-31-8220 induction of apoptosis. This activation of apoptosis is also accompanied by release of cytochrome c, but not by altered expression of Bcl-2 family protein or IAP family proteins. In R-U937 cells, Ro-31-8220 fails to cause release of cytochrome c, activation of caspase 3, or apoptosis. Activation of Akt occurs to a greater extent in the R-U937 cells than the U937 cells and thus might be related to protection from Ro-31-8220-induced apoptosis.
Insights
The protein kinase C (PKC) inhibitor Ro-31-8220 triggers apoptosis in leukemia cells via caspase activation and cytochrome c release. Resistance in derivative cells suggests Akt activation may protect against this PKC inhibitor-induced cell death.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Protein kinase C (PKC) inhibitors, such as Ro-31-8220, demonstrate anti-proliferative and apoptotic effects on human cancer cell lines.
- Understanding the precise molecular mechanisms underlying Ro-31-8220-induced apoptosis is crucial for its therapeutic development.
Purpose of the Study:
- To elucidate the molecular pathways involved in apoptosis induced by the PKC-specific inhibitor Ro-31-8220.
- To compare the cellular response to Ro-31-8220 in a human leukemia cell line (U937) and a resistant derivative (R-U937).
Main Methods:
- Treatment of U937 and R-U937 cells with Ro-31-8220.
- Assessment of apoptosis through caspase 3 activation, phospholipase C (PLC)-gamma1 cleavage, and cytochrome c release.
- Evaluation of Bcl-2 family protein and IAP family protein expression.
- Measurement of Akt activation levels.
Main Results:
- Ro-31-8220 induced apoptosis in U937 cells, evidenced by caspase 3 activation and cytochrome c release.
- The caspase inhibitor z-VAD-fmk blocked Ro-31-8220-induced apoptosis, confirming the role of caspases.
- R-U937 cells, resistant to Ro-31-8220, did not exhibit cytochrome c release or caspase 3 activation.
- Enhanced Akt activation was observed in R-U937 cells compared to U937 cells, potentially conferring resistance.
Conclusions:
- Ro-31-8220 induces apoptosis in U937 leukemia cells through a caspase-dependent pathway involving cytochrome c release.
- Resistance to Ro-31-8220 in R-U937 cells is associated with higher Akt activation and a lack of caspase-mediated apoptosis.
- Akt activation may play a protective role against Ro-31-8220-induced apoptosis.