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Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of
K Sugiyama1, T Akiyama, M Shimizu
1Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., Shizuoka, Japan.
Abstract:
7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor that is under development as an anticancer agent in the United States and Japan. Long-term exposure of human A549 non-small cell lung cancer cells to UCN-01 furnished cells (A549/UCN) with acquired resistance against UCN-01. In this study, the sensitivity of these cells toward the growth-arresting properties of certain conventional cytotoxic agents was explored. Cells were not cross-resistant against adriamycin, Taxol, staurosporine, and UCN-02, but they displayed 14- and 4.4-fold resistance against cisplatin and mitomycin C, respectively. Previous studies on the mechanism(s) of action of UCN-01 suggest that induction of apoptosis and G1 phase accumulation are important for its anticancer activity; therefore, we compared induction of apoptosis and cell cycle distribution caused by UCN-01 in wild-type A549 and A549/UCN cells using flow cytometry. UCN-01 (0.4 microM) induced apoptosis (62% terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells) in A549 cells, but not in A549/UCN cells. The percentages of cells that accumulated in G1 when exposed to UCN-01 (0.4 microM) were 22% in A549 cells and 67% in A549/UCN cells. These results suggest that acquired resistance of cancer cells against UCN-01 is characterized by attenuation of apoptosis induction associated with reinforcement of the G1 checkpoint and that apoptosis regulation is drastically altered in A549/UCN cells as compared with A549 cells. Cyclin-dependent kinase (CDK) inhibitor proteins p21 and p27 in A549/UCN cells were up-regulated, which was accompanied by overexpression of G1 cyclins D1 and E, but UCN-01 hardly affected levels of these proteins. In contrast, cyclin A, cyclin B1, retinoblastoma, and CDK2 proteins were apparently down-regulated, without changes in CDK4/6. UCN-01 hardly affected the expression level of cyclin B1 and induced dephosphorylation of retinoblastoma in both cell types. UCN-01 induced down-regulation of cyclin A level and CDK2 activity accompanied with its dephosphorylation in A549/UCN cells, but not in A549 cells. The antiapoptotic protein bcl-2 was apparently up-regulated in A549/UCN cells, however, bcl-xL, another antiapoptotic protein, was down-regulated, without changes in bak and bax. Taken together, these results are consistent with the notion that induction of apoptosis and block of cell cycle in G1 are important determinants of the sensitivity of cancer cells to UCN-01 and suggest that inhibition of CDK2 activity accompanied by its dephosphorylation and decrease of expression level of cyclin A might play an important role in the G1 phase accumulation induced by UCN-01.
Insights
Acquired resistance to 7-hydroxystaurosporine (UCN-01) in lung cancer cells involves reduced apoptosis and enhanced G1 cell cycle arrest. This resistance is linked to altered cyclin and CDK protein levels, impacting UCN-01
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- 7-Hydroxystaurosporine (UCN-01) is an investigational anticancer agent targeting protein kinases.
- Acquired resistance to UCN-01 was observed in human non-small cell lung cancer (A549) cells.
- Understanding resistance mechanisms is crucial for developing effective cancer therapies.
Purpose of the Study:
- To investigate the sensitivity of UCN-01-resistant A549 cells to conventional cytotoxic agents.
- To compare the effects of UCN-01 on apoptosis induction and cell cycle distribution in sensitive and resistant A549 cells.
- To elucidate the molecular alterations associated with acquired UCN-01 resistance.
Main Methods:
- Flow cytometry was used to assess apoptosis and cell cycle distribution.
- Western blotting was employed to analyze the expression and phosphorylation status of key cell cycle regulatory proteins (cyclins, CDKs, retinoblastoma).
- Sensitivity to various cytotoxic agents was evaluated in UCN-01-resistant cells.
Main Results:
- UCN-01-resistant cells (A549/UCN) showed cross-resistance to cisplatin and mitomycin C but not to other agents tested.
- UCN-01 failed to induce apoptosis in A549/UCN cells, whereas it induced significant apoptosis in sensitive A549 cells.
- A549/UCN cells exhibited a pronounced G1 phase cell cycle arrest in response to UCN-01 compared to A549 cells.
- UCN-01 resistance was associated with altered expression and phosphorylation of cyclins (e.g., cyclin A, B1) and cyclin-dependent kinases (e.g., CDK2).
- UCN-01-resistant cells showed increased p21 and p27, and altered levels of antiapoptotic proteins (bcl-2, bcl-xL).
Conclusions:
- Acquired resistance to UCN-01 in lung cancer cells is characterized by impaired apoptosis induction and enhanced G1 checkpoint activation.
- Alterations in cell cycle regulatory proteins, including CDK2 activity and cyclin A levels, play a significant role in UCN-01 resistance.
- Understanding these molecular changes provides insights into potential therapeutic strategies for overcoming UCN-01 resistance.