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Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines
Terence Kin-Wah Lee1, Tracy Ching-Man Lau, Irene Oi-Lin Ng
1Department of Pathology and Center for the Study of Liver Disease, University of Hong Kong, China.
Purpose:
Doxorubicin (DOX) is a commonly used anticancer drug which causes DNA damage and kills cancer cells mainly by apoptosis. However, the process leading to killing of cancer cells and the molecular basis of resistance to DOX are not well understood. To evaluate the role of p53 and the cellular effects of DOX on hepatoma cell lines, we examined three hepatoma cell lines with different p53 status--Huh-7 (mutated p53), HepG2 (wild-type p53) and Hep3B (deleted p53).
Methods:
The chemosensitivity of the three hepatoma cell lines was assessed using the MTT assay, and cell cycle distribution was evaluated by flow cytometry. Western blotting and immunostaining were employed to examine the protein alterations in response to DOX treatment, and a DNA fragmentation assay was performed to detect apoptosis.
Results:
Of the three cell lines, HepG2 was found to be most resistant to DOX, followed by Hep3B, and Huh-7 was most sensitive to DOX treatment. HepG2 showed G1 arrest 24 h after drug administration and upregulation of p53 protein level in a time-dependent manner. In Hep3B cells (deleted p53), G2/M phase arrest was observed soon after drug administration, accompanied by induced apoptosis that was p53-independent. In Huh-7 cells (mutated p53), which were most sensitive to DOX, there was neither G1 nor G2 arrest, and the level of p53 mutated protein was downregulated after DOX treatment. MDM2 and p27 proteins were downregulated in all cell lines independently of p53 status. p21 was upregulated following p53 activation at low doses of DOX in HepG2 cells, but at higher doses, p21 was downregulated in Huh-7 and HepG2 cells.
Conclusion:
DOX confers different chemosensitivity on different hepatoma cell lines with different p53 status. The contrasting relationships between chemosensitivity and p53 status and expression suggest that DOX-induced apoptosis and cell death involve pathways that are independent of p53.
Insights
Doxorubicin (DOX) sensitivity varies in hepatoma cells based on p53 status. DOX-induced cell death and apoptosis involve p53-independent pathways, revealing new insights into cancer drug resistance.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Doxorubicin (DOX) is a vital chemotherapy agent inducing cancer cell apoptosis via DNA damage.
- Understanding DOX resistance mechanisms and its impact on cancer cell death pathways is crucial for effective treatment.
- The role of the tumor suppressor protein p53 in DOX response remains incompletely understood.
Purpose of the Study:
- To investigate the differential chemosensitivity of hepatoma cell lines to DOX based on their p53 mutational status.
- To elucidate the cellular effects and molecular alterations induced by DOX in relation to p53.
- To explore the involvement of p53-dependent and independent pathways in DOX-induced apoptosis and cell death.
Main Methods:
- Chemosensitivity assessed using MTT assays across three hepatoma cell lines (Huh-7, HepG2, Hep3B) with distinct p53 statuses.
- Cell cycle distribution analyzed via flow cytometry.
- Protein expression changes (p53, MDM2, p27, p21) and apoptosis detected through Western blotting, immunostaining, and DNA fragmentation assays.
Main Results:
- HepG2 (wild-type p53) exhibited the highest DOX resistance, showing G1 arrest and p53 upregulation. Huh-7 (mutated p53) was most sensitive, with no cell cycle arrest and p53 downregulation.
- Hep3B (deleted p53) displayed G2/M arrest and p53-independent apoptosis.
- MDM2 and p27 were downregulated across all cell lines, irrespective of p53 status. p21 expression varied based on DOX dose and p53 activation.
Conclusions:
- Hepatoma cell chemosensitivity to DOX is significantly influenced by p53 status.
- DOX-induced apoptosis and cell death mechanisms can operate independently of p53.
- These findings highlight the complex interplay between p53 and DOX response, suggesting potential therapeutic strategies targeting p53-independent pathways.