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Characterization of a taxol-resistant human small-cell lung cancer cell line
1Pharmacology Division, National Cancer Center Research Institute, Tokyo.
Abstract:
Taxol is a novel anticancer agent with activity against a broad range of tumors. It has a unique ability to stabilize polymerized tubulin into microtubule bundles within the cell. We have established a taxol-resistant human small-cell lung cancer cell line (H69/Txl) by exposing H69 cells to stepwise increases in taxol concentration. The resistance of H69/Txl cells to taxol was 4.7-fold that of the original H69 cells: the IC50 values for H69 and H69/Txl were 113.7 +/- 56.54 nM and 538.7 +/- 214.7 nM by the tetrazolium dye assay, respectively. Removal of the drug from the medium resulted in a 38% decrease in the growth rate of H69/Txl as compared with that in the presence of 30 nM taxol, suggesting that the growth of H69/Txl was partially dependent on taxol. H69/Txl showed higher sensitivity to vinca alkaloids such as vindesine, vincristine and vinblastine than the parental H69. There was no significant difference in intracellular [3H]taxol content between H69 and H69/Txl cells. No MDR-1 mRNA was detected in H69/Txl by the reverse transcription polymerase chain reaction. There was no significant difference of total and polymerized tubulin content between H69 and H69/Txl cells. Altered mobility of one of the alpha-tubulin isoforms in H69/Txl was revealed by using isoelectric focusing and Western blotting with anti-alpha-tubulin antibody. In H69, two alpha-tubulin isoforms were observed, whereas three were evident in H69/Txl, two of them comigrating with the isoforms of H69 and the other being more acidic. We observed the increased acetylation of alpha-tubulin in H69/Txl cells as compared with that in H69 cells. The acetylation of alpha-tubulin may be responsible for the taxol resistance and/or taxol-dependent growth of H69/Txl.
Insights
Researchers developed a taxol-resistant lung cancer cell line (H69/Txl) showing increased alpha-tubulin acetylation, a potential mechanism for taxol resistance and dependent growth.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Taxol is a potent anticancer agent that stabilizes microtubules.
- Understanding mechanisms of taxol resistance is crucial for cancer therapy.
- Development of drug-resistant cancer cell lines aids resistance studies.
Purpose of the Study:
- To establish and characterize a taxol-resistant human small-cell lung cancer cell line.
- To investigate the molecular mechanisms underlying taxol resistance in this cell line.
- To explore potential alterations in tubulin dynamics and modifications.
Main Methods:
- Generation of taxol-resistant cells (H69/Txl) through stepwise drug exposure.
- Cell viability assays (tetrazolium dye) to determine IC50 values.
- Analysis of drug accumulation, MDR-1 mRNA expression, tubulin content, tubulin isoform mobility (isoelectric focusing, Western blotting), and alpha-tubulin acetylation.
Main Results:
- H69/Txl cells exhibited 4.7-fold resistance to taxol compared to parental H69 cells.
- H69/Txl cells showed increased sensitivity to vinca alkaloids and partially taxol-dependent growth.
- Increased acetylation of alpha-tubulin was observed in H69/Txl cells, with altered isoform mobility.
Conclusions:
- Alpha-tubulin acetylation is a potential contributor to taxol resistance and taxol-dependent growth in H69/Txl cells.
- The findings suggest novel mechanisms of taxol resistance independent of MDR-1 expression or altered drug accumulation.
- Further research into tubulin modifications could reveal new therapeutic strategies for overcoming taxol resistance.