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Published on: August 11, 2017
Establishment of pemetrexed-resistant non-small cell lung cancer cell lines
Dan Zhang1, Nobuaki Ochi, Nagio Takigawa
1Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Abstract:
Pemetrexed (PEM), a multitargeted antifolate with manageable toxicity, is active against non-squamous non-small cell lung cancer; however, most patients eventually acquire resistance to PEM. To elucidate the resistant mechanism, we established PEM-resistant lung adenocarcinoma cell lines. Two parental cell lines, PC-9 and A549, were treated with step-wise increasing concentrations of PEM. Growth inhibition was determined by the 3-[4,5-dimethyl-thizol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction. The four PC-9 sublines were more resistant than the PC-9 cell line to PEM (2.2-, 2.9-, 8.4-, and 14.3-fold, respectively). The four A549 sublines also showed more resistance to PEM (7.8-, 9.6-, 42.3-, and 42.4-fold, respectively) than the parent cell line. All resistant sublines showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine, 5-fluorouracil, or the active metabolite of irinotecan, SN-38. All PEM-resistant sublines expressed more TS than the parental cells, by polymerase chain reaction and Western blotting. DHFR was significantly increased in the four PEM-resistant A549 sublines. GARFT did not correlate with resistance to PEM. In summary, PEM-resistant cells remained sensitive to docetaxel, vinorelbine, 5-fluorouracil, and irinotecan. TS expression appeared to be associated with resistance to PEM.
Insights
Pemetrexed resistance in lung cancer cells is linked to increased thymidylate synthase (TS) expression. Resistant cells maintain sensitivity to other chemotherapies like docetaxel and 5-fluorouracil, offering alternative treatment options.
Area of Science:
- Oncology
- Pharmacology
- Molecular Biology
Background:
- Pemetrexed (PEM) is effective against non-squamous non-small cell lung cancer (NSCLC).
- Acquired resistance to PEM is a significant clinical challenge in NSCLC treatment.
- Understanding the mechanisms of PEM resistance is crucial for developing effective therapeutic strategies.
Purpose of the Study:
- To investigate the molecular mechanisms underlying pemetrexed resistance in non-small cell lung cancer.
- To establish and characterize pemetrexed-resistant lung adenocarcinoma cell lines.
- To identify potential biomarkers associated with pemetrexed resistance.
Main Methods:
- Development of pemetrexed-resistant cell lines (PC-9 and A549) through stepwise drug concentration increases.
- Assessment of cell growth inhibition using the MTT assay.
- Quantitative real-time PCR and Western blotting to analyze gene and protein expression of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT).
Main Results:
- Established PEM-resistant sublines of PC-9 and A549 cells exhibiting significantly increased resistance (2.2- to 42.4-fold).
- Resistant cells showed cross-resistance to cisplatin but remained sensitive to docetaxel, vinorelbine, 5-fluorouracil, and SN-38.
- Elevated expression of thymidylate synthase (TS) was observed in all PEM-resistant sublines.
- Increased dihydrofolate reductase (DHFR) expression was noted in resistant A549 sublines; GARFT expression did not correlate with resistance.
Conclusions:
- Thymidylate synthase (TS) expression is strongly associated with pemetrexed resistance in lung adenocarcinoma.
- PEM-resistant NSCLC cells retain sensitivity to alternative chemotherapeutic agents, including docetaxel, vinorelbine, 5-fluorouracil, and irinotecan.
- These findings suggest potential therapeutic strategies for patients who develop resistance to pemetrexed.
