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In Vitro schedule-dependent interactions between the multitargeted antifolate LY231514 and gemcitabine in human colon
Anna Tesei1, Luca Ricotti, Franca De Paola
1Istituto Oncologico Romagnolo, Forlì, Italy.
Purpose:
Multitargeted antifolate (MTA) and gemcitabine (GEM) have shown preclinical and clinical activity in tumor histotypes such as colon, renal, small and non-small cell lung cancers, hepatomas and carcinoid tumors. In our study, we investigated the cytotoxic activity of MTA alone or in combination with GEM using different exposure schedules in three different colon cancer cell lines (LoVo, WiDr, and LRWZ).
Experimental Design:
Cytotoxic activity was evaluated by sulforhodamine B assay, cell cycle perturbations and apoptosis were evaluated by flow cytometry, and thymidylate synthase expression was evaluated by immunohistochemical method.
Results:
A 48-h exposure to MTA caused a minimal and no-dose-response effect on the three cell lines used. Flow cytometric analysis showed a cell accumulation in S phase that completely resolved in LoVo and LRWZ cell lines and persisted in WiDr cells after a 48-h washout. Moreover, a significant increase in thymidilate synthase expression was observed in all of the cell lines after MTA exposure. Among the different combinations tested, the highest synergistic interaction, assessed using Kern's method and expressed as the synergistic ratio index, was produced by pretreatment with GEM followed by MTA (ratio index: 1.3- 6.7). It is possible that the depletion of nucleotide pools induced by MTA and required for DNA synthesis prevented cells from repairing DNA damage caused by GEM. The type and degree of drug interactions were not paralleled by apoptosis, which was almost always negligible, or by the type and persistency of the cell cycle perturbations.
Conclusions:
Our results indicate that the sequential administration of GEM --> MTA provides the greatest benefit in the clinical treatment of colon cancer.
Insights
Gemcitabine followed by multitargeted antifolate (GEM --> MTA) shows the greatest synergistic effect in colon cancer cell lines. This sequential administration is recommended for improved clinical treatment strategies.
Area of Science:
- Oncology
- Cancer Cell Biology
- Pharmacology
Background:
- Multitargeted antifolate (MTA) and gemcitabine (GEM) exhibit preclinical and clinical activity in various cancers.
- Colon cancer remains a significant health concern, necessitating novel therapeutic strategies.
Purpose of the Study:
- To investigate the cytotoxic activity of MTA alone and in combination with GEM.
- To evaluate different exposure schedules of MTA and GEM in colon cancer cell lines.
Main Methods:
- Sulforhodamine B assay for cytotoxic activity.
- Flow cytometry for cell cycle and apoptosis analysis.
- Immunohistochemistry for thymidylate synthase expression.
Main Results:
- MTA alone showed minimal dose-response effects but increased thymidylate synthase expression.
- GEM followed by MTA (GEM --> MTA) demonstrated the highest synergistic interaction.
- Cell cycle perturbations were observed, but apoptosis was generally negligible.
Conclusions:
- Sequential administration of GEM --> MTA offers the greatest benefit for colon cancer treatment.
- This combination strategy warrants further clinical investigation for colon cancer patients.
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