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A rapid mechanism-based screen to detect potential anti-cancer agents
D S Swaffar1, C M Ireland, L R Barrows
1Division of Pharmacology and Toxicology, School of Pharmacy, Northeast Louisiana University, Monroe 71209.
Abstract:
Several mutant Chinese hamster ovary (CHO) cell lines have been adapted to the microtiter tetrazolium assay in order to obtain useful mechanistic information relevant to the cytotoxic activity of marine natural products. The sensitivity of a DNA double-strand break repair deficient CHO line, xrs-6, was compared with that of a DNA repair competent CHO line, BR1, to several known drugs. The deficiency of the xrs-6 cells makes them overly sensitive to compounds [e.g. topoisomerase II (topo II) inhibitors] that produce DNA double-strand breaks. Described here is the validation of this unique cellular screen to detect such compounds. Those drugs thought to produce their effects by the inhibition of topo II, produced the largest differential cytotoxicity against the mutant CHO pair. Other agents that are known to either produce single-strand breaks, cross-links or to inhibit the synthesis of DNA did not possess appreciably enhanced cytotoxicity to the xrs-6 line. The usefulness of the screen was shown by its ability to detect topo II inhibitory activity in several new marine natural products. This activity was confirmed by an in vitro enzyme inhibition assay. In contrast, the screen predicted a lack of topo II inhibitory activity in some other structurally related marine natural products and this lack of activity was confirmed by an in vitro enzyme inhibition assay.
Insights
This study validates a new cell-based screen using Chinese hamster ovary (CHO) cells to identify topoisomerase II (topo II) inhibitors. The screen effectively detected topo II activity in marine natural products, confirming its utility for discovering cytotoxic compounds.
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Natural Product Chemistry
Background:
- Marine natural products are a rich source of potential therapeutic agents.
- Understanding the mechanism of action of cytotoxic compounds is crucial for drug development.
- Chinese hamster ovary (CHO) cell lines offer a valuable model for genotoxicity studies.
Purpose of the Study:
- To validate a microtiter tetrazolium assay using mutant Chinese hamster ovary (CHO) cell lines for mechanistic insights into cytotoxic activity.
- To establish a cellular screen for identifying topoisomerase II (topo II) inhibitors among marine natural products.
- To compare the sensitivity of DNA repair-deficient (xrs-6) and DNA repair-competent (BR1) CHO cells to cytotoxic agents.
Main Methods:
- Adaptation of mutant Chinese hamster ovary (CHO) cell lines (xrs-6 and BR1) to the microtiter tetrazolium assay.
- Comparative cytotoxicity testing of known drugs and novel marine natural products against xrs-6 and BR1 cells.
- In vitro enzyme inhibition assays to confirm topoisomerase II (topo II) inhibitory activity.
Main Results:
- The DNA double-strand break repair deficient CHO line (xrs-6) exhibited significantly higher sensitivity to topoisomerase II (topo II) inhibitors compared to the repair-competent line (BR1).
- The cellular screen successfully identified topoisomerase II (topo II) inhibitory activity in several new marine natural products.
- Agents causing single-strand breaks, DNA cross-links, or inhibiting DNA synthesis did not show enhanced cytotoxicity in the xrs-6 cells.
Conclusions:
- The developed cellular screen is a validated and effective tool for detecting topoisomerase II (topo II) inhibitory activity in marine natural products.
- This assay provides valuable mechanistic information for identifying cytotoxic compounds with potential therapeutic applications.
- The differential sensitivity of DNA repair-deficient CHO cells can be leveraged to discover specific classes of cytotoxic agents.