Most cancer cells acquire resistance to apoptosis-inducing drugs such as the widely-used drug doxorubicin. Brent Stockwell and colleagues used high-throughput cell-viability screening and a pathway-based analysis to find compounds that increase the lethality of doxorubicin in cancer cells that are resistant to it.

Two colon carcinoma cell lines were used for the screens. One cell line (RKO) expressed high levels of the DNA damage-response protein p53 and was sensitive to doxorubicin (a topoisomerase-II-based DNA-damaging agent). The other cell line (RKO-E6) expressed the viral oncoprotein E6, which induces p53 degradation, and was relatively resistant to doxorubicin. The authors reasoned that compounds that overcome E6-induced resistance should restore doxorubicin's lethality in RKO-E6 cells. Nearly 30,000 compounds were tested in the primary screen, and 278 of these were lethal. A secondary screen eliminated those compounds that did not depend on E6 and doxorubicin to induce cell death — 88 compounds remained.

So, what is the basis of the resensitization activity of these 88 compounds? Surprisingly, only one compound affected the expression level of p53, so the authors developed a pathway-based analysis using a co-treatment strategy to determine how the compounds functioned. In this analysis, compounds that increased the sensitivity of RKO-E6 cells to the microtubule inhibitor podophyllotoxin, the topoisomerase-I-based DNA-damaging agent camptothecin, and doxorubicin were assumed to act through general cell-death mechanisms and were eliminated. Compounds that increased sensitivity to both camptothecin and doxorubicin but not podophyllotoxin presumably act downstream of the DNA-damage response. Compounds that synergized only with doxorubicin were likely to operate at the level of, or upstream of, topoisomerase II proteins. These last two groups of compounds were studied in more detail.

The authors found several groups of compounds with the potential to restore doxorubicin lethality. These included quaternary ammonium compounds, protein-synthesis inhibitors, and a class of small molecules that included 1,3-bis(4-morpholinymethyl)-2-imidazolidinethione. The authors named another previously unexplored class of small molecules indoxins, for their ability to increase doxorubicin's lethality selectively. All of these classes of compounds upregulated topoisomerase IIα and/or induced S-phase arrest in RKO-E6 cells.

To further elucidate the mechanism of action of the indoxins, the authors synthesized a series of indoxin analogues and used indoxin affinity probes to identify their protein targets. One of the five proteins identified was myosin 1c. In keeping with its known functions, it is possible that nuclear myosin 1c mediates topoisomerase IIα transcription, whereas myosin 1c in the cytosol might mediate S-phase arrest.

Further investigation of these doxorubicin-enhancing small molecules for the adjuvant treatment of doxorubicin-resistant cancer is warranted.