Doxorubicin is one of the most important anticancer agents. However, the therapeutic index of doxorubicin is compromised by organ toxicity and resistance of tumor cells. Cellular resistance to doxorubicin is associated with altered expression of topoisomerase II, changes in glutathione levels or overexpression of P-glycoprotein. The major limiting factor of doxorubicin therapy is its cardiotoxicity involving acute myocardial injury and chronic cardiomyopathy. The molecular basis of doxorubicin-induced cardiomyopathy is not clarified. Promising approaches for the generation of expression profiles are cDNA microarrays and 2D gel electrophoresis with the benefit of rapid evaluation of large numbers of genes and proteins, respectively. Therefore, we have analyzed the influence of doxorubicin in the cervical carcinoma cell line HeLa using cDNA microarrays and 2D gel electrophoresis. Analysis of the cDNA microarray revealed that doxorubicin regulates genes of various different biological functions. Among these were genes encoding for numerous enzymes, proteins for cell structure and DNA replication, cell adhesion molecules as well as proteins with cardiovascular effects and immunomodulatory activity. In order to correlate these results with protein expression 2D gel electrophoresis has been performed. Cells treated with doxorubicin or DMSO were lysed and total protein extracts were focused isoelectrically within a 4 to 7 pH range. In a second step the focused proteins were separated by SDS-PAGE and silver stained. The resulting gels form three separate experiments as well as three different cell preparations have been analysed for consistent changes in the protein pattern using the Delta2D software (DECODON GmbH, Greifswald, Germany). Finally, a 2D DIGE experiment has been performed. Consistently regulated proteins were excised from the gel, digested and analyzed by matrix-assisted desorption and ionisation (MALDI) - time of flight (TOF) mass spectrometry. Among the about 1,200 separated protein spots 31 proteins were induced and 7 proteins repressed by doxorubicin including interferon-induced protein with tetratricopeptide repeats 4, vimentin, lamin, or N-myc-interactor. Several of these proteins were also identified by cDNA microarray analysis. In summary, our data demonstrate that the applied methods for detection of differences in gene and protein expression are useful for obtaining new insights into the molecular mechanisms of action of anticancer agents such as doxorubicin.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]