Epigenetic alterations such as aberrant DNA methylation and histone modifications contribute substantially to both the cause and maintenance of drug resistance. These epigenetic changes lead to silencing of tumor suppressor genes involved in key DNA damage-response pathways, making drug-resistant cancer cells nonresponsive to conventional anticancer drug therapies. Our hypothesis is that treating drug-resistant cells with epigenetic drugs could restore the sensitivity to anticancer drugs by reactivating previously silenced genes. To test our hypothesis, we used drug-resistant breast cancer cells (MCF-7/ADR) and two epigenetic drugs that act via different mechanisms--5-aza-2'-deoxycytidine (decitabine, DAC), a demethylating agent, and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor--in combination with doxorubicin. We show that the sequential treatment of resistant cells, first with an epigenetic drug (DAC), and then with doxorubicin, induces a highly synergistic effect, thus reducing the IC(50) of doxorubicin by several thousand fold. The sequential treatment caused over 90% resistant cells to undergo G2/M cell cycle arrest, determined to be due to upregulation of p21(WAF1/CIP1) expression, which is responsible for cell-cycle regulation. The induction of p21(WAF1/CIP1) correlated well with the depletion of DNA methyltransferase1 (DNMT1), an enzyme that promotes methylation of DNA, suggesting that the p21(WAF1/CIP1) gene may have been methylated and hence is inactive in MCF-7/ADR cells. Microarray analysis shows expression of several tumor suppressor genes and downregulation of tumor promoter genes, particularly in sequentially treated resistant cells. Sequential treatment was found to be significantly more effective than simultaneous treatment, and DAC was more effective than SAHA in overcoming doxorubicin resistance. Synergistic effect with sequential treatment was also seen in drug-sensitive breast cancer cells, but the effect was significantly more pronounced in resistant cells. In conclusion, the sequential treatment of an epigenetic drug in combination with doxorubicin induces a highly synergistic effect that overcomes doxorubicin resistance in breast cancer cells.