Combination therapy including antisense oligonucleotides (ODNs) with traditional chemotherapeutic agents offers potential benefits by increasing the effectiveness of the chemotherapeutics, reducing their effective dosage, and simultaneously reducing toxicity. Previously we have reported that antisense ODNs specific for transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR) (MR1 and MR2, respectively), are effective against the PC-3 in vitro and in vivo prostate cancer models. In this series we evaluated these antisense ODNs in various combinations and treatment cycles with paclitaxel (Taxol), cyclophosphamide (Cytoxan), mitoxantrone, carboplatin, cisplatin, and oxaliplatin in order to identify synergistic effects.We found that when either of the ODNs were simultaneously administered with Taxol, no synergistic activity was noted. However, when sequentially administered in a series 1 d apart, a pretreatment with the ODN directed against TGF-alpha (6.64 microm) followed by Taxol (5 nm) had significantly (p <0.001) greater activity than these agents similarly administered in the reverse order or simultaneously. When Cytoxan was administered in sequence with both ODNs significantly increased growth inhibition was obtained compared to when Cytoxan was administered alone. A 1 d treatment of PC-3 cells with Cytoxan followed the next day with MR1 was significantly more effective (p <0.0001). The reverse order, a pretreatment with MR1 followed by Cytoxan, also resulted in significant additional inhibition (p=0.0004). Similarly sequenced, MR2 followed by Cytoxan, was also significantly more effective (p=0.0014) than Cytoxan treatment alone. For mitoxantrone, which was administered in combination therapy with ODNs: mitoxantrone with MR1 was significantly more inhibitory than the combination of both MR1 and MR2 ODNs (p=0.006) and also mitoxantrone administered alone (p=0.0012). Mitoxantrone administered with MR1 was not significantly different from mitoxantrone given in combination with MR2. Although mitoxantrone and MR2 was statistically (p=00015) more inhibitory than mitoxantrone alone, this combination was barely more effective (p=0.04) than the MR1 ODN administered alone.