σ Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-independent apoptosis, and to down-regulate P-glycoprotein (P-gp) mRNA levels in some mouse and human models. In this study, we verified whether a mixed σ₂ agonist/σ₁ antagonist, PB28, was able to have antitumor activity and to enhance anthracycline efficacy in two human breast cancer cell lines, MCF7 and MCF7 ADR, both characterized by significant σ₂ receptor expression, by high and low σ₁ receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high σ₂ receptor affinity and low σ₁ receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC₅₀ in nanomolar range), a consistent time exposure-independent increase of G₀-G₁-phase fraction (of ∼20% of both cell lines) and caspase-independent apoptosis (15% increased after 1-day drug exposure). PB28 also reduced P-gp expression in a concentration- and time-dependent manner (∼60% in MCF7 and 90% in MCF7 ADR). We showed also a strong synergism between PB28 and doxorubicin by adopting either simultaneous or sequential schedules of the two drugs. We suggest that this synergism could depend on PB28-induced increase of intracellular accumulation of doxorubicin (∼50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the σ₂ agonist PB28 could be an interesting antitumor agent either in monotherapy or in combination with conventional drugs. [Mol Cancer Ther 2006;5(7):1807–16]