Piperlongumine (PL), a small molecule
alkaloid present in black pepper (Piper longum), has been reported to kill
tumor cells irrespective of their p53 gene status, however, the mechanisms involved are unknown. Since p53 is a redox-sensitive
protein, we hypothesized that the redox imbalance induced by PL may affect the structure and/or function of the mutant p53
protein and promote cell death. We used two human
colon cancer cell lines, the HT29 and SW620 which harbor the R273H
DNA contact abrogatory mutation in p53. PL treatment induced significant ROS production and
protein glutathionylation with a concomitant increase in Nrf-2 expression in both cell lines. Surprisingly, immunoprecipitation with wt-p53 specific
antibodies (PAb1620) or direct western blotting showed a progressive generation of wild-type-like p53
protein along with a loss of its mutant counterpart in PL-treated HT29 and SW620 cells. Moreover, the EMSA and
DNA-affinity blotting revealed a time-dependent restoration of
DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes, MDM2 and Bax. PL, while cytotoxic by itself, also increased the cell killing by many anticancer drugs. In nude mice bearing the HT29
tumors, PL alone (7.5 mg/kg daily) produced a 40% decrease in
tumor volume, which was accompanied by diminished intratumoral mutant p53
protein levels. The antitumor efficacy of
BCNU or
doxorubicin in HT29 xenografts was highly potentiated by PL, followed by expression of apoptotic
proteins. These clinically-relevant findings suggest that PL-induced oxidative milieu facilitates a weak functional restoration of mutant p53 through
protein glutathionylation and contributes to the increased drug sensitivity.