The Triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid and Its Derivatives Inhibit Mitochondrial Lon Protease Activity: A Potential Novel Pathway for the Induction of Lymphoma Cell Death.

Abstract 414

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its C(28) imidazole and methylester derivatives are novel oleanane triterpenoids exhibiting promise as both therapeutic and preventative agents for cancer. We have previously shown that these synthetic triterpenoids promote lymphoma cell death through a novel mitochondria-mediated mechanism whereby mitochondrial protein thiols are modified resulting in the formation of mitochondrial protein aggregates leading to apoptotic cell death (Cancer Res. 2007; 67:4). As mitochondrial Lon is a critical quality control protease that prevents the toxic accumulation and aggregation of damaged mitochondrial proteins we hypothesized that one effect of the triterpenoids might be to inhibit Lon-mediated proteolysis. Herein we show that CDDO and its derivatives inhibit purified Lon protease. In particular, the imidazole and methylester derivatives of CDDO (CDDO-Im and CDDO-Me) are more effective inhibitors of Lon than CDDO. Interestingly, these derivatives are also more potent inducers of lymphoma cell death than CDDO. We also show that CDDO-Me inhibits cellular mitochondrial Lon, as determined by its ability to inhibit the degradation of the Mitochondrial Transcription Factor A (TFAM), an endogenous Lon protein substrate. To exclude the possibility that the triterpenoid mediated inhibition of TFAM degradation is mediated by the cytoplasmic proteasome, we show that the triterpenoids have no effect on proteasome-mediated proteolysis. To determine the mechanism through which the triterpenoids block Lon activity, we next used a biotinylated-CDDO derivative and showed that this derivative forms adducts with Lon, suggesting that the triterpenoids directly inhibit the protease in cells. In contrast, the biotinylated-CDDO derivative did not form adducts with other proteins, such as the mitochondrial ClpP protease, TFAM, or the endoplasmic reticulum protein BiP, suggesting a level of specificity for triterpenoid adduct formation. Indeed, the formation of protein adducts with Lon is consistent with the fact that the triterpenoids contain a Michael Addition moiety which allows it to form adducts with both protein and non-protein thiols. Consistent with the inhibitory effects of the triterpenoids on mitochondrial Lon protease activity, CDDO-treated lymphoma cells exhibit striking electron dense inclusions within mitochondria, but not the cytosol or other cellular compartments. Similar inclusions within mitochondria are also seen in a Lon knockdown cell line, in which a short hairpin RNA targeting Lon mRNA is inducibly expressed in LS174 colon cancer cells upon treatment with doxycycline. This finding supports the notion that the CDDO-induced inclusions are due, in part, to inhibition of Lon protease activity. Furthermore, we show that Lon protein expression levels are higher in both malignant lymphoma cell lines and patient derived biopsy specimens, as compared to that seen in resting or activated normal B cells. Taken together, these results suggest that; (a) the mitochondrial ATP-dependent Lon protease is a new target of synthetic triterpenoids, and; (b) Lon may be a novel and viable target for cancer therapeutics. Supported by an NCI SPORE grant in lymphoma 1P50 CA130805.