The Triterpenoids 2-cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO) and Their Imidazole (CDDO-Im) and Dinitrile Derivatives (DI-CDDO) Elicit Apoptosis through a Novel Mitochondrial Pathway.
Authors: Brookes Paul , Tompkins Andrew , Morse Kimberly , Hilchey Shannon , Salim Suhail , Ray Denise , Phipps Richard , Bernstein Steven H. 
Published: April 2020

We have recently shown that B-cell non Hodgkin’s lymphoma express the transcription factor PPARγ and undergo apoptosis upon exposure to PPARγ ligands. The synthetic triterpenoid CDDO is a specific ligand for PPARγ, and CDDO and its derivatives, CDDO-Im and DI-CDDO, induce diffuse large cell lymphoma (DLCL) death (OCI Ly10 and OCI Ly19 cells), with a potency of DI-CDDO&gt;CDDO-Im&gt;CDDO, suggesting that such agents have therapeutic potential in lymphoma. The natural PPARγ ligand, 15d-PGJ2 (which also elicits DLCL death), has previously been shown to inhibit mitochondrial complex I, enhance mitochondrial reactive oxygen species (ROS) generation, and react with protein thiols. Given that CDDO is structurally similar to 15d-PGJ2 we hypothesized that CDDO-induced cell death may similarly be mediated via complex I inhibition, ROS generation, thiol oxidation, and opening of a large membrane pore complex in the mitochondrial membrane, termed the “permeability transition” (PT) pore. Studies on isolated rat liver mitochondria however showed that none of the CDDO-derivatives inhibited complex I activity or affected mitochondrial protein thiols. However, all three compounds did induce PT pore opening and mitochondrial swelling, with a concurrent loss of mitochondrial membrane potential, in a Ca2+ dependent manner (potency DI-CDDO&gt;CDDO-Im&gt;CDDO). This is consistent with a previously shown role for Ca2+ in CDDO-induced cell death. Interestingly, this mitochondrial swelling was not inhibited by the classical PT pore inhibitor cyclosporin A (CsA). This is supported by our findings that the induction of OCI-Ly19 cell death by CDDO was also not inhibited by CsA, or by another classical PT pore inhibitor, nortriptyline. These phenomena may be partially explained by invoking the “unregulated PT pore”. In addition to the classical PT pore, a non-CsA sensitive “unregulated PT pore” also exists, which is generated by the aggregation of misfolded mitochondrial membrane proteins that are induced by oxidants and thiol reactive agents. That exposure of mitochondria to CDDO results in the formation of “unregulated PT pores” is supported by our findings that the proteosome inhibitor PS341, potentiates CDDO-induced cell death, suggesting the involvement of a protein folding response. The temporal role of ROS in CDDO-induced cell death was also investigated, and it was found that the antioxidant N-acetyl-cysteine did not inhibit PT pore opening, but did inhibit cell death. This is consistent with our observation that ROS generation in isolated mitochondria was not immediately triggered by CDDO, but rather increased at delayed time points, placing it downstream of PT pore opening. This proposes the following novel model of a direct mitochondrial effect of CDDO and its derivatives:

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[CDDO{\rightarrow}\ mitochondrial\ protein\ misfolding\ {\rightarrow}\ unregulated\ PT\ pore\ formation\ {\rightarrow}\ ROS\ {\rightarrow}\ cell\ death\] \end{document}

In summary:

CDDO and its derivatives have direct effects on mitochondria, and represent novel therapeutic approaches for the treatment of patients with DLCL; and combinations of CDDO and its derivatives with proteosome inhibitors represent a rational combination to test in the context of clinical trials.