An Underlying Mechanism of Dual Wnt Inhibition and AMPK Activation: Mitochondrial Uncouplers Masquerading as Wnt Inhibitors

Abstract

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target β-catenin in this pathway. We now report that several “Wnt inhibitors” that allegedly target β-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known “Wnt inhibitor”, FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)­benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.

Mitochondrial uncoupling and the disruption of the metabolic network in hepatocellular carcinoma

“…These sulfonamides altered mitochondrial respiration activity through increased proton transport from the inner membrane space to the mitochondrial matrix. This dissipation of the proton and associate charge gradient ∆ψm across the inner mitochondrial membrane reduced ATP production [20]. These effects on cell proliferation of the hepatoma cell lines were in agreement with a recent report in which the OXPHOS uncoupling activity of FH535 and Y3 were severely impaired after the substitution of the active hydrogen in the sulfonamide bond that was involved in the proton translocation with an N-methyl group in the sulfonamide bond ( Figure 1).…”

“…In cancer cells, OXPHOS is not completely coupled and a small number of protons flow directly into the mitochondrial matrix across the inner mitochondrial membrane instead of passing through the ATP synthase complex, reducing the efficiency of ATP production. This proton leak phenomenon is enhanced upon FH535 and Y3 treatments and is linked to the functional disruption of the ETC and the uncoupling activity of FH535 in HCC and embryonic kidney cells [18,20]. Our OCR results in HCC cells showed significant changes in the proton leak upon FH535 or Y3 treatments, but not in the presence of the methylated analogs.…”

“…We analyzed the mechanism of action of FH535 and Y3 by generating methylated analogs, FH535-Me and Y3-Me, and studied their related effects on metabolic processes. As we described in a recent study [20], FH535 possessed a hydrophobic substructure and an ionizable nitrogen-hydrogen bond that participated in proton translocation in the mitochondrial inner space. After replacing the "active" hydrogen with a methyl group, the methylated analogs, FH535-Me and Y3-Me, lost their activity as proton uncouplers.…”

“…The resulting pro-oxidative state promoted by FH535 and Y3 cause apoptotic cell death and contribute to prevent tumor progression. In a recent study we reported the FH535-induced activation of AMPK and ensuing Acetyl-CoA carboxylase (ACC) inactivation [20]. ACC catalyzes malonyl-CoA synthesis, an inhibitor of CPT1.…”

“…Another group studied how sorafenib, a drug commonly used in the treatment of advanced HCC, acts as a mitochondrial uncoupler at low doses [19]. We demonstrated that the ability of FH535 to transport protons from the mitochondrial intermembrane space into the mitochondrial matrix altered the transmembrane potential and disrupted ATP generation [20,21]. We now report a study of the functional link between the uncoupler activities of FH535 and a new analog of FH535, 2,5-dichloro-N-(4-nitronaphthalen-1yl) benzenesulfonamide (Y3) and the methylated analogs (FH535-me) and Y3 (Y3-me) on HCC activity.…”