Guaiazulene derivative 1,2,3,4-tetrahydroazuleno[1,2-b] tropone reduces the production of ATP by inhibiting electron transfer complex II

Abstract

Molecularly targeted therapy has been used for treatment of various types of cancer. However, cancer cells often acquire resistance to molecularly targeted drugs that inhibit specific molecular abnormalities, such as constitutive activation of kinases. Even in cancer cells that have acquired resistance, enhanced anabolism, including the synthesis of nucleotides, amino acids and lipids, is common to normal cancer cells. Therefore, there is a renewed interest in effectively eliminating cancer cells by specifically targeting their abnormal energy metabolism. Multiple strategies are currently being developed for mitochondrial-targeted cancer therapy, with agents targeting oxidative phosphorylation, glycolysis, the tricarboxylic acid cycle and apoptosis. In this study, we found that one of the guaiazulene derivatives, namely, 1,2,3,4-tetrahydroazuleno[1,2-b] tropone (TAT), inhibited the proliferation of cancer cell lines stronger than that of normal cells. In addition, we showed that TAT inhibited energy production in cancer cell lines, resulting in apoptosis. Analyses done in cancer cell lines and in the animal model Caenorhabditis elegans suggested that TAT acts on the mitochondrial electron transfer complex II and suppresses cellular energy production by inhibiting oxidative phosphorylation across species. These results suggest that TAT could represent a novel anticancer agent that selectively targets mitochondria.

Keywords: C . elegans; OXPHOS; apoptosis; cancer; metabolism; mitochondria.

Fig. 1

Antioxidant activity and cell viability of TAT‐treated cells. (A) Structure of TAT. (B) FRAP assay to determine the antioxidant capacity of TAT and the positive controls guaiazulene and Trolox. (C–E) WST‐8 assay viability test of normal TIG‐1 fibroblasts, HeLa cancer cells and immortalized 293T cell lines, treated with or without 25 μm TAT. Means ± standard deviation are shown (n = 3; *P < 0.05, **P < 0.001, Student's t‐test).

Fig. 2

Increased cleavage of PARP and caspases in TAT‐treated cells. (A) Western blot of PARP and its cleavage products in HeLa cells treated with or without 25 μm TAT for each indicated time. Staurosporine was used as the positive control and tubulin as the loading control. (B) Western blots of caspase‐7 and caspase‐9 cleavage products after treatment of HeLa cells with 25 or 50 μm TAT for 48 h.

Fig. 3

TAT leads to decreased intracellular metabolism and ATP production. (A) Viable cells were measured using a Hoechst 33342 after 48 h at 25 μm TAT‐treated HeLa cells. (B–D) Cell metabolic activity (B, C) and ATP levels (D) of HeLa cells treated with 25 μm TAT were measured after 48 h and simultaneously normalized by the number of viable cells measured by the Hoechst 33342. WST‐8 measures intracellular dehydrogenase activity, and MTT measures mitochondrial reductase activity. (E) Measurement of intracellular ATP as an indicator in the C. elegans FeIS4 strain, which contains a luciferase transgene, after treatment with varying levels of TAT. Means ± standard deviation are shown (n = 3; *P < 0.05, **P < 0.001, Student's t‐test).

Fig. 4

Depolarization of mitochondrial membrane potential by TAT. (A) Mitochondrial membrane potential (ΔψM) was examined in HeLa cells with and without 25 μm TAT treatment at each indicated time using a cationic JC‐1 dye and confocal fluorescence microscopy. Apoptotic or unhealthy cells with a low ΔψM remained in the monomeric form as evidenced by the green fluorescence. (B) After incubation with or without 25 μm TAT for 24 h, the glucose analog 2‐NBDG (400 µm) was introduced for 30 min and uptake examined using confocal fluorescence microscopy. Fluorescence intensity was quantified using ImageJ (developed by Wayne Rasband in NIH, USA).

Fig. 5

Involvement of TAT in mitochondrial ROS production. (A) HeLa cells treated with or without 25 μm TAT were stained with a reduced form of MitoTracker Orange, CMH₂TMRos, to detect intracellular ROS using fluorescence microscopy. (B) C. elegans carrying the gfp‐tagged hsp‐6 transgene, which encodes a protein whose expression is up‐regulated in the UPR^mit, was treated with or without 50 μm TAT for 24 h, and the GFP expression levels were compared by western blotting. Tubulin represents the loading control. (C) 50 μm TAT treatment slightly, but significantly extended the average lifespan of wild‐type N2 C. elegans over that of control, untreated worms. (D) 50 μm TAT treatment significantly shortened the average lifespan of oxidative stress‐sensitive sek‐1 MAPK kinase mutant worms compared with that of controls (n = 3; *P < 0.1, **P < 0.05, Student's t‐test).

Fig. 6

TAT acts on electron transfer complex II. (A) TAT treatment reduced fumaric and malic acid levels in HeLa cells, while succinic acid was unchanged relative to that of untreated controls (n = 4; **P < 0.05, Student's t‐test). (B) The lifespan of the typically short‐lived and oxygen‐hypersensitive C. elegans mutant, mev‐1, which encodes the SDH subunit of electron transfer complex II, was measured after 50 μm TAT treatment and the average compared with that of control, untreated mev‐1 worms (n = 3; P = 0.99, Student's t‐test). (C) TAT cooperates with the electron transfer complex I inhibitor, rotenone, to inhibit cellular energy metabolism. HeLa cells were treated with or without TAT (25 μm) and rotenone (0.25 μm) for 48 h, and then ATP levels, WST‐8 and lactate dehydrogenase were measured, respectively. (D) TAT inhibits ATP synthesis in isolated mitochondria in cooperation with rotenone. Isolated mitochondria were treated with or without TAT (25 μm) and rotenone (0.5 μm) at 37°C for 10 min in reaction buffer. Then ATP levels were measured by adding ATP luminescence reagent.