A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation

Abstract

We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway.

Keywords: BCL-2 family members; apoptosis; cancer; mitochondria; prohibitins.

Figure 1. The presence of PHBs is required for fluorizoline-induced apoptosis

a. Chemical structure of fluorizoline (diaryl trifluorothiazoline compound 1a). b., c. Cre recombinase was transduced in WT and Phb2^fl/fl (Phb2^−/−) MEFs for 72 h. Then cells were untreated (UT) or treated with either 0.15 μg/mL Actinomycin D (ActD) or increasing doses of fluorizoline (F) for 24 h. d., e. Phb2^fl/fl MEFs were transfected with scramble (SC) or Phb2 (siPhb2) siRNA for 72 h. Afterwards, cells were treated with either 0.15 μg/mL Actinomycin D (ActD) or increasing doses of fluorizoline (F) for 24 h. b., d. Viability was measured by flow cytometry and it is expressed as the mean ± SEM (n ≥ 3) of the percentage of non-apoptotic cells (annexin V-negative). *p < 0.05, **p < 0.01, ***p < 0.001. c., e. Protein levels were analyzed by western blot. Tubulin and β-Actin were used as a loading control. These are representative images of at least three independent experiments.

Figure 2. Treatment with fluorizoline induces changes in the mitochondrial morphology and ultrastructure

a. HeLa cells were treated with DMSO (vehicle) or 10 μM fluorizoline for 4 h. Mitochondria were stained with 100 nM MitoTracker^® Red CMXRos and imaged using a confocal microscope. b. HeLa cells were incubated with DMSO (vehicle) or 2 μM fluorizoline for 24 h and changes in the mitochondrial morphology were visualized by transmission electron microscopy. Magnification at 6,000X (scale corresponding to 5 μm), 20,000X (scale corresponding to 1 μm), and 60,000X (scale corresponding to 0.5 μm). Arrowheads indicate the mitochondria.

Figure 3. Fluorizoline induces ROS production independently of PHBs and apoptosis induction

a. Jurkat cells were untreated (UT), or treated with 10 μM fluorizoline (F) for 1 h or 200 μM tert-butyl hydroperoxide (TBHP) for 30 min. b. Jurkat cells were untreated (CT) or pre-incubated with 150 μM MnTBAP for 1 h and then treated with DMSO (D) or 10 μM fluorizoline (F) for 1 h. c. Jurkat cells were untreated (CT) or pre-incubated with 150 μM MnTBAP for 1 h and then treated with DMSO (D) or 10 μM fluorizoline (F) for 24 h. Viability was measured by flow cytometry and it is expressed as the mean ± SEM (n = 3) of the percentage of non-apoptotic cells (annexin V-negative). d. Phb2^fl/fl MEFs were transfected with scramble (SC) or Phb2 (siPhb2) siRNA for 72 h. Cells were then untreated or treated with 20 μM fluorizoline (F) for 1 hour. a., b., d. Superoxide anion and hydroxyl radical production was analyzed by flow cytometry using CellROX^® Deep Red Reagent. Data show the mean values ± SEM relative to the mean of the control (a, n = 9; b, n = 3; d, n = 4). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Fluorizoline-induced apoptosis occurs through the mitochondrial pathway in a BAX- and BAK-dependent manner

a. WT, Bax^−/−, Bak^−/− or Bax^−/−/Bak^−/− MEFs were untreated (UT) or treated with 10 or 20 μM fluorizoline for 24 h. b., c. HeLa cells were transfected with scramble (SC) or BAX and BAK siRNA (siBAX/BAK) for 48 h. b. The efficiency of gene silencing was validated by western blot. β-Actin was used as a loading control. This is a representative image of at least three independent experiments. c. HeLa cells were untreated (UT) or treated with 5 or 10 μM fluorizoline (F) for 24 h. a., c. Viability was measured by flow cytometry and it is expressed as the mean ± SEM (n≥3) of the percentage of non-apoptotic cells (annexin V-negative).*p < 0.05, **p < 0.01.

Figure 5. Fluorizoline modulates the expression of BCL-2 family members by targeting PHBs in MEFs

a. Phb2^fl/fl MEFs were untreated (white bars) or treated with 20 μM fluorizoline (black bars) for 24 h. b. Phb2^fl/fl MEFs were transduced with Cre recombinase for 72 h (Phb2^−/−) and then untreated or treated with 20 μM fluorizoline for 24 h. a., b. mRNA levels were analyzed by RT-MLPA. White bars correspond to untreated cells, and black bars to fluorizoline-treated cells. Data show the mean values ± SEM of three independent experiments relative to the mean of the control. *p < 0.05, **p < 0.01, ***p < 0.001 untreated versus treated cells.

Figure 6. Fluorizoline changes mRNA and protein levels of various BCL-2 family members in HeLa cells

a. HeLa cells were untreated (white bars) or treated with 10 μM fluorizoline (black bars) for 24 h. mRNA levels were analyzed by RT-MLPA. Data show the mean values ± SEM of three independent experiments relative to the mean of the control. *p < 0.05, **p < 0.01, ***p < 0.001 untreated versus treated cells. b. HeLa cells were untreated or treated with 10 μM fluorizoline for 4, 8 and 24 h. c. HeLa cells were pre-incubated with 20 μM caspase inhibitor Q-VD-OPh for 30 min and then treated with 10 μM fluorizoline for 24 h. b., c. Protein levels were analyzed by western blot. Tubulin was used as a loading control. These are representative images of at least three independent experiments.

Figure 7. Loss of NOXA and BIM blocks fluorizoline-induced apoptosis

a. WT, Noxa^−/−, Bim^−/− or Noxa^−/−/Bim^−/− MEFs were untreated (UT) or treated with 10, 15 or 20 μM of fluorizoline for 24 h. b., c. HeLa cells were transfected with scramble (SC), NOXA siRNA (siNOXA), BIM siRNA (siBIM), or both NOXA and BIM siRNA (siNOXA/BIM) for 48 h and then untreated (UT) or treated with 5 or 10 μM fluorizoline (F) for 24 h. b. Protein levels were analyzed by western blot. Tubulin was used as a loading control. This is a representative image of at least three independent experiments. a., c. Viability was measured by flow cytometry and it is expressed as the mean ± SEM (n ≥ 3) of the percentage of non-apoptotic cells (annexin V-negative).*p < 0.05, **p < 0.01, ***p < 0.001.