A Bax-mediated mechanism for obatoclax-induced apoptosis of cholangiocarcinoma cells

Abstract

Apoptosis induction by BH3 mimetics is a therapeutic strategy for human cancer. These mimetics exert single-agent activity in cells "primed" for cell death. Primed cells are dependent upon antiapoptotic Bcl-2 proteins for survival and are characterized by the ability of the BH3 mimetic to induce cytochrome c release from their isolated mitochondria. Our aim was to examine the single-agent activity of obatoclax, a BH3 mimetic in cholangiocarcinoma cell lines. In clonogenic assays, inhibition of colony formation was observed by obatoclax treatment. Despite single-agent activity by obatoclax, the mitochondria from these cells did not release cytochrome c after incubation with this BH3 mimetic. However, immunofluorescence and cell fractionation studies identified Bax activation and translocation to mitochondria after treatment with obatoclax. shRNA targeted knockdown of Bax doubled the IC50 for obatoclax but did not abrogate its cytotoxicity, whereas knockdown of Bak did not alter the IC50. In a cell-free system, obatoclax induced an activating conformational change of Bax, which was attenuated by a site-directed mutagenesis of a previously identified protein activation site. Finally, the drug also elicited a significant in vivo response in a rodent model of this disease. In conclusion, single-agent obatoclax treatment results in Bax activation, which contributes, in part, to cell death in cholangiocarcinoma cells. These data indicate that BH3 mimetics may also function as direct activators of Bax and induce cytotoxicity in cells not otherwise primed for cell death.

Figure 1

Obatoclax has single agent activity against cholangiocarcinoma which is not associated with altered BCL-2 protein expression (A,B) Cell death was quantified by clonogenic assay (A) in four cholangiocarcinoma cell lines (HuCCT-1, Mz-ChA-1, BDEneu, KMCH-1) treated with obatoclax at doses ranging from 0 to 800 nM for 48 h. Results are colony forming units as a percentage of control and represent means from three separate trials. (B) Immunoblotting of three obatoclax-sensitive cholangiocarcinoma cell lines treated with vehicle or increasing doses of obatoclax (100nM-400nM) for 24 hours. (*) No commercially available antibody for rat Mcl-1.

Figure 2

Mitochondria from cholangiocarcinoma cells are not primed for death. Immunoblot for cytochrome c from supernatant and mitochondrial pellets indicating no release of cytochrome c with obatoclax treatment in isolated mitochondria. Mitochondria were incubated with tBid (20 nM) and lysed with detergent as positive controls.

Figure 3

Obatoclax treatment is associated with Bax activation, mitochondrial translocation, and membrane insertion. (A-D) Immunofluorescence for active Bax (red) (A) using a conformation specific antibody in HuCCT-1, Mz-ChA-1, and BDEneu cells treated with 400nM obatoclax for 24 hours demonstrated Bax activation in treated cells. Nuclei are stained with DAPI. (B) With obatoclax treatment of BDEneu cells, Bax was recruited to mitochondria. Normalized densitometric ratios are provided beneath the figure. (C) Bicarbonate treatment of mitochondria isolated from treated and untreated BDEneu cells demonstrated rapid membrane insertion of Bax with obatoclax treatment.

Figure 4

Obatoclax activation of Bax takes place through a direct mechanism, which is attenutated by site directed mutagenesis of the lysine 21 residue. Wild-type (A) recombinant human Bax or K21E mutant Bax was incubated with increasing doses of obatoclax. An increasing amount of active Bax was immunoprecipitated with obatoclax treatment in wild-type Bax, which was attenuated in the K21E mutant protein. Cross-linking of recombinant wild-type Bax protein (B) demonstrated increasing multimers with obatoclax treatment, which were not present in mutant protein. Normalized densitometric ratios are provided beneath the figure.

Figure 5

Targeted knockdown of Bak and Bax by shRNA demonstrated a Bax-dependent effect on cell death (A,B) shRNA specific for Bak and Bax were stably transfected into HuCCT-1 cells (A). shBak clones (1,3) and shBax clones (18,19) were subjected to increasing concentrations of obatoclax in clonogenic assays. Median dose-effect (D_m) was calculated using Calcusyn software and is plotted in a dose-effect plot (B), indicating an increase in D_m for the shBax clones, with unchanged D_m in the shBak clones as compared to controls. Results from the clonogenic assay were colony forming units as a percentage of control and included three separate trials for each clone.

Figure 6

Obatoclax has single agent activity in vivo. A significant decrease in tumor size was noted after intra-arterial injection of obatoclax for 5 days, *p<0.001 (A). Survival was improved in animals treated with obatoclax by intra-arterial injection (B), p=0.005.