Procaspase-3 activation as an anti-cancer strategy: structure-activity relationship of procaspase-activating compound 1 (PAC-1) and its cellular co-localization with caspase-3

Abstract

A goal of personalized medicine as applied to oncology is to identify compounds that exploit a defined molecular defect in a cancerous cell. A compound called procaspase-activating compound 1 (PAC-1) was reported that enhances the activity of procaspase-3 in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Experimental evidence indicates that PAC-1 activates procaspase-3 in vitro through chelation of inhibitory zinc ions. Described herein is the synthesis and biological activity of a family of PAC-1 derivatives where key functional groups have been systematically altered. Analysis of these compounds reveals a strong correlation between the in vitro procaspase-3 activating effect and their ability to induce death in cancer cells in culture. Importantly, we also show that a fluorescently labeled version of PAC-1 co-localizes with sites of caspase-3 activity in cancer cells. The data presented herein further bolster the hypothesis that PAC-1 induces apoptosis in cancer cells through the direct activation of procaspase-3, has implications for the design and discovery of next-generation procaspase-3 activating compounds, and sheds light on the anti-apoptotic role of cellular zinc.

Figure 1

PAC-1, and four classes of PAC-1 derivatives. Class I compounds (1a-k) have modifications on the phenolic ring, Class II compounds(2a-g) have modification on the benzyl ring, Class III compounds (3) have modifications on both the phenolic and benzyl rings, and Class IV compounds (4a-g) have modifications to the key heteroatoms and bonds indicated in red.

Figure 2

2h (pseudo-colored green) localizes to the cytoplasm in SK-MEL-5 cells and gives punctate staining similar to other fluorescent zinc chelators (see text). DNA was stained with SYBR green and pseudo-colored blue.

Figure 3

(Top) Activity of caspase-3 in vehicle-treated and PAC-1 treated SK-MEL-5 cells. Cells were treated with either DMSO or PAC-1 (25 μM), and then treated with FAM-DEVD-fmk (25 μM), a caspase-3/-7 inhibitor that will covalently append a fluorophore to enzymes that possess caspase-3/-7 activity. (Bottom) Visualization of the entire procaspase-3/caspase-3 population of SK-MEL-5 cells using an antibody to caspase-3.

Figure 4

Colocalization of 2h with sites of caspase-3/-7 enzymatic activity. SK-MEL-5 cells treated with DMSO show background levels of staining in both the red and green channels (A–C). Cells treated only with FAM-DEVD-fmk do not show any staining above background levels (D–F). Cells treated only with 2h shows punctate staining in the cytoplasm with no increase in the emission at 400 nm (G–I). Cells treated concurrently with 2h and FAM-DEVD-fmk show spots of intense caspase-3/-7 activity (pseudo-colored red) and punctate staining of 2h (pseudo-colored green), which exhibit good co-localization in the merged image (J–L).

Figure 5

Assessment of apoptotic induction by PAC-1 derivatives. U-937 cells were treated with PAC-1, 2d, and 4c (50 μM each) or vehicle control for 12 hours, then stained with propidium iodide (PI) and FITC-labeled Annexin V. All three compounds induce apoptosis as assessed by the Annexin V positive/PI negative populations. Results shown are representative of three replicate experiments with each compound.

Scheme 1

The condensation of hydrazides and aldehydes to create PAC-1 derivatives of Class I-III.

Scheme 2

The condensation of hydrazide 5 and aldehyde 20 provides 1g and its dimer, 1i.

Scheme 3

Syntheses of Class IV derivatives.

Scheme 4

The synthesis of 2h, a fluorescent version of PAC-1.