Differential effects of procaspase-3 activating compounds in the induction of cancer cell death

Abstract

The evasion of apoptosis is a key characteristic of cancer, and thus strategies to selectively induce apoptosis in cancer cells hold considerable promise in personalized anticancer therapy. Structurally similar procaspase activating compounds PAC-1 and S-PAC-1 restore procaspase-3 activity through the chelation of inhibitory zinc ions in vitro, induce apoptotic death of cancer cells in culture, and reduce tumor burden in vivo. Ip or iv administrations of high doses of PAC-1 are transiently neurotoxic in vivo, while S-PAC-1 is safe even at very high doses and has been evaluated in a phase I clinical trial of pet dogs with spontaneously occurring lymphoma. Here we show that PAC-1 and S-PAC-1 have similar mechanisms of cell death induction at low concentrations (less than 50 μM), but at high concentrations PAC-1 displays unique cell death induction features. Cells treated with a high concentration of PAC-1 have a distinctive gene expression profile, unusual cellular and mitochondrial morphology, and an altered intracellular Ca(2+) concentration, indicative of endoplasmic reticulum (ER) stress-induced apoptosis. These studies suggest strategies for anticancer clinical development, specifically bolus dosing for PAC-1 and continuous rate infusion for S-PAC-1.

Figure 1. Changes in intracellular zinc concentration upon PAC-1 and S-PAC-1 treatment

a) Structures of PAC-1 and S-PAC-1. FRET ratio changes in HeLa cells using the genetically-encoded zinc sensor ZapCY2. Graphs of a representative individual experiment show a decrease in intracellular free zinc in five cells treated with b)10 µM PAC-1 followed by 50 µM PAC-1, or c) 10 µM S-PAC-1 followed by 50 µM S-PAC-1. d) The decrease in intracellular zinc for both PAC-1 and S-PAC-1 is significant over multiple experiments. Error bars are standard error of the mean, p-values are from DMSO (10 cells or n=2 experiments), PAC-1 (21 cells or n=4 experiments), S-PAC-1 (14 cells or n=3 experiments).

Figure 2. Expression profiles for PAC-1 and S-PAC-1

a) HL-60 cells were treated with PAC-1 or S-PAC-1 at 25 µM for 6 hours. Shown are the most up- or down-regulated transcripts (p < 0.05) compared to DMSO control cells. White text: Fold-change values, black text: the ranks of transcripts in individual profiles. b) U-937 cells were treated with PAC-1 at 100 µM for 6 hours. Shown are the 30 most up- or down-regulated transcripts compared to DMSO control cells. White text: fold change values, red text: common genes with PAC-1 treatments at low (25 µM) concentrations. TNF (indicated with an *) is downregulated at high PAC-1 concentrations but shows upregulation at low PAC-1 concentrations.

Figure 3. Changes in cellular and mitochondrial morphology

T.E.M. images of U-937 cells treated with a) 100 µM PAC-1 for 1h, 5kx, b) 100 µM PAC-1 for 1h, 4kx (black arrows: large lysosome-like structures, white arrows: dilated ER), and c) 100 µM PAC-1 for 1h, 15kx (arrows: dark myelin-like mitochondrial inclusions). Images are representative of several cells, n = 3 experiments, all scale bars indicate 1 micron. Confocal images at 40× of HeLa cells stained with Mitotracker Red (false-colored red) and SYBR Green (false-colored blue) show no changes in mitochondria morphology at 60 min in d) untreated control, e) 100 µM S-PAC-1, f) 25 µM PAC-1, but punctate mitochondrial staining with g) 100 µM PAC-1, and h) 10µM thapsigargin (Tg). All confocal images are 40× magnification. Images are a single replicate, n ≥ 3 experiments, scale bar represents 10 microns.

Figure 4. Changes in cytosolic and ER calcium levels upon PAC-1 and S-PAC-1 treatment

Graphs of single representative experiments show a) an increase in the FRET ratio in four individual HeLa cells expressing genetically-encoded cytosolic calcium sensor D3cpV upon treatment with 100 µM PAC-1 but not S-PAC-1. Following treatment with PAC-1 or S-PAC-1, cells were washed and treated with ionomycin/Ca²⁺ to demonstrate the maximum FRET ratio and confirm sensor functionality in each cell. b) A greater decrease in the FRET ratio of genetically-encoded ER calcium sensor D1ER was observed upon treatment of 100 µM PAC-1 compared to S-PAC-1 (average of at least 9 cells or n=3 experiments) and c) the absolute change in FRET value is shown to demonstrate that 100 µM PAC-1 elicits a significantly different ER calcium response than S-PAC-1. Error bars represent standard deviation, p < 0.0001.

Figure 5. Neuronal cellular membrane and BBB permeabilities of PAC-1 and S-PAC-1

a) Mean concentrations of intracellular PAC-1 or S-PAC-1 within Neuro-2a cells following incubation with 50 µM PAC-1 or S-PAC-1 for 30 minutes. Experiment performed in triplicate, p > 0.5. To measure the BBB permeability of PAC-1 and S-PAC-1, C57BL/6 mice received PAC-1 or S-PAC-1 at 75mg/kg (dissolved in HPβCD) via lateral tail vein injection. Concentrations of PAC-1 and S-PAC-1 within b) serum (p < 0.005) and c) brain tissue (p < 0.005) 5 minutes post-injection. Cohort size per group (n=4). Error bars indicate standard deviation.

Figure 6. Short exposure to PAC-1 induces cell death

U-937 cells were treated with 100 µM PAC-1, 100 µM S-PAC-1, or DMSO for various exposure times, and cell viability was assessed by flow cytometry of the cells double stained AV/PI. Shown are the percent viable cells at 24h averaged over triplicate data. Error bars indicate standard error of the mean, * indicates p > 0.005, all other relationships between samples/exposure time, p < 0.005.