A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed - PubMed (original) (raw)

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed

Rahul Palchaudhuri et al. Cell Rep. 2015.

Abstract

Apoptosis is generally believed to be a process that requires several hours, in contrast to non-programmed forms of cell death that can occur in minutes. Our findings challenge the time-consuming nature of apoptosis as we describe the discovery and characterization of a small molecule, named Raptinal, which initiates intrinsic pathway caspase-dependent apoptosis within minutes in multiple cell lines. Comparison to a mechanistically diverse panel of apoptotic stimuli reveals that Raptinal-induced apoptosis proceeds with unparalleled speed. The rapid phenotype enabled identification of the critical roles of mitochondrial voltage-dependent anion channel function, mitochondrial membrane potential/coupled respiration, and mitochondrial complex I, III, and IV function for apoptosis induction. Use of Raptinal in whole organisms demonstrates its utility for studying apoptosis in vivo for a variety of applications. Overall, rapid inducers of apoptosis are powerful tools that will be used in a variety of settings to generate further insight into the apoptotic machinery.

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Raptinal Rapidly Induces Apoptosis

A) Structure of Raptinal. B) Scanning electron micrographs of U-937 cells show pronounced apoptotic blebbing after 1 hour of treatment with 10 μM Raptinal (right) versus vehicle control treated cells (left). C) AV/PI graphs of U-937 cells treated with 10 μM Raptinal for 2 hours show transition of cells through the early apoptotic AV+/PI− quadrant that is prevented by the pan-caspase inhibitor Q-VD-OPh. D) Immunoblots of U-937 cells treated with various toxins for 1 hour show more prominent activation of procaspase-3 (PC-3) to caspase-3 (C-3) and cleavage of PARP-1 (cPARP-1) by Raptinal (10 μM) versus 25 other toxins (all tested at 10 μM). E) Cell viability of U-937 cells assessed by AV/PI after 2 hour treatment with 10 μM Raptinal and 25 other small molecules (all tested at 10 μM). Data represent the mean ± SD from 3 independent experiments. F) Time course analysis of U-937 cell viability upon treatment with 10 μM of various anticancer agents and biological tool molecules. Cell viability was assessed by AV/PI analysis. G) Time course analysis of adherent cell viability upon treatment with Raptinal, 1541B, and Staurosporine (all tested at 10 μM). Cell viability was assessed by AV/PI analysis. See also Figure S1.

Figure 2

Figure 2. Raptinal Activates the Intrinsic Pathway and Requires Functional Mitochondria for Apoptosis Induction

A) Time course immunoblots of mitochondrial and cytosolic fractions of U-937 cells treated with 10 μM Raptinal show cytochrome c release and subsequent caspase-9 activation occur after 20 minutes of treatment. B) Time course immunoblot analysis of caspase-9, -3, and -8 activation in U-937 cells treated with 10 μM Raptinal. C) Relative percent caspase-3/-7 activity of MIA PaCa-2 cells treated with 10 μM Raptinal for 1 hour upon siRNA knockdown of apoptosis genes. D) Jurkat C8−/− are equally susceptible, Jurkat FADD−/− are more susceptible, while Jurkat Bcl-2 overexpressing cells are less susceptible than wild type cells to 10 μM Raptinal as assessed by AV/PI assay after 2 hours. Data represent the mean ± SD from 3 independent experiments. ** Indicates p values < 0.02 E) Transmission electron micrographs of U-937 cells treated with vehicle or 10 μM Raptinal for 5, 30 and 60 minutes. The images show rapid changes in mitochondrial morphology (arrows) after 5 minutes of treatment with Raptinal. At 30 minutes, mitochondria are largely devoid of cristae and at 60 minutes, peripheral nuclear condensation is apparent. Scale bars represent 1 micron. F) Raptinal does not induce cytochrome c release from the mitochondrial pellet into the supernatant of isolated mitochondria treated with Raptinal in vitro under non-respiring conditions. The positive control, pro-apoptotic Bid protein is able to induce cytochrome c release. G) U-937 cells pre-treated with various potential cytoprotective agents and inhibitors of various cellular pathways were treated with 10 μM Raptinal for 2 hours and protection from the effects of Raptinal was assessed by AV/PI. Data represent the mean % protection ± SD from 3 independent experiments. * Indicates p values < 0.05. H) Protective mitochondrial agents retard cytochrome c release and caspase 9 activation in U-937 cells. See also Figure S2.

Figure 3

Figure 3. Raptinal Exhibits Activity in vivo

A) Zebrafish embryos expressing secretory annexin V-YFP exhibit pronounced punctate YFP signal indicating phosphatidylserine externalization following 1.5 hours of treatment with 10 μM Raptinal. B) Quantification of apoptotic cells in Raptinal- versus DMSO-treated zebrafish under the conditions in A. Data represent the mean ± SD (n = 5 and n = 7 embryos for Raptinal and DMSO, respectively; *** indicates p value < 0.001). Raptinal inhibits subcutaneous B16-F10 melanoma tumor growth in vivo as measured by tumor volume (** indicates p value < 0.005) in C and tumor mass after tumor excision in D (* indicates p value < 0.05). E–F) Raptinal inhibits subcutaneous 4T1 breast cancer tumor growth in vivo as measured by tumor volume (* indicates p value < 0.05) in E and tumor mass after tumor excision (* indicates p value < 0.05) in F. Arrows in C and E indicate intraperitoneal Raptinal administration at 20 mg/kg once a day. Tumor images in D and F are representative of tumor size at the conclusion of the studies. Data in C, D, E and F represent the mean ± SEM (n = 7 mice/group). See also Figure S3.

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