A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed

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 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 to study 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.

The Combination of Vemurafenib and Procaspase-3 Activation Is Synergistic in Mutant BRAF Melanomas

“…Cells were treated with 1 µM of staurosporine for 24 h or with 13 µM of raptinal(37) for 3 h as positive control, DMSO as negative control and indicated concentrations of PAC-1 and vemurafenib for 0, 2, 4, 7, 10, 12, 16, 20 or 24 h. Plates were then assessed for caspase-3/7 activity via addition of bifunctional lysis and activity buffer (200 mM HEPES, 400 mM NaCl, 40 mM DTT, 0.4 mM EDTA, 1% Triton-X, pH 7.4) with 20 µM of Ac-DEVD-AFC (Cayman Chemicals) as the fluorogenic substrate (λ ex =400 nm, λ em =505 nm). Plates were pre-incubated at 37 °C at 30 min in the Synergy multi-mode reader (BioTek) then read for 30 min at 3 min intervals.…”

Systematic Quantification of Population Cell Death Kinetics in Mammalian Cells

“…For reasons that remain poorly understood, cell death kinetics vary considerably between lethal stimuli, cell lines and cell death pathways, as well as between individual cells within the same population (Bernheim et al, 1977; Biton and Ashkenazi, 2011; Dixon et al, 2012; Lu et al, 2014; Shimizu et al, 2004; Spencer et al, 2009; Vanden Berghe et al, 2010). A major goal of the present work was to develop means to quantify this variability in cell death kinetics at the population level, as this knowledge may enhance our understanding of different lethal pathways, improve the classification of lethal perturbations and help identify new drugs that act through unique mechanisms (Grootjans et al, 2016; Hafner et al, 2016; Harris et al, 2016; Palchaudhuri et al, 2015; Tyson et al, 2012). …”

Tricking a Cancer Cell into Committing Suicide or Apoptosis

“…A landmark research by Paul Hergenrother and his team (Nature Clinical Biology) nearly found a way around this natural biological process that kick starts apoptosis [7]. They found a synthetic molecule that directly activates procaspase-3 to caspase-3 thus initiating apoptosis in the tumour.…”

“…So it was postulated that therapy could be tailored to the cancer, the organ and patient's condition by measuring the levels of pro-caspase-3. This selective treatment was expected to minimize collateral damage and toxic effects of chemotherapy [5][6][7].…”

“…There are other examples like Epstein-Barr virus producing a protein similar to anti-apoptotic gene BcL-2 leading to cell proliferation. Melanoma cells are also known to avoid apoptosis by inhibiting gene encoding Apaf-1 [2][3][4][5][6][7].…”