Mitochondria-targeting drugs arsenic trioxide and lonidamine bypass the resistance of TPA-differentiated leukemic cells to apoptosis

Abstract

IntroductionChemotherapeutic drugs can kill cultured leukemic cell lines by inducing apoptosis. 1 This mode of cell death may be clinically relevant because apoptotic blast cells have been detected in the peripheral blood of patients with acute leukemia who received chemotherapeutic drugs. 2 Hallmarks of apoptosis in leukemic cells include characteristic morphologic changes such as cell shrinkage, membrane blebbing, and cell fragmentation into apoptotic bodies, internucleosomal DNA fragmentation, and limited proteolytic cleavage of selective intracellular proteins such as the nuclear enzyme poly(ADP-ribose)polymerase (PARP). This cell death phenotype is induced by activation of a constitutively expressed apoptotic machinery that involves a family of cysteine proteases known as caspases, organized in a branched proteolytic cascade. These enzymes are synthesized as inactive proenzymes (procaspases) whose proteolysis at internal aspartate residues generates large and small subunits. 3 Heterodimerization of these subunits is required to form the active enzyme (caspase) that cleaves intracellular substrates, either a downstream procaspase or other cellular proteins. 4 Activation of the caspase cascade in response to specific damage induced by chemotherapeutic drugs is the consequence of a disruption of the mitochondrial membrane barrier function. 5 Although the exact mechanism remains controversial, mitochondrial changes lead to the release of soluble apoptogenic proteins such as cytochrome c, 6 apoptosis-inducing factor (AIF), 7 and Smac/Diablo 8,9 from the mitochondrial intermembrane space to the cytosol. Once in the cytosol, cytochrome c, in the presence of adenosine triphosphate (ATP), induces conformational changes of an adaptor molecule designated Apaf-1 that recruits and activates procaspase-9 molecules. 10,11 In turn, caspase-9 activates the downstream caspase cascade that involves caspase-3 and other effector enzymes. 12 Smac/Diablo was recently shown to suppress the inhibition of caspases by proteins known as inhibitor of apoptosis proteins (IAPs), thereby increasing their sensitivity to the cytochrome c/ATP pathway of activation. 8,9 Once activated, effector caspases cleave a limited set of essential cellular proteins, leading to cell dismantling. [13][14][15] Mitochondrial function during apoptosis is controlled by the Bcl-2 family of proteins that could prevent the opening of the permeability transition pore or stabilize the barrier function of the outer mitochondrial membrane and prevent the release of apoptogenic molecules. 5,16 How the specific damage induced by chemotherapeutic drugs leads to mitochondrial changes and activation of the caspase cascade under the control of Bcl-2-related proteins remains poorly understood. We have shown previously that differentiation of human myeloid leukemia cell lines along a macrophagic pathway induced by exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) correlated with a resistance phenotype to apoptosis induced by a series of anticancer dru...

Effects of caspase inhibitors (z‐VAD‐fmk, z‐VDVAD‐fmk) on Nile Red fluorescence pattern in 7‐ketocholesterol‐treated cells: Investigation by flow cytometry and spectral imaging microscopy

“…These different data were validated by those obtained with VP16 (Figs. 2C-2F), which is known to activate numerous caspases, including caspase-2 (38), and which are in agreement with previous investigations (39,40). So, taken together, our data strongly support an activation of caspase-2 under treatment with 7KC.…”

Molecular Targets of Arsenic Trioxide in Malignant Cells

“…ATO-induced apoptosis is associated with a loss of inner mitochondrial transmembrane potential and release of cytochrome c into the cytosol [7][8][9]. Experiments with purified mitochondria show that administration of ATO promotes opening of the permeability transition pore, releasing intermembrane proteins, which ultimately cause caspase activation [10]. Caspase induction and activation were also observed in APL cells from patients treated with ATO [1].…”

Caspase-10 involvement in cytotoxic drug-induced apoptosis of tumor cells

“…Overexpression of Bcl-2, that modulates the activity of BH3-only proteins before or at the mitochondrial level and prevents the release of cytochrome c from the mitochondria in etoposidetreated cells (Kamesaki et al, 1993), and overexpression of heat shock protein (HSP) 27, that interferes with cytochrome c released from the mitochondria in the cytosol to prevent caspase-9 activation (Bruey et al, 2000), both prevented caspase-10 activation in etoposide-treated cells (Figure 6a), suggesting that cytochrome c is critical for etoposide-induced caspase-10 activation. Exposure of HeLa, 293T and U937 cells to Caspase-10 in drug-induced apoptosis R Filomenko et al arsenic trioxide, a compound that was shown to directly target the mitochondria to trigger the release of cytochrome c and downstream caspase cascade activation (Sordet et al, 2001), induced a decrease in caspase-10 proform level in these three cell types. Similar results were obtained with caspase-9 proform that served as a control (Figure 6b).…”

“…Cell viability was monitored using trypan blue exclusion. Mitochondrial and cytosolic fractions were prepared as described (Sordet et al, 2001). The different fractions collected were frozen at À801C until use.…”