Involvement of mitochondria and caspase-3 in ET-18-OCH3-induced apoptosis of human leukemic cells

Abstract

The induction of cell death in leukemic HL‐60 cells by the ether lipid 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3; edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET‐18‐OCH3 induced disruption of the mitochondrial transmembrane potential (ΔΨm) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET‐18‐OCH3 also induced caspase‐3 activation in human leukemic cells, as assessed by cleavage of caspase‐3 into the p17 active form and cleavage of the caspase‐3 substrate poly(ADP‐ribose) polymerase (PARP). ET‐18‐OCH3 analogues unable to induce apoptosis failed to disrupt ΔΨm and to activate caspase‐3. ET‐18‐OCH3‐resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase‐3 activation and did not undergo ΔΨm disruption upon ET‐18‐OCH3 incubation. Cyclosporin A partially inhibited ΔΨm dissipation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic cells. Overexpression of bcl‐2 by gene transfer prevented ΔΨm collapse, ROS generation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic T cells. Pretreatment with the caspase inhibitor Z‐Asp‐2,6‐dichlorobenzoyloxymethylketone prevented ET‐18‐OCH3‐induced PARP proteolysis and DNA fragmentation, but not ΔΨm dissipation. ET‐18‐OCH3 did not affect the expression of caspases and bcl‐2‐related genes. ET‐18‐OCH3‐induced apoptosis did not require protein synthesis. Our data indicate that ΔΨm dissipation and caspase‐3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET‐18‐OCH3, and that the sequence of events in the apoptotic action of ET‐18‐OCH3 on human leukemic cells is: ΔΨm disruption, caspase‐3 activation and internucleosomal DNA degradation. Int. J. Cancer 86:208–218, 2000. © 2000 Wiley‐Liss, Inc.

Mn‐SOD antisense upregulates in vivo apoptosis of squamous cell carcinoma cells by anticancer drugs and γ‐rays regulating expression of the BCL‐2 family proteins, COX‐2 and p21

“…[41][42][43][44] These Bcl-2 activities appear to potently inhibit ROIinduced apoptosis. The decrease of Bcl-2 expression by Mn-SOD antisense, therefore, appears to explain the enhanced apoptosis in tumors of the Mn-SOD antisense group.…”

Edelfosine lipid nanosystems overcome drug resistance in leukemic cell lines

“…However, this mechanism might be diminished in the case of encapsulated drug, as it could be directed to different cell targets once inside the cell, promoting other drug actions and therefore overcoming resistance to the drug. In fact, it has also been described that ET also triggers the intrinsic apoptotic signaling pathway [23,28]. Besides, there are more mechanisms implicated such as endoplasmic reticulum stress and the production of reactive oxygen species [29,30].…”

Involvement of mitochondria and recruitment of Fas/CD95 signaling in lipid rafts in resveratrol-mediated antimyeloma and antileukemia actions

“…Quantitation of apoptotic cells was calculated by flow cytometry as the percentage of cells in the sub-G 1 region (hypodiploidy) in cell-cycle analysis as previously described (Gajate et al, 2000b). The possible implication of Fas/CD95-FasL/CD95L interaction in resveratrol-induced apoptosis was evaluated by using the blocking anti-Fas/CD95 SM1/23 IgG 2b monoclonal antibody (Bender MedSystems, Vienna, Austria) as described previously (Gajate et al, 2003).…”

“…Cytofluorimetric analysis of mitochondrial transmembrane potential and generation of reactive oxygen species To evaluate DC m and the generation of ROS, cells (10 6 /ml) were incubated in PBS with 20 nM 3,3 0 -dihexyloxacarbocyanine iodide (DiOC 6 (3)); green fluorescence; Molecular Probes, Leiden, The Netherlands) and 2 mM dihydroethidine (HE; red fluorescence after oxidation; Sigma) for 40 min at 37 1C, followed by analysis on a Becton Dickinson (San Jose, CA, USA) fluorescence-activated cell sorting FACSCalibur flow cytometer as previously described (Gajate et al, 2000b).…”