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Caspases participation in cell death induced by the GD2-specific antibodies

Abstract

Contribution of the main caspases in cytotoxic effects induced by the monoclonal antibody 14G2a specific to the tumor-associated ganglioside GD2 was studied in the EL-4 mouse lymphoma cells. The constitutive expression of the procaspase genes was found in the EL-4 cells. Incubation of the cells with the 14G2a antibodies did not result in increasing of the procaspase synthesis. We also demonstrated with the use of fluorescently labeled substrates of caspases that the procaspase enzymatic activity was not enhanced. At an equal level of cell death, activities of caspase-3 and caspase-9 in the cells which were incubated with the 14G2a antibodies were 7.5 and 3 times lower, respectively, than those in the staurosporine-treated cells. The pan-caspase inhibitor (Z-VAD-FMK) and the caspase-3 inhibitor decreased the cytotoxic effects induced by the 14G2a antibodies by 9–16 and 6–13%, respectively. The staurosporine-induced level of the apoptosis decreased by 55–65% under the same conditions. Inhibitors of the initiation caspase-8 and caspase-9 had no influence on the antibody-induced cell death. The inhibitory analysis also demonstrated that the caspases were not involved in the triggering of the initial stages of the antibody-induced cell death such as apoptotic volume decrease and permeabilization of the cell plasma membrane. Thus, caspases did not play a key role in the cell death induced by the anti-GD2 antibodies, and their slight enzymatic activity did not determine the main mechanism of cell death mediated through the tumor-associated ganglioside GD2.

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Abbreviations

7-AAD:

7-aminoactinomycin D

AVD:

apoptotic volume decrease

FBS:

fetal bovine serum

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

FITC:

Fluorescein isothiocyanate

HEPES:

2-[4-(2-hydroxyethyl)piperazine-1-yl]ethylsulfonic acid

PI:

propidium iodide

siRNA:

small interfering RNA

Z-VAD-FMK:

pan-caspase inhibitor

Z-DEVD-FMK/AFC:

inhibitor/substrate of caspase-3

Z-IETD-FMK/AFC:

inhibitor/substrate of caspase-8

Z-LEHD-FMK/AFC:

inhibitor/substrate of caspase-9

anti-GD2-mAb:

GD2-specific monoclonal antibody

PBS:

phosphate buffered saline

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Correspondence to R. V. Kholodenko.

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Original Russian Text © P.A. Vishnyakova, I.I. Doronin, I.V. Kholodenko, D.Yu. Ryazantsev, I.M. Molotkovskaya, R.V. Kholodenko, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 3, pp. 305–314.

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Vishnyakova, P.A., Doronin, I.I., Kholodenko, I.V. et al. Caspases participation in cell death induced by the GD2-specific antibodies. Russ J Bioorg Chem 40, 279–287 (2014). https://doi.org/10.1134/S1068162014030157

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  • DOI: https://doi.org/10.1134/S1068162014030157

Keywords

  • GD2
  • tumor-associated gangliosides
  • caspases
  • cell death
  • apoptosis
  • monoclonal antibodies