open access

Vol 56, No 4 (2018)
Original paper
Submitted: 2018-02-12
Accepted: 2018-09-30
Published online: 2018-10-05
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Semispecific TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) displays cytotoxic activity by induction of apoptosis, autophagy and protein aggregation in U937 cells

Lukasz P. Bialy1, Jean Fayet2, Grzegorz M. Wilczynski3, Izabela Mlynarczuk-Bialy1
DOI: 10.5603/FHC.a2018.0020
·
Pubmed: 30294774
·
Folia Histochem Cytobiol 2018;56(4):185-194.
Affiliations
  1. Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, Warsaw, Poland
  2. Department of Ophthalmology, First Medical Faculty, Medical University of Warsaw, Poland
  3. Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology PAS Warsaw, Poland

open access

Vol 56, No 4 (2018)
ORIGINAL PAPERS
Submitted: 2018-02-12
Accepted: 2018-09-30
Published online: 2018-10-05

Abstract

Introduction. The main component of extralysosomal proteolysis is the ubiquitin-proteasome system (UPS), which is supplemented by tripeptidyl peptidase II (TPPII). That system is a target for anticancer strategies by using proteasome inhibitors. Data from several studies on leukemic cells share evidence for the beneficial and potential role of TPPII in cell survivability. Therefore, the aim of this work was to analyze the effect of AAF-cmk, a membrane permeable semi-specific TPPII inhibitor, on human monocytic leukemic cells U937 for translational research.

Material and methods. We studied the viability of U937 cells incubated with AAF-cmk using tetrazolium salt reduction assay (MTT) and apoptosis induction by assessing caspase activation by Western blotting and Annexin V binding assays. Transmission electron microscopy (TEM), a gold standard for apoptosis and autophagy detection, was used to assess the ultrastructure of U937 cells.

Results. Incubation of cells with AAF-cmk reduced their viability and induced apoptosis by intrinsic pathway. In groups treated with AAF-cmk, activation of caspases 9 and 3 was observed and caspase inhibition by zVDA restored cell viability. TEM revealed the presence of ultrastructural features of apoptosis and authophagy. Moreover, we identified two types of protein aggregates. The first one was found in close proximity to the endoplasmic reticulum (ER) and corresponds to Aggresome-Like Structure (ALIS); however, the second novel type of aggregate was not related to ER elements, but rather to free cytosolic ribosomes. This type did not correspond to the aggresome neither in localization nor the structure, thus we referred these aggregates as ALiSNER (Aggresome-Like Structure Not Associated With the ER).

Conclusions. Our results provide novel and important findings about the role of TPPII in protein homeostasis and cell survival. Since semispecific TPPII inhibitor AAF-cmk displays cytotoxic activity against leukemic U937 cells in vitro it can be considered as a potential anticancer agent.

Abstract

Introduction. The main component of extralysosomal proteolysis is the ubiquitin-proteasome system (UPS), which is supplemented by tripeptidyl peptidase II (TPPII). That system is a target for anticancer strategies by using proteasome inhibitors. Data from several studies on leukemic cells share evidence for the beneficial and potential role of TPPII in cell survivability. Therefore, the aim of this work was to analyze the effect of AAF-cmk, a membrane permeable semi-specific TPPII inhibitor, on human monocytic leukemic cells U937 for translational research.

Material and methods. We studied the viability of U937 cells incubated with AAF-cmk using tetrazolium salt reduction assay (MTT) and apoptosis induction by assessing caspase activation by Western blotting and Annexin V binding assays. Transmission electron microscopy (TEM), a gold standard for apoptosis and autophagy detection, was used to assess the ultrastructure of U937 cells.

Results. Incubation of cells with AAF-cmk reduced their viability and induced apoptosis by intrinsic pathway. In groups treated with AAF-cmk, activation of caspases 9 and 3 was observed and caspase inhibition by zVDA restored cell viability. TEM revealed the presence of ultrastructural features of apoptosis and authophagy. Moreover, we identified two types of protein aggregates. The first one was found in close proximity to the endoplasmic reticulum (ER) and corresponds to Aggresome-Like Structure (ALIS); however, the second novel type of aggregate was not related to ER elements, but rather to free cytosolic ribosomes. This type did not correspond to the aggresome neither in localization nor the structure, thus we referred these aggregates as ALiSNER (Aggresome-Like Structure Not Associated With the ER).

Conclusions. Our results provide novel and important findings about the role of TPPII in protein homeostasis and cell survival. Since semispecific TPPII inhibitor AAF-cmk displays cytotoxic activity against leukemic U937 cells in vitro it can be considered as a potential anticancer agent.

Get Citation

Keywords

U937 cells; AAF-cmk; TPPII; apoptosis; autophagy; proteasome; aggresome; TEM; ALiSNER

About this article
Title

Semispecific TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) displays cytotoxic activity by induction of apoptosis, autophagy and protein aggregation in U937 cells

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 56, No 4 (2018)

Article type

Original paper

Pages

185-194

Published online

2018-10-05

DOI

10.5603/FHC.a2018.0020

Pubmed

30294774

Bibliographic record

Folia Histochem Cytobiol 2018;56(4):185-194.

Keywords

U937 cells
AAF-cmk
TPPII
apoptosis
autophagy
proteasome
aggresome
TEM
ALiSNER

Authors

Lukasz P. Bialy
Jean Fayet
Grzegorz M. Wilczynski
Izabela Mlynarczuk-Bialy

References (1)
  1. Bialy LP, Kuckelkorn U, Henklein P, et al. Changes in spatio-temporal localization of tripeptidyl peptidase II (TPPII) in murine colon adenocarcinoma cells during aggresome formation: a microscopy study based on a novel fluorescent proteasome inhibitor. Histol Histopathol. 2018 [Epub ahead of print]: 18042.

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