Prolyl oligopeptidase participates in the cytosine arabinoside-induced nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase in a human neuroblastoma cell line

https://doi.org/10.1016/j.bbrc.2021.07.094Get rights and content

Highlights

  • Ara-C-induced nuclear translocation of GAPDH involves its interaction with POP.

  • Ara-C is cytotoxic to cells arrested at G0/G1 after serum starvation.

  • SUAM-14746, a POP inhibitor, acts protectively against Ara-C-induced cell death.

  • Ara-C-induced nuclear translocation of GAPDH is reduced in POP-KO cells.

Abstract

Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP.

Introduction

Prolyl oligopeptidase (POP) is a post-proline cleaving serine peptidase that is highly similar among several species [1,2]. We searched for proteins that bind to POP and identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [3]. In human neuroblastoma NB-1 cells, cytosine arabinoside (Ara-C) was found to induce cell death by translocating GAPDH into the nucleus. Further, an interaction between POP and GAPDH during this process may occur [3].

GAPDH is widely regarded as a ubiquitous glycolytic enzyme and is a simple ‘housekeeping’ protein. However, this enzyme has been shown to be involved in many cellular processes. Due to the non-glycolytic roles of GAPDH, this enzyme is considered a moonlight protein in cells. GAPDH plays an important role in stress response leading to apoptosis [4], with the cytoplasmic to nuclear translocation of GAPDH preceding the onset of apoptosis [5]. A general mechanism of Ara-C cytotoxicity is the incorporation of nucleoside analogs into DNA, catalyzed by DNA polymerase [6]. However, Ara-C has been reported to induce apoptosis in postmitotic cerebellar granular cells cultured from postnatal rats [6,7]. Further, GAPDH nuclear translocation has been demonstrated to be involved in the apoptosis of non-neuronal and neuronal cells [8]. However, the mechanism of nuclear translocation of GAPDH induced by Ara-C has not been elucidated. Interestingly, the POP inhibitor, ONO-1603, was found to suppress the overexpression of GAPDH mRNA in cultured central nervous system neurons undergoing age-induced apoptosis and prevented GAPDH nuclear translocation [9]. Puttonen et al. [10] also reported that another POP inhibitor, Z-Pro-Prolinal, inhibited 6-hydroxydopamine-induced GAPDH nuclear translocation in monkey fibroblast CV1–P cells. The aim of this study was to compare POP-knockout (POP–KO) cells with wild-type cells and to demonstrate that the interaction between POP and GAPDH is involved in Ara-C-induced GAPDH nuclear translocation and cell death. Our results show that the interaction between POP and GAPDH is required for Ara-C-induced GAPDH nuclear translocation and cell death.

Section snippets

Cell culture and reagents

NB-1 (IFO 50295) human neuroblastoma cells were obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan) and maintained in RPMI-1640 (Sigma-Aldrich) containing 10% fetal calf serum (FCS, Gibco) and 50 μg/ml kanamycin at 37 °C in a humidified atmosphere of 5% CO2/95% air. POP-KO NB-1 cells were generated using CRISPR/Cas9 technology through the guide-it CRISPR/Cas9 system (Takara Bio Inc.), in accordance with the manufacturer's instructions. sgRNAs were designed using

Ara-C-induced nuclear translocation of GAPDH involves its interaction with POP in NB-1 cells

Using confocal microscopy, we monitored the distribution of GAPDH between cellular compartments in NB-1 cells before and after Ara-C treatment. Fluorescence immunostaining showed that GAPDH immunoreactivity primarily displayed cytosolic localization and was excluded from the nucleus in untreated control cells; however, after exposure to 20 μM Ara-C for 24 h, GAPDH immunoreactivity was significantly accumulated in the nucleus. Furthermore, pre-treatment with 40 μM SUAM-14746 was found to

Discussion

Ara-C induces the nuclear translocation of GAPDH and causes cell death [8]. Previously, we reported that GAPDH is a binding partner of POP, SUAM-14746 protects against Ara-C-induced cytotoxicity but does not affect the cytotoxicity of doxorubicin, and SUAM-14746 enhances the interaction between POP and GAPDH in the cytoplasm and inhibits the nuclear translocation of GAPDH [3]. In the present study, we generated POP-KO cells to demonstrate the involvement of POP in the Ara-C-induced nuclear

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

We would like to thank Editage (www.editage.com) for English language editing.

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