Early apoptotic features of K562 cell death induced by 5-aminolaevulinic acid-based photodynamic therapy

https://doi.org/10.1016/j.jphotobiol.2003.07.007Get rights and content

Abstract

5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) is used to eliminate cancerous cells through photoactivation of endogenously formed protoporphyrin IX (PPIX) following the administration of PPIX precursor, 5-aminolaevulinic acid (ALA). We report on the kinetics of PPIX accumulation and the mechanism of cytotoxic effects of ALA-PDT studied in the chronic myelogenous leukaemia derived cell line K562. The PPIX distribution and, consequently, cytotoxic effects were found to be heterogenous. A subpopulation of K562 cells accumulating PPIX to a lesser extent exhibits only transient cell cycle arrest. A fraction of cells, probably those with higher PPIX accumulation, are irreversibly damaged by ALA-PDT. We detected several signs of an early apoptosis: lowering of Bcl-xL expression, decrease of the mitochondrial and plasma membrane potential, the cytochrome c release into the cytoplasm, and the unmasking of the mitochondrial antigen 7A6. However, late apoptotic events were lacking: neither caspase-3 activation nor DNA fragmentation occurred. Instead, rapidly progressing cell necrosis resulting from plasma membrane damage was observed. We suggest that the high level of the antiapoptotic heat-shock proteins HSP70 and HSP27 found by us in the K562 cells is responsible for the inhibition of the apoptotic process upstream of caspases activation.

Introduction

The cell line K562 which is derived from the patient with chronic myelogenous leukaemia (CML) [1] is commonly employed as the ‘in vitro’ model of the blast phase of this disease. It bears the Philadelphia chromosome (Ph+) resulting from the reciprocal translocation between chromosomes 9 and 22 [2]. As a result of this translocation K562 cells express chimeric fusion protein Bcr-Abl whose constitutive Abl kinase activity is elevated [3], [4]. On the other hand, the principle cellular antioncogenic compound, the protein p53, is not expressed in this cell line [5]. These two factors are considered responsible for the known resistance of K562 cells to drugs and ionising radiation-induced cell death. Several lines of evidence show that Bcr-Abl exerts its antiapoptotic effects against diverse apoptotic stimuli through blockage of the mitochondrial release of cytochrome c and other preapoptotic mitochondrial perturbations [6], [7], [8]. Moreover, Bcr-Abl also prolongs cell cycle arrest at the G2/M checkpoint, which effect can be promoted by the treatment with genotoxic compounds [9]. The mechanism by which Bcr-Abl protein promotes the antiapoptotic effects is not entirely clear. It has been proposed on the basis of experiments based on the specific inhibition of Bcr-Abl tyrosine kinase activity with Imatinib mesylate (STI 571), that Bcr-Abl antiapoptotic effects are in part caused by the phosphorylation-mediated activation of signal transducer and activator of transcription STAT-5, leading to the increased expression of the antiapoptotic protein Bcl-xL [10], [11]. Inhibition of Bcr-Abl kinase activity blocks the proliferation of Bcr-Abl positive leukaemic cells and induces apoptosis [12].

In our previous reports we dealt with the mechanism of cytotoxic effects produced by photodynamic treatment (PDT) based on photoactivation of endogenously formed protoporphyrin IX (PPIX) following incubation of leukaemic cells with 5-aminolaevulinic acid (ALA-PDT) [13], [14], [15], [16]. By this procedure the feedback control of haem biosynthesis is bypassed [17] and high amounts of ultimate heme precursor, the photosensitising compound PPIX, are accumulated in cancerous compared to normal cells [18], [19], [20], [21]. Subsequent irradiation of ALA-treated cells with visible light leads by energy transfer to the formation of reactive oxygen species, predominantly singlet oxygen, 1O2, which initiates cytotoxic processes [22], [23]. We have recently shown that in promyelocytic leukaemia derived HL60 cell line ALA-PDT activated two apoptotic pathways, the mitochondrial and endoplasmic reticulum stress induced apoptotic pathways, resulting in highly efficient cell killing [24].

In this communication we report on the mechanism of cytotoxic effects imposed on the chronic myelogenous leukaemia cell line K562 by ALA-PDT. Although with the K562 cell line the ALA-PDT principle has first been demonstrated [18], the detailed mechanism of ALA-PDT cytotoxic effects on this cell line has never been described. It followed from our studies that, although apoptosis is triggered in K562 cells by ALA-PDT and early proapoptotic events like dissipation of mitochondrial membrane potential Δψm as well as mitochondrial cytochrome c release occur, caspase-3 is not activated and apoptosis does not proceed to the execution phase. Instead, rapidly progressing cell necrosis resulting from plasma membrane damage was observed.

Section snippets

Chemicals

5-Aminolaevulinic acid (ALA), propidium iodide (PI), RPMI-1640 medium, fluorogenic substrate Ac-DEVD-AFC, bongkrekic acid (BKA), the mouse monoclonal antibodies to Bcl-xL and heat shock protein 60 (HSP60) and the sheep monoclonal antibody to cytochrome c were purchased from Sigma (Prague, Czech Republic). The mouse monoclonal antibodies to heat shock proteins HSP27 and HSP70 were obtained from ALEXIS Biotechnology (Grünberg, Germany). The APO 2.7 Kit was purchased from Coulter/Immunotech

Protoporphyrin IX synthesis

The efficiency of ALA-PDT strongly depends on the level of PPIX which is attained in the cells at the end of their incubation with ALA. We studied in detail kinetics of PPIX accumulation in the cells as well as of its release into the surrounding medium. Fig. 1 (representative example from four experiments) shows levels of PPIX found in the cell lysate and in the supernatant at different time intervals after addition of ALA. The cell density was determined concurrently by cell counting (panel

Discussion

Few studies have been published to date concerning the photodynamic treatment of K562 cell line established from a chronic myeloid leukaemia patient. In the recent work [31] comparing different cell lines, K562 cells were found to be markedly less sensitive to ALA-PDT than the other lines examined. This finding was attributed to the observed higher ferrochelatase activity. The authors supposed that due to an efficient conversion to haemin, the intracellular PPIX level did not achieve the

Acknowledgements

This work was supported by Grant NL 7681-3 of the Grant Agency of the Ministry of Health, Czech Republic. The authors wish to thank J. Souček for the [3H]thymidine measurements and H. Pilcová for the excellent technical assistance. Thanks are due to P. Jones for the revision of the text.

References (37)

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