Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544

doi:10.1016/j.taap.2007.10.004

Toxicology and Applied Pharmacology

Volume 227, Issue 1, 15 February 2008, Pages 84-96

https://doi.org/10.1016/j.taap.2007.10.004 Get rights and content

Abstract

Despite strong evidence concerning the high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes (NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives, Angelicin (ANG) and 4,6,4′-trymetyl angelicin (TMA). All derivatives photoinduce cellular death, TMA being the most active compound. The cell cycle analysis showed that the four derivatives induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis. The G1 arrest is correlated to an increase in the expression of p21^Waf1/Cip1, a protein associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544 resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at serine-15. The levels of p21^Waf1/Cip1 paralleled p53 protein staining pattern suggesting that p53 activation correlated with p21^Waf1/Cip1 induction. Simultaneous to p53 activation, psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria.

Introduction

Nowadays human skin diseases such as psoriasis, T-cell lymphoma (cutaneous T-cell lymphoma, CTCL) and vitiligo are commonly treated with a combination of psoralens and UVA radiation commonly referred to as PUVA therapy (Dall'Acqua et al., 2004).

Psoralens, also known as furocoumarins, are naturally occurring or synthetic tryciclic aromatic compounds deriving from the condensation of a coumarine nucleus with a furan ring which can be photoactivated by UVA to become a potent photosensitizing agent. They are a tricyclic aromatic compound with a planar structure that helps them to intercalate between nucleic acid base pairs. Following UVA irradiation, the intercalated complex becomes activated resulting in the formation of photoadducts with pyrimidines in cellular DNA (Dall'Acqua et al., 2004). The psoralen monoadducts formed in the DNA can further react photochemically with a pyrimidine base on the complementary strand of the DNA thus leading to an interstrand cross-link (ICL), believed to be the primary cause of PUVA-induced cell killing. New psoralen derivatives have been synthesized, including angelicin that is an angular psoralens. It allows only monofunctional photobinding thus reducing undesirable side effects, especially long-term ones such as genotoxicity and skin cancer risk (Bordin et al., 1991).

Although many aspects of PUVA therapy have been extensively studied, its action mechanism remains unclear. Previous studies have reported that human epidermal keratinocytes and peripheral blood lymphocytes show an inhibition of proliferation in response to 8-methoxypsoralen (8-MOP) and UVA in vitro (Yoo et al., 1996, Vallat et al., 1994).

In lesioned psoriatic tissue treated with PUVA, the growth of epidermal keratinocytes is suppressed, while tissue-infiltrating lymphocytes are largely depleted from disease skin (Vallat et al., 1994).

These findings suggest that resident skin cells and infiltrating cellular elements might be the direct target of PUVA actions in vivo. Recently it has been shown that in a lymphoblastoid cell line, upon irradiation with psoralen the prevailing mode of cell death was apoptosis (Canton et al., 2002). These results have been recently confirmed by our group in a human promyelocytic leukemia cell line HL-60 in which psoralens along with UVA irradiation induced apoptotic cell death involving the activation of caspase-3 (Viola et al., 2004a).

The present study was aimed at both assessing the prevailing form of cell death and the mechanism of action in a human cell line of keratinocytes (NCTC-2544) irradiated in the presence of psoralen derivatives. We evaluated four derivatives: two linear ones, 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP), which are able to form DNA interstrand cross-link and two angular ones, angelicine (ANG) and 4,6,4′-trimethylangelicine (TMA) (Fig. 1) which due to their structure can only form monoadduct to DNA, thus allowing for an interesting comparison between the different biological effects of molecules belonging to the same class of compounds but endowed with different properties to form only single or bifunctional adducts to DNA.

We extended our investigation to the examination of the activation of p53 and mitochondrial activity providing evidence for the simultaneous activation of p53 and the mitochondrial apoptotic pathway and for the involvement of cell cycle deregulation during PUVA mediated apoptosis in a human cell line of keratinocytes.

Section snippets

Chemicals

All the psoralen derivatives used in this study belong to the collection of the Department of Pharmaceutical Sciences, University of Padova. The pancaspase inhibitor Z-VAD.fmk was purchased from Vinci-Biochem (Firenze, Italy).

Irradiation procedure

Two HPW 125 Philips lamps, mainly emitting at 365 nm, were used for irradiation experiments as previously reported (Viola et al., 2004b). The spectral irradiance of the source was 4.0 mW cm^− 2 as measured at the sample level by a Cole-Parmer Instrument Company radiometer

PUVA treatment induce decreases of viability of NCTC-2544

In order to establish a range of psoralen concentrations and UVA dosages that would allow to study cellular PUVA effects, viability of PUVA-treated cells was analyzed by MTT assay. We used 1.25 and 2.5 J cm^− 2 as UVA dosages and different concentrations of psoralens starting from 10 μM to 1.25 μM. Viability was monitored at 24, 48 and 72 h from irradiation. Cells treated with psoralen derivatives or UVA alone did not induce cell death. As shown in Fig. 2, PUVA treatment caused a remarkable UVA

Discussion

In this study we have examined four psoralen derivatives that differ for their ability to form ICL. A first result that is evidenced in this study is that the two angular derivatives used ANG and TMA are stronger inducers of apoptosis in comparison to the two linear derivatives. This is in agreement with our previous finding. It is important to note that only the linear derivatives are able to form bifunctional covalent adduct with DNA, while the angular derivatives are able to form only

Acknowledgments

This work was generously financed by the “Fondazione Città della Speranza”. The English revision of the manuscript by Dr. Colette Case was also gratefully acknowledged.

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Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544

Abstract

Introduction

Section snippets

Chemicals

Irradiation procedure

PUVA treatment induce decreases of viability of NCTC-2544

Discussion

Acknowledgments

Pharmac. Ther.

FEBS Lett.

Cell

Cell

Mol. Cell

FEBS Lett.

Biophys. J.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Immunol. Methods

Toxicol. In Vitro

Am. J. Pathol.

J. Invest. Dermatol.

FEBS Lett.

Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture

Biochem. J.

Stress signals utilize multiple pathways to stabilize p53

Mol. Cell. Biol.

Enhanced phosphorylation of p53 by ATM in response to DNA damage

Science

Apoptosis induced by exposure to a low steady-state concentration of H₂O₂ is a consequence of lysosomal rupture