Cell death by reactive oxygen species generated from water-soluble cationic metalloporphyrins as superoxide dismutase mimics

doi:10.1016/S0162-0134(01)00187-8

Journal of Inorganic Biochemistry

Volume 85, Issues 2–3, June 2001, Pages 201-208

https://doi.org/10.1016/S0162-0134(01)00187-8 Get rights and content

Abstract

We investigated the effect on cell death of reactive oxygen species induced by water-soluble cationic metalloporphyrins with superoxide dismutase (SOD) activity. The SOD activity of 5,10,15,20-tetrakis(4-N-methylpyridyl)]porphine (MPy₄P) containing Fe, Mn or Cu was measured using a cytochrome c assay by the xanthine/xanthine oxidase system and stopped-flow kinetic analysis. Cell viability of four cell lines treated with metalloporphyrins, mitomycin c (MMC), or cisplatin was estimated by a trypan blue exclusion assay. FeMPy₄P with a high SOD activity showed a significant cytotoxicity compared with MMC and cisplatin, while CuMPy₄P without SOD activity exhibited no cytotoxicity. However, MnMPy₄P showing an SOD activity as high as that of FeMPy₄P did not indicate cytotoxicity. These findings suggest that FeMPy₄P as SOD mimic converts intracellular O₂^⋅− to H₂O₂ and that it rapidly reacts with H₂O₂ to form ^⋅OH, causing DNA damage and inducing cell death. On the other hand, MnMPy₄P did not participate in the Fenton reaction, so that DNA damage in the cells treated with MnMPy₄P was not observed. In addition, the cytotoxicity by the metalloporphyrin was inversely correlated with the SOD activity of the cells and the selective damage at cellular and DNA levels was confirmed. We believe that for an anticancer drug with antioxidant ability O₂^⋅− is useful as a target molecule to induce selective cell death between cancer and normal cells and that metalloporphyrins showing SOD activity and Fenton-like reaction are a new class of anticancer agents.

Introduction

Reactive oxygen species (ROS) such as superoxide radical anion (O₂^⋅−) or hydrogen peroxide (H₂O₂) are important regulators of cell death [1], [2], [3]. H₂O₂ is known to induce a mitochondrial membrane permeability and the drop of the mitochondrial membrane potential.

Such events can cause the release of cytochrome c from mitochondria and the release initiates the activation of caspase-3, resulting in apoptosis. Although H₂O₂ is implicated as a mediator of apoptosis, it is also reported that low H₂O₂ levels trigger apoptosis, while high levels lead to necrosis [4], [5]. Thus, oxidative stress induced by H₂O₂ plays an important role in cell death.

Many anticancer drugs are known to induce generation of ROS in cancer cells, and the induction of cancer cell apoptosis by ROS is considered to be potentially useful therapeutic strategies [6], [7], [8], [9]. Low molecular antioxidants having a superoxide dismutase (SOD) activity can convert intracellular O₂^⋅− to H₂O₂ and O₂, suggesting that they may increase the concentration of H₂O₂ in cancer cells.

In this paper, we investigated the effect of ROS generated by antioxidants with SOD activity on cell death. Water-soluble cationic metalloporphyrins as SOD mimics were used as the antioxidant [10], [11], [12]. Porphyrin derivatives have widely been used as photosensitizers for photodynamic therapy in the treatment of cancer, and the compounds are known to exhibit a marked anticancer activity with relatively low toxicity. We also examined the effect of oxidative damage of ROS generated by the metalloporphyrins as SOD mimics on four cell lines without phototoxic effects.

Section snippets

Synthesis of metalloporphyrins

Cationic porphyrin, [5,10,15,20-tetrakis(4-N-methylpyridyl)]porphine (MPy₄P), was synthesized as reported in the literature [13]. FeMPy₄P, MnMPy₄P, and CuMPy₄P were purified by an ion-exchange HP-20 and the residual metals in their porphyrins were completely removed. Their structures were determined by ¹H-NMR spectroscopy and elemental analysis.

These metalloporphyrins were stable from pH 1 to 9 and no dissociation of metal from porphyrin ring was seen in the presence of EDTA.

Cytochrome c assay

Catalysis of

SOD activity of water-soluble cationic metalloporphyrins

In this study, the water-soluble cationic metalloporphyrins containing Fe, Mn or Cu were used as antioxidants. The SOD activity of these porphyrins was measured using a cytochrome c assay by the xanthine–XOD system. In addition, the direct monitoring of O₂^⋅− decay by stopped-flow kinetic analysis was also measured to obtain accurate and reliable SOD activity.

The Fe-porphyrin-induced decay of O₂^⋅− was found to follow first-order kinetics in O₂^⋅−, as shown in Fig. 1, and the rate constants (k_cat)

Discussion

The results obtained in this study demonstrated that FeMPy₄P exhibited a selective cytotoxicity toward the four cell lines and further that the cytotoxicity was strongly dependent on the SOD activity of the cells. However, MnMPy₄P had no cytotoxic effect on the cells, although its SOD activity was about the same as that of FeMPy₄P. CuMPy₄P showing no SOD activity also caused no cell death. We also found that all the cells used in this study showed no morphological signs of apoptosis and died by

Abbreviations

SOD

superoxide dismutase

DMSO

dimethylsulfoxide

HEPES

[2-{4-(2-hydroxyethl)-1-piperazinyl}ethanesulfonic acid]

MPy₄P

meso-tetrakis(4-N-methylpyridiniumil)porphyrin

EDTA

dihydrogen ethylenediaminetetraacetate

EMEM

Eagle’s minimum essence medium

FBS

fetal bovine serum

XOD

xanthine oxidase

PBS

phosphate buffered saline

CLA

2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-α]pyrazin-3-one

Acknowledgements

This work was partially supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

References (24)

P.-F. Li et al.
FEBS Lett.
(1999)
J.M. Dypbukt et al.
J. Biol. Chem.
(1994)
M.B. Hampton et al.
FEBS Lett.
(1997)
I. Muller et al.
Biochem. Biophys. Res. Commun.
(1997)
K.M. Faulkner et al.
J. Biol. Chem.
(1994)
J.M. McCord et al.
J. Biol. Chem.
(1969)
J. Butler et al.
J. Biol. Chem.
(1982)
R.H. Weiss et al.
J. Biol. Chem.
(1993)
H. Kimura et al.
FEBS Lett.
(1988)
M. Nakano et al.
Anal. Biochem.
(1990)

S.I. Liochev et al.

Free Radic. Biol. Med.

(1998)

O.I. Aruoma et al.

J. Biol. Chem.

(1989)

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Cell death by reactive oxygen species generated from water-soluble cationic metalloporphyrins as superoxide dismutase mimics

Abstract

Introduction

Section snippets

Synthesis of metalloporphyrins

Cytochrome c assay

SOD activity of water-soluble cationic metalloporphyrins

Discussion

Abbreviations

Acknowledgements

FEBS Lett.

J. Biol. Chem.

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Anal. Biochem.

Free Radic. Biol. Med.

J. Biol. Chem.