Elsevier

Free Radical Biology and Medicine

Volume 113, December 2017, Pages 406-412
Free Radical Biology and Medicine

Original article
Synergistic enhancement of topotecan-induced cell death by ascorbic acid in human breast MCF-7 tumor cells

https://doi.org/10.1016/j.freeradbiomed.2017.10.377 Get rights and content

Highlights

  • One-electron oxidation of topotecan generates topotecan phenoxy radical.

  • Topotecan radical rapidly reacts with glutathione to generate glutathiyl radical.

  • Ascorbic acid (AA) significantly enhances topotecan cytotoxicity in MCF-7 tumor cells.

  • Ascorbic acid significantly increased topoisomerase I-dependent DNA damage.

  • Synergistic cytotoxicity of topotecan and AA may result from increased DNA damage.

Abstract

Topotecan, a derivative of camptothecin, is an important anticancer drug for the treatment of various human cancers in the clinic. While the principal mechanism of tumor cell killing by topotecan is due to its interactions with topoisomerase I, other mechanisms, e.g., oxidative stress induced by reactive free radicals, have also been proposed. However, very little is known about how topotecan induces free radical-dependent oxidative stress in tumor cells. In this report we describe the formation of a topotecan radical, catalyzed by a peroxidase-hydrogen peroxide system. While this topotecan radical did not undergo oxidation-reduction with molecular O2, it rapidly reacted with reduced glutathione and cysteine, regenerating topotecan and forming the corresponding glutathiyl and cysteinyl radicals. Ascorbic acid, which produces hydrogen peroxide in tumor cells, significantly increased topotecan cytotoxicity in MCF-7 tumor cells. The presence of ascorbic acid also increased both topoisomerase I-dependent topotecan-induced DNA cleavage complex formation and topotecan-induced DNA double-strand breaks, suggesting that ascorbic acid participated in enhancing DNA damage induced by topotecan and that the enhanced DNA damage is responsible for the synergistic interactions of topotecan and ascorbic acid. Cell death by topotecan and the combination of topotecan and ascorbic acid was predominantly due to necrosis of MCF-7 breast tumor cells.

Introduction

Topoisomerase I (topo I) is a nuclear enzyme responsible for maintaining DNA topology and functions [1], [2], [3], [4], [5]. Camptothecin and its analog topotecan (TPT) are important anticancer agents for the treatment of various human malignancies in the clinic [6], [7], [8]. The major mechanism of action of camptothecin and TPT (Fig. 1) is due to the stabilization of transient complexes formed between topo I and DNA (cleavable complexes), resulting in the formation of highly toxic double-strand breaks in tumor cells, causing cell death [2], [3], [5]. While other mechanisms of actions of TPT that are independent of topo I have also been suggested, including induction of oxidative stress [9], [10] and inhibition of hypoxia-inducible factor [11], [12], there is very little known about how TPT induces oxidative stress in tumor cells. Akbas et al. [9] have shown that treatment of MCF-7 tumor cells with TPT leads to increased formation of reactive oxygen species (ROS) and nitrite, indicating increased oxidative stress. In addition, Timur et al. [13] have shown that there is a significant decrease in glutathione levels following TPT treatment of MCF-7 breast cancer cells with concomitant increases in lipid peroxidation and levels of antioxidant enzymes, superoxide dismutase, glutathione peroxidase and catalase, suggesting TPT-induced oxidative stress in MCF-7 tumor cells. It is interesting to note that Sordet et al. [14], [15] have shown that ROS generated by certain compounds, e.g., arsenic trioxide, induce formation of DNA-topo I complexes, which may play a role in the mechanism of drug cytotoxicity. Moreover, Daroui et al. [16] have shown that H2O2 cytotoxicity is partly mediated by topo I. Thus, it is possible that ROS formed from TPT may also contribute to topo I-induced DNA damage and cytotoxicity.

It has been shown that ascorbic acid (AA) induces tumor cell death by generating extracellular hydrogen peroxide (H2O2) which is taken up by tumor cells and leads to the formation of reactive hydroxyl radical (OH) in the presence of metal ions [17], [18], [19]. The role of ascorbic acid (commonly known as Vitamin C) in cancer is extremely controversial [20], [21], [22], [23]. Many investigators have found that ascorbic acid is highly toxic to tumor cells and combinations of ascorbic acid with several anticancer drugs are more than additive in tumor cell killing [19], [24], [25], [26], while other investigators have found no effect in cell culture and animal models [27], [28].

Because of the lack of understanding of how TPT produces reactive free radical species in tumor cells that can significantly affect topo I-induced DNA cleavage formation and modulate the toxicity of topo I drugs, we have investigated the mechanisms of free radical formation from TPT and examined the effects of ascorbic acid on TPT cytotoxicity in MCF-7 breast cancer cells. We have found that in the presence of H2O2 and peroxidases, TPT generates a TPT radical (TPT), which is very stable and does not react with O2 to redox cycle and generate superoxide/H2O2. However, TPT is extremely reactive with glutathione and cysteine, forming the corresponding thiyl (GSand Cys) radicals and regenerating TPT. Moreover, we found that ascorbic acid (a cellular H2O2 generator) modulates the cytotoxicity of TPT, and the combinations are highly synergistic in MCF-7 breast cancer cells.

Section snippets

Materials and methods

Camptothecin was a gift of the Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program of NCI, NIH. Topotecan hydrochloride was obtained from Cayman Chemicals (Ann Arbor, MI). A stock solution of camptothecin was prepared in DMSO, TPT was dissolved in doubly distilled water, and solutions were stored at −80 °C. Ascorbic acid and horseradish peroxidase (HRP) were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). Ascorbic acid (100 mM) was dissolved in chelex-treated

ESR studies

Incubation of TPT with rat microsomes in the presence or absence of NADPH gave no ESR detectable radical. However, when TPT was incubated with HRP in the presence of H2O2, a strong ESR signal was detected (Fig. 2 Panel A). This single line spectrum had a g value of 2.006. It was found to be stable under aerobic conditions, demonstrating that the radical did not react with molecular O2. The same radical was also detected when human myeloperoxidase was used in the presence of H2O2. While the

Discussion

Studies described in this report show that one-electron oxidation of TPT, catalyzed by a peroxidase-hydrogen peroxide system, readily forms a TPT radical. Because camptothecin, the parent drug of TPT, lacks a 10-OH group in the quinolone ring, and did not form any radical under the same experimental conditions that generated TPT, we believe that the 10-OH of the TPT is required for this free radical formation, and the radical detected, therefore, is a phenoxyl radical-derived polymer radical

Acknowledgements

We thank Ms. Mary Mason for her invaluable help in editing the manuscript. We also thank Drs. Maria Kadiiska and Douglas Ganini for their critical evaluation of the manuscript. We also thank Brett Wagner for his helpful discussions.

Funding

This research was supported by the intramural research program of the National Institute of Environmental Health Sciences, NIH (ZIA E505013922). Statements contained herein do not necessarily represent the statements, opinions, or conclusions of NIEHS, NIH, or the US Government.

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