Original article
Synergistic enhancement of topotecan-induced cell death by ascorbic acid in human breast MCF-7 tumor cells
Graphical abstract
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 (GS•and 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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