Cancer Letters

Cancer Letters

Volume 298, Issue 2, 8 December 2010, Pages 231-237
Cancer Letters

Anti-tumor and radiosensitization activities of the iron chelator HDp44mT are mediated by effects on intracellular redox status

https://doi.org/10.1016/j.canlet.2010.07.010Get rights and content

Abstract

A novel iron chelator, HDp44mT, has been reported to have potent anti-proliferative effects on cancer cells; however, the underlying mechanism of action is not well understood. In this study, we characterized the cytotoxic effect of HDp44mT in a chemo- and radio-resistant cell line (PC-3) of prostatic cancer origin. The activity of HDp44mT at nM concentrations was dependent on the intracellular GSH and atmospheric O2 concentration, rather than iron deprivation. HDp44mT also radiosensitized PC-3 cells in a GSH-dependent manner. Interestingly, this radiosensitizing effect was observed under aerobic and, to a larger extent, hypoxic conditions, suggesting its potential utility as a radiosensitizer for some radioresistant tumors.

Introduction

Iron is essential for DNA synthesis and a variety of other biochemical and cellular functions. Studies have found that several iron chelators have anti-cancer properties in vitro and in preclinical tumor models, and therefore are of potential utility for the treatment of cancer [1]. In addition to the iron deprivation, some iron chelators (triapine [2], tachypyr [3], and PKIH [4], [5]) are redox-active, which may contribute to their cytotoxicity. Such redox-active chelators allow for redox cycling of bound iron between the ferric and ferrous states (Fe III  II) [6]. In addition, unlike other Fe chelators, such as desferrioxamine (DFO, which occupies each of the seven Fe coordination sites), the redox-active chelators have at least one of these sites open or occupied by a readily dissociable ligand such as water, and thereby provide access for other ligands that react with iron [7]. By juxtaposing iron and other reactants, the chelators catalyze biochemical reactions that produce various reactive oxygen species (ROS), such as the hydroxyl radicals (HOradical dot) [6], [8]. In addition, redox-active chelators may bind to iron from the cellular storage pool, thereby increasing the cellular labile iron available for redox reactions.

HDp44mT (di-2-pyridyl ketone 4,4-dimethyl-3-thiosemicarbazone, Fig. 1A) is a novel redox-active iron chelator that has been developed recently by Yuan et al. [9]. The affinity of HDp44mT for iron has been verified by its effect on iron uptake and efflux [9]. HDp44mT is uncharged at physiological pH and moderately lipophilic, which enables it to be water soluble, yet permeable across cell membranes [10]. In the presence of iron, HDp44mT oxidizes ascorbic acid and benzoic acid and increases the cellular level of ROS [10]. With an IC50 of less than 100 nM for various cancer cell lines [9], HDp44mT has higher anti-proliferative potency than DFO and other cytotoxic chelators. In vivo experiments have shown HDp44mT-mediated growth inhibition of xenograft tumors (melanoma, lung, and ovarian cancer) in nude mice, with no apparent hematological toxicity or systemic Fe depletion [11]. The precise mechanism of action of the HDp44mT-induced anti-tumor effect is not well understood. Both iron depletion and redox effects have been hypothesized to play a role in the killing of tumor cells. Here we describe studies performed to elucidate the mechanism of action of HDp44mT.

Radiation therapy requires the presence of oxygen (O2) for effective killing of tumor cells, and the hypoxic microenvironment in solid tumors makes the tumor cells radioresistant. However, some redox-active agents are known to have radiosensitizing activities under hypoxia. Such agents include the electron-affinic O2 mimics (e.g., misonidazole), redox cyclers that deplete radioprotective thiols (e.g., Motexafin gadolinium), and agents that modify hypoxia-induced redox signaling [12]. Given that the redox activity of HDp44mT enhances Fe(II  III) redox-cycling and HOradical dot generation, in this study, we tested the potential of HDp44mT as a radiosensitizer under normoxic as well as hypoxic conditions.

Section snippets

Cell culture, reagents, and equipment

To test the cytotoxic and radiosensitizing effect of HDp44mT, we used a chemo- and radio-resistant prostate cancer cell line (PC-3). PC-3 cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) unless otherwise specified. Primary epithelial cell cultures from normal prostate were established by methods previously described [13]. HDp44mT was synthesized according to the method described

HDp44mT induces growth inhibition and cell death in PC-3 cells

Incubation of PC-3 cells with HDp44mT resulted in dose- and time-dependent inhibition of cell proliferation measured by MTT assay (Fig. 1B). The IC50 for HDp44mT after ⩾8 h treatment was less than 20 nM. This dose- and time-dependent cytotoxicity of HDp44mT was also evident in cell survival measured by clonogenic survival assay (Fig. 1C) and cell viability measured by Trypan Blue exclusion assay (Fig. 1D). No significant cell cycle arrest was observed in HDp44mT-treated PC-3 cells. The

Discussion

The intracellular labile Fe concentration is estimated to be ∼10−6 M [15], [18] and is tightly controlled by the Fe absorption and storage machinery. Given the large pool of Fe in cellular storage and the medium (4.37 μM in DMEM with 10% FBS [19]), we hypothesized that HDp44mT at nM concentrations would not have a significant impact on cellular labile Fe. Indeed, iron supplementation did not abrogate HDp44mT toxicity and, compared with HDp44mT, 2,2′-dipyridyl (another well-known and

Conflicts of interest

None declared.

Acknowledgement

The work was supported by American Urological Association Postdoctoral Fellowship. We appreciate Dr. Haige Lu for performing structural NMR analysis after HDp44mT synthesis.

References (20)

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