Elsevier

Biochemical Pharmacology

Volume 66, Issue 2, 15 July 2003, Pages 225-237
Biochemical Pharmacology

DNA strand breaks and apoptosis induced by oxaliplatin in cancer cells

https://doi.org/10.1016/S0006-2952(03)00260-0Get rights and content

Abstract

Platinum anticancer drugs, such as cisplatin, are thought to exert their activity by DNA damage. Oxaliplatin, a clinically active diaminocyclohexane platinum compound, however, requires fewer DNA-Pt adducts than cisplatin to achieve cell growth inhibition. Here we investigated whether secondary DNA damage and apoptotic responses to oxaliplatin compensate for the reduced formation of DNA adducts. Oxaliplatin treatment of leukemic CEM and ovarian A2780 cancer cells resulted in early (4 hr) induction of DNA single-strand breaks measured by nucleoid sedimentation. These infrequent early lesions progress with time into massive double-stranded DNA fragmentation (fragments >50 kbp) paralleled by characteristic apoptotic changes revealed by cell morphology and multivariate flow cytometry. Profound oxaliplatin-induced apoptotic DNA fragmentation was detectable following a 24 hr treatment of A2780 and CEM cells with 2 and 10 μM oxaliplatin, respectively. This DNA fragmentation was inhibited completely by the broad-spectrum caspase inhibitor Z-VAD-fmk. Cisplatin, which forms markedly more DNA-Pt adducts in CEM and A2780 cells than equimolar oxaliplatin, was similarly potent as oxaliplatin in terms of early strand breaks and later apoptotic responses. Oxaliplatin was also profoundly apoptotic in several other tumor cell lines of prostate origin but had only a marginal effect in normal prostate PrEC cells. Collectively, the results demonstrate that, relative to the magnitude of the primary DNA-Pt lesions, oxaliplatin is disproportionately more potent than cisplatin in the induction of apoptosis. Apoptosis induction, possibly enhanced by a contribution of targets other than DNA, seems to be an important factor in the mechanism of action of oxaliplatin.

Introduction

Oxaliplatin, l-OHP, oxalatoplatinum, eloxatin (Fig. 1) is a third generation platinum antitumor analog in which 1,2-diaminocyclohexane (DACH) ligand substitutes for the amine groups of cisplatin (for review see [1], [2]). Oxaliplatin has demonstrated a broad spectrum of activity in a wide range of human tumors in vitro and in vivo. Oxaliplatin also exhibits a safe toxicity profile and in various clinical situations is superior to cisplatin by being less toxic and retaining activity against cisplatin-resistant tumors [3], [4], [5]. Oxaliplatin is approved for use in the treatment of advanced colorectal cancer in the United States, Europe, Asia, and Latin America.

Oxaliplatin shares various mechanistic properties with the parent platinum drug, cisplatin. Both drugs react with the same GC-rich sites in naked DNA and similarly prefer GC-enriched regions of cellular DNA [6]. Like cisplatin, oxaliplatin induces mainly intrastrand crosslinks but forms also interstrand crosslinks (ISC) and DNA-protein crosslinks (DPC) in cellular DNA [6], [7]. All these oxaliplatin-induced DNA lesions are likely to play a role in cell growth inhibition. Oxaliplatin tends to be at least as cytotoxic as cisplatin but is often more active in various cisplatin-refractory cell lines [7], [8], [9], [10], [11], [12].

Several studies repeatedly show that oxaliplatin is markedly less reactive with naked DNA [6], [13] and forms fewer adducts with cellular DNA than equimolar cisplatin [6], [7], [12]. For example, our investigations demonstrated that total platination, ISC, and DPC induced by oxaliplatin in human leukemic CEM cells and ovarian carcinoma A2780 cells under various treatment schemes amount to approximately one-half of the respective lesions induced by equimolar cisplatin [7]. Yet, oxaliplatin is as potent as cisplatin in growth inhibition of these cell lines. Recent studies by Pendyala and coworkers using several models of cisplatin and oxaliplatin-sensitive and -resistant cell lines concluded that, compared to cisplatin, lower intracellular concentration and fewer DNA-Pt adducts are sufficient for oxaliplatin to exert its cytotoxicity [12].

It is not always apparent whether and how different responses to DNA adducts might compensate for the reduced initial platination levels by oxaliplatin. Although differential adduct repair plays a role in cellular responses to platinum drugs [14], [15], [16], [17], oxaliplatin-induced DNA damage appears to be no more difficult to repair than cisplatin-induced damage in cellular systems [7], [12] and in cell-free excision repair systems [18]. A potentially important difference between cisplatin and oxaliplatin is that only cisplatin-DNA adducts are recognized by MMR proteins, probably reflecting the different structure of the oxaliplatin adduct, in which the bulky and hydrophobic DACH ring protrudes directly outward into the major groove [19], [20]. Cisplatin cytotoxicity is thought to be actually enhanced by the MMR system due to futile cycles of trans-lesion synthesis [21], [22]. Loss of MMR that desensitizes cells to cisplatin but not to oxaliplatin, might thus explain the higher oxaliplatin cytotoxicity in some MMR-deficient cells, although cisplatin cytotoxicity showed no correlation with the levels of MMR proteins in the NCI panel of 60 cell lines [23]. The differences in MMR-proficient cells must, on the other hand, reflect other factors, possibly including a contribution of targets other than DNA.

For various anticancer drugs, regardless of their specific mechanism of action, diverse initial signals are integrated by apoptotic machinery, giving rise either to cell commitment to death or to cell survival. The higher cytotoxicity of oxaliplatin in various MMR-proficient cell systems might reflect death responses beyond the level anticipated when only the magnitude of DNA adducts is considered. Little is known, however, about the apoptotic potential of oxaliplatin and, especially, how it compares to the apoptotic potential of cisplatin.

In this study, we determined the timing of early cellular responses to oxaliplatin, including secondary DNA lesions, and their progression to massive apoptosis. Oxaliplatin and cisplatin were compared using two human neoplastic cell lines, leukemic CEM and ovarian carcinoma A2780, for which the levels of drug-induced lesions have been characterized previously [7]. The results demonstrate that oxaliplatin is disproportionately more potent than cisplatin, given the lower levels of DNA adducts, in inducing early secondary DNA strand breaks and massive apoptosis. These findings are further expanded by documenting that oxaliplatin promotes apoptosis in other cancer cell lines but not in normal cells.

Section snippets

Drugs

Oxaliplatin and DACH-PtCl2 were obtained from Sanofi-Synthelabo Research. Cisplatin was purchased from Sigma. Stock solutions of oxaliplatin and cisplatin were made in water and in saline, respectively, and stored at −20°.

Cell culture

Human cell lines, ovarian carcinoma A2780 (from Dr. S. Chaney, originally provided by Dr. T. Hamilton) and leukemia CEM (from Dr. W.T. Beck), were cultured as described previously [7] using RPMI 1640 (Gibco) and minimal essential medium Eagle, Joklik’s modification (Sigma),

Model cell lines and drug cytotoxic activities

In the main part of this study, we compared oxaliplatin and cisplatin for various effects pertinent to apoptosis induction, with the focus on drug-induced DNA fragmentation. These experiments were carried out in MMR-proficient human leukemic CEM and ovarian carcinoma A2780 cells, for which we have previously determined the levels of total DNA adducts and specific types of DNA lesions and their localization at the genomic level [6], [7]. While A2780 cells are more sensitive than CEM cells, in

Discussion

Cytotoxic effects of platinum compounds, such as cisplatin, are believed to be exerted through drug-induced DNA adducts. Oxaliplatin, a third generation clinical platinum analog, is also definitely a DNA-reactive drug. It is puzzling, however, that oxaliplatin needs to form markedly fewer primary DNA lesions than cisplatin for equitoxic effects, even though the types of Pt-DNA adducts formed by oxaliplatin, their location and their removal closely resemble the respective attributes of

Acknowledgements

This study was supported in part by a grant from Sanofi-Synthelabo Research and an NIH Grant CA78706. Skilful technical assistance of Ms. B. Arnett, A. Hidgon, and B. Leal is acknowledged. We thank also Dr. Paul E. Juniewicz (Sanofi-Synthelabo) for encouragement, numerous helpful discussions, and support of these investigations, and Dr. Maryanne Herzig for the critical reading of the manuscript.

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    Preliminary account of this study was presented in part at the 89th Annual Meeting of the American Association for Cancer Research, New Orleans, LA, March 28–April 1, 1998, Proceedings, p. 158, and 90th Annual Meeting of the American Association for Cancer Research, Philadelphia, PA, April 10–14, 1999, Proceedings, p. 488.

    1

    Present address: Departement de Medecine, Institut Gustave-Roussy, 39 rue Camille-Desmoulins, 94800 Villejuif, France.

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