Elsevier

Leukemia Research

Volume 34, Issue 7, July 2010, Pages 925-931
Leukemia Research

Arsenic trioxide and ascorbic acid demonstrate promising activity against primary human CLL cells in vitro

https://doi.org/10.1016/j.leukres.2010.01.020 Get rights and content

Abstract

The compromised antioxidant defense system in chronic lymphocytic leukemia (CLL) suggested a potential use for reactive oxygen species (ROS) generating arsenic trioxide (ATO) and ascorbic acid. While both ATO and ascorbic acid mediate cytotoxicity in CLL B cells as single agents, the efficacy of ATO is enhanced by ascorbic acid. This effect is dependent on increased ROS accumulation, as pretreatment of B-CLL cells with a glutathione reducing buthionine sulfoximine or catalase inhibiting aminotriazole, enhanced ATO/ascorbic acid-mediated cytotoxicity. Pretreatment with reducing agents such as catalase, or thiol antioxidant, N-acetyl cysteine or GSH also abrogated ATO/ascorbic acid-mediated cytotoxicity. Furthermore, Hu1D10-mediated cell death was enhanced with ATO and ascorbic acid, thus justifying potential combination of ATO/arsenic trioxide therapy with antibodies such as Hu1D10 that also cause accumulation of ROS.

Introduction

B-cell chronic lymphocytic leukemia (CLL) is one of the most common adult leukemias and has a high mortality rate. In recent years monoclonal antibody therapies, like Rituximab, have shown some promise in combating this disease; however the search for a more effective therapy is still continuing. In 1984, Farber et al. [1] showed that B-lymphocytes from CLL patients lost viability rapidly compared to T-cells when incubated with a H2O2 generating system. Several reports have suggested that CLL cells have a compromised antioxidant defense system evident from the low activities of the major antioxidant enzymes superoxide dismutase and catalase and the accumulation of degradation products like malonaldialdehyde and 8-oxo-deoxyguanosine [2], [3], [4]. However there were no documented pre-clinical reports attempting to exploit this phenomenon with therapeutic agents relevant to clinical investigation.

Arsenic trioxide, a potent carcinogen and environmental toxin, has the ability to induce apoptosis in several tumors/leukemias [5], [6], [7], [8], [9], [10]. It is highly active in acute promyelocytic leukemia (APL) [11], [12], [13], [14], [15], [16]. In APL, its cytotoxic effect is thought to be mediated via its ability to generate reactive oxygen species (ROS) [17], possibly through the membrane bound NADPH oxidases [18]; and also due to its binding affinity to thiol groups which results in a decreased glutathione pool in the cells [19], [20]. The cytotoxic effect of arsenic trioxide has been shown to increase with the addition of ascorbic acid which presumably aids in its redox cycling [21], [22]. Based upon the susceptibility of CLL cells to oxidative stress previously demonstrated, we sought to determine the effect of arsenic trioxide with or without ascorbic acid treatment in CLL [1].

Section snippets

Cells

Blood was obtained from patients with B-cell CLL with informed consent under a protocol approved by the hospital internal review board. All patients examined in this series had immunophenotypically defined CLL as outlined by the modified 96 National Cancer Institute criteria (Cheson et al. [23]). Enriched B-lymphocyte fractions were prepared by using MACS negative selection kit (Miltenyi Biotec, Auburn, CA) or by “Rosette-Sep” kit (Stem Cell Technologies, Vancouver, British Columbia, Canada)

Arsenic trioxide- and ascorbic acid-mediated cytotoxicity in CLL cells in vitro

To systematically analyze the cytotoxic effects of arsenic trioxide (ATO) and ascorbic acid in B-CLL cells, we isolated purified CD19+ B cells from B-CLL patients. The demographics of the patient samples used are shown in Table 1. Cells from 10 patients were incubated with increasing concentrations of ATO for 24 and 72 h cell viability was analyzed by flow cytometry using propidium iodide (PI) and Annexin-V conjugated FITC reagents. Populations excluding both PI and Annexin-V–FITC staining were

Discussion

In the present work we have shown that the susceptibility of CLL B-lymphocytes to ROS can be exploited with arsenic trioxide, a therapeutic agent currently approved for clinical use in acute promyelocytic leukemia. Furthermore, we have demonstrated that CLL is similar to multiple myeloma where the cytotoxic effect of arsenic trioxide is greatly enhanced by the addition of ascorbic acid. Diverse forms of ROS (O2, radical dotOH, H2O2, 1O2, etc.) can be formed due to ATO/ascorbic acid. We have demonstrated

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

NM and JCB. contributed equally to this work as co-senior authors. This work was supported by National Cancer Institute P01 CA95426, The Leukemia and Lymphoma Society of America and The D. Warren Brown Foundation (JCB and NM).

Contributions. SB contributed to the design of the study and acquisition of data, and analysis and drafted the first draft of the article. XBZ and APM contributed to the acquisition of data. XM and DJ performed the statistical analysis. MV contributed to patient

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    1

    Present address: Department of Surgery, The Ohio State University, Columbus, OH, USA.

    2

    Present address: R43D, AP31-2, Global Pharmaceutical and Analytical Sciences, Abbott Laboratories, Abbott Park, IL, USA.

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