Cancer Letters

Cancer Letters

Volume 179, Issue 2, 28 May 2002, Pages 151-156
Cancer Letters

Transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells

https://doi.org/10.1016/S0304-3835(02)00005-8Get rights and content

Abstract

Multiple drug resistance is a significant problem in small-cell lung cancer (SCLC). Artemisinin (ART) is a natural product used to treat drug-resistant malaria. The drug is effective because the Fe2+ present in infected erythrocytes acts non-enzymatically to convert ART to toxic products. We tested the effects of ART on drug-sensitive (H69) and multi-drug-resistant (H69VP) SCLC cells, pretreated with transferrin (TF) to increase the intracellular Fe2+ level. Antibody staining followed by flow cytometry analysis showed twice the level of TF receptors on the H69VP as compared to the H69 cells. Low doses of ART were cytotoxic to SCLC cells. The cytotoxicity of ART for H69VP cells (IC50=24 nM) was ten-fold lower than for H69 cells (IC50=2.3 nM), indicating that ART is part of the drug resistance phenotype. Pretreatment of H69 cells with 220–880 nM TF did not alter the IC50 for ART. However, in the ART-resistant H69VP cells, pretreatment with TF lowered the ART IC50 to near drug-sensitive levels (IC50=5.4 nM after 4 h pretreatment with 880 nM TF). Desferrioxamine (5 μM) inhibited the effect of TF on the IC50 for ART in drug-resistant cells but did not have an effect on ART cytotoxicity in drug-sensitive cells. DNA fragmentation as measured by ELISA occurred within ART-treated cells, with kinetics indicating apoptosis rather than necrosis. This was confirmed by TUNEL staining. These data indicate the potential use of ART and TF in drug-resistant SCLC.

Introduction

Small-cell lung cancer (SCLC) accounts for about 20% of all lung cancers and is particularly aggressive, with a 5-year survival rate at diagnosis rarely exceeding 10% [1]. Treatment of SCLC usually involves chemotherapy, with platinum-based combinations including etoposide, doxorubicin, 5-fluorouracil and taxol most common [2]. Unfortunately, drug resistance often develops and this makes treatment ineffective [3]. Because there are numerous resistance mechanisms [4], overcoming drug resistance remains a major clinical challenge.

Extracts of the plant Artemesia annua have been used in China and elsewhere for over 1600 years to treat fevers associated with malaria. The active ingredient, artemisinin (ART), has been isolated from the plant and its structure determined [5]. ART is a sesquiterpene trioxane lactone with an endoperoxide bridge that is essential for its activity [6]. High concentrations of iron in red blood cells infected with the malarial parasite, Plasmodium falciparum, react with the endoperoxide to form free radicals, which kill the parasites [7], [8]. The low toxicity of ART and its derivatives, and its ability to kill parasites resistant to other anti-malarials, has led to its widespread use to treat malaria [8], [9], [10].

ART is cytotoxic in micromolar concentrations to Ehrlich ascites tumor cells [11], [12], [13] and in nanomolar amounts, several ART derivatives are active against P388 leukemia and A540 lung carcinoma cells [14]. Two studies, one in vivo with an implanted rat fibrosarcoma [15] and the other in vitro with leukemia cells [16] have shown that preloading tumor cells with iron can lead to enhanced toxicity by ART derivatives. Receptors for the iron-carrying protein, transferrin (TF), occur in only a few types of human cells, including basal epidermal keratinocytes, pancreatic islet cells, and liver parenchyma [17]. However, the receptor is more abundant in tumor cells that are rapidly proliferating [18], in particular drug-resistant cells [19], [20]. Since the ligand-bound TF receptor is internalized [21], tumor cells exposed to TF may have increased cellular Fe2+ concentrations. Since Fe2+ is necessary for activation of ART cytotoxicity, this suggests a hypothetical strategy for the use of ART in drug-resistant cells: preload the cells with TF by virtue of their increased level of TF receptor, and then expose the cells to ART. We report here a study testing this hypothesis in drug-sensitive and drug-resistant SCLC cells.

Section snippets

Cell lines

H69 human small-call lung carcinoma cells were grown as a suspension in AIM-V serum-free medium (Life Technologies, Rockville, MD) in a 5% CO2 atmosphere at 37 °C. A drug-resistant cell line (H69VP) was selected in etoposide [22] and grown in AIM-V. This cell line shows resistance to etoposide (nine-fold), doxorubicin (ten-fold) and vincristine (ten-fold) [23].

Quantitation of TF receptors

Logarithmically growing cells (2×105) were suspended in Dulbecco's PBS without Ca2+ or Mg2+, washed twice and resuspended in the PBS.

TF receptors on drug-sensitive and drug-resistant cells

H69 drug-sensitive and H69VP drug-resistant SCLC cells were stained with FITC-labeled anti-TF receptor monoclonal antibodies and analyzed by flow cytometry. The mean fluorescence for the H69VP cells (148 arbitrary units) was greater than that for the H69 cells (104) (Fig. 1). Subtracting autofluorescence controls for each (58 and 51, respectively), the fluorescence due to antibody staining was 90 for H69VP and 53 for H69. FITC beads were used to generate a standard curve for fluorescence vs.

Discussion

Drug-resistant tumor cells express a number of upregulated proteins. Our data, showing double the concentration of TF receptors on the surfaces of drug-resistant SCLC cells as compared to sensitive cells (Fig. 1), are consistent with similar data obtained by the same methods on drug-resistant leukemia cells [20]. Others have used the unusual expression of this receptor on tumor cells as a starting point for targeted therapy, in which TF is coupled with a chemotherapeutic drug [18] or the drug

Acknowledgements

We thank L. Brown for invaluable assistance with flow cytometry. This work was supported by the Pritzker Family Foundation and Alpern Foundation.

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