Transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells
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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2017, European Journal of Medicinal ChemistryCitation Excerpt :Despite of the stimulating antitumor effects, some preliminary clinical trials showed that ARS just have weak to moderate efficacy [2,3]. However, ARS-like derivatives are still attractive anticancer candidates under development due to their excellent safety [4,5]. Hybridization strategy is a classical structure modification strategy to improve the activity of natural products and extensively applied in the modification of ARS.
- 1
Present address: University of Chicago School of Medicine, Chicago, IL 60637, USA.
- 2
Present address: Stanford University, Stanford, CA 94305, USA.