Elsevier

Life Sciences

Volume 76, Issue 11, 28 January 2005, Pages 1267-1279
Life Sciences

Effects of artemisinin-tagged holotransferrin on cancer cells

https://doi.org/10.1016/j.lfs.2004.08.020Get rights and content

Abstract

Artemisinin reacts with iron to form free radicals that kill cells. Since cancer cells uptake relatively large amount of iron than normal cells, they are more susceptible to the toxic effect of artemisinin. In previous research, we have shown that artemisinin is more toxic to cancer cells than to normal cells. In the present research, we covalently attached artemisinin to the iron-carrying plasma glycoprotein transferrin. Transferrin is transported into cells via receptor-mediated endocytosis and cancer cells express significantly more transferrin receptors on their cell surface and endocytose more transferrin than normal cells. Thus, we hypothesize that by tagging artemisinin to transferrin, both iron and artemisinin would be transported into cancer cells in one package. Once inside a cell, iron is released and can readily react with artemisinin close by tagged to the transferrin. This would enhance the toxicity and selectivity of artemisinin towards cancer cells. In this paper, we describe a method to synthesize such a compound in which transferrin was conjugated with an analog of artemisinin artelinic acid via the N-glycoside chains on the C-domain. The resulting conjugate (‘tagged-compound’) was characterized by MALDI-MS, UV/Vis spectroscopy, chemiluminescence, and HPLC. We then tested the compound on a human leukemia cell line (Molt-4) and normal human lymphocytes. We found that holotransferrin-tagged artemisinin, when compared with artemisinin, was very potent and selective in killing cancer cells. Thus, this ‘tagged-compound’ could potentially be developed into an effective chemotherapeutic agent for cancer treatment.

Introduction

An important aspect of cancer chemotherapy is to design drugs that have high potency and specificity in killing cancer cells. In this paper, we describe the synthesis of a compound that has these properties. This involves the covalent tagging of artemisinin analogs to the N-glycoside moiety of holotransferrin.

Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua L. The compound and its analogs are being used as an antimalarial and their pharmacology and pharamcokinetics have been well studied (Dhingra et al., 2000, Li and Wu, 2003, Navaratnam et al., 2000). Artemisinin contains an endoperoxide that could react with an iron atom to form a carbon-based free radical. Such free radical, when formed intracellularly, could cause macromolecular damages and lead to cell death. Since cancer cells uptake a large amount of iron compared to normal cells, they are more vulnerable to the cytotoxic effect of artemisinin than normal cells. Our previous research (Lai and Singh, 1995, Singh and Lai, 2001) have shown that, in vitro, Molt-4 cells, a human leukemia cell line, and human breast cancer cells are more susceptible to the cytotoxic effect of artemisinin than their normal counterparts (i.e., human lymphocytes and normal breast cells, respectively). The LD50 for Molt-4 cells is approximately 100 times less than that of lymphocytes. Further research has shown that artemisinin induces mainly apoptosis in cancer cells (Singh and Lai, 2004). Various other researchers have also reported the potential anticancer properties of artemisinin and its analogs (Beekman et al., 1997, Beekman et al., 1998, Chen et al., 2003, Chen et al., 2004, Efferth et al., 2001, Efferth et al., 2002, Efferth and Oesch, 2004, Jeyadevan et al., 2004, Lee et al., 2000, Li et al., 2001, Mukanganyama et al., 2002, Posner et al., 1999, Posner et al., 2003, Posner et al., 2004, Reungpatthanapong and Mankhetkorn, 2002, Sadava et al., 2002, Sun et al., 1992, Woerdenbag et al., 1993, Wu et al., 2001).

In mammalian cells, iron is transported into the cytoplasm via a receptor-mediated endocytosis process (Andrews, 2000). Binding of the plasma iron-carrying protein transferrin to cell surface transferrin receptors triggers endocytosis. A drop in pH in the endosome causes the release of iron from transferrin. Iron is then actively pumped out into the cytoplasm. Transferrin and transferrin receptors are recycled back to the cell surface. Since cancer cells require a large amount of iron, e.g., as a cofactor in the synthesis of deoxyriboses before cell division, they express a high number of transferrin receptors on their surface. For example, breast cancer cells have 5–15 times more transferrin receptors on their cell surface than normal breast cells (Reizenstein, 1991), and transferrin receptors are expressed on cell surface of breast carcinoma cells but not on benign breast tumor cells (Raaf et al., 1993). Breast cancer cells do take up more iron than normal breast cells (Shterman et al., 1991).

We speculate that if artemisinin is covalently attached to holotransferrin (iron-loaded transferrin), it would be transported in the same package into cells and react with the iron within the endosome where iron would be released from holotransferrin. This may enhance the cytotoxic potency and selectivity of artemisinin on cancer cells.

Transferrin is a glycoprotein. Its protein moiety is mainly involved in its binding to cell surface transferrin receptors, whereas the carbohydrate chains are not involved in receptor binding (Mason et al., 1993). Transferrin has two N-glycosides attached to Asn residues in the C-terminal domain (Van Halbeek et al., 1981). Periodate oxidation of these carbohydrate chains generate reactive aldehyde groups that can be modified with a variety of hydrazine or aminoxy derivatives of artemisinin. Assuming that all 1,2-diol moieties are oxidized to the corresponding aldehyde group, we estimate that as many as 10 artemisinin derivatives could be tagged to a molecule of transferrin. Thus, we have tagged an artemisinin analog artelinic acid to the gycosylate-moiety of holotransferrin using a relatively simple process. Holotransferrin was first treated with NaIO4 to oxidize the N-glycoside chains to expose aldehyde groups on the surface. Artelinic acid hydrazide was then reacted with the oxidized holotransferrin to form a covalent conjugate (the ‘tagged-compound’). Mass spectral analysis showed that the ‘tagged-compound’ contained on an average of 4 artelinic acid moieties per molecule.

In this paper, we report a method to synthesize this ‘tagged-compound’ and the results of testing the compound on Molt-4 cells (a human leukemia cell line) and normal human lymphocytes. We compared the potency of the ‘tagged-compound’ with dihydroartemisinin, an artemisinin analog.

In addition, we tested the potency of a compound in which artelinic acid was attached to lysine residues in holotransferrin. To prepare this compound, holotransferrin was reacted with artelinic acid and N-ethyl-N′-dimethylaminopropylcarbodiimide (EDC). Thus, reactive lysine residues on the protein surface could be acylated by artelinic acid. It is expected that this latter compound would be less potent than the ‘tagged-compound’ because attachment of artelinate to lysines would interfere with the binding of holotarnsferrin to transferrin receptors.

Section snippets

General

All starting materials and reagents for organic synthesis were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Dihydroartemisinin was a gift from Holley Pharmaceuticals (Fullerton, CA). All reactions were carried out in oven-dried glassware under an inert atmosphere of nitrogen. Flash column chromatography was carried out with EM type 60 (230–400 mesh) silica gel. 1H and 13C NMR spectra were recorded on a Bruker 500 MHz DRX Avance FT-NMR spectrometer at

Synthesis of artelinic acid hydrazide and tagged transferrin

Overall steps of synthesis of artelinic acid hydrazide is shown in Fig. 1. The first coupling step gave a reasonably high yield of the artelinate ester by using a new TMSOTf-mediated reaction (Kim and Sasaki, 2004). In the second step, the endoperoxide bond in artemisinin did not react with hydrazine, a strong reducing agent, even with an elevated temperature and a prolonged reaction time. The overall yield of artelinic acid hydrazide is 64%, starting from dihydroartemisinin.

The tagging

Discussion

Artemisinin and its derivatives react rapidly with iron when they are mixed in solution. We were initially concerned about the stability of the’ tagged-compound’ because both iron and artemisinin are held closely in the compound. The UV/Vis and chemiluminescence data show that the tagged transferrin contains both iron and active artemisnin moieties. The partial loss of iron in the ‘tagged-compound’ occurs during the oxidation step. After the tagging reaction, the ‘tagged-compound’ is very

Acknowledgments

This research was supported by the Artemisinin Research Foundation and Chongging Holley Holdings. We thank Dr. Catalin Doneanu of the Department of Medicinal Chemistry, University of Washington, Seattle for recording the MALDI-MS spectra of proteins, and Himani Singh for assistance in the preparation of this manuscript.

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