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

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Cancer Letters 179 (2002) 151–156

Transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells David Sadava a,b,*, Tiphanie Phillips a,1, Cindy Lin a,2, Susan E. Kane b b

a Keck Science Center, 925 N. Mills Avenue, Claremont, CA 91711, USA Division of Molecular Medicine, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA

Received 3 August 2001; received in revised form 12 December 2001; accepted 13 December 2001

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 Fe 21 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 Fe 21 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 ¼ 24 nM) was ten-fold lower than for H69 cells (IC50 ¼ 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 ¼ 5:4 nM after 4 h pretreatment with 880 nM TF). Desferrioxamine (5 mM) 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. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human lung cancer; Drug resistance; Artemisinin; Transferrin

1. Introduction Small-cell lung cancer (SCLC) accounts for about 20% of all lung cancers and is particularly aggressive,

* Corresponding author. Keck Science Center, 925 N. Mills Avenue, Claremont, CA 91711, USA. Tel.: 11-909-607-3949; fax: 11-909-621-8588. E-mail address: [email protected] (D. Sadava). 1 Present address: University of Chicago School of Medicine, Chicago, IL 60637, USA. 2 Present address: Stanford University, Stanford, CA 94305, USA.

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

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00005-8


D. Sadava et al. / Cancer Letters 179 (2002) 151–156

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 [8–10]. ART is cytotoxic in micromolar concentrations to Ehrlich ascites tumor cells [11–13] 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 Fe 21 concentrations. Since Fe 21 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.

2. Materials and methods 2.1. 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 8C. 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].

2.2. Quantitation of TF receptors Logarithmically growing cells (2 £ 10 5) were suspended in Dulbecco’s PBS without Ca 21 or Mg 21, washed twice and resuspended in the PBS. Mouse FITC-labeled anti-human TF receptor (CD71, 200 mg/ml, Santa Cruz Biotechnology) was diluted 1:20 and added to the cell suspension for a final volume of 250 ml. Following incubation at 4 8C for 1 h in the dark, the cells were washed twice in PBS as above and then stained in propidium iodide. The mean fluorescence of the cells was measured by flow cytometry. A standard curve using FITC-beads was generated by flow cytometry to relate the cell fluorescence measurements to actual FITC concentration, and therefore to the number of receptors per cell. 2.3. Cytotoxicity experiments ART and/or holotransferrin and/or desferrioxamine mesylate (all obtained from Sigma, St. Louis, MO) were added as indicated to logarithmically growing SCLC cells in 1 ml suspensions at 6000 cells/ml. After 4 days of continuous exposure, cells were counted in a Coulter Z-1 counter. Counts were validated microscopically by a hemocytometer after staining with Trypan Blue. All experiments were done in triplicate and repeated at least three times. IC50 was calculated as compared to solvent controls. Means were calculated and compared by two-tailed ttest for significance between H69 and H69VP for that treatment. 2.4. DNA fragmentation test The mechanism of cytotoxicity by ART and/or TF was investigated by the kinetics of cellular DNA fragmentation (Boehringer Mannheim kit). Briefly, 2 £ 10 5 cells/ml were incubated for 14 h at 37 8C and 5% CO2 in AIM-V medium with 10 mM BUdR. The cells were washed and resuspended to 10 5 cells/ ml in BUdR-free medium. A total of 200 ml of the culture was incubated with drug(s) (10 nM ART, 880 nM TF) for the indicated time, after which 100 ml of the culture medium was removed for quantitation of labeled DNA fragments by ELISA. This is a measure of cell necrosis. DNA fragments inside the cells (a measure of apoptosis) were measured following cell

D. Sadava et al. / Cancer Letters 179 (2002) 151–156

lysis in BSA-Tween 20 at 21 8C for 30 min. Following centrifugation, the lysate was used for ELISA. For ELISA, a round-bottom microtiter plate was coated with anti-DNA antibody (mouse anti-human DNA monoclonal, clone MCA-33) overnight at 4 8C. Following blocking with BSA, 100 ml extract with the labeled DNA fragments was added to the coated wells and incubated for 90 min at room temperature. The DNA was fixed and denatured by microwave irradiation at 500 W for 5 min. Then anti-BUdR (mouse monoclonal BMG 6HB, F 0 ab fragment) conjugated with peroxidase was added and the wells were incubated for 90 min at room temperature. Peroxidase substrate (TMB) was then added and following incubation in the dark for 120 min, the reaction was stopped with 500 ml of concentrated H2SO4. The resulting color was quantitated by absorbance at 450 nm.


H69VP and 53 for H69. FITC beads were used to generate a standard curve for fluorescence vs. FITC and the log-log slope (0.87) was used to calculate the actual FITC concentration, and therefore receptor concentration, on the cells. These were 22,000 TF receptors/H69VP cell and 13,000/H69 cell. 3.2. Cytotoxicity of ART and TF ART was cytotoxic to H69 cells at low doses and the IC50 for ART was ten-fold higher in H69VP cells (Table 1). TF alone was not cytotoxic for either cell line. When H69 cells were preincubated with increasing concentrations of TF, there was no effect on the cytotoxicity of ART. However, when H69VP cells

2.5. TUNEL analysis for apoptosis Logarithmically growing cells were washed twice in PBS-1% BSA and resuspended in the buffer to 5 £ 10 6 cells/ml. To 500 ml cells, 100 ml of 4% paraformaldehyde was added and fixation proceeded at room temperature for 1 h. Following washing in PBS, the cells were resuspended in 100 ml cold permeabilization solution (0.1% Triton X-100, 0.1% Na-citrate) for 2 min at 4 8C. The cells were washed twice in PBS and then resuspended in 50 ml TUNEL mixture containing TdT and fluorescein-dUTP (Boehringer Mannheim kit). Following incubation in the dark at 37 8C for 1 h, the cells were counterstained with PI and analyzed by flow cytometry. 3. Results 3.1. 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

Fig. 1. Evaluation of TF receptors on H69 drug-sensitive and H69VP drug-resistant cells. Cells (5–8 £ 10 5 cells/ml) were suspended and stained with FITC-goat anti-human TF-receptor antibody and analyzed by flow cytometry.


D. Sadava et al. / Cancer Letters 179 (2002) 151–156

Table 1 Cytotoxicity of ART and ART-TF in drug-sensitive and drug-resistant cells a Condition

H69-sensitive (IC50 nM)

H69VP-resistant (IC50 nM)

ART ART 1 220 nM TF ART 1 440 nM TF ART 1 880 nM TF

2.3 ^ 0.8 2.4 ^ 1.0 2.8 ^ 0.5 2.6 ^ 0.9

24.3 ^ 4.7* 17.5 ^ 3.9* 13.4 ^ 3.3* 5.4 ^ 1.1*

a Cells (6 £ 10 3/ml) were preincubated for 4 h without or with TF, and then ART was added. Cytotoxicity was measured by cell counts after 4 days. Controls (no drug) had 2 £ 10 5 cells/ml after 4 days. *Significantly different (P , 0:05) from the IC50 for H69 cells.

were preincubated with 220–880 nM TF for 4 h, the IC50 for ART was reduced; at the highest concentration of TF, the IC50 for ART was only two-fold greater than that for H69 cells. To determine whether the TF effect was due to chelatable Fe 21, cells were incubated in 5 mM desferrioxamine mesylate following incubation in TF but before the addition of ART. This chelator had little effect on the IC50 for ART in H69 or H69VP cells, but largely blocked the effect of TF on reducing the IC50 in H69VP cells (Table 2).

into the medium by necrotic cells (Fig. 2). Preincubation of both cell lines with 880 nM TF for 4 h followed by the IC50 concentration of ART for 4 h (2 nM in H69 and 20 nM in H69VP) led to DNA fragmentation patterns identical to those shown for ART alone. Incubation in TF alone for 4 h did not result in DNA fragmentation. 3.4. TUNEL assays DNA breaks due to apoptosis were measured directly by the TUNEL method (Fig. 3). When H69 or H69VP cells were incubated with 20 nM ART for 3 h, there was extensive TUNEL positivity. This did not occur with 880 nM TF alone. 4. 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

3.3. Kinetics of DNA fragmentation To determine the mechanism of cytotoxicity, we treated either H69 or H69VP cells with 10 nM ART and measured DNA fragmentation by ELISA. These fragments were largely formed within the cells, since the bulk of the fragments were released only when cells were lysed rather than being directly released Table 2 Effect of desferrioxamine on ART and ART-TF cytotoxicity a Condition

H69-sensitive (IC50 nM)

H69VP-resistant (IC50 nM)

ART ART 1 220 nM TF ART 1 440 nM TF ART 1 880 nM TF

2.7 ^ 1.1 2.9 ^ 1.0 3.3 ^ 0.8 2.7 ^ 0.5

20.7 ^ 6.4* 19.3 ^ 5.5* 18.5 ^ 6.6* 18.0 ^ 7.2*

a Cells (6 £ 10 3/ml) were preincubated for 4 h without or with TF and then 5 mM was added for an additional 2 h. Then, ART was added. Cytotoxicity was measured by cell counts after 4 days. Controls (no drug) had 2 £ 10 5 cells/ml after 4 days. *Significantly different (P , 0:05) from the IC50 for H69 cells.

Fig. 2. Kinetics of DNA fragmentation in SCLC cells exposed to ART. Triplicate 5 ml cultures (5 £ 10 5 cells/ml) were prelabeled with BUdR and then exposed to ART for 1 or 4 h. DNA fragments released to the medium or within the cells (lysate) were determined by ELISA (Roche Boehringer Mannheim). Error bars indicate SD.

D. Sadava et al. / Cancer Letters 179 (2002) 151–156


TF effect may be due to an increase in intracellular chelatable Fe 21 is indicated by the inhibition of the effect by the chelating agent, desferrioxamine mesylate (Table 2). The concentration of the chelator was low enough not to be cytotoxic to the cells and did not inhibit ART cytotoxicity in either cell line. These results indicate that the ART-TF combination may be useful in treating drug-resistant SCLC.

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

References Fig. 3. Apoptosis of H69 cells treated with ART. Cells (10 6/ml) were incubated for 4 h in 3 nM ART and then fixed, permeabilized and stained with a fluorescent TUNEL reaction (Boehringer Mannheim). Following counterstaining with PI, the cells were analyzed by flow cytometry.

therapy, in which TF is coupled with a chemotherapeutic drug [18] or the drug is encapsulated within a liposome with TF on its surface [24]. We have taken a different approach, using the known effects of Fe 21 in activating the anti-malarial, ART [8]. This drug was cytotoxic at low concentrations to SCLC cells (Table 1). Because ART induced the gradual formation of DNA fragments in the cells, rather than their rapid release into the culture medium (Fig. 2), it is probable that ART is pro-apoptotic. This was confirmed by TUNEL staining (Fig. 3). These data indicate that ART, a drug with minimal toxicity and widespread use, may be appropriate for treating drug-sensitive SCLC. Drug-resistant SCLC cells had a ten-fold higher IC50 for ART than the drug-sensitive cells (Table 1). This indicates that ART is part of the general drug resistance phenomenon in these cells. Our hypothesis was that this resistance to ART could be overcome if the drug-resistant cells were preloaded with internalized TF by virtue of their elevated levels of TF receptor. Our data showed that as the TF concentration given to the resistant cells increased, the IC50 for ART was significantly reduced (Table 1). That the

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