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. 2007 Aug 1;2(8):e693.
doi: 10.1371/journal.pone.0000693.

Artesunate induces ROS-mediated apoptosis in doxorubicin-resistant T leukemia cells

Thomas Efferth  1 , Marco Giaisi, Annette Merling, Peter H Krammer, Min Li-Weber
Affiliations

Artesunate induces ROS-mediated apoptosis in doxorubicin-resistant T leukemia cells

Thomas Efferth et al. PLoS One. .

Abstract

Background: A major obstacle for successful cancer treatment often is the development of drug resistance in cancer cells during chemotherapy. Therefore, there is an urgent need for novel drugs with improved efficacy against tumor cells and with less toxicity on normal cells. Artesunate (ART), a powerful anti-malarial herbal compound, has been shown to inhibit growth of various tumor cell lines in vitro and of xenografted Kaposi's sarcoma in mice in vivo. However, the molecular mechanisms by which ART exerts its cytotoxicity have not been elucidated. The ART-class of anti-malarial compounds is attractive due to their activity against multidrug-resistant Plasmodium falciparum and Plasmodium vivax strains. Another salient feature of these compounds is the lack of severe side effects in malaria patients.

Methodology and principal findings: In this study, we used T-cell leukemias as a model system to study the molecular mechanisms of ART-induced apoptosis. The most typical anticancer drugs are DNA intercalators such as Doxorubicin. To investigate drug sensitivity and resistance, we chose a Doxorubicin-resistant leukemia cell line and investigated the killing effect of ART on these cells. We show that ART induces apoptosis in leukemic T cells mainly through the mitochondrial pathway via generation of reactive oxygen species (ROS), a mechanism different from Doxorubicin. This is confirmed by the fact that the antioxidant N-Acetyle-Cysteine (NAC) could completely block ROS generation and, consequently, inhibited ART-induced apoptosis. Therefore, ART can overcome the Doxorubicin-resistance and induce the Doxorubicin-resistant leukemia cells to undergo apoptosis. We also show that ART can synergize with Doxorubicin to enhance apoptotic cell death in leukemic T cells. This synergistic effect can be largely explained by the fact that ART and Doxorubicin use different killing mechanisms.

Conclusions: Our studies raise the possibility to develop ART in combination with other established anticancer drugs which induce apoptosis through the pathways or mechanisms different from ART.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ART induces apoptosis in malignant T cells.
Jurkat, Hut-78, Molt-4 and CEM leukemic T cells were incubated with different doses of ART for 24 or 48 h as indicated. The apoptotic cell death was determined either by FSC/SSC index for changes in cell size and granularity (A) or by DNA fragmentation (B). Results are representative of three independent experiments determined in triplicates.
Figure 2
Figure 2. ART induces apoptosis through the intrinsic (mitochondria) pathway.
(A) The death receptor system is not required for ART-induced apoptosis. Jurkat cells deficient in FADD (FADD−/−), caspas-8 (casp-8−/−), or over-expressing Bcl-2 and parental (A3) Jurkat cells were treated with different does of ART. Apoptotic cell death was determined by FSC/SSC in triplicates. (B) ART-induced apoptosis involves caspases. Jurkat cells were treated with ART (4 µg/ml) in the presence or absence of 50 µM of pan-caspase inhibitor zVAD-fmk for 48 h. Apoptotic cell death was determined by DNA fragmentation in triplicates. (C) ART induces cytochrome c release and activation of caspase-2, 3 and 9. CEM leukemia cells were treated with 1 µg/ml ART for different times as indicated. Cell lysates were subjected to Western blotting with antibodies against cytochrome c, caspase-2, 3, 8, 9, PARP, and control antibodies against tubulin. Data are representative of three independent experiments.
Figure 3
Figure 3. ROS mediate ART-induced apoptosis in leukemia cells.
(A) Jurkat cells were treated with 4 µg/ml of ART for different times as indicated and the redox status was monitored by the oxidation-sensitive fluorescent dyes for H2O2. (B) CEM cells were treated with 0.5 µg/ml doses ART. After 30 min, the redox status was measured as in (A). (C) and (D) ART-induced apoptosis was blocked by the antioxidant NAC. Jurkat (C) and CEM (D) cells were treated with ART in the presence (15 mM) or absence of NAC for 24 h. Apoptotic cell death was analyzed by FSC/SSC in triplicates. Results are representative of four independent experiments.
Figure 4
Figure 4. ART induces Doxorubicin-resistant leukemic cells to undergo apoptosis by a mechanism different from the one induced by Doxorubicin.
(A) ART induces apoptosis in Doxorubicin-resistant leukemic cells. Doxorubicin-resistant (CEM-DoxR) and parental (CEM-parental) CEM cells were treated with either 1 µg/ml of ART or 0.5 µg/ml of Doxorubicin for 24 h. Apoptosis was determined by FSC/SSC (left panel) and DNA fragmentation (right panel). Results are representative of two independent experiments. (B) NAC inhibits ART-induced apoptosis in Doxorubicin-resistant leukemic cells. CEM-DoxR cells were treated with different doses of ART in the presence or absence of NAC (15 mM). Apoptotic cell death was determined by DNA fragmentation in triplicates. (C) and (D) NAC does not inhibit Doxorubicin-induced apoptosis. CEM-parental (C) and Jurkat (D) cells were treated either with ART or Doxorubicin in the presence or absence of NAC. Apoptotic cell death was determined by DNA fragmentation. Results are representative of four independent experiments. The p value was determined by the statistic program of Microsoft Excel. (E) ART induces ROS generation in CEM-DoxR cells. CEM-DoxR cells were treated with 4 µg/ml of ART in the presence or absence of NAC (15 mM) for 30 min. The redox status was measured as in figure 3. (F) Doxorubicin does not induce ROS in leukemic T cells. CEM-DoxR, CEM-parental and Jurkat cells were treated with 0.5 µg/ml of Doxorubicin in the presence or absence of NAC for 30 min. The redox status was measured as in (E). Results are representative of three independent experiments. (G) Doxorubicin does not interfere with the redox assay. CEM cells were treated with Doxorubicin (2 µg/ml) or ART (4 µg/ml) alone or in combination for 30 min. The redox status was measured as in F.
Figure 5
Figure 5. ART sensitizes malignant T cells to Doxorubicin-induced apoptosis.
Jurkat (A) and CEM (B) cells were treated with combinations of different doses of ART and Doxorubicin for 24 h as indicated. The drugs were added at the same time. Apoptotic cell death was quantified by DNA fragmentation in triplicates. Results are representative of three independent experiments. The p value was determined by a two-way ANOVA test with factors Dox-, ART-, and their interaction. For Jurkat cells, the p values are p<0.0001 for Dox- and ART-treatment and p = 0.0006 for the combination treatment. For CEM cells, the p values are p<0.0001 for Dox-, ART- as well as the combination treatment.
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