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.
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.
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.
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.
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.