Curcumin Enhanced Busulfan-Induced Apoptosis through Downregulating the Expression of Survivin in Leukemia Stem-Like KG1a Cells

Abstract

Leukemia relapse and nonrecurrence mortality (NRM) due to leukemia stem cells (LSCs) represent major problems following hematopoietic stem cell transplantation (HSCT). To eliminate LSCs, the sensitivity of LSCs to chemotherapeutic agents used in conditioning regimens should be enhanced. Curcumin (CUR) has received considerable attention as a result of its anticancer activity in leukemia and solid tumors. In this study, we investigated the cytotoxic effects and underlying mechanisms in leukemia stem-like KG1a cells exposed to busulfan (BUS) and CUR, either alone or in combination. KG1a cells exhibiting BUS-resistance demonstrated by MTT and annexin V/propidium iodide (PI) assays, compared with HL-60 cells. CUR induced cell growth inhibition and apoptosis in KG1a cells. Apoptosis of KG1a cells was significantly enhanced by treatment with CUR+BUS, compared with either agent alone. CUR synergistically enhanced the cytotoxic effect of BUS. Seven apoptosis-related proteins were modulated in CUR- and CUR+BUS-treated cells analyzed by proteins array analysis. Importantly, the antiapoptosis protein survivin was significantly downregulated, especially in combination group. Suppression of survivin with specific inhibitor YM155 significantly increased the susceptibility of KG1a cells to BUS. These results demonstrated that CUR could increase the sensitivity of leukemia stem-like KG1a cells to BUS by downregulating the expression of survivin.

Figure 1

CD34⁺CD38⁻KG1a cells were insensitive to BUS. (a) KG1a cells were stained with FITC-conjugated CD38 antibody and PE-conjugated CD34 antibody and subjected to flow cytometry to analyze the purity of the CD34⁺CD38⁻ cells population. (b, c) KG1a cells were exposed to different concentrations of BUS for 24 or 48 h (c). MTT assay was performed (b) and apoptosis (c) was detected by annexin V/PI assay. Cells in the lower right quadrant represent early apoptosis and cells in the upper right quadrant represent late apoptosis. The graph displays the means ± SD of three independent experiments. ^∗∗ P < 0.01, ^∗∗∗ P < 0.001 (compared with untreated KG1a cells).

Figure 2

CUR suppressed cell growth, induced S phase arrest, and induced cell apoptosis in KG1a cells. (a) KG1a cells were treated with different concentrations of CUR for 24 or 48 h. MTT assays were performed. (b) KG1a cells were treated with different concentrations of CUR for 48 h and analyzed for DNA content by flow cytometry. (c) KG1a cells were treated with CUR and inoculated in methylcellulose for 14 days and then observed under a right microscope (magnification ×40). The graph displays means ± SD of three independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 (compared with control). (d) KG1a cells were treated with different concentrations of CUR for 48 h and analyzed by flow cytometry. The graph displays means ± SD of three independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01 (compared with control).

Figure 3

CUR increased BUS-induced apoptosis by downregulating procaspase-3 followed by PARP degradation in KG1a cells. (a, b) KG1a cells were treated with different concentrations of CUR or BUS alone or CUR + BUS for 48 h and analyzed by flow cytometry (a) and western blot (b). The graphs represent means ± SD of three independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 4

CUR synergistically enhanced the cytotoxic effect of BUS in KG1a cells. KG1a cells were exposed to CUR + BUS at different doses but in a constant ratio (CUR to BUS: 8 μM to 80 μM, 16 μM to 160 μM, and 32 μM to 320 μM, resp.) for 48 h examined by MTT assay. (a, b) CI-effect plots and median-effect plots were generated using Compusyn software. The points A, B, and C represent CI values for the three combination groups, respectively. (c) The graph displays means ± SD of three independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. (d) KG1a cells were treated with CUR or BUS alone or CUR + BUS for 48 h and analyzed with flow cytometry. The percentages of cells in S and G2/M phases were significantly higher in CUR + BUS group compared with the CUR- or BUS-alone group.

Figure 5

Expression of antiapoptosis protein survivin in KG1a cells. (a, b) KG1a cells were treated with CUR (16 μM), BUS (80 μM), or CUR + BUS for 48 h tested by protein arrays kit (a) as described in “methods.” The intensities of green fluorescence spots represent survivin expression. Survivin expression was significantly decreased in CUR and CUR + BUS groups, compared with controls, the same as the results analyzed by western blot analysis.

Figure 6

Suppression of survivin with YM155 could induce apoptosis and increase the sensitivity to BUS in KG1a cells. (a) KG1a cells were treated with different concentrations of YM155 for 24 and 48 h and examined by MTT assay. ^∗∗ P < 0.01 and ^∗∗∗ P < 0.001 (compared with control) and ^# P < 0.05 (compared with 48 h group). (b) KG1a cells exposed to YM155 (2.43 ng/mL) and BUS (80 μM) alone or CUR + BUS were analyzed by flow cytometry. The graph displays means ± SD of three independent experiments. ^∗∗ P < 0.01, ^∗∗∗ P < 0.001.

Figure 7

Mechanisms of CUR-induced apoptosis and enhanced sensitivity to BUS in KG1a cells, indicating the potential role of survivin.