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.
Hematopoietic stem cell transplantation (HSCT) is currently one of the most effective methods of curing hematopoietic malignances [
The alkylating agent BUS is commonly applied in different conditioning regimens for HSCT, to eliminate the underlying leukemia cells and exert an immunosuppressive effect. However, BUS is associated with severe toxicities, including liver, lung, and skin toxicities, hemorrhagic cystitis, diarrhea, and mucositis [
Curcumin (CUR) is a polyphenol derived from the rhizomes of turmeric, which has received considerable attention as a result of its chemopreventive, chemotherapeutic, and chemosensitizing activities in leukemia and various solid tumors, via targeting multiple signaling pathways [
In this study, we explored the cytotoxic efficiencies and molecular mechanisms of CUR and BUS alone and in combination in KG1a cells.
Reagents include RPMI-1640 (Hyclone, SH30809.01B), fetal bovine serum (Hyclone, SH30084.03), penicillin and streptomycin (PAA, P11-010), CUR (Sigma, 458-37-7), DMSO (Amresco, 67-68-5), BUS (Sigma, 55-98-1), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Seebio, 298-93-1), hydroxypropyl methylcellulose (Amresco, 9004-65-3), anti-CD34-PE/CD38-FITC (BD Biosciences, USA), FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA), CycleTEST Plus DNA Kit (BD Biosciences, USA), anti-PARP (BD, USA, 1 : 500), anti-caspase-3 (CST, USA, 1 : 5000), anti-survivin (BD, USA, 1 : 5000), ym155 (SELLECK, 781661-94-7), Human Apoptosis Antibody Array Kit (RayBio, USA), electrophoresis apparatus trophoresis (Tanon EPS200), and LI-COR Odyssey Scanner (USA).
Human acute myeloid leukemia KG1a cells and human acute promyelocyte leukemia HL-60 cells were cultured in RPMI-1640 with 10% inactivated fetal bovine serum, penicillin, and streptomycin at 37°C under 5% CO2, which were kindly presented by Miaorong She (Department of Hematology, Guangdong General Hospital, Guangzhou, China).
Cells viability was estimated by MTT assay. KG1a and HL-60 cells in logarithmic phase at 5 × 105 cells/mL were incubated in 96-well plates in the presence or absence of the indicated test samples in a final volume of 0.2 mL for 24 h or 48 h at 37°C under 5% CO2. 20
Single-cell suspensions of 1.0 × 106 of KG1a and HL-60 cells were washed in PBS containing 2% fetal calf serum (FCS). The cells were resuspended in PBS and incubated for 30 min at 4°C with antibodies to surface antigens CD34 and CD38. Mouse IgG isotype was used as a control. The cells were then analyzed by flow cytometry.
Approximately 500 treated or untreated cells per well were cultured in RPMI 1640 medium supplemented with 0.9% methylcellulose and 20% fetal bovine serum (FBS) in a final volume of 1 mL at 37°C under 5% CO2. Colonies (
The apoptotic rates of KG1a and HL-60 cells were determined by annexin V binding assays, according the manufacturer’s instructions. Briefly, approximately 1.0 × 106 cells in 6-well plates were treated with various concentrations of the indicated test samples at 37°C under 5% CO2 for 48 h. The cells were then harvested to analyze apoptosis. Cells were washed twice with cold PBS and then resuspended in 1x Binding Buffer at a concentration of 1 × 106 cells/mL and 100
Approximately 1.0 × 106 cells in 6-well plates were treated with various concentrations of the indicated test samples at 37°C under 5% CO2 for 48 h. Cell cycle analysis was performed by flow cytometry using the CycleTEST Plus DNA Kit (BD Biosciences), according to manufacture’s instructions.
Total cellular proteins were isolated with lysis buffer (RIPA). Equal amounts of protein were subjected to 10% or 15% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies (anti-PARP, anti-caspase-3, and anti-survivin) over night at 4°C and then incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody at room temperature for 1-2 h. The protein bands were imaged using a chemiluminescence reagent (CTB, USA) and densities value of the bands was analyzed using Image J software, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH; HC301; 1 : 5000) as the internal reference.
The expression of 43 apoptosis-related proteins was analyzed using a Human Apoptosis Antibody Array Kit (RayBio, USA). Briefly, according to instructions, each of the capture antibodies was printed on the membranes, followed by addition of the treated or untreated cell lysate. After extensive washing, the membranes were incubated with a cocktail of biotin-conjugated anti-apoptotic protein antibodies. After incubation with the infrared fluorescent agent-streptavidin, the fluorescence signals were visualized using a LI-COR Odyssey Scanner.
The data ware represented as the mean ± standard deviation (SD) and analyzed using SPSS 13.0 and Graphpad Prism 5 software. Means of different groups were compared using one-way ANOVA followed by Bonferroni multiple comparison to evaluate the differences between two groups under multiple conditions. If the date failed the normality test, the Kruskal-Wallis one-way ANOVA on ranks was used for data that failed the normality test. A value of
The percentages of CD34+CD38− cells were 92.3% in KG1a cells, but no CD34+CD38− cells were detected among the HL-60 cells (Figure
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.
KG1a cells were treated with various concentrations of CUR (0–32
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.
We determined if CUR could increase BUS-induced apoptosis in KG1a cells by examining proapoptotic effects of CUR and BUS alone and in combination (CUR + BUS) using annexin V/PI. Apoptosis was significantly increased in CUR + BUS group, compared with CUR- or BUS-alone groups (Figure
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.
We investigated the ability of CUR to enhance the cytotoxic effect of BUS by treating KG1a cells with combinations of the two drugs at different doses but in a constant ratio (CUR to BUS: 8
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
We investigated the molecular mechanisms responsible for CUR-induced apoptosis and enhanced BUS-induced apoptosis in KG1a cells treated with 16
Expression of apoptosis-related proteins in various treated groups.
Name | Control | CUR | BUS | CUR + BUS | CUR/control | BUS/control | CUR + BUS/control |
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(fold-change) | (fold-change) | (fold-change) | |||||
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BAX | 11665.057 | 11534.453 | 11072.484 | 10875.680 | 0.989 | 0.949 | 0.932 |
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Bcl-w | 3934.754 | 3354.182 | 3464.131 | 3789.188 | 0.852 | 0.880 | 0.963 |
BID | 1241.608 | 1159.421 | 1473.127 | 1702.301 | 0.934 | 1.186 | 1.371 |
BIM | 8791.171 | 7763.556 | 8225.960 | 8295.705 | 0.883 | 0.936 | 0.944 |
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Caspase-8 | 6636.880 | 7681.166 | 7505.017 | 7338.462 | 1.157 | 1.131 | 1.106 |
CD40 | 7229.618 | 5707.767 | 6661.514 | 5874.868 | 0.789 | 0.921 | 0.813 |
CD40L | 16087.024 | 12956.135 | 15884.779 | 14392.403 | 0.805 | 0.987 | 0.895 |
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CytoC | 8302.835 | 6758.989 | 8156.269 | 7832.756 | 0.814 | 0.982 | 0.943 |
DR6 | 3370.080 | 2669.250 | 3008.736 | 2958.532 | 0.792 | 0.893 | 0.878 |
Fas | 25854.867 | 22694.082 | 24157.601 | 23693.574 | 0.878 | 0.934 | 0.916 |
FasL | 7155.526 | 6020.454 | 7420.907 | 7047.913 | 0.841 | 1.037 | 0.985 |
HSP27 | 2395.653 | 2109.392 | 2279.382 | 2807.833 | 0.881 | 0.951 | 1.172 |
HSP60 | 24408.943 | 20936.090 | 30040.496 | 23881.647 | 0.858 | 1.231 | 0.978 |
HSP70 | 6055.367 | 7532.267 | 6146.040 | 6971.961 | 1.244 | 1.015 | 1.151 |
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IGF-I | 1772.604 | 1407.585 | 1857.630 | 1717.974 | 0.794 | 1.048 | 0.969 |
IGF-II | 7991.872 | 8066.316 | 10003.085 | 9495.273 | 1.009 | 1.252 | 1.188 |
IGFBP-1 | 3060.239 | 2144.135 | 2680.707 | 2416.014 | 0.701 | 0.876 | 0.789 |
IGFBP-2 | 3750.645 | 2977.966 | 3901.504 | 3569.769 | 0.794 | 1.040 | 0.952 |
IGFBP-3 | 5321.179 | 3985.511 | 5547.657 | 5313.061 | 0.749 | 1.043 | 0.998 |
IGFBP-4 | 2012.843 | 1573.358 | 1736.271 | 1873.496 | 0.782 | 0.863 | 0.931 |
IGFBP-5 | 11366.442 | 9202.906 | 10929.497 | 10428.404 | 0.810 | 0.962 | 0.917 |
IGFBP-6 | 2354.116 | 1950.567 | 2340.662 | 2413.603 | 0.829 | 0.994 | 1.025 |
IGF-1sR | 5755.629 | 4286.285 | 5791.576 | 5056.269 | 0.745 | 1.006 | 0.878 |
Livin | 7478.838 | 6829.468 | 7818.628 | 7778.504 | 0.913 | 1.045 | 1.040 |
p21 | 17207.390 | 15850.718 | 18463.352 | 17993.517 | 0.921 | 1.073 | 1.046 |
p27 | 8486.943 | 7790.358 | 8890.430 | 8879.213 | 0.918 | 1.048 | 1.046 |
p53 | 9829.587 | 9303.164 | 11354.853 | 11263.882 | 0.946 | 1.155 | 1.146 |
SMAC | 9838.568 | 10157.840 | 11915.987 | 12549.047 | 1.032 | 1.211 | 1.275 |
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sTNF-R1 | 3284.761 | 2747.670 | 3781.346 | 3136.960 | 0.836 | 1.151 | 0.955 |
sTNF-R2 | 3504.793 | 2428.035 | 3118.079 | 2815.066 | 0.693 | 0.890 | 0.803 |
TNF-alpha | 2641.505 | 1771.889 | 2802.065 | 2499.200 | 0.671 | 1.061 | 0.946 |
TNF-beta | 6946.720 | 4871.952 | 6715.585 | 6430.648 | 0.701 | 0.967 | 0.926 |
TRAILR-1 | 3835.964 | 3031.569 | 4114.182 | 4102.643 | 0.790 | 1.073 | 1.070 |
TRAILR-2 | 7488.942 | 6315.272 | 7505.017 | 7691.701 | 0.843 | 1.002 | 1.027 |
TRAILR-3 | 4649.857 | 3717.494 | 4614.036 | 4605.376 | 0.799 | 0.992 | 0.990 |
TRAILR-4 | 4613.933 | 3900.142 | 4694.541 | 4706.646 | 0.845 | 1.017 | 1.020 |
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KG1a cells were treated with CUR (16
Expression of antiapoptosis protein survivin in KG1a cells. (a, b) KG1a cells were treated with CUR (16
We clarified the role of CUR-induced survivin downregulation in sensitization of KG1a cells to BUS by suppressing survivin expression using the specific inhibitor YM155. The proapoptotic effect and sensitivity to BUS were evaluated by flow cytometry. The cytotoxic activity of YM155 in KG1a cells was detected by MTT assays. YM155 exhibited time- and dose-dependent growth-inhibitory effects in KG1a cells (Figure
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.
LSCs were a rare population of cells in patients with leukemia. They possess characteristics of self-renewal, chemotherapy resistance, and immune resistance [
CUR and its analogs have been showed to suppress the growth of various leukemia cells, including U937 cells [
Insensitivity of LSCs to conditioning chemotherapeutic drugs such as BUS is a major reason for leukemia relapse after HSCT. In this study, KG1a cells displayed resistance to BUS, indicated by a lack of apoptosis induction. We there explored the effects of the combination of CUR and BUS on apoptosis in KG1a cells. Encouragingly, CUR markedly enhanced BUS-induced apoptosis, as confirmed by annexin V/PI and western blot analysis. Similarly, the combination of various concentrations of CUR and BUS produced a synergistic antiproliferation effect in KG1a cells. Accumulating evidence suggests that CUR potentiates the effect, including enhancing the antiapoptotic effects of chemotherapeutic drugs such as 5-fluorouracil, bortezomib, FOLFOX, and paclitaxel
The results of apoptosis arrays showed that seven apoptosis-related proteins were significantly modulated in KG1a cells treated with CUR and CUR + BUS (Figure
Mechanisms of CUR-induced apoptosis and enhanced sensitivity to BUS in KG1a cells, indicating the potential role of survivin.
This study also demonstrated that survivin expression was downregulated by CUR and CUR + BUS (Figures
In summary, this study demonstrated underlying new mechanisms whereby CUR may overcome BUS insensitivity by downregulating survivin in leukemia stem-like KG1a cells. CUR, alone or in combination with BUS, could be a potential anti-LSCs agent for preventing leukemia relapse and reducing the NRM after HSCT. BUS is currently still widely used in the pretreatment of HSCT, but it shows significant side effects and carcinogenicity in patients undergoing HSCT, resulting in danger of being replaced by other conditioning regimens. CUR may solve these issues by combining BUS in the conditioning regimen.
The authors have no competing interests to declare.
Guangyang Weng and Yingjian Zeng contributed equally to this work.
The authors would like to thank Yanjie HE (Laboratory of Hematology, Zhujiang Hospital) for assistance with experimental techniques, Pingfang Xia for revising the paper, and Miaorong She for KG1a cells. This work was supported by the National Natural Science Foundation of China (Grant no. 30973454).