Determination of cytotoxicity of doxorubicin (Dox), idarubicin (Ida), and curcumin (Cur) on leukemic cell viability by MTT assay. From the cell viability curve, it can be seen that Dox (Fig. 1A) and IDa (Fig. 1B) showed the highest cytotoxicity for EoL-1 cells, followed by U937, KG-1, and KG-1a cells. The cytotoxicity of all the treatments was assessed using an inhibitory concentration at a 50% growth (IC50) value. Ida demonstrated the greatest cytotoxic effects on KG-1a, KG-1, EoL-1, and U937 cells with IC50 values of 19.82 ± 1.80, 5.45 ± 0.89, 2.57 ± 0.32, and 4.73 ± 2.38 ng/mL, followed by Dox with IC50 values of 0.69 ± 0.12, 0.21 ± 0.02, 0.02 ± 0.01, and 0.08 ± 0.02 µg/mL, respectively. The IC50 values of all the chemotherapeutic drugs for the leukemic cell line models are shown in Table 1.
Table 1
The IC50 values of chemotherapeutic drugs and curcumin on KG-1a, KG-1, EoL-1, and U937 cells.
Treatment | IC50 value |
KG-1a | KG-1 | EoL-1 | U937 |
Idarubicin (Ida) (ng/mL) | 19.82 ± 1.80### | 5.45 ± 0.89*** | 2.57 ± 0.32*** | 4.72 ± 2.38*** |
Doxorubicin (Dox) (µg/mL) | 0.69 ± 0.12### | 0.21 ± 0.02*** | 0.02 ± 0.01***,## | 0.08 ± 0.02***,# |
Curcumin (Cur) (µg/mL) | 9.19 ± 0.49# | 7.31 ± 1.45* | 5.55 ± 0.46**,# | 3.55 ± 0.54***,## |
The significance of mean differences was assessed using one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to KG-1a cells. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to KG-1 cells. |
Cur was chosen to study the combination effect in order to improve the efficacy of Dox and Ida in AML treatment. For single treatment, Cur exhibited the highest cytotoxicity for EoL-1 cells, followed by U937, KG-1, and KG-1a cells (Fig. 1C). The IC50 values of Cur for KG-1a, KG-1, EoL-1, and U937 cells were 9.19 ± 0.49, 7.31 ± 1.45, 5.55 ± 0.46, and 3.55 ± 0.54 µg/mL, respectively (Table 1).
The IC50 values of Dox in leukemic stem cells (KG-1a and KG-1) were found to be significantly higher than for leukemic cells, EoL-1 and U937 cells. However, in the group of the leukemic stem cell line, KG-1 cells were substantially more responsive to Dox and Ida than KG-1a cells were, indicating that a high number of LSCs affected the chemotherapeutic treatment’s sensitivity. Furthermore, the IC50 values of Cur in KG-1a cells were considerably higher than those in the other cells. These findings demonstrated the drug resistance in LSCs against LCs and suggest that it might be possible to use to improve the potency of AML treatment.
Determination of cytotoxicity of combined doxorubicin–curcumin (Dox–Cur) and idarubicin–curcumin (Ida–Cur) on leukemic cell viability by MTT assay. In this study, various doses of Dox and Ida (ranging from 0 to IC80 values), and Cur from the previous results, were used to investigate the combination effects on the cell viability of AML cell lines. Dox was used to treat KG-1a (0–1.30 µg/mL), KG-1 (0–0.84 µg/mL), EoL-1 (0–0.08 µg/mL), and U937 (0–0.16 µg/mL) cells, and Ida was used to treat KG-1a (0–40.0 ng/mL), KG-1 (0–20.0 ng/mL), EoL-1 (0–8.0 ng/mL), and U937 (0–9.2 ng/mL) cells; these cells were cotreated with Cur at concentrations of the IC20 (condition 1), IC30 (condition 2), IC40 (condition 3), and IC50 (condition 4), respectively. The co-treatments of Dox–Cur and Ida–Cur exhibited higher cytotoxicity for KG-1a, KG-1, EoL-1, and U937 cells than single drug treatments did in dose dependent manners, according to the cell viability (see supplementary Fig. S1, S2). The IC50 value of each co-treatment demonstrated that curcumin did not only enhance the efficacy of doxorubicin and idarubicin in leukemic stem cells and non-leukemic stem cells, but it also decreased the doses of drugs in co-treatment when compared to a single treatment due to the decrease in IC50 values of drugs used in each treatment. The IC50 values of Dox-Cur and Ida-Cur at different conditions in each cell line are shown in the supplementary Table S1 – S4.
Synergistic effects of combination treatment. From the combination index (CI) calculation, formulations 3 and 5 of Dox–Cur showed a synergistic effect (CI < 1) on KG-1a and EoL-1 cells and an additive effect (CI = 1) on U937 cells at the IC50 values, while most of the Ida–Cur exhibited an antagonist effect (CI > 1) in all the leukemic cell lines as shown in Table 2. These data might have resulted from the fact that curcumin could not achieve high cytotoxicity for idarubicin, which had a very low usage dose. Thus, most of the Ida–Cur-treated samples exhibited an antagonist effect in AML leukemic cell lines.
Table 2
IC50 values of co-treatment of Dox-Cur and Ida–Cur on KG-1a, KG-1, EoL-1, and U937 cells
Cell line | Dox–Cur | CI value | Ida–Cur | CI value |
KG-1a | Dox + Cur 1 (4.5 µg/mL) | 1.08 | Ida + Cur 1(4.5 µg/mL) | 1.16 |
| Dox + Cur 2 (5.5 µg/mL) | 1.12 | Ida + Cur 2 (5.5 µg/mL) | 1.27 |
| Dox + Cur 3 (7.0 µg/mL) | 0.97 | Ida + Cur 3 (7.0 µg/mL) | 1.21 |
| Dox + Cur 4 (9.0 µg/mL) | 1.02 | Ida + Cur 4 (9.0 µg/mL) | 1.09 |
KG-1 | Dox + Cur 1 (3.5 µg/mL) | 1.36 | Ida + Cur 1 (3.5 µg/mL) | 1.44 |
| Dox + Cur 2 (4.5 µg/mL) | 1.07 | Ida + Cur 2 (4.5 µg/mL) | 1.55 |
| Dox + Cur 3 (6.0 µg/mL) | 1.04 | Ida + Cur 3 (6.0 µg/mL) | 1.28 |
| Dox + Cur 4 (7.5 µg/mL) | 1.07 | Ida + Cur 4 (7.5 µg/mL) | 1.11 |
EoL-1 | Dox + Cur 1 (3.0 µg/mL) | 1.23 | Ida + Cur 1 (3.5 µg/mL) | 1.27 |
| Dox + Cur 2 (4.0 µg/mL) | 1.12 | Ida + Cur 2 (4.0 µg/mL) | 1.24 |
| Dox + Cur 3 (4.5 µg/mL) | 0.92 | Ida + Cur 3 (4.5 µg/mL) | 0.85 |
| Dox + Cur 4 (5.5 µg/mL) | 1.03 | Ida + Cur 4 (5.5 µg/mL) | 1.03 |
U937 | Dox + Cur 1 (2.0 µg/mL) | 1.46 | Ida + Cur 1 (2.0 µg/mL) | 1.35 |
| Dox + Cur 2 (2.5 µg/mL) | 1.55 | Ida + Cur 2 (2.5 µg/mL) | 1.40 |
| Dox + Cur 3 (3.0 µg/mL) | 1.42 | Ida + Cur 3 (3.0 µg/mL) | 1.26 |
| Dox + Cur 4 (3.5 µg/mL) | 1.00 | Ida + Cur 4 (3.5 µg/mL) | 1.04 |
Effects of various conditions of combined treatment of Dox–Cur at concentration value of IC 20 on cell number and cell viability in FLT-3 protein expressing leukemic cells. Due to the synergistic and additive effect of Dox–Cur, all the conditions of co-treatment were chosen to investigate the effects on cell number and viability of FLT-3 protein- expressing AML leukemic cells, including KG-1a, KG-1 and EoL-1 cells. The IC20 values of the Dox treatments obtained from the previous section were used to cotreat for 48 h with Cur. Dox–Cur conditions 1, 2, 3, and 4 (Dox (ng/mL) + Cur (µg/mL)) of KG-1a cells were 15.0 + 4.5, 16.0 + 5.5, 12.0 + 7.0, and 8.0 + 9.0, respectively. The conditions for KG-1 cells were 22.0 + 3.5, 10.0 + 4.5, 7.0 + 6.0, and 6.0 + 7.5, while the conditions of EoL-1 cells were 3.0 + 3.0, 0.7 + 4.0, 0.5 + 4.5, and 0.4 + 5.5, respectively. The results show that Dox concentrations at IC20 values and all co-treatment conditions consistently reduced the cell number of all leukemic cell lines (see supplementary Fig. S3A, S4A, S5A). The total cell number of KG-1a cells (control group) was 2.59 × 105 cells/mL, and they gradually decreased to 1.85 × 105, 1.59 × 105, 1.42 × 105, 1.24 × 105, and 0.98 × 105 cells/mL in response to Dox and Dox–Cur treatment conditions 1, 2, 3, and 4, respectively. The total cell number of KG-1 cells decreased from 3.39 × 105 cells/mL (control group) to 2.58 × 105, 2.18 × 105, 2.00 × 105, 1.70 × 105, and 1.44 × 105 cells/mL in Dox and Dox–Cur treatment conditions 1, 2, 3, and 4, respectively. According to the cell number of EoL-1 cells after Dox and Dox–Cur conditions 1, 2, 3, and 4, the treatments reduced to 6.62 × 105, 5.77 × 105, 4.69 × 105, 4.01 × 105, and 3.27 × 105 cells/mL, respectively, compared to 12.07 × 105 cells/mL (control group). Cell viability of each sample was higher than 80% of the total cell count (see supplementary Fig. S3B, S4B, S5B).
The total cell number of Cur treatments at the IC20, IC30, IC40, and IC50 values were observed to gradually decrease in all the cell lines in a dose dependent manner (see supplementary Fig. S3C, S4C, S5C). Moreover, these data also corresponded to the decline in the cell numbers during the Dox–Cur treatment. The cell viability for each concentration of Cur was also higher than 80% of the total cell count (see supplementary Fig. S3D, S4D, S5D).
Effects of combined treatments of Dox-Cur at concentration value of IC 20 on FLT3 protein expressions in FLT-3 protein expressing leukemic stem cells and leukemic cells. FLT3 protein is a member of the class III receptor tyrosine kinase (RTK) family32. It is mostly found to overexpress on the cell surface of AML leukemic stem cells and leukemic cells and plays an important role in cell survival and proliferation of leukemic cell blasts33. Cur has previously been shown to have an inhibitory effect on FLT3 protein expression in many types of FLT-3 expressing leukemic cell lines, such as EoL-1 and MV4-1127. Thus, it was selected as a target protein for Dox–Cur treatment.
In this study, KG-1a, KG-1, and EoL-1 cells were treated with Dox and all conditions of co-treatment at a concentration value of IC20, and FLT3 protein expression levels were detected by Western blotting. The results show that Dox and Dox–Cur co-treatment could decrease FLT-3 protein expression. In KG-1a cells, the FLT3 protein levels of Dox and Dox–Cur treatment conditions 1, 2, 3, and 4 were decreased by 20.4 ± 8.8%, 51.6 ± 14.5%, 54.6 ± 12.2%, 80.2 ± 5.7%, and 92.2 ± 8.2%, respectively (Fig. 2A, 2C). For KG-1 cells, FLT3 proteins were gradually decreased by 2.7 ± 6.5%, 42.3 ± 2.9%, 55.0 ± 5.4%, 52.0 ± 7.3%, and 57.9 ± 11.5% in respond to Dox and Dox–Cur conditions 1, 2, 3, and 4, respectively (Fig. 3A, 3C). Similarly to KG-1a and KG1 cells, FLT3 protein expression in EoL-1 cells was reduced to 2.7 ± 5.6%, 10.7 ± 4.2%, 29.9 ± 6.8%, 35.2 ± 6.4%, and 43.7 ± 15.7% in response to Dox and Dox–Cur conditions 1, 2, 3, and 4, respectively, compared to the control group (100% expression level) (Fig. 4A, 4C). Additionally, the ability of each concentration of Cur in the combination treatment to suppress FLT3 protein expression was evaluated. The results demonstrate that all concentrations of single Cur treatments were able to decrease the protein expression levels compared to the control group in a dose-dependent manner (Fig. 2B, 2D, 3B, 3D, 4B, 4D).
Effects of combination treatments of various concentrations of Cur and a fixed concentration of Dox on cell number and viability in leukemic stem cells and leukemic cells. According to the results presented in previous section, Cur and Dox–Cur treatments could inhibit AML LSC and LC cell proliferation more effectively than Dox treatment alone. Three non-toxic concentrations within the range of Cur's IC20 value and a fixed concentration of Dox from Dox–Cur condition 1 were tested with KG-1a, KG-1, and EoL-1 cells for 48 h to confirm the impact of Cur on cell proliferation inhibition of Dox. The results demonstrate that the co-treatments of Dox–Cur significantly decreased the cell number of both cell lines in a dose dependent manner when compared to a single Dox treatment and control (Fig. 5). The cell number of KG-1a cells in the control group was 3.44 ⋅ 105 cells/mL, and decreased to 2.96 × 105, 2.21 × 105, 1.91 × 105, and 1.46 × 105 cells/mL in response to Dox, Dox + Cur at 4 µg/mL, Dox + Cur at 4.5 µg/mL, and Dox + Cur at 5 µg/mL, respectively (Fig. 5A). In addition, the cell number of KG-1 cells decreased from 4.18 × 105 cells/mL in the control group to 3.48 × 105, 2.93 × 105, 2.47 × 105, and 2.22 × 105 cells/mL in response to Dox, Dox + Cur at 3 µg/mL, Dox + Cur at 3.5 µg/mL, and Dox + Cur at 4 µg/mL, respectively (Fig. 5C). Moreover, the number of EoL-1 cells also decreased from 10.43 × 105 cells/mL in the control group to 8.99 × 105, 6.82 × 105, 6.08 × 105, and 4.94 × 105 cells/mL in response to the treatments of Dox, Dox + Cur at 2.5 µg/mL, Dox + Cur at 3 µg/mL, and Dox + Cur at 3.5 µg/mL, respectively (Fig. 5E). All samples demonstrated percentage of viable cells higher than 80% of the total cell count (Fig. 5B, 5D, 5F).
Effects of combination treatments of various concentrations of Cur and a fixed concentration of Dox on FLT3 protein expressions in leukemic stem cells. From the previous section, it can be observed that Cur and Dox-Cur co-treatments were more effective in suppressing FLT3 protein expression in all AML leukemic cell lines than single Dox treatment was. Moreover, the percentages of FLT3 protein in all conditions of co-treatments were also similar to those of Cur treatment alone. As the result, to confirm the effect of Cur in increasing the inhibitory effect of Dox on FLT3 expression, the co-treatment of Dox–Cur condition 1 was used. From the Western blotting, it was observed that all the non-toxic concentrations of Cur used in the study remarkably increased the efficacy of Dox in reducing the FLT3 protein expression in all three AML cell lines. The FLT3 expression level of KG-1a cells after treatment with Dox (15 ng/mL), Dox + Cur (4 µg/mL), Dox + Cur (4.5 µg/mL), and Dox + Cur (5 µg/mL) were decreased by 14.1 ± 5.2%, 35.8 ± 8.5%, 38.2 ± 3.3%, and 37.8 ± 7.0%, respectively, compared to the vehicle control (100% FLT3 protein expression level) (Fig. 6A, 6B). For KG-1 cells, the FLT3 protein levels were decreased by 17.9 ± 7.6%, 38.2 ± 13.0%, 39.7 ± 11.4%, and 47.2 ± 5.4% in response to Dox (22 ng/mL), Dox + Cur (3 µg/mL), Dox + Cur (3.5 µg/mL), and Dox + Cur (4 µg/mL), respectively (Fig. 6C, 6D) while the FLT3 protein expression levels in EoL-1 cells were reduced to 3.5 ± 8.9%, 10.2 ± 8.1%, 15.6 ± 7.1%, and 34.6 ± 8.9% in response to the treatments of Dox (2.8 ng/mL), Dox + Cur (2.5 µg/mL), Dox + Cur (3 µg/mL), and Dox + Cur (3.5 µg/mL), respectively, from 100% protein expression level of the vehicle control (Fig. 6E, 6F).