Co-Treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells

This study aims to enhance efficacy and reduce toxicity of the combination treatment of a drug and curcumin (Cur) on leukemic stem cell and leukemic cell lines, including KG-1a and KG-1 (FLT3+ LSCs), EoL-1 (FLT3+ LCs), and U937 (FLT3− LCs). The cytotoxicity of co-treatments of doxorubicin (Dox) or idarubicin (Ida) at concentrations of the IC10–IC80 values and each concentration of Cur at the IC20, IC30, IC40, and IC50 values (conditions 1, 2, 3, and 4) was determined by MTT assays. Dox–Cur increased cytotoxicity in leukemic cells. Dox–Cur co-treatment showed additive and synergistic effects in several conditions. The effect of this co-treatment on FLT3 expression in KG-1a, KG-1, and EoL-1 cells was examined by Western blotting. Dox–Cur decreased FLT3 protein levels and total cell numbers in all the cell lines in a dose-dependent manner. In summary, this study exhibits a novel report of Dox–Cur co-treatment in both enhancing cytotoxicity of Dox and inhibiting cell proliferation via FLT3 protein expression in leukemia stem cells and leukemic cells. This is the option of leukemia treatment with reducing side effects of chemotherapeutic drugs to leukemia patients.


Introduction
Leukemia is among the top 10 cancers diagnosed globally. It is a group of cancers of early blood-forming cells, which are characterized by the uncontrolled production and accumulation of blast or immature abnormal blood cells in the peripheral blood and bone marrow. Leukemia can be divided into four major types according to the stage and cell of origin: acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL). AML is the most common type of acute leukemia in adults, with the highest incidence and death rate in both sexes. It can be distinguished by clonal expansion of abnormal myeloid blasts in bone marrow, peripheral blood, or other tissues. According to recent data, 15-25% of AML patients fail to achieve complete remission (CR) due to chemotherapy resistance and may show relapse, with the overall 5-year survival rate of approximately 40% [1,2]. Moreover, between 10 and 40% of newly diagnosed AML patients do not achieve CR with intensive KG1), FLT3-overexpressing leukemic cells (EoL-1), and non FLT3-expressing leukemic cells (U937). FLT3 protein is a member of the class III receptor tyrosine kinase (RTK) family [38]. It is overexpressed 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 blasts [39]. 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 MV4-11 [32]. Thus, it was selected as a target protein for Dox-Cur treatment. Moreover, the effects of co-treatments on FLT3 protein expression and total cell numbers were determined.

Determination of Cytotoxicity of Doxorubicin (Dox), Idarubicin (Ida), and Curcumin (Cur) on Leukemic Cell Viability by MTT Assay
A cell viability curve demonstrated Dox ( Figure 1A) and IDa ( Figure 1B) exhibited 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 (IC 50 ) value. Ida demonstrated the greatest cytotoxic effects on KG-1a, KG-1, EoL-1, and U937 cells with IC 50 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 IC 50 values of 0.69 ± 0.12, 0.21 ± 0.02, 0.02 ± 0.01, and 0.08 ± 0.02 µg/mL, respectively. The IC 50 values of all the chemotherapeutic drugs for the leukemic cell line models are shown in Table 1. KG1), FLT3-overexpressing leukemic cells (EoL-1), and non FLT3-expressing leukemic cells (U937). FLT3 protein is a member of the class III receptor tyrosine kinase (RTK) family [38]. It is overexpressed 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 blasts [39]. 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 MV4-11 [32]. Thus, it was selected as a target protein for Dox-Cur treatment. Moreover, the effects of co-treatments on FLT3 protein expression and total cell numbers were determined.
The IC 50 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, KG-1 leukemic stem cells were substantially more responsive to Dox and Ida than KG-1a cells, indicating a high number of LSCs affected the chemotherapeutic treatment's sensitivity. Furthermore, the IC 50 values of Cur in KG-1a cells were considerably higher than those in the other cells. These findings demonstrated the drug resistance in LSCs compared with LCs and suggested a possible route to improve the potency of traditional AML chemotherapeutics.

Synergistic Effects of Combination Treatment
Using a combination index (CI) calculation, formulations 3 and 4 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 IC 50 values, while most of the Ida-Cur treatments exhibited an antagonist effect (CI > 1) in all the leukemic cell lines ( Table 2). It seemed Cur could not achieve high cytotoxicity in the Ida-Cur combination since Ida had an extremely low effective dose. Thus, most of the Ida-Cur-treated samples exhibited an antagonistic effect in AML leukemic cell lines.  Figures S3A-S5A). The total cell number of KG-1a cells (control group) was 2.59 × 10 5 cells/mL, and cell number gradually decreased to 1.85 × 10 5 , 1.59 × 10 5 , 1.42 × 10 5 , 1.24 × 10 5 , and 0.98 × 10 5 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 × 10 5 cells/mL (control group) to 2.58 × 10 5 , 2.18 × 10 5 , 2.00 × 10 5 , 1.70 × 10 5 , and 1.44 × 10 5 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 × 10 5 , 5.77 × 10 5 , 4.69 × 10 5 , 4.01 × 10 5 , and 3.27 × 10 5 cells/mL, respectively, compared with 12.07 × 10 5 cells/mL (control group). Cell viability of each sample was higher than 80% of the total cell count (see supplementary Figures S3B-S5B). The total cell number of Cur treatments at the IC 20 , IC 30 , IC 40 , and IC 50 values were observed to gradually decrease in all the cell lines in a dose dependent manner (see supplementary Figures S3C-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 Figures S3D-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) family [38]. It is overexpressed 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 blasts [39]. 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 MV4-11 [32]. Thus, it was selected as a target protein for Dox-Cur treatment.

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
Thus far, Cur and Dox-Cur treatments were found to inhibit AML LSC and LC cell proliferation more effectively than Dox treatment alone. Thus, three non-toxic concentrations within the range of Cur IC 20 value and a fixed concentration of Dox from Dox-Cur condition 1 were tested using KG-1a, KG-1, and EoL-1 cells for 48 h. Co-treatments of Dox-Cur significantly decreased the cell number of both cell lines in a dose-dependent manner when compared with a single-Dox treatment and control ( Figure 5). The cell number of KG-1a cells in the control group was 3.44 × 10 5 cells/mL, and decreased to 2.96 × 10 5 , 2.21 × 10 5 , 1.91 × 10 5 , and 1.46 × 10 5 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 ( Figure 5A). In addition, the cell number of KG-1 cells decreased from 4.18 × 10 5 cells/mL in the control group to 3.48 × 10 5 , 2.93 × 10 5 , 2.47 × 10 5 , and 2.22 × 10 5 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 ( Figure 5C). Moreover, the number of EoL-1 cells also decreased from 10.43 × 10 5 cells/mL in the control group to 8.99 × 10 5 , 6.82 × 10 5 , 6.08 × 10 5 , and 4.94 × 10 5 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 ( Figure 5E). All samples exhibited viable cells higher than 80% of the total cell count ( Figure 5B,D,F).     Cur (4 µg/mL), Cur (4.5 µg/mL), and Cur (5.5 µg/mL) for 48 h. The data are shown as Mean ± SD from 3 independent experiments. The significance of mean differences was assessed using one-way ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with VC. ## p < 0.01 and ### p < 0.001 compared with single-Dox treatment.

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
Thus far, Cur and Dox-Cur treatments were found to inhibit AML LSC and LC cell proliferation more effectively than Dox treatment alone. Thus, three non-toxic concentrations within the range of Cur IC20 value and a fixed concentration of Dox from Dox-Cur condition 1 were tested using KG-1a, KG-1, and EoL-1 cells for 48 h. Co-treatments of Dox-Cur significantly decreased the cell number of both cell lines in a dose-dependent manner when compared with a single-Dox treatment and control ( Figure 5). The cell number of KG-1a cells in the control group was 3.44 × 10 5 cells/mL, and decreased to 2.96 × 10 5 , 2.21 × 10 5 , 1.91 × 10 5 , and 1.46 × 10 5 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 ( Figure 5A). In addition, the cell number of KG-1 cells decreased from 4.18  10 5 cells/mL in the control group to 3.48 × 10 5 , 2.93 × 10 5 , 2.47 × 10 5 , and 2.22 × 10 5 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 ( Figure 5C). Moreover, the number of EoL-1 cells also decreased from 10.43  10 5 cells/mL in the control group to 8.99 × 10 5 , 6.82 × 10 5 , 6.08 × 10 5 , and 4.94 × 10 5 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 ( Figure 5E). All samples exhibited viable cells higher than 80% of the total cell count ( Figure 5B,D,F). The significance of mean differences was assessed using one-way ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with VC. # p < 0.05 and ## p < 0.01 compared with single-Dox treatment. The data are shown as mean ± SD from 3 independent experiments. The significance of mean differences was assessed using one-way ANOVA. * p < 0.05 and *** p < 0.001 compared with VC. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with single-Dox treatment.

Discussion
Doxorubicin (Dox) and idarubicin (Ida) are the standard chemotherapy treatments for AML patients. These compounds can destroy leukemic cells by binding to DNA and inhibiting topoisomerase II activity in DNA transcription, thereby triggering apoptosis or autophagy [16,40,41]. The cytotoxic activity of these anthracyclines was determined in each leukemic cell line by MTT assays. Both drugs showed the greatest cytotoxicity for EoL-1 cells, followed by U937, KG-1, and KG-1a cells. The inhibitory concentrations at cell growth values of 50 (IC50) for Ida on KG-1a and KG-1 cells were 19.82 ± 1.80 and 5.45 ± 0.89 ng/mL, respectively. In contrast, Dox showed lower cytotoxicity than Ida with IC50 values of 0.65 ± 0.13 and 0.21 ± 0.02 μg/mL, respectively. Ida and Dox doses in a previous report in vitro were 1100 ng/mL and 0.11.5 μg/mL, respectively, in normal and leukemic human bone marrow progenitors [42]. Drug doses were within the range of our studies. However, doses used in this study presented activity at low levels when compared with The data are shown as mean ± SD from 3 independent experiments. The significance of mean differences was assessed using one-way ANOVA. * p < 0.05 and *** p < 0.001 compared with VC. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with single-Dox treatment.

Discussion
Doxorubicin (Dox) and idarubicin (Ida) are the standard chemotherapy treatments for AML patients. These compounds can destroy leukemic cells by binding to DNA and inhibiting topoisomerase II activity in DNA transcription, thereby triggering apoptosis or autophagy [16,40,41]. The cytotoxic activity of these anthracyclines was determined in each leukemic cell line by MTT assays. Both drugs showed the greatest cytotoxicity for EoL-1 cells, followed by U937, KG-1, and KG-1a cells. The inhibitory concentrations at cell growth values of 50 (IC 50 ) for Ida on KG-1a and KG-1 cells were 19.82 ± 1.80 and 5.45 ± 0.89 ng/mL, respectively. In contrast, Dox showed lower cytotoxicity than Ida with IC 50 values of 0.65 ± 0.13 and 0.21 ± 0.02 µg/mL, respectively. Ida and Dox doses in a previous report in vitro were 1-100 ng/mL and 0.1-1.5 µg/mL, respectively, in normal and leukemic human bone marrow progenitors [42]. Drug doses were within the range of our studies. However, doses used in this study presented activity at low levels when compared with the previous report. Looking at intravenous injections of Ida and Dox in a rat model, the maximum tolerated doses of both drugs were 3 mg/kg and 0.75 mg/kg per injection. Moreover, the cardiac toxicity of Ida remained significantly lower than that of Dox [43].
Next, studies were conducted to determine the effect of combining the natural product curcumin with these AML chemotherapeutics. Cur tended to increase the cytotoxicity of Dox and Ida on all leukemic cell lines; however, several conditions of Dox + Cur cotreatment showed additive and synergistic manners, whereas most conditions of Ida + Cur co-treatment suggested that Cur was antagonistic. Ida appears to have a mechanism similar to Dox in that it can intercalate DNA and block topoisomerase II activity [44], thus differences may be due to the chemical structures of Ida and Dox. The absence of the methoxyl group at position 4 of idarubicin's structure increased the lipophilicity and rate of cellular uptake, leading to greater toxicity than that of daunorubicin or doxorubicin [45]. Furthermore, Ida is relatively more potent than doxorubicin in suppressing the growth of low or nonproliferating progenitor cells [42]. Thus, it is likely that the concentrations of Cur used in the combination treatment were too low to achieve a synergistic effect when combined with Ida. As a result, increasing the concentration of Cur combined with Ida may be explored in future research.
In addition, the cytotoxicity of curcumin (Cur), a natural substance with chemosensitizing and chemoprotective activities [26], was also examined with four leukemic cell lines by MTT assay. Cur demonstrated the highest cytotoxic effect on U937 cells, followed by EoL-1, KG-1, and KG-1a cells. Thus, Cur was selected as a supplementary substance for enhancing the efficiency and decreasing the toxicity of anthracycline drugs in this study.
Appropriate concentrations of Dox, Ida, and Cur were chosen for the combination effect, based on the results of the preliminary screening study. The co-treatments of Ida and Dox at concentrations in the range of IC 10 to IC 50 values and low concentrations of Cur at 1, 2, and 3 µg/mL did not show a different effect on cell viability of KG-1a and KG-1 cells, when compared with a single treatment (data not shown). Thus, KG-1a, KG1-a, EoL-1, and U937 cells were treated with Dox and Ida at the concentration values of IC 10 -IC 80 combined with Cur at concentrations of IC 20 , IC 30 , IC 40 , and IC 50 values, respectively. To investigate the combination effect, the percentage of cell viability of each treatment was calculated and compared with that for the single drug and vehicle control, and the inhibitory concentrations at 20% (IC 20 ) and 50% (IC 50 ) were determined.
Co-treatment of Dox-Cur and Ida-Cur tended to increase the cytotoxicity for KG-1a, KG-1, EoL-1, and U937 cells in dose-dependent manners as compared with single-drug treatment. Moreover, Cur also enhanced the cytotoxic efficacy for both chemotherapeutic drugs in dose dependent manners, based on the lower IC 50 values of anthracyclines used in co-treatment in each cell line.
Dox and Ida are usually ineffective due to an increase in LSCs, drug resistance, and relapse in AML patients. In this study, the natural substance Cur was found to improve the cytotoxicity of Dox and Ida in all the cell lines due to its anti-leukemic (apoptotic induction) [46] and chemosensitizing (decreasing MDR-1 gene expression) [34] activities. For these reasons, Cur improved both chemotherapies by lowering IC 50 values for Dox and Ida in co-treatments when compared with single drug treatments.
It is notable that effective doses of the co-treatments used to treat KG-1a cells were higher than those for KG-1, EoL-1, and U937 cells. KG-1a and KG-1 cells are leukemic stem cell lines with a high percentage of leukemic stem cells (~95% and~55%, respectively). These cells are well-known for their chemotherapy resistance, which includes a prolonged stage G 0 of the cell cycle and high expression of the drug efflux pump. Since the EoL-1 and U937 cells lack these stem cell features, they were more vulnerable to the co-treatments.
The combination treatment of Dox-Cur showed synergistic and additive cytotoxic effects on both AML leukemic stem cell lines (KG-1a and KG-1 cells) and AML leukemic cell lines (EoL-1 and U937 cells). Even though only Dox-Cur condition 3 showed synergism on KG-1a and EoL-1 cells, Cur was able to lower chemotherapeutic agent doses. The combination treatment also reduced the concentration at the IC 50 value of Dox in each cell line which could be a useful formulation to decrease the cytotoxicity of Dox on normal cells. However, the poor solubility and short biological half-life of Cur, as well as the non-specific activity of Dox, may result in low absorption and cytotoxicity of these drugs in tumor cells [16,27].
FLT3 is a key driver of AML, and its mutations are associated with the development of high risk of relapse in patients. Previous studies demonstrated that Cur has an inhibitory effect on FLT3 protein expression in leukemic cells [32]. Thus, the combination of Dox and Cur for AML treatment may lead to FLT3 protein expression reduction, which in turn could denote reduced proliferation of leukemic cells.
In this study, non-toxic doses at IC 20 of the four conditions of Dox-Cur co-treatments decreased the cell number and showed a higher inhibitory effect on FLT3 protein expression than single Dox in both stem cell and leukemic cell lines. However, when compared with Cur alone, none of the co-treatments showed any differences. To confirm, Dox-Cur condition 1 was selected for the inhibitory effect of various concentrations of Cur with a fixed concentration of Dox on cell number and FLT3 protein expression. Dox-Curtreated samples exhibited a lower cell number and FLT3 protein level than treatment with Dox alone in all cell lines. It is possible that Cur was the main compound in cotreatment that could suppress the FLT3 protein expression in a dose-dependent manner, leading to a decrease in cell proliferation, while the main functions of Dox, in order to eradicate tumor cells, involved inhibiting cell cycle progression, producing oxidation, and inducing apoptosis, which is unrelated to the inhibition of FLT3 protein expression [16]. In addition, these functions of Dox could affect cell proliferation, resulting in the decrease in cell number in single-Dox-treated samples. The co-treatment had an inhibitory effect on FLT3 protein expression. Notably, the IC 20 values of Dox in Dox-Cur condition 1 (Dox 15 ng/mL + Cur 4.5 µg/mL) and 2 (Dox 16 ng/mL + Cur 5.5 µg/mL) in KG-1a cells were not reduced in a dose-dependent response to the Cur increase. This might be because the chosen Cur concentration was too low, making the lowering of the Dox concentration in the reaction impractical, as shown by the cell number and FLT3 protein expression level.
Co-treatment likely assists in enhancing the cytotoxic effects of Dox by inhibiting the cell proliferation activity of AML leukemic stem cells and leukemic cells as a result of the decrease in the cell proliferation rate of each co-treatment condition compared with the single treatment and vehicle control. These findings are consistent with a previous study in which the combination of Dox with SU11657, a FLT3 inhibitor, increased the survival rate of APL mice and overcame resistance to traditional chemotherapies in AML [47].

Cytotoxicity of Single Doxorubicin, Idarubicin, and Curcumin (Curcuminoid Mixture) on Leukemic Stem Cell and Leukemic Cell Viability by MTT Assay
KG-1a and KG-1 cell lines were adjusted to 1.5 × 10 4 cells, while EoL-1 and U937 cells were adjusted to 3.0 × 10 4 and 1.0 × 10 4 cells in 100 µL of complete medium, and then seeded into flat-bottom 96-well plate and incubated at 37 • C under 5% CO 2 atmosphere for 24 h. Following that, doxorubicin (Dox), idarubicin (Ida), and curcumin (Cur) were diluted in 100 µL of medium with the 2-fold dilution technique and applied to the cells to obtain the final concentrations from 0.001 to 2 µg/mL for Dox and Ida and 1.56 to 50 µg/mL for Cur for 48 h. Complete medium and DMSO were used as cell control and vehicle control, respectively. Afterwards, 100 µL of medium was removed, and 15 µL of MTT dye solution was added and further incubated for 4 h. After removing the supernatant, 200 µL of DMSO were added to dissolve the formazan crystals (cell viability indication). The optical density was measured using an ELISA plate reader at 578 nm with the reference wavelength at 630 nm. The percentage of cell survival was calculated from the absorbance of test and control wells using the equation below, and the inhibitory concentration at 20% (IC 20 40 , and IC 50 values, as well as DMSO, to prepare the combination and single drug treatments, respectively. All the treatments were added to the cells and incubated for 48 h. The cell viability in each treatment was determined by the MTT assay, as described in Section 4.3.

Synergistic Effects of Combination Treatment
The combination index (CI) is used to quantitatively define the synergistic (CI < 1), additive (CI = 1), and antagonist effect (CI > 1) of a drug-drug interaction [48]. It can be calculated by using the following equation:

CI =
Dose of drug in combination at ICx Dose of sin gle drug at ICx × Dose of Cur in combination at ICx Dose of sin gle Cur at ICx ICx = The concentrations required to produce the given effect, such as IC 50 .

Cell Number and Cell Viability of FLT3-Exprssing Cells Determined by the Trypan Blue Exclusion Method
KG-1a, KG-1, and EoL-1 cells were adjusted to 1.5 × 10 5 and 3.0 × 10 5 cells/mL, respectively, and incubated with non-toxic concentrations (IC 20 ) of Dox, Cur, and combination treatment at 37 • C under 5% CO 2 atmosphere for 48 h. Then, the treated cells were collected, and their cell number and percent of cell viability were estimated using the trypan blue exclusion method by mixing the cells and the 0.4% trypan blue solution in a ratio of 1:1; following this, the cells were counted in a hemacytometer under a light microscope.

Western Blotting
KG-1a, KG-1, and EoL-1 cells were prepared and treated with Dox, Cur, and the co-treatment as discussed in Section 4.6. After that, the cells were harvested after 48 h of incubation, and the whole proteins were extracted using RIPA buffer. The protein concentration was measured with the Folin-Lowry method. The protein lysates were separated through 7.5% SDS-PAGE and then transferred to PVDF membranes. For the antibody-protein reaction step, the membrane was cut to separate FLT3 (target protein) and GAPDH (internal control protein), and then blocked in 5% skim milk. The part of the membrane containing FLT3 protein was probed with rabbit polyclonal anti-FLT3 at a dilution 1:1000, whereas the part containing GAPDH was probed with rabbit polyclonal anti-GAPDH antibody at a dilution of 1:1000. After that, the reaction was followed by a 1:15,000 dilution of HRP-conjugated goat anti-rabbit IgG. The proteins were visualized using Luminata™ Forte Western HRP substrate. Finally, the protein band signal (chemiluminescence) was detected by X-ray film or Fluorchem E Western blot and gel imager (ProteinSimple, San Jose, CA, USA) and quantified using a scan densitometer (Bio-Rad, Hercules, CA, USA) or Fluorchem Q program (ProteinSimple, CA, USA).

Statistical Analysis
The average of triplicate experiments and standard derivation (SD) were used for quantification. The levels of target protein expressions were compared with those of the vehicle control in each experiment. The results are shown as mean ± SD. The differences between the means of each sample were analyzed by one-way analysis of variance (one-way ANOVA). Statistical significance was considered at p < 0.05, p < 0.01, and p < 0.001.

Conclusions
Overall, anthracyclines (Dox and Ida) and Cur, a natural phenolic compound with antitumor activity, were shown to be effective AML chemotherapeutic agents. The combination of Dox and Cur had a synergistic effect and could improve Dox anti-tumor activity in AML cells, particularly leukemic stem cells, by inhibiting cell proliferation through FLT-3 protein suppression. This study demonstrated the benefit of co-treatment combining the natural product curcumin and chemotherapeutic drugs (Dox and Ida) in leukemia therapy as a potential approach to decrease chemotherapy dose and thereby reduce associated side effects. Adding nontoxic doses of edible Cur to chemotherapeutic drugs enhanced the cytotoxicity of Dox and suppressed leukemic stem cell proliferation. This finding presents an alternative choice that may be useful in the development of a promising regimen for the treatment of AML relapse in the future.
Supplementary Materials: The following are available online, Figure S1: Cytotoxic effects of cotreatment of doxorubicin and curcumin (Dox-Cur) on KG-1a, KG-1, EoL-1, and U937 cell lines, Figure S2: Cytotoxic effects of co-treatment of Idarubicin and curcumin (Ida-Cur) on KG-1a, KG-1, EoL-1, and U937 cell lines, Figure S3: Cell number and cell viability of KG-1a cells after treatment with Dox, Cur, and combined treatment of Dox-Cur for 48 h, Figure S4: Cell number and cell viability of KG-1 cells after treatment with Dox, Cur, and combined treatment of Dox-Cur for 48 h, Figure S5: Cell number and cell viability of EoL-1 cells after treatment with Dox, Cur, and combined treatment of Dox-Cur for 48 h, Table S1: IC 50 values of single and co-treatment of Dox or Ida and Cur on KG-1a