Prestoblue Cell Viability reagent, RPMI-1,640 and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and methanol was purchased from Avantor Performance Materials (Center Valley, PA, USA). Primary antibodies against caspase-3 (no. 9662), caspase-8 (no. 4927) and caspase-9 (no. 9508), poly adenosine diphosphate ribose polymerase (PARP; no. 9532), B-cell lymphoma-extra large (Bcl-xL; no. 2764), survivin (no. 2808), Bak (no. 12105), Bid (no. 2003), death receptor 4 (DR4; no. 42533) and β-actin (no. 3700) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against B-cell lymphoma 2 (Bcl-2; sc-509) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against death receptor 5 (DR5; ab8416) were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)G (AP124P) and goat anti-rabbit IgG (AP132P) secondary antibodies were obtained from EMD Millipore (Billerica, MA, USA).

HDW plants were dried and ground into a fine powder. HDW (500 g) was extracted with 5,000 ml of 50% ethanol using the refluxing method, and was then filtered. The ethanol solvent was then evaporated on a rotary evaporator in a water bath at 60°C for 24 h. In addition, the dried powder of HDW was obtained via a spraying desiccation method using a spray dryer. Stock solutions of HDW were prepared by dissolving the powder in dimethyl sulfoxide (DMSO) to a concentration of 100 mg/ml and stored at −20°C. The working concentrations of HDW were subsequently prepared by diluting the stock solution in RPMI-1640 medium, and the final concentration of DMSO in the medium was stored at <0.2%.

The WEHI-3 murine myelomonocytic leukemia cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in RPMI-1640 medium containing 10% FBS, penicillin (100 IU/ml) and streptomycin (100 µg/ml) at 37°C under a humidified 5% CO₂ atmosphere until use in further experiments. The morphology of cells was observed by a photomicroscope (21).

The cell viability assay was performed using Prestoblue Cell Viability reagent (Invitrogen; Thermo Fisher Scientific, Inc.). WEHI-3 cells (2×10⁴ cells/well) were seeded in 96-well plates. The cells were incubated with increasing concentrations (0.1, 0.2, 0.4, 0.8, 1.6 mg/ml) of HDW and ATO (0.8, 1.6, 3.2, 6.4 and 12.8 µM) for 24 and 48 h at 37°C. Following incubation, 10 µl Prestoblue Cell Viability reagent was added to each well and incubated for 3 h at 37°C, following which the media was discarded. Violet formazan precipitate was dissolved in 100 µl of DMSO and the color absorbance was recorded at 570 nm using a microplate reader. The control value corresponding to untreated cells was taken as 100% and the viability of treated samples were expressed as a percentage of the control.

Male BALB/c mice, ~22–28 g in weight and 4–6 weeks of age, were obtained from the National Laboratory Animal Center (Taipei, Taiwan). A total of 54 mice were randomly divided into 9 groups (n=6 each) and subsequently received different treatments as described below. WEHI-3 cells (1×10⁵ cells/0.1 ml/mouse) were intraperitoneally (i.p.) administered to mice and two weeks later, the mice were successfully induced as a model of leukemia. Group I mice served as untreated controls; group II mice were i.p. injected with WEHI-3 cells to form the leukemia group; group III mice were treated with crude extracts of HDW (100 mg/kg) in double distilled water (DDW) following i.p. injection of WEHI-3 cells; group IV mice were treated with crude extracts of HDW (250 mg/kg) in DDW following i.p. injection of WEHI-3 cells; group V mice were treated with crude extracts of HDW (500 mg/kg) in DDW following i.p. injection of WEHI-3 cells; group VI mice were treated with ATO (5 mg/kg) following i.p. injection of WEHI-3 cells; group VII mice were treated with ATO (5 mg/kg) and HDW (100 mg/kg) following i.p. injection of WEHI-3 cells; group VIII mice were treated with ATO (5 mg/kg) and HDW (250 mg/kg) following i.p. injection of WEHI-3 cells and group IX mice were treated with ATO (5 mg/kg) and HDW (500 mg/kg) following i.p. injection of WEHI-3 cells. The crude extract of HDW was administered via oral gavage to each mouse from the treatment groups with the above doses on a daily basis for 2 weeks, and each mouse was subsequently weighed. Mice were maintained at a constant temperature of 25±1°C in 55% humidity with a 12 h light/dark cycle, and were regularly provided with food and water. Ethical approval was granted by the Institutional Animal Care and Use Committee (no. 2016-091). The mice were sacrificed with CO₂ for further studies.

Spleen and liver samples were isolated from all mice and weighed individually, following sacrifice as previously described (22).

WEHI-3 cells were plated in a 10 cm dish and exposed to the indicated concentrations of HDW and ATO for 48 h. Cells were harvested and lysed in ice-cold radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM dithiothreitol and 10 µl/ml protease inhibitor cocktail; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Whole cell lysates (50 µg) with equal protein concentrations were obtained using the Protein Assay kit from Bio-Rad Laboratories, Inc. (Hercules, CA, USA) according to the manufacturer's protocol and further separated by SDS-PAGE (8–12%) and transferred onto a polyvinylidene fluoride membrane (EMD Millipore) as previously described (23–25). After blocking the membrane with blocking buffer [Tris-buffered saline with Tween-20 (TBST) containing 5% non-fat milk and 0.1% NaN₃], they were hybridized separately with primary antibodies. All primary antibodies were diluted with antibody buffer (TBST containing 5% bovine serum albumin and 0.1% NaN₃) to a dilution of 1:1,000. Finally, the membrane was hybridized with secondary antibody (HRP-conjugated goat anti-mouse IgG or goat-rabbit IgG at a dilution of 1:5,000), and protein bands were presented using enhanced chemiluminescence western blot analysis detection reagents (GE Healthcare Life Sciences, Chalfont, UK). The density of each band was quantified by ImageJ version 1.47 (National Institutes of Health, Bethesda, MA, USA).

Each experiment was repeated at least three times and data are presented as the mean ± standard deviation (n=6). All data were analyzed using IBM SPSS software (version 22.0 for Windows; IBM SPSS, Armonk, NY, USA). Differences between the control and experimental groups were analyzed using independent Student's t-test when P<0.05 was considered to indicate a statistically significant difference.

The combined effect of HDW and ATO exposure compared with individual exposure to both agents were initially observed at 24 and 48 h to elucidate whether combination therapy may result in a longer-lived restriction of cell proliferation compared with individual therapies. As shown in Fig. 1, following treatment with an indicated concentration of ATO or HDW, the cell viability of WEHI-3 cells was markedly decreased in a dose- and time-dependent manner. Notably, the combination of ATO and HDW synergistically reduced the viability of WEHI-3 cells, demonstrating a lower cell survival rate compared with those of single ATO- or HDW-treated groups. These results indicate that a combination of ATO with HDW is capable of increasing the sensitivity of ATO for killing leukemia cells.

The combined effect of HDW and ATO on BALB/c mice was examined following i.p. injection with WEHI-3 cells. The procedure of leukemic mice establishment and treatment are presented in Fig. 2A. All mice were weighed and the mean weight of each group is presented in Fig. 2B, which indicates that oral treatment of HDW and ATO alone or a combination of HDW and ATO did not significantly affect the body weight of mice with leukemia. Furthermore, the survival rate of tumor-bearing mice was markedly reduced in the 500 mg/kg/day HDW group and 5 mg/kg/day ATO group (Fig. 2C), respectively. Based on these observations, HDW and ATO alone may have effective anti-leukemic activity in WEHI-3 cells in vivo.

Mice were sacrificed following treatment with the indicated agents, and the spleen and liver was harvested from each mouse. Representative spleens are presented in Fig. 3A, which indicate that HDW and ATO treatments alone were able to reduce the size of the spleen, and that increasing the dose of HDW led to a greater reduction in the spleen size. The combination of 5 mg/kg/day ATO with 250 or 500 mg/kg/day HDW was also shown to be capable of reducing the size of the spleen (P<0.05 and P<0.01, respectively; Fig. 3B). Liver tissues were weighed, and the results are presented in Fig. 3C. The combination of 250 mg/kg/day HDW and 5 mg/kg/day ATO and 500 mg/kg/day HDW and 5 mg/kg/day ATO significantly reduced the weights of spleen when compared with the leukemia control group (P<0.05 and P<0.01, respectively; Fig. 3B). However, oral treatment of HDW or ATO alone, or combination of HDW and ATO did not significantly affect the weights of liver samples (Fig. 3C).

Our recent study demonstrated that HDW-induced apoptosis in HL-60 cells may be due to the induction of the DR pathways (26). The present study evaluated whether HDW was able to induce apoptosis through the induction of the DR pathways in WEHI-3 cells. HDW treatment induced an increase in DR4 and DR5 expression (Fig. 4A) in a dose-dependent manner; indicating that DR4 and DR5 upregulation are important in HDW-mediated apoptosis in leukemia cells.

The synergistic effects of HDW and ATO on the activation of DR4 and DR5 was examined. As shown in Fig. 4B, the expression levels of DR4 and DR5 were markedly increased following treatment with HDW or ATO alone, compared with control cells. Furthermore, the combination treatment of HDW and ATO induced a higher expression of DR4 and DR5 compared with those in the single HDW or ATO-treated group. The results revealed that the synergistic effects of HDW and ATO on cell viability are via the activation of the DR4 and DR5 signaling pathway in WEHI-3 cells.

Caspase activation is crucial in the initiation and execution of apoptosis. In order to determine whether HDW-stimulated apoptosis went through the activation of caspase, WEHI-3 cells were treated with or without the indicated concentration of HDW for 48 h prior to assessment of PARP, caspase-3, −8 and −9 protein levels. Fig. 5A demonstrates that HDW upregulated the levels of cleaved PARP, cleaved caspase-8 and cleaved caspase-9 expression, whereas levels of pro-caspase-3 were downregulated in WEHI-3 cells. These results suggest that the caspase cascade contributed to HDW-induced apoptosis in WEHI-3 cells.

The association between HDW and the levels of Bcl-2 family proteins was subsequently examined. Fig. 5B demonstrates that HDW administration decreased anti-apoptotic protein levels in a dose-dependent manner. Furthermore, treatment with HDW decreased the total Bcl-2, Bcl-xL and survivin protein levels, respectively. In addition, our data revealed that HDW increased the expression of Bak in a dose-dependent manner, and that Bid, a substrate of caspase-8, was also activated by HDW (Fig. 5C).

As shown in Fig. 6A, following treatment with HDW or ATO alone, the activation of PARP and caspase-3, −8 and −9 was increased compared with the control cells. The combination treatment of HDW and ATO induced a higher activation of PARP, caspase-3, −8 and −9 than those in the single HDW or ATO-treated groups (Fig. 6A). As depicted in Fig. 6B and C, the cells exposed to HDW or ATO alone markedly decreased the expression levels of Bcl-2, Bcl-xL and survivin, and increased the expression levels of Bak and t-Bid when compared with control cells. Notably, co-treatment of cells with HDW and ATO enhanced the effect in comparison with cells treated with HDW or ATO alone. These observations collectively suggest that combination treatment of WEHI-3 cells with HDW and ATO activated the intrinsic mitochondrial and extrinsic DR pathways to induce cell apoptosis.