Rheum turkestanicum Induced Apoptosis Through ROS Without a Differential Effect on Human Leukemic Cells

3.1. Plant Materials

R. turkestanicum roots were gathered from Chenar village, Razavi Khorasan, Iran. Experts at the Herbarium Department of Ferdowsi University of Mashhad identified the plant (No., 21377). The dried root (60 g) was ground into fine powder; then, 50 g of the powder was extracted in a Soxhlet system using 70% ethanol during 48 hours. The hydroalcoholic extract was dried on a water bath and kept frozen (-20°C) for further use (yield, 19% w/w). Phosphate-buffered saline (PBS) was used to dissolve the extract to a final dose of 50 mg/mL before further use in cytotoxicity and apoptosis assays.

3.2. Cell Lines and Reagents

Pasteur Institute of Iran provided HL60 and NB4 cells. Resazurin reagent (300 μM of resazurin, 78 μM of methylene blue, 1 mM of potassium hexacyanoferrate III, and 1 mM of potassium hexacyanoferrate II), 2,7-dichlorofluorescein diacetate (DCFH-DA), propidium iodide (PI), nitro blue tetrazolium (NBT), phorbol myristate acetate (PMA), and arsenic trioxide (stock concentration of 1 mM) were provided by Sigma Co. (St. Louis, MO, USA). In addition, Gibco BRL (NY, USA) provided high-glucose RPMI 1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin solution. Giemsa stain was purchased from Merck (Darmstadt, Germany).

3.3. Human Normal Cell Isolation and Cell Culture

Polymorphonuclear cells (PMNs) from healthy volunteers were isolated under sterile conditions by two consecutive Ficoll-Hypaque (Pharmacia) density gradient centrifugations (21). PMN was purified to more than 97%, based on the morphological assessment of Wright-stained smears. After rinsing with sterile PBS, RPMI medium was used to resuspend the cells. HL60, NB4, and PMN cells were stored at a temperature of 37°C in a 90% humidified atmosphere (5% CO₂).

RPMI 1640 medium was supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL). Afterwards, incubation of cells was performed with various doses of R. turkestanicum (60 - 500 µg/mL) and As₂O₃ (50 µM) for 24, 48, and 72 hours, respectively. For every dose and time interval, we used an untreated control, receiving 0.5% PBS medium (an equal volume). All treatments were performed in triplicate.

3.4. Cell Viability

A resazurin reagent was used for measuring cell viability. To each well of the culture plates (96-well), 100 µL of NB₄, HL₆₀, and PMN cells (1 × 10⁵ cells) was added. R. turkestanicum extract (60 - 500 µg/mL) or As₂O₃ (50 µM) was used to treat the cells for 24, 48, and 72 hours. Afterwards, 20 µL of the resazurin reagent was added to each well, and incubation was performed during four hours. The intensity of fluorescence emission from the resorufin product was examined (relative to the viable cell count in each well), using a fluorescence Victor X5 2030 Multilabel Plate Reader (PerkinElmer, Shelton, Connecticut) at excitation/emission wavelengths of 485 nm/530 nm (27).

3.5. ROS Assessment

In brief, HL₆₀ and NB4 cells (105/well) were incubated with DCF-DA (20 µM) for 30 minutes in the dark and then treated with R. turkestanicum extract (60 - 500 µg/mL) or As₂O₃ (50 µM) for two hours. Then, the cells were transferred to a 75-mm Falcon™ polystyrene tube. After rinsing twice with PBS, a FLUOstar Galaxy fluorescence plate reader (PerkinElmer 2030, Finland) was used to determine DCF fluorescence intensity at excitation/emission wavelengths of 485 nm/530 nm (28).

3.6. Apoptosis

PI staining of the treated cells was performed to detect the apoptotic cells; then, flow cytometry was applied to determine the sub-G1 peak. In short, R. turkestanicum extract (125 - 500 µg/mL) or As₂O₃ (50 µM) was used to treat HL60 and NB4 cells during 48 hours. Incubation of cells was performed at 4°C in darkness overnight, using 500 µL of hypotonic PI buffer (50 µg/mL). Afterwards, flow cytometry was applied to examine the samples. Overall, 10,000 events were reported per sample, and WINMDI software was used to assess the data (10).

3.7. Morphological and Differentiation Assay

Diagnosis and morphological typing were determined by standard techniques using Giemsa-stained smears, and differentiation test was performed for NBT reduction. The NBT reaction acts as a sensitive and simply quantified marker for differentiation of leukemic cells; moreover, it can eliminate observer bias caused by morphological evaluation alone.

The cells (1 × 10⁶ cells/mL) were treated in a 6-well plate with R. turkestanicum extract (60, 125 µg/mL) or As₂O₃ (0.5 µM) (29) for three days. Then, PBS was used to wash the cells twice, and the pellets were resuspended in 0.2% NBT (with an equal volume) and dissolved in Dulbecco’s phosphate-buffered saline (consisting of 200 ng/mL of PMA freshly diluted). After 25 minutes of incubation at a temperature of 37°C in darkness, cytospin was prepared on the slides, and Giemsa staining was carried out; then, presence of blueblack formazan granules was assessed in 300 cells (30).

3.8. Statistics

GraphPad Prism version 6 (GraphPad Software Inc., CA, USA) was used for analyzing the data. ANOVA test as well as Tukey’s multiple comparison test were applied for comparison among the groups. The significance level was set at 0.05. In this study, the data are presented as mean ± SEM of three independent analyses, which were performed in triplicate.

4.1. Dose-Dependent Reduction of Cell Viability Using R. turkestanicum

To evaluate the toxic effects of R. turkestanicum, HL60 and NB4 cells were incubated with different doses of R. turkestanicum (60 - 500 µg/mL) and As₂O₃ (50 µM, as the positive control) over 72 hours; the resazurin reagent was used to analyze cell viability (Figure 1). The PMN cells were incubated with R. turkestanicum (200 - 800 µg/mL) for 24 hours. In comparison with PMN cells, R. turkestanicum-mediated toxicity was significantly higher in HL60 and NB4 cells. The findings are graphically presented in Figure 1 and are summarized in Table 1.

Figure 1.

Anti-proliferative effects of R. turkestanicum on leukemic cells. HL60 andNB4 cells were treated with different concentrations of R. turkestanicum (60 - 500 µg/mL) or As₂O₃ (50µM) for 24 - 72 hours. Normal polymorph nuclear (PMN) cells were also treated with R. turkestanicum (200 - 800 µg/mL) for 24 hours. The percentage cell viability (quantitated by resazurin assay) was normalized against the negative controls for each cell type. Data are expressed as mean ± SEM of 3 separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with the control value.

As shown in Figure 1, treatment with R. turkestanicum could reduce cell viability in both cell lines dose- and time-dependently. The IC₅₀ values of R. turkestanicum in HL60 cells were respectively 518.6, 336.2, and 99.0 µg/mL at 24, 48, and 72 hours following treatment. However, at 24, 48, and 72 hours following treatment, IC₅₀ of R. turkestanicumin NB4 cells was found to be 597.8, 506.0, and 460.2 µg/mL, respectively (Table 1).

Treatment of HL60 cells with 250 and 500 µg/mL of R. turkestanicum for 72 hours could decrease cell viability more than As₂O₃ (P < 0.001). The viability of R. turkestanicum treated HL60 cells at 250 and 500 µg/mL significantly reduced to 15% and 12.1%, respectively versus 30.9% in the As₂O₃ group (in comparison with the vehicle-treated cells; Figure 1; P < 0.001). On the other hand, viability of R. turkestanicum-treated NB4 cells at 500 µg/mL significantly reduced to 44.4% versus 50.7% in the As₂O₃ group (50 µM) at 72 hours following treatment (Figure 1, P < 0.001). In contrast, the extract did not have any cytotoxic effects up to 800 µg/mL in PMN cells after 24 hours (Figure 1).

4.2. R. turkestanicum Dose-Dependent Increase in ROS Generation

Our findings showed a regular time- and dose-dependent rise in the ROS generation in HL60 and NB4 cells at two hours after treatment with R. turkestanicum, compared to the control group (Figure 2). On comparing with control HL60 cells, 250 and 500 µg/mL R. turkestanicum induced the formation of ROS during three hours of incubation (165 ± 1.1% and 172 ± 4.2% vs. 147 ± 4.1% in the As₂O₃ group; P < 0.001 in comparison with the controls). The ROS content majorly increased in NB4 cells two hours after treatment with 500 µg/mL R. turkestanicum, compared to the control group (148 ± 7.2% in the experimental group versus 140 ± 8.1% in the As₂O₃ group; P < 0.001).

Figure 2.

Effect of R. turkestanicum on intracellular reactive oxygen species (ROS) in leukemic cells. NB4 and HL60 cells were treated with different concentrations of R. turkestanicum (60 - 500 µg/mL) or As₂O₃ (50 µM) for two hours. Data are expressed as mea ± SEM of three independent experiments. **P < 0.01 and ***P < 0.001 as compared with control value.

4.3. R. turkestanicum Concentration-Dependent Induction of Apoptotic Cell Death

Figure 3 shows that R. turkestanicum at high concentrations induces apoptosis in NB4 and HL60 cells, significantly. After 48 hours of treatment, R. turkestanicum treated HL60 cells showed 63.2% (250 µg/mL) and 65.9% (500 µg/mL) against 55.5% in the As₂O₃ group (50 µM) (P < 0.001 in comparison with the controls). In addition, 48.0% of R. turkestanicum-treated NB4 cells underwent apoptosis, versus 45.5% of cells in the As₂O₃ group (P < 0.001 in comparison with the vehicle-treated cells). The apoptotic cell count increased concomitantly with concentration in the experimental group. The extract increased apoptosis in HL60 more than NB4 cells, especially at low concentrations (P < 0.001, Figure 3).

Figure 3.

Effect of R. turkestanicum on apoptosis in leukemic cells. A, NB4 and HL60 cells were incubated with different concentrations of R. turkestanicum (125 - 500 µg/mL) or As₂O₃ (50 µM) for 48 hours. Apoptosis was assayed by PI staining and analyzed by flow cytometry. B, Apoptosis rate is shown by bar graph. The data are mean ± SEM of three independent experiments. ***P < 0.001 as compared with the control value.

4.4. R. turkestanicum Did Not Induce Differentiation of Leukemic Cells Toward Granulocyte Pattern

The Giemsa and NBT tests showed that R. turkestanicum did not induce maturation of cells to neutrophils. In the morphological Giemsa assay, the cells showed promyelocytes characteristic with cytoplasmic granules, like control cells. In functional NBT assay, cells did not have any intracellular blue-black formazan deposits (Figure 4).

Figure 4.

Effect of R. turkestanicum on morphological and functional properties of granulocyte in leukemic cells. NB4 cells were treated with R. turkestanicum (60 µM) or As₂O₃ (0.5 µM) for 72 hours. Morphological and functional properties of granulocyte were determined by Gimsa and NBT assays, respectively. In Giemsa-stained slides, As₂O₃-treated cells (as positive control) showed polymorphonuclear morphology of granulocyte, and R. turkestanicum-treated cells showed promyelocytes characteristic, same as the control. On NBT slides, As₂O₃-treated cells (as the positive control) showed intracellular blue-black formazan deposits, and R. turkestanicum-treated cells did not have any intracellular blue-black formazan deposits (bar represents 0.01 mm).