Anti-cancer effects of ginsenoside compound k on pediatric acute myeloid leukemia cells

The pediatric AML cell lines Kasumi-1 and MV4-11 were purchased from American Type Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), plus 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Compound K (99%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). All experiments were performed with the approved of the Central South University Institutional Care and Use Committee (IACUC).

Cell viability was determined using the Sulforhodamine B (SRB) method as described [16]. Briefly, at the point of sample collection, the cells cultured in 96-well plates were fixed in 10% trichloroacetic acid (TCA) and incubated for 3 hr at 4°C. The fixed cells were washed with tap water for 4 times followed by staining with 0.4% SRB (Fluka) for 30 min at room temperature. The excess dye was washed off with 1% acetic acid for 4 times and the cells were dried by blow dryer. 150 μl 10 mM Tris-base (pH10.5) was added to each well to solubilize SRB by shaking the plates for 5 min on a shaker and the absorbance of SRB was measured at 550 nm.

The cell proliferation was assayed based on the measurement of 5-bromo-2-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation process by using the Cell Proliferation ELISA BrdU Kit (Roche Applied Science) as per manufacturer’s protocol. In brief, the BrdU labeling solution was added at the final concentration of 10 μM to the cell culture in 96-well plates during the last 4 hr of compound K treatment. After removal of labeling medium, FixDenat solution was added to the cells and incubated for 30 min at room temperature. The FixDenat solution was flicked off and the cells were incubated with anti-BrdU-POD solution for 90 min at room temperature. The cells were then washed with PBS for 3 times and incubated with Substrate solution for 15 min at room temperature, and the absorbance was measured at 405 nm (reference wavelength at 490 nm).

Cells treated with compound K at indicated duration were trypsinized and fixed in 70% ethanol for ≥2 hr on ice. After a brief centrifugation, the cell pellet was suspended in propidium iodide staining solution containing 0.1% (v/v) Triton X-100, 0.2 mg/ml DNase-free RNase A, and 20 μg/ml propidium iodide. After incubation at room temperature for 30 min, cells were analyzed for DNA content by flow cytometry.

Cell apoptosis was detected based on assessing cytoplasmic histone-associated DNA fragments by using the Cell Death Detection ELISA^PLUS Kit (Roche Applied Science) as per manufacturer’s instruction. In brief, at the point of sample collection, detached cells in 96-well plates were precipitated by centrifugation and pooled with attached cells. Supernatant containing cytoplasmic fraction was transferred to the streptavidin-coated plate. Immunoreagent was then added to each well in the plate and incubated on a shaker for 2 hr at room temperature. The solution was removed and the plate was washed with incubation buffer for 3 times, followed by ABTS solution incubation until sufficient color was developed. The reaction was stopped by addition of ABTS Stop Solution and the absorbance was measured at 405 nm (reference wavelength at 492 nm).

Soluble protein extracts or immunoprecipitated protein were boiled in sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. Separated protein bands were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (Thermal Fisher Inc.). Primary antibodies used were γH2AX, cleaved PARP, cleaved caspase-3 (Cell Signaling Technology, Cat. 9718, 5625 and 9664, respectively), and β–actin (Santa Cruz, sc-47778). The intensity of immunoreactive protein bands was measured using the Odyssey Infrared Imaging System (Li-Cor).

Data are presented as the mean ± SEM from three independent experiments. The Student’s two-tailed t test was used to determine the difference between treatment group and control group.

We first examined the effect of compound K on the growth of Kasumi-1 and MV4-11 cells using the SRB assay as described in Materials and Methods. As presented in Figure 2A and 2B, the cells were treated with 5, 10 or 20 μM compound K for indicated time and compound K showed significant growth inhibition in a dose- and time-dependent manner in both cell lines. We then assessed the effect of compound K on Kasumi-1 cell proliferation as determined by DNA synthesis using the Cell Proliferation ELISA BrdU Kit. We found that the BrdU incorporation was significantly suppressed at 12 hr by 20 μM compound K, and this suppression became more dramatic at 20 hr of treatment (Figure 3). Notably, the inhibition of proliferation at 20 hr was already ~65%, while the inhibition of growth at 24 hr was only ~30%, indicating that the effect of compound K on proliferation is not likely a secondary result of growth inhibition.

In order to further demonstrate the effects of compound K on earlier cellular events, we then evaluated Kasumi-1 cell cycle distribution upon compound K treatment. The cells were treated with 20 μM compound K for indicated duration and then processed and subjected to flow cytometry analysis. The percentage of DNA content at different cell cycle phases was presented in Table 1, showing that starting from 12 hr of compound K treatment, significant G1 arrest was observed. The effect continued to escalate with time. We next examined the effect of compound K on apoptosis in Kasumi-1 cells by using the Cell Death Detection ELISA^PLUS Kit that quantitatively determines cytoplasmic histone-associated DNA fragments induced by cell death. We found that compound K induced significant apoptosis at 12 hr of treatment and this induction became more evident at 20 hr (Figure 4A). Compound K-induced apoptosis was further validated by Western blotting analysis of γH2AX, the phosphorylated form of H2AX as the cellular response to DNA double strand breaks [17]. As shown in Figure 4B, compound K treatment led to increased expression of γH2AX in a dose-dependent manner. In support of this finding, the expression of cleaved PARP and caspase-3 was also significantly elevated in response to compound K treatment (Figure 4C), suggesting compound K-induced DNA double strand breaks triggered apoptosis. This is consistent with our cell growth inhibition result. Taken together, these observations suggest that the growth inhibitory effect of compound K on the pediatric AML cells could be attributable to suppression of cell proliferation and induction of G1 cell cycle arrest and apoptosis.