Plant extraction

The bitter leaves were harvested from Xuyen Moc district, Ba Ria–Vung Tau province, Vietnam. Dang Le Anh Tuan, Ph.D., of the Botany Laboratory in the Department of Ecology and Evolutionary Biology, Faculty of Biology and Biotechnology, University of Science, Vietnam National University, Ho Chi Minh City, performed the botanical identification (voucher: PHH0004908; Supplementary Figure 1). After washing and thoroughly drying at 40C, the leaves were ground to a powder, which was then suspended in 96% ethanol (1:10 w/v). The plant extract was collected and rotary evaporated to obtain a crude extract. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) was used to dissolve the crude extract (VAE) into a solution for use, which was stored at −20C until needed.

Cell culture

The human leukemia cell line K562 was obtained from Prof. Yuko Sato (Tokyo, Japan)22, 23. The K562 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium (Sigma-Aldrich, USA) supplemented with 10% inactivated fetal bovine serum (Thermo Fisher Scientific, USA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, USA) at 37C with 5% CO. Fibroblast cells were cultured in Dulbecco’s modified Eagle’s medium (StemCell, Singapore) prepared similarly to RPMI 1640.

Cytotoxicity effect of VAE extract

The toxicity of the VAE on K562 cells was evaluated using the trypan blue exclusion method in a six-well plate24. Briefly, 1500 µL of K562 cells at a density of 2×10 cells/mL was added to each experimental well before the same volume of VAE at 0 to 100 µg/mL was added. The plates were incubated for 72 hours at 37C with 5% CO. Then, cell viability was calculated as the percentage difference between the treated and negative control groups.

The toxicity of the VAE on fibroblasts was determined using the 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, USA). Briefly, 100 µL of fibroblasts at a density of 2×10 cells/mL was added to each experimental well and incubated in a cell culture incubator. After 24 hours, 100 µL of the VAE at 0 to 200 µg/mL was added. After 72 hours, the viability of the fibroblasts was measured using the MTT assay.

Untreated cells were used as negative controls. Moreover, the effect of the DMSO (Protide, USA) was evaluated at 0.1%, corresponding to the solvent content in the highest VAE treatment.

Annexin V/PI analysis

K562 cells at a density of 10 cells/mL were exposed to the VAE at 50 and 100 µg/mL. After 24 hours, the K562 cells were collected and washed twice with phosphate-buffered saline (PBS; TBR company, Vietnam). Then, the cells were stained according to the ANNEX100B protocol (BioRad, USA). Briefly, cell pellets were resuspended in 195 µL of 1× binding buffer before adding 5 µL of Annexin V. After incubation for 15 minutes in the dark, the cells were washed with 200 µL of binding buffer and resuspended in 190 µL of binding buffer before adding 10 µL of propidium iodide (PI). The stained cells were analyzed using a BD Accuri C6 Plus Flow Cytometer (BD Biosciences, USA).

mRNA expression analysis

K562 cells at a density of 10 cells/mL were exposed to the VAE at 50 and 100 µg/mL. After 16 hours, the K562 cells were collected and washed with PBS. Next, their RNA was extracted according to the TRIzol reagent guidelines (Thermo Fisher Scientific, USA). Then, mRNA expression was detected using the SensiFAST SYBR No-ROX One-Step Kit (Meridian Bioscience, USA) with the primers listed in Table 1. Gene expression was determined using reverse transcription quantitative PCR and the 2 method25.

Data Analysis

Experiments were repeated at least three times, and data are presented as mean ± standard deviation. Statistical analyses were conducted using GraphPad Prism software (version 9.0.0) with α = 0.05.

VAE strongly inhibited myelocytic leukemia cells

K562 cell viability was greater with (116.90% ± 16.92%) than without 0.1% DMSO ( = 0.0002). In contrast, 0.1% DMSO did not significantly affect fibroblast viability ( = 0.0786). Therefore, 0.1% DMSO was considered benign for evaluating cell growth (Figure 1). The VAE significantly decreased leukemia cell proliferation but did not significantly affect fibroblast proliferation (Figure 2andSupplemental Figure 2). The IC values for the VAE were 8.78 ± 2.22 µg/mL for K562 cells and > 200 µg/mL for fibroblasts. The effects of VAE on K562 cells were classified as selective based on an estimated selective index (SI) of 22.78.

VAE induced apoptosis in K562 cells

K562 cells were further examined by staining with PI and Annexin V after 24 hours of VAE exposure. Cell death increased with VAE concentration. The percentages of Annexin V-positive and PI-positive cells were higher among cells treated with 50 or 100 µg/mL VAE than among untreated control cells ( < 0.0001). Most cells died due to apoptosis (6.10% ± 0.10% for 50 µg/mL and 6.50% ± 0.44% for 100 µg/mL VAE) or necrosis (5.52% ± 0.50% for 50 µg/mL and 6.59% ± 0.52% for 100 µg/mL VAE; Figure 3). In addition, the VAE tended to arrest cells at G2/M (9.95% ± 0.84% for 50 µg/mL and 10.67% ± 0.40% for 100 µg/mL VAE) or S (2.08% ± 0.07% for 50 µg/mL and 3.26% ± 0.03% for 100 µg/mL VAE) checkpoints since the percentage of cells in these phases increased after exposure in a dose-dependent manner (Figure 4).

VAE regulates mRNA expression in K562 cells

We examined the expression of genes involved in apoptosis, the cell cycle, and breakpoint cluster region (BCR)-Abelson (ABL) pathway signaling (Figure 5). The control group comprised healthy cells at the same density as the experimental groups. When 2 values were compared between the 50 and 100 µg/mL VAE experimental groups, the mRNA levels of the following target genes decreased with increasing VAE concentration: retinoblastoma transcriptional corepressor 1 (/; from 5.59 ± 2.63 to 1.24 ± 0.88), B cell lymphoma-leukemia 2 (; from 1.00 ± 0.46 to 0.60 ± 0.15), BCL2-like 1 (/; from 2.44 ± 0.51 to 1.16 ± 0.61), BCL2-associated X apoptosis regulator (; from 1.75 ± 0.17 to 1.23 ± 0.12), caspase 9 (; from 9.57 ± 1.40 to 2.84 ± 0.32), and the fusion gene (from 2.73 ± 0.13 to 1.84 ± 0.33). In contrast, the mRNA levels of the following target genes increased with increasing VAE concentration: baculoviral IAP repeat containing 5 (/; from 1.04 ± 0.15 to 2.00 ± 0.67), caspase 3 (; from 1.21 ± 0.29 to 1.78 ± 0.51), andfrom 0.71 ± 0.40 to 1.00 ± 0.33). However, VAE did not affect tumor protein p53 () and mRNA levels.