Abstract

Introduction: This study aimed to demonstrate the cytotoxic effect of a bitter leaf (Vernonia amygdalina Del.) ethanol extract on myeloid leukemia cells.

Methods: The plant extract was prepared using the maceration method. The toxicity assays used the trypan blue exclusion method. Flow cytometry and reverse transcription PCR methods were used to deduce the mechanism of action.

Results: The V. amygdalina Del. extract strongly affected K562 cells, with a half-maximal inhibitory concentration of 8.78 ± 2.224 µg/mL. The extract could induce apoptosis and arrest the cell cycle in K562 cells. The extract increased the mRNA levels of caspase 3 (CASP3), baculoviral IAP repeat containing 5 (BIRC5/survivin), and phosphatidylinositol 3-kinase (PI3K) and decreased the mRNA levels of retinoblastoma transcriptional corepressor 1 (RB1/pRB), B cell lymphoma/leukemic 2 (BCL2), BCL2-like 1 (BCL2L1/BCL-XL), caspase 9 (CASP9), and the breakpoint cluster region (BCR)-Abelson (ABL) fusion gene.

Conclusion: The V. amygdalina Del. extract strongly inhibited the acute myeloid leukemia cell line K562. It was found to arrest the cell cycle and induce apoptosis by regulating the expression of related genes that predicted targeting BCR-ABL downregulation.

Introduction

Cancer is a leading cause of mortality worldwide, with >19 million new cases and nearly 10 million deaths1, 2. Unfortunately, cancer cases are expected to increase significantly over the next decade3. The economic burden on patients and their families is enormous, significantly affecting public health, the national economy, and social security4. Therefore, medical research is racing to develop effective cancer treatments to prolong patient lives. However, current treatments remain largely ineffective5. One of the least treatable cancers is leukemia, which causes > 250,000 deaths and nearly 500,000 new diagnoses1, 2. Despite advances in knowledge and medical techniques, leukemia-related mortality remains high6.

Phytochemical compounds are being explored as potential treatments for blood cancer7, 8. Various plant compounds have shown inhibitory effects on leukemia cell proliferation. For example, maytansinoids and their derivatives extracted from Maytenus serrata inhibited tubulin, alvocidib extracted from Dysoxylum binectariferum inhibited cyclin-dependent kinase 9 (CDK9) activity, and omacetaxine mepesuccinate extracted from Cephalotaxus harringtonia has been approved by the US Food and Drug Administration9, 10, 11.

Bitter leaf (Vernonia amygdalina Del.) is among the major sources of compounds with scientifically demonstrated anticancer activity. Bitter leaf extract disrupts the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway, the mitogen-activated protein kinase (MAPK) pathway, and fms-related receptor tyrosine kinase 3 (FLT3) phosphorylation, inhibiting cancer cell proliferation12, 13, 14, 15. Studies have found the bitter leaf to be cytotoxic in breast cancer (half-maximal inhibitory concentration [IC50]: MCF-7 = 50.36 µg/mL, 4T1 = 25.04 ± 0.36 µg/mL, and T47D = 59.19 ± 0.55 µg/mL), neuroblastoma (IC50: U-87 = 18.80 ± 1.11 µg/mL), prostate cancer (IC50: PC-3 = 196.60 µg/mL and DU145 = 40.10 ± 4.30 µg/mL), and acute myeloblastic leukemia (IC50: HL-60 = 5.58 µg/mL, THP-1 = 24.17 ± 3.33 µg/mL, MOLM-13 = 11.45 ± 2.12 µg/mL, and MV4-11 = 16.08 ± 1.21 µg/mL) cells16, 17, 18, 19, 20, 21. However, few studies have examined bitter leaf’s effect on leukemia cells, especially chronic myeloid leukemia, one of the four main leukemia groups. Therefore, this study aimed to investigate the cytotoxicity of a bitter leaf ethanol extract on chronic myeloid leukemia cell line K562 and determine its mechanism of action.

Methods 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 40oC, 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 V. amygdalina extract (VAE) into a solution for use, which was stored at −20oC 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 37oC with 5% CO2. 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 2×105 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 37oC with 5% CO2. 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 2×105 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 105 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 105 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−ΔΔCT method25.

Table 1.

Primers used for analysis

Gene Sequences (5’ – 3’) Reference TP53 TGTGGAGTATTTGGATGACA Kang Pa Lee, et al . 26 GAACATGAGTTTTTTATGGC pRB ACTCCGTTTTCATGCAGAGACTAA Deborah L. Burkhart, et al . 27 GAGGAATGTGAGGTATTGGTGACA Bcl-XL TTGGACAATGGACTGGTTGA Suresh Kumar, et al . 28 GTAGAGTGGATGGTCAGTG Bcl-2 AAGATTGATGGGATCGTTGC M. Jaberipour, et al . 29 GCGGAACACTTGATTCTGGT Bax TGGCAGCTGACATGTTTTCTGAC Kostas V Floros, et al . 30 TCACCCAACCACCCTGGTCTT Survivin GTTGCGCTTTCCTTTCTGTC Sang Il Kim, et al . 31 TCTCCGCAGTTTCCTCAAAT Caspase-3 GAACTGGACTGTGGCATTGA Sadia Perveen, et al . 32 CCTTTGAATTTCGCCAAGAA Caspase-9 GGTGATGTCGGTGCTCTTGA IDT, Inc. CGACTCACGGCAGAAGTTCA BCR-ABL CGGGAGCAGCAGAAGAAGTTGTTC Nga Nguyen, et al. 33 CAGGCACGTCAGTGGTGTCTCTGTG MAPK TGAAATGACAGGCTACGTGG Liping Jiang, et al . 34 GACTTCATCATAGGTCAGGC Pi3K GGTTGTCTGTCAATCGGTGACTGT Ismael Riquelme, et al . 35 GAACTGCAGTGCACCTTTCAAGC