Anticancer Activities In Vitro And Possible Mechanism Of EM23, A Natural Sesquiterpene Lactone From Elephantopus Mollis, Against Human Myeloid Leukemia Cells

Objectives: Elephantopus mollis(EM) is a traditional herbal medicine with multiple pharmacological activities. In our previous study, a series of sesquiterpene lactones(SLs) was isolated and purified from EM. Among them, EM23 exhibited better proliferation inhibitory activity in human chronic myeloid leukemia K562 cells and acute myeloid leukemia HL-60 cells. However, the intracellular molecular targets of EM23 and the underlying molecular mechanisms have not been elucidated. Thus, the objectives of the present study were to investigate the in vitro anti-leukemia activity of EM23, explore it’s intracellular targets and mechanisms, and provide scientific evidences for the development of EM23 to be a drug candidate for the treatment of myeloid leukemia. Methods: 1. EM23 inhibits cell proliferation and cell cycle progression: effects of EM23 on human myeloid leukemia cell and normal mammalian cells proliferation were assessed using CCK-8 and MTT assay, respectively; effects of EM23 on cell cycle distribution were determined using PI staining assay. 2. EM23 induced cell apoptosis: apoptosis induction by EM23 was examined using Annexin-V-FITC/PI assay, TUNEL and DAPI co-staining assay; expression levels of caspases associated with apoptosis were analyzed by western blotting; fluorescent probe JC-1 were used to detect mitochondrial membrane potential(MMP). 3. EM23 interfered with cellular redox homeostasis: DCFH-DA assay was used to detect ROS production in EM23 treated cells; western blotting and real-time quantitative PCR(QT-PCR) assay was carried out to detect the m RNA and expression levels of thioredoxin(Trx) and thioredoxin reductase(Trx R). 4. Effects of EM23 on ASK1 and p38/JNK/ERK MAPK activation: the activation of ASK1 and its downstream regulatory targets, the p38, JNK, and ERK MAPKs, in EM23-treated K562 and HL-60 cells was detected by western blotting; effects of ERK inhibitor FR180204 on EM23-mediated proliferation inhibitory activities were investigated by CCK-8 assay. 5. Inhibitory effects of EM23 on tumor necrosis factor alpha(TNF-α)-mediated NF-κB activation: the p65 localization in EM23-treated K562 and HL-60 cells was investigated by immunofluorescence analysis and western blotting; effect of EM23 on TNF-α-mediated degradation of IκBα was detected by western blotting; the cytotoxicity of the combined treatment of EM23 and TNF-α was detected by CCK-8 assay.Results: In the current study, we report that EM23, a natural sesquiterpene lactone isolated from EM, inhibits the proliferation of human chronic myeloid leukemia K562 cells and acute myeloid leukemia HL-60 cells by inducing apoptosis. Translocation of membrane-associated phospholipid phosphatidylserines(PS), changes in cell morphology, activation of caspases and cleavage of PARP were concomitant with this inhibition. The involvement of the mitochondrial pathway in EM23-mediated apoptosis was suggested by observed disruptions in MMP. Mechanistic studies indicated that EM23 caused a marked increase in the level of reactive oxygen species(ROS). Pretreatment with N-acetyl-L-cysteine(NAC), a ROS scavenger, almost fully reversed EM23-mediated apoptosis. In EM23-treated cells, the m RNA and expression levels of Trx and Trx R, two components of the Trx system involved in maintaining cellular redox homeostasis, were significantly down-regulated. EM23 may attenuate Trx R activity in in cell-free systems by alkylation of C-terminal redox-active site of Trx R. Concomitantly, Trx regulated the activation of apoptosis signal-regulating kinase 1(ASK1) and its downstream regulatory targets, the p38, JNK, and ERK MAPKs. EM23-mediated activation of ASK1/MAPKs was significantly inhibited in the presence of NAC. Furthermore, TNF-α-mediated activation of nuclear factor-κB(NF-κB) was suppressed by EM23, as suggested by the observed blockage of p65 nuclear translocation, phosphorylation and reversion of IκBα degradation following EM23 treatment.Conclusion: In summary, EM23 showed the ability to inhibit the proliferation of human myeloid leukemia K562 and HL-60 cells by inducing apoptosis via mitochondrial pathways. Suppression of the Trx system following treatment with EM23 could result in ROS accumulation and subsequently activate a number of Trx-dependent pathways, including the ASK1, p38, JNK and ERK MAPK pathways, which may contribute to EM23-mediated apoptosis. Furthermore, EM23 also effectively blocks TNF-α/NF-κB axis signaling, implying that EM23 has a potential role in preventing NF-κB-mediated anti-apoptotic pathways. This report represents the first comprehensive analysis of the cellular signaling events that are affected by treatment with EM23, a compound found in EM, in human myeloid leukemia cells. The findings contained here in detail a possible anticarcinogenic molecular mechanism for EM23.