Low concentration of formononetin stimulates the proliferation of nasopharyngeal carcinoma cell line CNE2 by upregulating bcl-2 and p-ERK1/2 expression

Abstract

Context Formononetin is a typical phytoestrogen, which is a bioactive component found in red clover plants. Previous studies have shown that formononetin inhibits the proliferation of several types of cancer cells, including prostate cancer and osteosarcoma. However, how formononetin affects the proliferation of CNE2 is not clear. Objective The objective of this study is to investigate the effects of formononetin on nasopharyngeal carcinoma cells in vitro, along with the underlying mechanism. Materials and methods CNE2 cells were incubated with various concentrations of formononetin (0, 0.1, 0.2, 0.3 and 1 μM) for 48 h. Cell proliferation was measured by [3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay, while the rate of apoptosis was measured by flow cytometry. Bcl-2 and bax mRNA expression levels were determined by real time polymerase chain reaction (RT-PCR), while p-ERK1/2 and bcl-2 protein expression levels were quantified by Western blotting. Results Formononetin promoted the proliferation of CNE2 cells at low concentrations (0, 0.05, 0.1, 0.2, 0.5, 1, 2 and 5 μM), OD values increased from 0.27 ± 0.01 to 0.30 ± 0.01, 0.30 ± 0.01,0.36 ± 0.01, 0.35 ± 0.01, 0.34 ± 0.01, 0.34 ± 0.01 and 0.32 ± 0.01, respectively. The percentage of late apoptosis declined from 6.77% ± 0.73% (0 μM group) to 6.2% ± 0.4% (0.1 μM group), 3.83% ± 0.71% (0.3 μM group) and 5.1% ± 0.52% (1M group). The mRNA levels of bax and bcl-2 were down- and upregulated, respectively, by formononetin. Bcl-2 and p-ERK1/2 protein levels were also upregulated. Conclusions Formononetin stimulates CNE2 cell proliferation and has an inhibitory effect on CNE2 cells apoptosis, which is mediated by the activation of the ERK1/2 signaling pathways.

Formononetin: A Review of Its Anticancer Potentials and Mechanisms

“…It inactivates signaling pathways, namely, JAK/STAT pathway, PI3K/AKT pathway, as well as MAPK (ERK1/2) pathways. Formononetin also modulates several miRNA expressions as well as supresses cell migration, invasion, and angiogenesis (Li et al, 2014; Liu et al, 2014b; Yang et al, 2014; Hu and Xiao, 2015; Guo et al, 2016; Qi et al, 2016; Wu et al, 2017; Kim et al, 2018a; Park et al, 2018; Wang et al, 2018; Zhang et al, 2018a). NAG-1, nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1; IAP, inhibitors of apoptosis proteins; IL-6, interleukin-6; PARP, poly(ADP-ribose) polymerase; GSH, glutathione; GSSG, oxidized glutathione; GR, glutathione reductase; PTEN, phosphatase and tensin; HIFα, hypoxia inducible factor α; miR, microRNA; TIMP, tissue inhibitors of metalloproteinases; ER, estrogen receptor; MMP, matrix metalloproteinases; CDK, cyclin dependent kinases; STAT, signal transducer and activator of transcription; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; IGF-1R, insulin-like growth factor 1 receptor; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; ROS, reactive oxygen species.…”

“…Although most studies demonstrated the anticancer effect of formononetin, there were two studies suggesting a potential cancer-promoting effect of formononetin at low concentration (<6 μM). Formononetin was reported to stimulate proliferation of nasopharyngeal CNE2 cells at 0.3 μM by activating ERK1/2 signaling pathway and upregulation of Bcl-2 anti-apoptotic protein (Guo et al, 2016). Another recent study reported that formononetin at 1–6 μM stimulated proliferation of estrogen receptor alpha (ERα)-positive breast cancer cell lines (MCF-7 and BT474) but did not affect the proliferation of ERα-negative breast cancer cell (MDA-MB-231) (Chen et al, 2018a).…”

“…This result reflects the weaker effect of formononetin on cell proliferation and anti-apoptosis in breast cancer cells as compared to HUVECs, suggesting that long-term use of formononetin could be used by postmenopausal women to alleviate symptoms of estrogen deficiency while conferring a lower risk of postmenopausal breast cancer as compared to estrogen (Chen et al, 2018a). In contrast, however, a low dose of formononetin (0.1 and 0.3 μM) was found to accelerate the proliferation of nasopharyngeal carcinoma by upregulating miR-375 expression and leading to the upregulation of ERα as well as downregulation of PTEN (Guo et al, 2016). These studies indicate that treatment with formononetin at low concentrations results in complex, cell type-specific effects in ER-positive cancer cells via the modulation of miRNA.…”

Low concentration of formononetin promotes proliferation of estrogen receptor-positive cells through an ERα-miR-375-PTEN-ERK1/2-bcl-2 pathway

“…Our previous study demonstrated that low concentrations of formononetin (< 0.3 μM) were capable of stimulating cell proliferation and inhibiting cell apoptosis in CNE2 cells by up-regulating bcl-2 and p-ERK1/2 expression [ 21 ]. This suggests that formononetin is potentially involved in an ER-MAPK/ERK-bcl-2 signaling pathway that promotes growth.…”

Potential Anticancer Properties and Mechanisms of Action of Formononetin

“…Secondary metabolites isolated from natural products encompassing terpenes, alkaloids, isoflavones, and polyphenols have been reported as anticancer agents [64, 65]. Anticancer properties of formononetin have been documented against several types of cancer [66] such as breast [67], colon [41], glioma [54], osteosarcoma [68], multiple myeloma [52], adrenal medulla [51], nasopharyngeal [50], prostate [49], bladder [45], laryngeal [23], lung [43], and cervical cancer [42] (Figure 3).…”