The sesquiterpene lactone eupatolide induces apoptosis in non-small cell lung cancer cells by suppressing STAT3 signaling

https://doi.org/10.1016/j.etap.2020.103513Get rights and content

Highlights

  • Eupatolide inhibits STAT3 activation to induce NSCLC cell apoptosis.

  • Sustained activation or knockdown of STAT3 suppressed or enhanced the activity of eupatolide.

  • This paper firstly reported that eupatolide could effectively inhibit NSCLC progression.

Abstract

We aimed to evaluate the role of a natural sesquiterpene lactone, eupatolide, in non-small-cell lung cancer (NSCLC) and further explore its underlying mechanism on regulating the activation of signal transducer and activator of transcription 3 (STAT3), which is thought to have carcinogenic function in a variety of malignancies including lung cancer. Cell survival was measured by Cell Counting Kit-8 assay. in vivo experiments were performed by inoculating NSCLC cells into nude mice. Western blot and qRT-PCR were applied to detect the activation level of STAT3 and the mRNA levels of anti-apoptotic markers. The cell apoptosis was measured by Annexin V-FITC/PI Apoptosis Detection Kit. Our results showed that eupatolide suppressed the survival of NSCLC cells in a dose and time dependent manner. Furthermore, eupatolide increased the anti-tumor activity of the chemotherapeutic drugs cisplatin and 5-Fluoracil (5-FU). The xenograft study revealed that eupatolide suppressed tumor growth of NSCLC cells in vivo. Furthermore, eupatolide induced apoptosis by suppressing the activation of STAT3 in NSCLC cells. Sustained activation or knockdown of STAT3 suppressed and enhanced the activity of eupatolide, respectively. This paper is the first to report that eupatolide could effectively inhibit NSCLC progression, suggesting that eupatolide might be utilized as a novel STAT3 inhibitor for treating NSCLC.

Introduction

Lung cancer, with a high incidence and mortality rate in China, is the most common malignant tumor in the world (Jiang et al., 2017). According to different histological behaviors and pathological manifestations, primary lung cancer is mainly divided into small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) (Osmani et al., 2018). Among them, NSCLC accounts for about 85 % of lung cancer cases, which is a disease characterized by molecular subtypes of genetic mutations (Wang et al., 2016). Cisplatin and 5-Fluorouracil (5-FU) are the most commonly used drugs in current combination chemotherapy. With the in-depth study of the molecular mechanism of lung cancer, targeted therapy has become a hot spot in NSCLC treatment (Langhammer and Scheerer, 2017). Exploring novel tumor-associated genes and possible molecular pathways to understand their impact on the occurrence and development of lung cancer are in urgent need for finding new effective drugs.

Signal transducer and activator of transcription 3 (STAT3) is a signal transduction protein involved in a large number of cytokine and growth factor responses in cells, which is responsible for regulating several important physiological processes including cell differentiation, growth, proliferation, and apoptosis (Hillmer et al., 2016). Numerous studies have confirmed that tumor occurrence, development, and prognosis have strong correlations with STAT3 (Yu et al., 2014). Upon activation, STAT3 directly induces the expression of target factors Bcl-xL, Bcl-2, Mcl-1 and survivin, thereby blocking the process of tumor cell apoptosis. Studies have shown that Bcl-2 could inhibit the synthesis and activation of caspase-3, which participates in the cleavage of various substrates in the cell, leading to cell apoptosis. Poly ADP-ribose polymerase (PARP) is an enzyme that is located in the nucleus and is closely related to DNA repair under stress conditions. PARP cleavage is considered to be an important indicator of cell apoptosis, and is generally considered to be an indicator of caspase-3 activation.

The sesquiterpene lactone eupatolide is a novel terpenoid, which could be isolated from Inula helenium and Inula japonica (Boldbaatar et al., 2017). Currently, a variety of sesquiterpene lactones are confirmed to play several different biological functions such as anti-inflammation, anti-tumor, de-worming, anti-bacterial, and hypoglycemic and analgesic activities (Adekenov, 2017). However, current studies on eupatolide are very limited. Lee et al. reported that eupatolide sensitized human breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis (Lee et al., 2010). Furthermore, eupatolide inhibits the TGF-β1-induced migration of breast cancer cells via downregulation of SMAD3 phosphorylation and transcriptional repression of ALK5 (Boldbaatar et al., 2017). These previous studies suggest that eupatolide might play a role in promoting apoptosis in tumors. Therefore, our study aimed to evaluate the role of eupatolide in NSCLC progression when treated with cisplatin or 5-FU and further explore its underlying mechanism in regulating the activation of STAT3.

Section snippets

Plasmids and reagents

The empty vector (#37,632) or constitutively active STAT3 (#24,983) were purchased from Addgene (Shanghai, China). The shNC, shSTAT3#1 and shSTAT3#2 plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA). Eupatolide was purchased from Shanghai YuanYe Biotechnology (Shanghai, China). Cisplatin and 5-FU were purchased from Selleck Chemicals (Shanghai, China). Human epidermal growth factor (EGF) was purchased from Macgene (Shanghai, China).

Cell culture

Human lung adenocarcinoma cell lines (A549, H1975

Eupatolide inhibited cell survival in NSCLC cell lines

To investigate whether eupatolide could exert anti-tumor effects in NSCLC, we first-tested the effects of eupatolide treatment at different concentrations and times on the cell survival of NSCLC cell lines. Fig. 1A showed the eupatolide chemical structure. We found that eupatolide suppressed the cell survival of A549, H460, H1299 and H1975 cells in a dose-dependent manner (Fig. 1B). As shown in Fig. 1B, the IC50 values of eupatolide in A549, H460, H1299 and H1975 were about 10 μM, 18 μM, 16 μM

Discussion

Lung cancers are the most common malignant cancer type with the highest mortality rate in the world (Brody, 2014). Among them, NSCLC accounts for 80 %–85 % of all lung cancer cases (Pastorkova et al., 2016). As a broad-spectrum anti-tumor drug, cisplatin mainly plays a role in inhibiting DNA replication of cancer cells, and has the characteristics of exact curative effect, wide anti-tumor spectrum and high anti-cancer activity (Xiao et al., 2017). In NSCLC, cisplatin is a first-line treatment

Funding

The Study was supported by the Joint construction project of Henan Province medical science and technology research plan (SB201901036); Key scientific research project plan of Henan Provincial Department of Education (19A320015).

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgments

None.

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    These authors contributed equally to this work.

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