Cucurbitacin IIb induces apoptosis and cell cycle arrest through regulating EGFR/MAPK pathway

https://doi.org/10.1016/j.etap.2020.103542Get rights and content

Highlights

  • Cucurbitacin IIb (CuIIb) exhibited the proliferation inhibitory activity in A549 cells.

  • CuIIb induced apoptosis via STAT3 pathway.

  • CuIIb suppressed the cell cycle and resulted G2/M phase cell cycle arrest.

  • Leu694 and Met769 are key amino acids for EGFR-CuIIb binding.

  • CuIIb inhibited the kinase activity of EGFR and may serve as a potential EGFR TKI.

Abstract

Epidermal growth factor receptor (EGFR) is considered as a valid target in the clinical trials of anticancer therapy and tyrosine kinase inhibitors (TKIs) of EGFR are approved for cancer treatments. In present work, cucurbitacin IIb (CuIIb) was confirmed to exhibit the proliferation inhibitory activity in A549 cells. CuIIb induced apoptosis via STAT3 pathway, which was mitochondria-mediated and caspase-dependent. CuIIb also suppressed the cell cycle and induced G2/M phase cell cycle arrest. CuIIb was capable of suppressing the signal transmitting of the EGFR/mitogen-activated protein kinase (MAPK) pathway which was responsible for the apoptosis and cell cycle arrest. Homogeneous time-resolved fluorescence (HTRF) analysis demonstrated that the kinase activity of EGFR was inhibited by CuIIb. Molecular docking suggested that the CuIIb-EGFR binding fundamentally depends on the contribution of both hydrophobic and hydrogen-bonding interactions. Hence CuIIb may serve as a potential EGFR TKI.

Introduction

The epidermal growth factor receptor (EGFR) belongs to the erbB family which consists of four closely related receptor tyrosine kinases, erbB1 (also known as EGFR), erbB2, erbB3, and erbB4 (Bethune et al., 2010; Roskoski, 2019; Saki et al., 2013). All of the erbB family proteins are transmembrane receptor tyrosine kinases consisting of three functional regions, designated as an extracellular ligand-binding region, a transmembrane region with the single hydrophobic anchor sequence, and an intracellular tyrosine kinase region (Chiou et al., 2015; Liang et al., 2020; Tang et al., 2015). Binding of the specific ligand, such as the epidermal growth factor (EGF), causes the dimerisation of the EGFR with the consequent initiation of the divergent intracellular signaling pathways cascade (Scartozzi et al., 2007; Xu et al., 2017). In the downstream of EGFR, there are two major signaling pathways, phosphatidylinositol 3-kinase (PI3K)/AKT and Ras/MEK/ERK (Hu et al., 2017; Tsai et al., 2012; Zhang et al., 2020b). It is well known that EGFR plays a crucial role in cell migration, apoptosis, and proliferation (Yarden, 2001; Zhang et al., 2020d). Overactivation of EGFR signaling pathways has been demonstrated as a significant step in the pathogenesis and progression of multiple malignant tumors (Bodey et al., 2005; Zhang et al., 2019c). Meanwhile, EGFR is observed to be overexpressed or dysregulated in many solid tumors and confirmed as a valid target in the clinical trials of anticancer therapy (Ciardiello et al., 2003). Therefore, the tyrosine kinase inhibitors (TKIs) of EGFR are approved in the treatment of non-small-cell lung cancer (NSCLC) throughout the world (Becker et al., 2011; Zhang et al., 2019b). The first-generation EGFR TKIs, such as erlotinib and gefitinib, were designed to reversibly compete for the ATP-binding sites and thus blocked the activation of EGFR downstream signaling pathways (Liang et al., 2020; Lin et al., 2014). Unfortunately, these synthetic EGFR TKIs may result in side effects, such as diarrhea and papulopustular rash (Welch and Moore, 2007).

As a group of highly oxidized tetracyclic triterpenes with biological activities and medicinal properties, cucurbitacins exhibited strong cytotoxicity in EGFR-overexpressed NSCLC cell lines by blocking the activation of EGFR and its downstream signaling pathways (Cheng et al., 2017; Ciardiello and Tortora, 2003; Peters et al., 2003). Based on the characteristics of their structures, cucurbitacins were arbitrarily divided into twelve categories and classified as cucurbitacins A–T (Alsayari et al., 2018; Jing et al., 2020b; Zhu et al., 2018). Cucurbitacins were investigated widely for their pharmacological activities, such as cytotoxic, anti-inflammatory, and anticancer activities (Graziose et al., 2013; Jayaprakasam et al., 2003). Certain cucurbitacins were shown to exhibit antiproliferative activity on several human cancer cell lines, including lung, breast, and brain cancers (Alghasham, 2013; Zhang et al., 2019a). Furthermore, cucurbitacin B, D, E, and I were reported to inhibit cancer cells proliferation respectively in HCT-116, MCF-7, NCI-H460, and SF-268 cell lines (Alghasham, 2013; Hussain et al., 2019). In a murine model, cucurbitacin E could not only inhibit the Yes‑associated protein (YAP) signaling pathway, but also suppress the brain metastasis of human NSCLC (Hsu et al., 2019). It was also demonstrated that cucurbitacin B significantly blocked cell cycle at the G2/M phase and induced mitochondrial apoptosis in NSCLC cell lines (Kausar et al., 2013; Khan et al., 2017). In addition, cucurbitacin IIb (Fig. 1A) was found to exert therapeutic efficacy in inflammation-related diseases through regulating multiple cellular behaviors (Wang et al., 2014). Although the anti-inflammatory activity of cucurbitacin IIb (CuIIb) has been reported, the cytotoxic effect of CuIIb towards A549 cells and the related molecular mechanism remain unclear (Zhang et al., 2020a).

In this work, CuIIb was observed to be a potential TKI of EGFR by homogeneous time-resolved fluorescence (HTRF) kinase inhibition analysis. In addition, CuIIb inhibited the proliferation of A549 cells by regulating the EGFR/mitogen-activated protein kinase (MAPK) signaling pathway which eventually resulted in apoptosis and cell cycle arrest. Moreover, the binding interaction of CuIIb with EGFR was evaluated by molecular docking.

Section snippets

Reagent

CuIIb was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Stock solution of CuIIb was prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. DMSO was purchased from Sigma-Aldrich (St Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Bellefonte, PA, USA). HTRF KinEASE-TK kit was purchased from Cisbio (Codolet, France). GAPDH antibody was purchased from Gene Tex (San Antonio, TX, USA). Other primary

Cytotoxicity of CuIIb to A549 cells

Mitochondrial succinate dehydrogenase exits in viable cell and is capable of reducing MTT to Formazan which is soluble in DMSO and exits an absorption at 570 nm. Therefore, MTT reduction tested by the 570 nm absorption is a representation of cell viability. MTT assay has been developed to assess the cytotoxicity of CuIIb to A549 cells. As shown in Fig. 1B, CuIIb significantly inhibited the proliferation of the cells. The inhibition showed a dose-dependent manner with the tested concentrations.

Discussion

Although the cytotoxicity of cucurbitacins to various cancer cells have well been reported previously (Cai et al., 2015; Chen et al., 2012; Garg et al., 2018; Kaushik et al., 2015), the cytotoxicity of CuIIb was rarely reported (Ren et al., 2012). In this work, the IC50 value of A549 cells was putatively to be approximately 70 μM (Fig. 1B) after 24 h-treatment with an initial cell population of 1 × 104/well, which is similar to its analogue CuIIa (Zhang et al., 2019a). However, the value of the

Conclusion

In conclusion, CuIIb exerted the proliferation inhibitory activity in A549 cells through acting as a TKI of EGFR. By inhibiting the kinase activity of EGFR, CuIIb suppressed the signal fall of EGFR/MAPK pathway, leading to caspase-dependent mitochondria-mediated apoptosis via STAT3 pathway, as well as cell cycle arrested at G2/M phase. The results of molecular docking suggested that the CuIIb-EGFR binding fundamentally depends on the contribution of both hydrophobic and hydrogen-bonding

Declaration of Competing Interest

The authors declare no conflict of interest.

CRediT authorship contribution statement

Yuan Liang: Investigation, Writing - original draft. Tiehua Zhang: Methodology, Project administration. Li Ren: Investigation, Methodology. Siyuan Jing: Investigation. Zhuolin Li: Validation. Peng Zuo: Investigation, Software. Tiezhu Li: Funding acquisition, Resources. Yongjun Wang: Funding acquisition, Supervision. Jie Zhang: Conceptualization, Funding acquisition, Writing - review & editing. Zhengyi Wei: Data curation, Formal analysis, Writing - review & editing.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31871717, 31972160, and U19A2035), the National Key Research and Development Program of China (2018YFD0300201), the Science and Technology Development Project Foundation of Jilin Province (20200201204JC), and the Science and Technology Projects of Jilin Provincial Department of Education in the 13th Five-Year Plan (JJKH20190112KJ).

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