Elsevier

Toxicology

Volume 427, 1 November 2019, 152298
Toxicology

Omethoate induces pharyngeal cancer cell proliferation and G1/S cell cycle progression by activation of Akt/GSK-3β/cyclin D1 signaling pathway

https://doi.org/10.1016/j.tox.2019.152298Get rights and content

Highlights

  • Omethoate increased cell proliferation of FaDu cells in a dose- and time- dependent manner.

  • Omethoate induced Akt/GSK-3β/cyclin D1 signaling pathway activation of FaDu cells.

  • Omethoate induced cell cycle process transition from G1 to S phase in FaDu cells.

  • Akt1/2 kinase inhibitor and siAkt suppressed omethoate-induced activation of Akt/GSK-3β/cyclin D1 signaling pathway.

Abstract

Omethoate is a broad category of organophosphorous pesticides (OPs) and has toxic effects on human health under long-term, low-dose exposure. However, the role of omethoate in cancer development remains elusive. The incidence of global head and neck squamous cell carcinomas (HNSCC) has markedly increased in recent years. Thus, we examined whether omethoate induced the proliferation of FaDu cells (a cell line of HNSCC) and if so, what the underlying mechanism was. The study revealed that omethoate induced FaDu cell growth in a dose- and time-dependent manner. Omethoate stimulated FaDu cell proliferation was mainly due to enhancing the G1 to S phase transition by flow cytometry analysis. We also found that omethoate up-regulated cyclin D1, a key gene controlling the G1-S transition. Furthermore, we showed that omethoate was capable of activating the Akt/GSK-3β signaling pathway. Blockage of Akt by siRNA or small molecule inhibitor significantly suppressed omethoate-induced cyclin D1 expression and cell proliferation. Collectively, these findings demonstrated for the first time that omethoate could induce the pharyngeal cancer cell proliferation by activation of the Akt/GSK-3β/cyclin D1 signaling pathway.

Introduction

As an economical and efficient insecticide and acaricide, omethoate is one of the most frequently used organophosphorus pesticides (OPs) around the world, especially in China. If exposed to humans, the omethoate can accumulate for a long period of time after entering the body, which becomes harmful to human health (Rong et al., 2015; Sieke et al., 2018). This compoundIt has been caused public concern due to its toxic effects on non-target organisms after application (Pan et al., 2015) and is currently on the list of “Priority Monitoring Pesticides” published by the Ministry of Environmental Protection of the People’s Republic of China. Omethoate has toxic effects on non-target organisms after application (Pan et al., 2015). The public are easy to expose to omethoate, as residual omethoate has been detected in vegetables, fruits, grains and tea (Hao et al., 2011; Pan et al., 2015; Zhang et al., 2014). In the process of its use and production, it can also cause acute and chronic harm to the health of a person who come into contact with it, while the effects fromits low-dose exposure may go unnoticed (Duan et al., 2017a). Although, acute organophosphorous poisoning incidents have been gradually reduced in recent years, the toxic effects on human health caused by low-dose exposure to omethoate is attracting the public’s attention. Accumulating studies have concluded that long-term, low-dose exposure to OPs shares a close relationship with human tumorigenesis, adverse reproductive outcomes, and neurological and neurobehavioral function abnormalities (Duan et al., 2017a; Weichenthal et al., 2010). Presently, however, there is no direct experimental evidence of omethoate in cancer development thus far.

Head and neck squamous cell carcinoma (HNSCC) represents 6% of all cancer cases worldwide (Mourad et al., 2017; Vigneswaran and Williams, 2014). HNSCC is often found in the pharynx and larynx (Rothenberg and Ellisen, 2012) and is thought to account for 350,000 cancer deaths worldwide per year, with males affected significantly more than females at a ratio of 4:1 (Ferlay et al., 2018, 2010). Furthermore, for individuals involved in the production and pesticide spraying of omethoate, the absence of personal protective equipment could make it easier to inhale the compound, which may also increase the incidence of HNSCC. Moreover, due to its rapid growth and infiltration, the prognosis of HNSCC is not optimistic. Combination of operative and nonoperative treatment not only affects the patient’s survival, but also greatly influences the patient’s quality of life (Bai et al., 2018). As the molecular mechanism of HNSCC is still poorly understood, it becomes urgent to study its pathogenesis and development. Uncontrolled cellular proliferation is the hallmark of cancer, and tumor cells typically acquire damaged genes that directly regulate the cell cycle (Sherr, 1996).

Cyclins are essential regulators of the cell cycle machinery. Among them, cyclin D1 is one of the most frequently altered cell cycle regulators in cancers. Deregulated function of cyclin D1, often resulting from overexpression of its protein level, has been documented in numerous human cancers (Balandaram et al., 2016; He et al., 2013). Cyclin D1 is a multifunctional oncoprotein that regulates the G1 to S phase transition by binding to CDK4 or CDK6 and phosphorylating pRb (Pestell, 2013; van Caloen and Machiels, 2019). Akt also plays an important role in the regulation of cell growth, survival, differentiation, migration and metabolism by phosphorylating a number of proteins (Hlobilkova et al., 2007; Choi et al., 2018). It has been shown that Akt phosphorylates glycogen synthase kinase-3β (GSK-3β) at Ser9 residue and subsequently inhibits its kinase activity (Qin et al., 2015). Normally, GSK-3β negatively regulates cyclin D1 through enhancing proteasomal degradation of cyclin D1 by phosphorylating its Thr286 and inhibiting cyclin D1 gene transcription by increasing degradation of β-catenin (Ye et al., 2013). Thus, Akt phosphorylation and inhibition of GSK-3β will lead to the upregulation of cyclin D1.

In this study, we demonstrated that exposure of omethoate induced HNSCC cell proliferation and G1/S transition. Mechanistically, omethoate activated Akt, which led to the inhibition of GSK-3β and upregulation of cyclin D1. Our findings might provide direct evidence of omethoate promoting HNSCC growth.

Section snippets

Chemicals

Omethoate (Product Number: 36181) and Akt1/2 kinase inhibitor (1,3-Dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate) (Product Number: A6730) were purchased from Sigma–Aldrich (St. Louis, MO).

Cell culture

The FaDu (human pharyngeal squamous carcinoma cell) cell line was purchased from Cell Bank of the Chinese Academy of Sciences in Shanghai and cultured in DMEM medium (10-013-CVR, Corning) supplemented with 10% fetal

Omethoate stimulates proliferation of FaDu cells

FaDu cells were treated with various concentrations (0, 0.1, 1, 10, 50, 100, 200 and 500 nM) of omethoate for 24 h. CCK-8 assay revealed that the number of FaDu cells increased in a dose-dependent manner, with the 100 nM dose demonstrating the highest rate of proliferation (Fig. 1A). We then used the 100 nM dosage for our time course studies, where FaDu cells were exposed to 100 nM of omethoate for different durations (0, 12, 24, 48 and 72 h). The results showed that FaDu cell growth was

Discussion

Omethoate is a broad category of organophosphorous pesticides (OPs) with increasing domestic use (Duan et al., 2017a). In recent years, acute poisoning incidents caused by OPs have been reduced, while the chronic toxic effect on human health caused by low-dose exposure to OPs has attracted the public’s attention (Celik et al., 2011). Omethoate exposure affects different organs including the lung, gastrointestinal tract, liver, brain and cardiomyocytes (Wang et al., 2016; Yan et al., 2012), and

Declaration of Competing Interest

The authors have no conflicts to disclose.

Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (No. 81873249, No. 81602010) and the Natural Science Foundation of Shandong Province (ZR2019MH058, ZR2016HP16).

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