Mechanisms underlying dimethyl sulfoxide-induced cellular migration in human normal hepatic cells

https://doi.org/10.1016/j.etap.2020.103489Get rights and content

Highlights

  • DMSO induces cell migration when cells are arrested in S or G0/G1 phase.

  • Unbalance of MMPs and TIMPs leads to cell migration in DMSO-treated cells.

  • Signaling pathways of p38/HSP27 and TGF-β/p-Smad3 modulate DMSO-induced migration.

  • Cellular migration in G0/G1 arrested cells is associated with NRP1 expression.

Abstract

Numerous studies have reported that low-dose dimethyl sulfoxide (DMSO, <1.5%, v/v) can interfere with various cellular processes, such as cell proliferation, differentiation, apoptosis, and cycle. By contrast, minimal information is available about the effect of low-dose DMSO on cell migration. Here, we report the effect of DMSO (0.0005%−0.5%, v/v) on cellular migration in human normal hepatic L02 cells. We used the Cell Counting Kit-8 assay to measure cell viability, scratch wound healing assay to observe cellular migration, flow cytometry to analyze cell cycle and death pattern, reverse transcription quantitative polymerase chain reaction to evaluate mRNA expression, and Western blot to detect protein levels. After treatment with 0.0005% (v/v) DMSO, more cells entered S phase arrest, the MMP1/TIMP1 ratio increased, and HSP27 expression was elevated. After treatment with 0.1% (v/v) DMSO, more cells entered G0/G1 phase arrest, the MMP2/TIMP2 ratio increased, the p-p38 and p-Smad3 signaling pathways were activated, and neuropilin-1 expression was elevated. These results showed that cells migrate when their MMP1/TIMP1 and MMP2/TIMP2 ratios are imbalanced. Such migration is modulated by the p38/HSP27 signaling pathway and TGF-β/Smad3 dependent signaling pathway.

Introduction

Dimethyl sulfoxide (DMSO) is commonly used as a solvent for hydrophobic substances (Takeda et al., 2016). Low-dose (0.1%–1.5%) DMSO, the common concentration range for cellular treatments, has been reported to penetrate into the cell membrane, induce alterations in membrane phospholipid composition, decrease nucleic acid content, delay cell cycle progression, perturb protein secondary structure, and substantially enhance the Z form of DNA (Tunçer et al., 2018). Consequently, numerous cellular processes, such as cell proliferation (de Abreu Costa et al., 2017; Sharma et al., 1998), cycle (Chetty et al., 2013; Tunçer et al., 2018), differentiation (Alizadeh et al., 2016; Pal et al., 2012; Tunçer et al., 2018), and apoptosis (Banič et al., 2011; Hanslick et al., 2009; Trubiani et al., 1996; Yuan et al., 2014), are disturbed in cells treated with low-dose DMSO. Compared with the aforementioned cell processes, information about the effect of low-dose DMSO on cell migration is minimal, and thus, we aim to explore this topic.

Extracellular matrix degradation is crucial for cell migration (Bonnans et al., 2014; Lu et al., 2011). Matrix metalloproteinases (MMPs) comprise a family of calcium-dependent zinc-containing endopeptidase enzymes that are inhibited by the tissue inhibitors of metalloproteinases (TIMPs) once they are activated (Brew and Nagase, 2010; Cui et al., 2017; Pittayapruek et al., 2016). MMPs degrade extracellular matrices, such as triple-helical fibrillar collagens, type IV collagen, and gelatin (Apte and Parks, 2015; Chung et al., 2004). Cells frequently migrate when MMP and TIMP expression levels are imbalanced (Brayton, 1986; Tjomsland et al., 2016). Low-dose DMSO has been demonstrated to affect the expression of gelatinase MMP2 (Davis et al., 1998), leading to imbalance between MMP2 and TIMP2. Low-dose DMSO has also been reported to change MMP9 (Koizumi et al., 2003; Tjäderhane et al., 2013) and TIMP1 (Young et al., 2004) expression, resulting in an imbalance between MMP9 and TIMP1. Thus, low-dose DMSO may drive cellular migration by disturbing the balance between MMPs and TIMPs.

Transforming growth factor-β (TGF-β)/Smad3-dependent signaling and mitogen-activated protein kinase (MAPK) p38/heat-shock protein (HSP) signaling are well-documented signaling cascades that involve the modulation of the balance between MMPs and TIMPs. MMP1 is a target for negative regulation by TGF-β through cellular Smad3 (Yuan and Varga, 2001). MMP2 is a target for positive regulation by TGF-β in a Smad3-dependent manner (Wiercinska et al., 2011). HSP27-overexpressing cells have a less invasive phenotype, reduced levels of MMP2 (Lee et al., 2008), and increased levels of MMP9 (Hansen et al., 2001). Thus, we propose that the TGF-β/Smad3 and p38/HSP27 signaling pathways may be involved in DMSO-induced MMP/TIMP imbalance. In the present study, we evaluated the effects of DMSO (0.0005%−0.5%, v/v) on cellular migration in human normal hepatic L02 cells. Furthermore, we studied the roles of the TGF-β/Smad3 and p38/HSP27 signaling pathways in cell migration simulated by different DMSO concentrations.

Section snippets

Materials

DMSO was purchased from Sigma-Aldrich (St. Louis, MO, USA). L02 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was supplied by GIBCO BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was produced by Tianhang Biotechnology (Hangzhou, China). The Cell Counting Kit-8 (CCK-8) was produced by Dojindo (Kumamoto, Japan). The cell cycle analysis kit and the Annexin V/PI Apoptosis Detection Kit I were obtained

Cells migrated under the treatments of 0.0005% (v/v) and 0.01% (v/v) DMSO

As shown in Fig. 1A, the viability exceeded 80% in the cells treated with DMSO concentrations of <1% (v/v) for 24 h. The 1%, 2%, and 5% DMSO concentrations reduced the cell viability to 83.7%, 18.4%, and 14.9%, respectively. Subsequently, five DMSO concentrations were selected to study the cell viability for 24 and 48 h. The one-way ANOVA with Dunnett’s test indicated that DMSO concentrations of 0.0005% at 24 h (Fig. 1B) and 0.0005%, 0.005%, and 0.01% at 48 h (Fig. 1C) exhibited more preserved

Discussion

Here we found that 0.0005% and 0.01% DMSO could induce S phase or G0/G1 phase arrest and imbalance MMPs and TIMPs, which caused cell migration modulated by the p38/HSP27 and TGF-β1/Smad3 signaling pathways. As we know, this is the first report demonstrating that DMSO induces two forms of cells migration, setting the basis for further analysis of cells migration under cycle arrest.

Disturbs of the MMP/TIMP balance by MMP expression increase provoke cells migration and invasion (Lin et al., 2019).

Conclusion

The present study indicates that 0.0005% and 0.1% DMSO-treated cell migration occurs by disturbing the balance between MMPs and TIMPs. This process is modulated by p38/HSP27 and the TGF-β signaling pathway. Further studies are required to explore the differences in cell movement driven by cell proliferation and epithelial-to-mesenchymal transition and the roles of phosphorylated and unphosphorylated p38 and HSP27 in cell cycle arrest induced by DMSO.

CRediT authorship contribution statement

Fengmei Wei: Conceptualization, Methodology, Data curation, Writing - original draft. Long Zhao: Methodology, Resources, Project administration. Yuhong Jing: Methodology, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research is supported by the Special Fund of the Chinese Central Government for Basic Scientific Research Operations (lzujbky-2017-129).

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