Elsevier

Life Sciences

Volume 90, Issues 21–22, 6 June 2012, Pages 851-861
Life Sciences

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

https://doi.org/10.1016/j.lfs.2012.03.039Get rights and content

Abstract

Aims

Formation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.

Main methods

Colon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.

Key findings

Increased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.

Significance

Our experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.

Introduction

Cell migration depends mainly on dynamic reorganization of the actin cytoskeleton controlled by numerous actin binding proteins (ABPs) (Lambrechts et al., 2004, Olson and Sahai, 2009). ABPs regulate the polymerization of monomeric (G) to the filamentous (F) actin form and determine the supramolecular organization of actin filaments.

Gelsolin is a multifunctional actin binding protein controlling the length of actin filaments by its severing and capping activity. In addition, gelsolin can bind one or two actin molecules and then act as a polymerization nucleator thus promoting formation of new microfilaments (Yin and Stossel, 1979). Immunofluorescence observations have shown that gelsolin in different cells is localized mainly within the perinuclear cytoplasm and along stress fibers (Mazur et al., 2010, Paddenberg et al., 2001). However, gelsolin has been found also in the cell periphery within presumed ruffles and lamellipodia where it co-localizes with other ABPs e.g. cofilin and actin depolymerizing factor (ADF) (Mazur et al., 2010). The functional analysis of fibroblasts isolated from gelsolin knock-out mice has indicated that gelsolin mediates cell migration, since the gelsolin deficiency resulted in the formation of excessive stress fibers and defective ruffling and cell motility (Witke et al., 1995, Azuma et al., 1998). In view of the biological activities of gelsolin these data suggested that it plays an essential role in the regulation of actin polymerization and cycling — necessary for cell migration.

Gelsolin has been reported to play an unique role in podosome formation in osteoclasts (Chellaiah et al., 2000, Chellaiah, 2006). Podosomes are highly dynamic, actin-rich structures present in many motile cells, implicated in adhesion and matrix degradation (Chellaiah et al., 2000, Linder and Aepfelbacher, 2003). Several signaling molecules including phosphatidylinositol 3-phosphate, c-Src, PYK2-kinase and cytoskeletal proteins such as vinculin, α-actinin and gelsolin were found to be involved in integrin-dependent formation of podosomes (Chellaiah, 2006, Linder and Aepfelbacher, 2003). It has been shown that gelsolin deficiency blocks podosome assembly and affects the α(v)β(3) integrin signaling pathway controlling cell motility (Chellaiah et al., 2000, Chellaiah, 2006).

Changes in gelsolin expression are observed in a number of human tumor cells and are often connected with development and progression of cancer. Some studies have shown a correlation between the level of gelsolin expression and cell migration potential. Gelsolin overexpression in cultured fibroblasts resulted in their increased motility (Cunningham et al., 1991), whereas in cells isolated from gelsolin‐null mice exhibited a decrease in cell motility. Such studies demonstrated that neuronal growth cones, osteoclasts and fibroblasts of these mice are characterized by reduced migration abilities and delayed retraction of lamellipodia and filopodia (Lu et al., 1997). However the exact role of gelsolin in carcinogenesis is still controversial (Cunningham et al., 1991, Li et al., 2010). Gelsolin down-regulation has been described for tumor cells isolated from breast (Dong et al., 2002, Winston et al., 2001), colon (Furuuchi et al., 1997, Gay et al., 2008), ovarium (Noske et al., 2005), prostate (Lee et al., 1999) and bladder cancer (Tanaka et al., 1995). On the contrary, a number of studies have reported that upregulation of gelsolin is associated with poor prognosis and higher risk of cancer recurrence. Indeed, elevated levels of gelsolin expression have been detected in non-small cell lung (Yang et al., 2004), oral squamous (Shieh et al., 2006), urothelial (Rao et al., 2002), pancreatic (Thompson et al., 2007) and recently also in cervical carcinomas (Liao et al., 2010). In non-small lung cancers high gelsolin levels correlated with lymphatic invasion and were associated with poor survival (Yang et al., 2004). Depletion of gelsolin using small interfering RNAs (siRNAs) in pancreatic (Thompson et al., 2007), prostate and breast cancer cell lines caused a marked reduction in cells motility (Van den Abbeele et al., 2007).

We have previously observed a correlation between the level of gelsolin expression and cancer cell migration ability and attributed this effect to an altered organization of the actin cytoskeleton (Litwin et al., 2008). The aim of these studies was to specify the role of gelsolin in determining migration abilities of cancer cells. Here we used the human colon adenocarcinoma LS180 and melanoma A375 cell lines, which we previously characterized with a low and a high level of gelsolin expression, respectively. The LS180 cells were transfected with pEGFP-N1 plasmid encoding human cytoplasmic gelsolin fused C-terminally to enhanced green fluorescent protein (EGFP). Transfection with small interfering RNAs (siRNAs) specific for human gelsolin was performed in the case of human melanoma A375 cells. The goal of both molecular procedures was to gain further evidence for the involvement of gelsolin in the migration process of cancer cells.

Section snippets

Cell culture

The human colon adenocarcinoma cell line LS180 was obtained from the Institute of Immunology and Experimental Therapy, Polish Academy of Science in Wroclaw, Poland. The LS180 cells were grown in OptiMEM® medium (Invitrogen) containing 5% foetal bovine serum (FBS), 2 mM glutamine and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin). The human melanoma A375 cell line (CRL-1619™, ATCC) was obtained from the American Type Culture Collection. The cells were cultured in Dulbecco's modified

Gelsolin overexpression in the LS180 colon cancer cells

Our previous studies have shown the correlation between gelsolin level in different cancer cell lines and their migration ability (Litwin et al., 2008). In this study we used the human colon adenocarcinoma LS180 and melanoma A375 cell lines, characterized with a low and a high level of gelsolin expression, respectively (Fig. 1A, B). Therefore, transfection with small interfering RNAs (siRNAs) specific for human gelsolin was performed in the case of human melanoma A375 cells. Human colon

Discussion

Aberrant regulation of cell migration underlies progression of many diseases, including cancer cell invasion and metastasis. Cancer cell migration through tissue barriers requires the degradation of particular components of the extracellular matrix (ECM) followed by an altered dynamic interaction between actin cytoskeleton and the ECM proteins. These processes are influenced by growth factors, but are crucially dependent on adhesion receptors and regulated by specific actin binding molecules (

Conclusions

The changes in gelsolin level correlate with different migration abilities of human adenocarcinoma and melanoma cells. Gelsolin overexpression in human colon adenocarcinoma cell line — LS180 affects the state of actin polymerization (F:G), actin organization and cell migration. Vinculin redistribution and its colocalization with gelsolin suggest that the higher migration ability of the LS180 cells overexpressing gelsolin might be due to the formation of podosome-like structures. Downregulation

Conflict of interest statement

None of the authors of the manuscript has declared any conflict of interest.

Acknowledgments

This work was supported by grant no. N N301 241936, from the Ministry of Science and Higher Education, Poland.

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