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Tumor Biology

, Volume 36, Issue 10, pp 7947–7960 | Cite as

γ-Synuclein confers both pro-invasive and doxorubicin-mediated pro-apoptotic properties to the colon adenocarcinoma LS 174T cell line

  • Kai-Wey Goh
  • Yee-How Say
Research Article

Abstract

γ-synuclein, a neuronal protein of the synuclein family, is involved in carcinogenesis. To investigate its role in colorectal cancer carcinogenesis, we overexpressed γ-synuclein in LS 174T colon adenocarcinoma cell line (termed LS 174T-γsyn). When compared with untransfected/mock transfectants, LS 174T-γsyn had higher mobility in scratch wound assay, tend to scatter more in cell-scattering assay, and had enhanced lamellipodia and filopodia formation in cell-spreading assay. Enhanced adhesion of LS 174T-γsyn to fibronectin and collagen and significantly higher proliferation rate showed that γ-synuclein was able to increase extracellular matrix interaction and promoted proliferation of LS 174T. Higher invasiveness of LS 174T-γsyn was evidenced by enhanced invasion to the bottom of the basement membrane in Boyden chamber assay. However, LS 174T-γsyn were significantly more vulnerable to doxorubicin, vincristine and hydrogen peroxide insults, via apoptotic cell death. LS 174T-γsyn also had reduced anchorage-independent growth as shown by reduced colony formation and reduced anoikis resistance. We found that overexpression of γ-synuclein confers both pro-invasive and doxorubicin-mediated pro-apoptotic properties to LS 174T, where the former was mediated through enhanced cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation, while the latter involved hepatocyte growth factor (HGF) downregulation and subsequent downstream signalling pathways possibly involving extracellular signal-regulated kinases (ERK)1/2, p38α, c-Jun N-terminal kinase (JNK) pan and Signal Transducers and Activators of Transcription (STATs). This unexpected contrasting finding as compared to other similar studies on colon cancer cell lines might be correlated with the degree of tumour advancement from which the cell lines were derived from.

Keywords

Synuclein-gamma Colon cancer Invasion Metastasis Apoptosis 

Notes

Acknowledgments

This work was supported by grants from the UTAR Research Fund - IPSR/RMC/UTARRF/C210/S1 and IPSR/RMC/UTARRF/2012-C2/G05.

Conflicts of interest

None

Supplementary material

13277_2015_3455_Fig8_ESM.gif (38 kb)
Fig. S1

γ-synuclein overexpression had no effect on HEK 293 cell motility and extracellular matrix interaction. a Stable expression of γ-synuclein was determined by Western blot analysis using anti-γ-synuclein, a 16 kDa band corresponding to the molecular mass of human γ-synuclein protein was detected in HEK 293-γsyn. Lane 1 Untransfected HEK 293; Lane 2 HEK 293–3.1; Lane 3 HEK 293-γsyn. Cell migration was analyzed by using the scratch wound assay. b Scratch wound micrograph for HEK 293 viewed under ×40 magnification with Nikon Eclipse TS100 inverted microscope and open area of HEK 293 analysed with TScratch software. c Data of wound open area was expressed in percent and representing the mean ± SEM of three independent experiments. Mean values were compared using ANOVA followed by LSD’s post-hoc test. d Cell scattering and cell spreading morphology HEK 293 (Magnification power ×200). (I) Control (II) Cell scattering (III) Cell spreading. Cells showed no scattering pattern and spider-like pattern after serum starvation for 24 h in cell scattering; HEK 293 cells were incubated with various concentrations of fibronectin (e) for 30 min and rat tail collagen I (f) for 15 min. Adherent cells were stained, lysed and quantified at OD570. Qualitative variations in adhesion were measured as changes in OD values. Data of OD expressed in mean ± SEM obtained from three independent experiments. Mean values were compared using ANOVA followed by LSD’s post hoc test. (GIF 38 kb)

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High resolution image (TIFF 17.7 MB)
13277_2015_3455_Fig9_ESM.gif (7 kb)
Fig. S2

γ-synuclein overexpression did not enhance HEK 293 cell proliferation and had no effect on anoikis. a HEK 293 were seeded into 96-well plates. The cells were cultured for 24 h (day 0) prior to MTT assay. The procedures were repeated for days 1, 3 and 5. The absorbance values at 550 nm in MTT assay were then normalised to day 0. Data of percentage of cells expressed in mean ± SEM obtained from three independent experiments. Mean values were compared using ANOVA followed by LSD’s post-hoc test. b Anoikis assay. HEK 293 were seeded in poly-HEMA-coated wells for 3 days. Viability of the cells was then determined by MTT assay. The absorbance values at 550 nm in MTT assay were then normalised to untransfected cells. Data of percentage of vial cell expressed in mean ± SEM obtained from three independent experiments. Mean values were compared using ANOVA followed by LSD’s post-hoc test. (GIF 7 kb)

13277_2015_3455_MOESM2_ESM.tif (34.1 mb)
High resolution image (TIFF 34.0 MB)
13277_2015_3455_Fig10_ESM.gif (18 kb)
Fig. S3

γ-synuclein overexpression in HEK 293 cells had no effect in sensitising LS 174T cells towards cancer drug- and oxidative stress- induced apoptotic cell death. Cells were exposed to doxorubicin hydrochloride - DOX a at concentrations of 20, 10 and 5 μM, vincristine sulphate - VIN; b at concentrations of 0.1, 0.05 and 0.025 μM and H2O2 c at concentrations of 0.8, 0.4 and 0.2 mM for 48 h before MTT assay was performed and absorbance was read at 550 nm. These results represent mean ± S.E.M. of three independent experiments. Mean values were compared using ANOVA followed by LSD’s post-hoc test. d ROS generated within cells was detected using fluorescent probe DCFH-DA upon treatment with 3 mM of H2O2 for 30 mins. Nikon Eclipse TS100 fluorescence microscope image with enhanced fluorescence revealed no ROS elevation in HEK 293-γsyn. (magnification power ×200). (GIF 18 kb)

13277_2015_3455_MOESM3_ESM.tif (62.9 mb)
High resolution image (TIFF 62.8 MB)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  1. 1.Department of Science and Engineering, Centre for Foundation StudiesUniversiti Tunku Abdul Rahman (UTAR) Perak CampusKamparMalaysia
  2. 2.Department of Biomedical Science, Faculty of ScienceUniversiti Tunku Abdul Rahman (UTAR) Perak Campus, Jalan UniversitiKamparMalaysia

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