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

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.

Peer Review #1 of "Cellular prion protein and γ-synuclein overexpression in LS 174T colorectal cancer cell drives endothelial proliferation-to-differentiation switch (v0.1)"

“…Western blot confirmed the overexpression of γ-Syn and PrP C in LS 174T-γ-Syn and LS 174T-PrP cells, respectively, as established previously (Chieng & Say, 2015;Goh & Say, 2015) ( Figure 1 A&B, respectively). MTT proliferation assay was then carried out to evaluate the effects of LS 174T CM on EA proliferation rate and to determine the optimal treatment duration for the rest of the study ( Figure 1C).…”

“…All cell lines have been checked to ensure they are free of contamination and have been used from young stock (less than 7 passages). LS 174T overexpressing γ-Syn (LS 174T-γ-Syn) and PrP (LS 174T-PrP) were previously established in our laboratory (Chieng & Say, 2015;Goh & Say, 2015), and overexpression of γ-Syn and PrP C in LS 174T cells was confirmed once again in this study by Western blotting, as detailed previously (Chieng & Say, 2015;Goh & Say, 2015). In this study, untransfected LS 174T, LS…”

“…Substantial studies illustrate the role of these proteins in fostering tumorigenesis, including promoting cancer cell migration, adhesion, invasion, and survival (Liu et al, 2005;Ye et al, 2009;Yap & Say, 2011;Yap & Say, 2012). Previously, we showed that the overexpression of γ-Syn in LS 174T colorectal adenorcarcinoma cell line could confer both pro-invasive and doxorubicin-mediated pro-apoptotic properties to the cells (Goh & Say, 2015). In addition, we also showed that PrP C could promote LS 174T cells carcinogenesis by increasing invasiveness PeerJ reviewing PDF | (2018:02:23859:1:1:NEW 23 Feb 2018)…”

“…The secretion of interleukin-8 (IL-8) from LS 174T-γ-Syn was exceptionally heightened, whereas moderate secretions of urokinase-type plasminogen activator (uPA) and angiogenin were found in LS 174T-PrP.4. DiscussionContinuing our research in elucidating the roles of neuronal proteins γ-Syn and PrP C in colorectal cancer cell biology(Yap & Say, 2011;Yap & Say, 2012;Chieng & Say, 2015;Goh & Say, 2015), we investigated the angiogenic effects of these two proteins in LS 174T cells by performing in vitro angiogenesis assay. Angiogenesis is a complex process which involves the proliferation, invasion, migration, and tube formation of ECs(Bussolino, Mantovani & Persico, PeerJ reviewing PDF | (2018:02:23859:1:1:NEW 23 Feb 2018)…”

Zebularine Promotes Hepatic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells by Interfering with p38 MAPK Signaling

“…In addition to inflammation and stress responses, p38 is involved in the control of cell cycle and proliferation [ 13 ]. Previous studies have indicated the reciprocal regulation of HGF and activated p38 in cell proliferation [ 14 – 16 ]. Transforming growth factor- β (TGF- β ) downregulated activation of HGF precursors and influenced hepatocyte plasticity via p38 signal pathway [ 15 , 17 , 18 ].…”