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Abstract
The synuclein family consists of three distinct genes, α-synuclein, β-synuclein, and γ-synuclein. The α-synuclein and β-synuclein are predominately expressed in brain and especially α-synuclein is related with Parkinson's disease, Alzheimer's disease, and dementia with Lewy bodies. The γ-synuclein was first identified as breast cancer specific gene 1. It is expressed in the peripheral nervous system and also detected in breast and ovarian cancers. The γ-synuclein is also known to mediate metastasis of breast and ovarian cancer cells. Insulin-like growth factor 1 (IGF-I) is one of the growth factors that plays an important role in cell proliferation and migration in cancer cells, as well as in normal cells. In this study, we investigated the migrations of SKOV-3, MDAMB-231, and HeLa cells by the recombinant synuclein proteins (α-, β-, and γ-synucleins) and IGF-I and the molecular mechanism. Furthermore, we investigated the membrane ruffle formation of SKOV-3 cells by recombinant synuclein proteins and IGF-I. As a result, synucleins and IGF-I were found to induce cancer cell migrations. Simultaneous synucleins and IGF-I treatment on the cancer cells induced more migrations than the individual synuclein or IGF-I treatments. The synucleins or IGF-I treatments increased the expressions of membrane-type1 matrix metalloproteinase (MT1-MMP) and cluster of differentiation 44 (CD44). Moreover, simultaneous synucleins and IGF-I treatments further increased the expressions of MT1-MMP and CD44. The synucleins and IGF-I promoted the conformational change of actin filaments, and then this led to the membrane ruffle formation.
Figures and Tables
Figure 1
SDS-PAGE analysis of the purified recombinant synucleins. The purified recombinant synucleins were separated by 15% SDS-PAGE and stained with Coomassie brilliant blue. α-synuclein (α-syn), β-synuclein (β-syn), and γ-synuclein (γ-syn).
Figure 2
The recombinant synucleins induced cancer cell migration. SKOV-3, MDAMB-231, HeLa cells were treated with 100 nM and 500 nM of the recombinant synucleins for 24 h. (A) The wound gaps of SKOV-3 cells at 0 h and 24 h. The gaps of 0 h and 24 h were compared by ImageJ. (B) SKOV-3, (C) MDAMB-231, (D) HeLa cells were quantified (n = 3). Scale bar, 500 µm. *p < 0.05, **p < 0.001.
Figure 3
IGF-I induced cancer cell migration. SKOV-3, MDAMB-231, HeLa cells were treated with 1 to 100 ng/ml of IGF-I for 24 h. (A) The wound gaps of SKOV-3 cells at 0 h and 24 h. The gaps of 0 h and 24 h were compared by ImageJ. (B) SKOV-3, (C) MDAMB-231, (D) HeLa cells were quantified (n = 3). Scale bar, 500 µm. *p < 0.05.
Figure 4
The recombinant synucleins and IGF-I induced cancer cell migration. SKOV-3, MDAMB-231, HeLa cells were treated with 5 ng/ml of IGF-I and 500 nM of the recombinant synucleins for 24 h. (A) The wound gaps of SKOV-3 cells at 0 h and 24 h. The gaps of 0 h and 24 h were compared by ImageJ. (B) SKOV-3, (C) MDAMB-231, (D) HeLa cells were quantified (n = 3). Scale bar, 500 µm. *p < 0.05.
Figure 5
The recombinant synucleins induced the expression of MT1-MMP. For investigation of the expression of MT1-MMP, FACS analysis was accomplished. SKOV-3 cells were treated with 500 nM of (A) α-synuclein, (B) β-synuclein, and (C) γ-synuclein for 24 h. MDAMB-231 cells were treated with 500 nM of (D) α-synuclein, (E) β-synuclein, and (F) γ-synuclein for 24 h. HeLa cells were treated with 500 nM of (G) α-synuclein, (H) β-synuclein, and (I) γ-synuclein for 24 h. Solid gray, unstained (uns). Tinted gray with line, control (con). Bold black line, recombinant synucleins.
Figure 6
The recombinant synucleins and IGF-I induced the expression of MT1-MMP. For investigation of the expression of MT1-MMP, FACS analysis was accomplished. SKOV-3 cells were treated with (A) 5 ng/ml of IGF-I, 5 ng/ml of IGF-I and 500 nM of (B) α-synuclein, (C) β-synuclein, and (D) γ-synuclein for 24 h. MDAMB-231 cells were treated with (E) 5 ng/ml of IGF-I, 5 ng/ml of IGF-I and 500 nM of (F) α-synuclein, (G) β-synuclein, and (H) γ-synuclein for 24 h. HeLa cells were treated with (I) 5 ng/ml of IGF-I, 5 ng/ml of IGF-I and 500 nM of (J) α-synuclein, (K) β-synuclein, and (L) γ-synuclein for 24 h. Solid gray, unstained. Tinted gray with line, control (con). Gray line, IGF-I. Bold black line, recombinant synucleins.
Figure 7
The recombinant synucleins induced the expression of CD44. For investigation of the expression of CD44, FACS analysis was accomplished. SKOV-3 cells were treated with 500 nM of (A) α-synuclein, (B) β-synuclein, and (C) γ-synuclein for 24 h. MDAMB-231 cells were treated with 500 nM of (D) α-synuclein, (E) β-synuclein, and (F) γ-synuclein for 24 h. HeLa cells were treated with 500 nM of (G) α-synuclein, (H) β-synuclein, and (I) γ-synuclein for 24 h. Solid gray, unstained. Tinted gray with line, control (con). Bold black line, recombinant synucleins.
Figure 8
The recombinant synucleins and IGF-I induced the expression of CD44. For investigation of the expression of CD44, FACS analysis was accomplished. SKOV-3 cells were treated with (A) 5 ng/ml of IGF-I, 5 ng/ml of IGF-I and 500 nM of (B) α-synuclein, (C) β-synuclein, and (D) γ-synuclein for 24 h. MDAMB-231 cells were treated with (E) 5 ng/ml of IGF-I, 5 ng/ml of IGF-I and 500 nM of (F) α-synuclein, (G) β-synuclein, and (H) γ-synuclein for 24 h. HeLa cells were treated with (I) 5 ng/ml of IGF-I, 5 ng/ml of IGF-I and 500 nM of (J) α-synuclein, (K) β-synuclein, and (L) γ-synuclein for 24 h. Solid gray, unstained. Tinted gray with line, control (con). Gray line, IGF-I. Bold black line, recombinant synucleins.
Figure 9
The recombinant synucleins and IGF-I induced the membrane ruffling formation of SKOV-3. For investigation of the membrane ruffling formation, confocal microscopy was accomplished. (A) as control. SKOV-3 cells were treated with 500 nM of (B) α-synuclein, (C) β-synuclein, and (D) γ-synuclein, (E) 5 ng/ml of IGF-I, 5 ng/ml of IGF-I and 500 nM of (F) α-synuclein, (G) β-synuclein, and (H) γ-synuclein for 24 h. For F-actin staining, cells were stained with rhodamine phalloidin. Scale bar, 20 µm.
Table 1
Endotoxin contents of before and after removing the endotoxin from the purified recombinant synucleins. For removing the endotoxin, the recombinant synucleins were reacted in Detoxi-Gel Endotoxin Removing Column. Before and after removing the endotoxin, endotoxin contents were quantified by LAL test.
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