Investigation of proliferation and migration of tongue squamous cell carcinoma promoted by three chemokines, MIP-3α, MIP-1β, and IP-10

Figures & data

Table 1 Relative intensity of fluorescence signals between UM-1, CAL-27, TCA-8113 and squamous cells

Cytokine	UM-1/squamous cella	CAL-27/squamous cell	TCA-8113/squamous cell
IP-10	4.36	3.78	0.84
MIP-1β	3.38	2.13	0.68
MIP-3α	36.27	69.99	0.81
IL-1β	10.65	15.44	6.77
MCP-4	0.05	0.46	0.26
IL-6	6.13	8.46	2.62
IL-8	8.40	11.09	4.00
TNF-α	8.53	7.14	2.73
OPG	6.20	837.39	12.09

Note:

a Squamous cell: the keratinized squamous cells from the masticatory mucosa distributed in hard palate.

Abbreviations: IL, interleukin; IP-10, interferon gamma-induced protein 10; MIP-1β, macrophage inflammatory protein-1β; MIP-3α, macrophage inflammatory protein-3α; TNF, tumor necrosis factor; OPG, osteoprotegerin.

Table 2 Expression of IP-l0, MIP-1β, and MIP-3α detected by ELISA

Cytokine	UM-1	CAL-27	TCA-8113	Epithelial cells
MIP-1βa	305.4±13.2	236.4±19.1	89.9±8.8	91.1±11.9
MIP-3α	406.8±17.8	486.2±13.1	95.5±10.5	88.2±7.6
IP-10	311.1±18.0	214.9±18.2	89.1±9.7	95.6±10.6

Note:

a MIP-1β (F1: 183.7, p1: 0.0), MIP-3α (F2: 789.8, p2: 0.0), IP-10 (F3: 156.8, p3: 0.0).

Abbreviations: ELISA, enzyme-linked immunosorbent assay; IP-10, interferon gamma-induced protein 10; MIP-1β, macrophage inflammatory protein-1β; MIP-3α, macrophage inflammatory protein-3α.

Figure 1 High expression of MIP-3α in the cytoplasm and nucleus of TSCC.

Notes: Immunohistochemistry of MIP-3α in normal tissues (A) and squamous cell carcinoma (B). The relatively high expression of MIP-3α in the cytoplasm and nucleus of TSCC was indicated by a dark yellow staining, whereas in the squamous cells, the staining was lighter. Scale bar =100 μm.
Abbreviations: MIP, macrophage inflammatory protein; TSCC, tongue squamous cell carcinoma.

Table 3 Expression of MIP-3α in the TSCC and squamous cells

MIP-3α
Squamous cells	20	14	6	30

Abbreviations: MIP, macrophage inflammatory protein; TSCC, tongue squamous cell carcinoma.

Table 4 Expression of MIP-3α in the TSCC cells was correlated with the lymph node metastasis and the clinical stages (P<0.05), but not with the pathologic grade of the tongue cancer

	Case number	MIP-3α		χ^2	P-value
		−	(+, ++, +++)
Clinic stage					0.029
I–II	24	13	11	4.763
III–IV	22	5	17
Lymph node metastasis					0.022
No	29	15	14	5.225
Yes	17	3	14
Pathologic grade					0.615
I	20	7	13	0.253
II	26	11	15

Abbreviations: MIP, macrophage inflammatory protein; TSCC, tongue squamous cell carcinoma.

Figure 2 Expression of CCR5, CCR6, and CXCR3 in three human tongue squamous cell carcinoma cell lines (CAL-27, UM-1, and TCA-8113) by Western blot.

Abbreviations: CCR5, C-C chemokine receptor type 5; CCR6, C-C chemokine receptor type 6; CXCR3, C-X-C motif chemokine receptor 3.

Figure 3 Expression of CCR5, CCR6, and CXCR3 in both cell membrane and cytoplasm of CAL-27, UM-1 and TCA-8113 by immunofluorescence staining.

Abbreviations: CCR5, C-C chemokine receptor type 5; CCR6, C-C chemokine receptor type 6; CXCR3, C-X-C motif chemokine receptor 3.

Figure 4 Viability assays show the proliferation of CAL-27 cells by IP-10 (A), MIP-3α (B), and MIP-1β (C).

Note: The proliferation was MIP-1β concentration dependent (F=23.476, P=0.000), but not time dependent (F=1.761, P=0.150).
Abbreviations: IP-10, interferon gamma-induced protein 10; MIP-1β, macrophage inflammatory protein-1β; MIP-3α, macrophage inflammatory protein-3α.

Figure 5 Apoptosis in CAL-27 cells induced by MIP-1β.

Abbreviations: FITC, fluorescein isothiocyanate; MIP-1β, macrophage inflammatory protein-1β.

Figure 6 Apoptosis in Cal-27 cells induced by IP-10.

Abbreviations: FITC, fluorescein isothiocyanate; IP-10, interferon gamma-induced protein 10.

Figure 7 Invasive capability of TSCC cells by Transwell invasion assay.

Notes: Transwell invasion assay shows the effects of MIP-1β (B), MIP-3α (C), and IP-10 (D) on the invasive capability of TSCC cells, in comparison with the PBS control (A). The invasiveness was quantified as the percentage of cells invaded per group (E). The results show MIP-3α and IP-10 increased the invasive capability of TSCC cells (P<0.05), while MIP-1β did not (P>0.05).
Abbreviations: IP-10, interferon gamma-induced protein 10; MIP, macrophage inflammatory protein; TSCC, tongue squamous cell carcinoma.

Figure 8 MMP-2 and MMP-9 activities were significantly increased in the MIP-3α-and IP-10-treated cells.

Notes: Expression (A) and the enzymatic activities (B) of MMP-2 and MMP-9 in the TSCC cells.
Abbreviations: IP-10, interferon gamma-induced protein 10; MIP-1β, macrophage inflammatory protein-1β; MIP-3α, macrophage inflammatory protein-3α; MMP, matrix metalloproteinase; TSCC, tongue squamous cell carcinoma.