Overexpression of chemokine ligand 7 is associated with the progression of canine transmissible venereal tumor

Abstract

Background: Chemokines play multiple roles in the development and progression in a variety of tumors. Chemokine (C-X-C motif) ligand 7 (CXCL7) has been found associated with pro-inflammatory responses, but its role in cancer growth remains unclear. Our previous study showed that R phase tumor infiltrating lymphocytes (TILs) produced large amounts of interleukin (IL)-6 which antagonized transforming growth factor (TGF)-β derived from CTVT to diminish the immune-suppressive microenvironment. Now we intend to determine the expression pattern of CXCL7 and the role of IL-6/TGF-β in CXCL7 induction during spontaneous progressive (P) and regressive (R) phases in canine transmissible venereal tumor (CTVT).

Results: We have demonstrated that CXCL7 expressed at high level in P phase and down-regulated in R phase by western blot and real-time PCR. This suggested that CXCL7 expression was negatively correlated with the tumor growth. Co-culturing TILs with CTVT cells was found to reduce CXCL7 expression, while adding IL-6 blocking antibody reversed it. Moreover, in P phase CTVT, while IL-1β and TGF-β had no obvious effect on CXCL7 expression, IL-6 was found significantly to reduce CXCL7 expression in a dose-dependent manner. The mRNA expression results of CXCL7 receptor, CXCR2, further confirmed the effects of IL-6 concentration on the CXCL7 expression.

Conclusion: CXCL7 overexpression might be associated with the progressive growth of CTVT. The results shown here also suggest the role of CXCL7 in cancer development and the potential as the anti-cancer therapeutic target.

Figure 1

CXCL7 expression is concordant with the CTVT progression phase.A. Levels of mRNA expression of CXCL7 from CTVT tumor masses (n = 4) measured by real-time RT-PCR. The amounts of mRNA are expressed relative to the amount of β-actin mRNA in each sample and are shown as the mean ± S.D. (**, P < 0.01). B. Western blot analysis of CXCL7 protein expressed in CTVT. CXCL7 proteins were recognized by polyclonal rabbit antibody against canine CXCL7 in the lysates of CTVT tumor masses. Actin was used as the internal control.

Figure 2

R-phase TIL inhibited the expression of CXCL7 in P-phase CTVT cells. A coculture system was analyzed using a Corning transwell system, in which TIL cells (0.1–1×10⁶ cells/well) were seeded in the upper chamber, with a 0.4-μm-pore filter between that and a lower chamber containing CTVT cells (1×10⁶ cells/well). After 12 h of incubation, the gene expression of CXCL7 in the CTVT cells was evaluated. Significant differences in the various stimulators are expressed as * (P < 0.05) and ** (P < 0.01) as compared with CTVT alone. The results represent the mean ± S.D. of three independent experiments.

Figure 3

CXCL7 and CXCR2 expression in P-phase CTVT cells was regulated by IL-6. Levels of mRNA expression of CXCL7 (A) and CXCR2 (B) after freshly prepared P phase CTVT cells were treated with 10, 30 or 90 ng/mL IL-6 for 12 h, measured by real-time RT-PCR. The results represent the mean ± S.D. of three independent experiments (* P < 0.05 as compared with the CTVT alone group).

Figure 4

CXCL7 expression in CTVT under IL-1β, TGF-β and IL-6 stimulation. Levels of mRNA expression of CXCL7 after freshly prepared P-phase CTVT cells were treated with IL-1β, TGF-β and/or IL-6 (90 ng/mL) for 12 h, measured by real-time RT-PCR. The amounts of mRNA are expressed relative to the amount of β-actin mRNA in each sample and are shown as the mean ± S.D. (B) 1 μg/ml of anti-IL-6 antibody was adding into the TIL/CTVT cocultured transwell system. TIL cells (0.3 × 10⁶ cells/well) were seeded in the upper chamber and the lower chamber containing CTVT cells (1 × 10⁶ cells/well). After 12 h incubation, the CXCL7 gene expression in the CTVT cells was evaluated. Significant differences in the various stimulators are expressed as ** (P < 0.01) as compared with CTVT alone.