Metastasis-Associated Protein 1 Is Involved in Angiogenesis after Transarterial Chemoembolization Treatment

Abstract

Background: Transarterial chemoembolization (TACE), a well-established treatment for unresectable hepatocellular carcinoma (HCC), blocks the arterial blood supply to the tumor, which can be short-lived as development of collateral neovessels, leading to the failure of treatment. Metastasis-associated protein 1 (MTA1) is involved in development of tumors and metastases. However, the role of MTA1 in angiogenesis is still obscure.

Methods: We detected the expression of MTA1 and hypoxia-inducible factor-1α (HIF-1α) and microvessel density (MVD) value in liver tumor tissues and tumor periphery before and after TACE treatment. Hepatocellular carcinoma cell line HepG2, tube formation assay, and chorioallantoic membrane (CAM) assay were applied to explore the mechanism of MTA1 in angiogenesis.

Results: We found that expression of MTA1 increased after TACE treatment, especially in tumor periphery, which was accompanied by markedly elevated MVD value, indicating a significant correlation between MTA1 and MVD value. Moreover, MTA1 contributed to neovascularization of residual tumors. Cellular experiments further revealed that MTA1 increased the stability and the expression of HIF-1α, and overexpression of MTA1 enhanced tube formation and neovessels of chick embryos.

Conclusions: MTA1 is an active angiogenic regulator; our results shed light on better understanding in neovascularization, which are helpful to predict prognosis of TACE, and provide evidences for intervention to improve therapeutic effects on HCC.

Figure 1

Ultrasound-guided liver biopsy and grade of immunohistochemical staining. (a) The percutaneous liver biopsies were performed by a radiologist using real-time ultrasound guidance. (b) Intensity of immunohistochemical staining was graded as none, +, ++, and +++, respectively (magnification = ×400, scale bar = 25 μm).

Figure 2

Immunohistochemistry and western blot analysis in liver tumor tissues. (a) MTA1 and HIF-1α staining in tumor tissues before and after TACE treatment (magnification = ×200, scale bar = 100 μm). (b) Western blot results of MTA1 and HIF-1α in patients' samples of tumor periphery (P1 to P6). Lanes 1, 4, and 7 indicated pre-TACE treatment; Lanes 2, 5, and 8 indicated one month after TACE treatment; Lanes 3, 6, and 9 indicated two months after TACE treatment.

Figure 3

Expression of MTA1, HIF-1α and MVD in tumor peripheries, before TACE treatment and one month and two months after TACE treatment, respectively. Arrows indicated microvessels (magnification = ×40, and ×200, scale bar = 100 μm). (a), (d), (g) MTA1 staining. (b), (e), (h) HIF-1α staining. (c), (f), (i) MVD (CD34 staining).

Figure 4

Correlation between MTA1 expression and MVD value. (a) Before TACE (r = 0.19, P > 0.1). (b) One month after TACE (r = 0.46, P < 0.01). (c) Two months after TACE (r = 0.77, P < 0.001).

Figure 5

MTA1 increased HIF-1α stability and interacted with HIF-1α in vitro and in vivo. (a) HepG2 cells were stably transfected with the indicated expression vector and untreated or exposed to 1% O₂ (hypoxia) for 4 h. (b) Mock-HepG2 and MTA1- Flag-HepG2 cells were transiently transfected with -HA or HIF-1α-HA as indicated, and cells were then untreated or exposed to hypoxic conditions for 4 h. (c) HIF-1α was translated in the presence of [³⁵S]methionine and mixed with GST- or GST-MTA1-bound beads. Ten percent of the material is used as a control in this assay.

Figure 6

MTA1 overexpression enhanced angiogenesis. (a) CMs were collected from nontransfected (Con), mock-transfected (Mock), or MTA1-transfected HepG2 cells (MTA1). HUVECs on matrigel were grown in the CM for 24 h. VEGF (10 ng/mL) was employed as a positive control. (i) Representative images showing tube formation (magnification = ×40, scale bar = 100 μm). (ii) Tube length was quantified and expressed as the means ± SD (n = 5). ^##P < 0.01 (VEGF versus Con); ^∗∗P < 0.01 (MTA1 versus Con). (b) The chick embryos were treated with CMs collected from nontransfected (Con), mock-transfected (Mock), or MTA1-transfected HepG2 cells (MTA1). VEGF (30 ng/mL) was employed as a positive control (magnification = ×20, scale bar = 200 μm).