TGF beta1 and related-Smads contribute to pulmonary metastasis of hepatocellular carcinoma in mice model

Abstract

Background: Recent studies indicate that Transforming Growth Factor beta (TGF β) correlated with pulmonary metastasis of cancers. However, the correlation between TGF β and pulmonary metastasis of hepatocellular carcinoma (HCC) is till unknown.

Methods: We detected the in vitro and in vivo expression levels of TGF β1/Smads by Real-time PCR and Western blot in MHCC97-H and MHCC97-L cell lines, which are HCC cell lines and have higher and lower pulmonary metastatic potential respectively.

Results: TGF β1 mRNA level in MHCC97-L tumors were higher than that in MHCC97-H tumors, (2.81±1.61 vs. 1.24±0.96, P=0.002), TGF β1 protein level in MHCC97-L tumors were also higher than that in MHCC97-H tumors (1.37±0.95 vs. 0.32±0.22, P<0.001). In addition, the TGF β1 mRNA level positively correlated with pulmonary metastasis, and the relations between TGF β1 and Smads were also found (R2=0.12 and 0.40, respectively).

Conclusions: Our results suggest that TGF β/ Smads promote pulmonary metastasis of HCC.

Figure 1

Comparison of Growth and pulmonary metastsis in mice models. A) Growth curve of MHCC97-H and MHCC97-L models; B) Average days which were spent for getting to tumor size. * denoted P<0.05, Error bar represent the standard errors of the mean. C,D) MHCC97-L models (C) had smaller pulmonary metastatic loci than MHCC97-H models (D). Arrows denote metastatic loci.

Figure 2

The TGF β/Smads levels in different cell lines and animal models. A) The different expression levels of TGF β in MHCC97-H and MHCC97-L by western blot analysis. (B). Western blot analysis for tumors. TGF β1 (25KD) and β-actin(43KD) bands of samples from two models. Ratio means: ratio of TGF β1 to β-actin bands intensity. C). The different expression levels of TGF β in MHCC97-H and MHCC97-L by cytoimmunochemistry. The brown-yellow color means positive staining, a: MHCC97-L, b: MHCC97-H. (×20 objective field). D) The expression of TGF β1 in MHCC97-H and MHCC97-L models by immunohistochemisty staining, the brown-yellow color means positive staining. a: MHCC97-L model, b: MHCC97-H model. (×20 objective field).

Figure 3

The expression of TGF β correlated with pulmonary metastasis. A) MHCC97-L model had a high expression levels than MHCC97-H model by ELASA. * denoted P<0.05. B) TGF β1 in metastasis group have higher levels than in non- metastasis group. C-D) The correlations between TGF β1 mRNA and Smad2, as well as Smad7. Dot denoted the each samples; Lines represent regression line, R: correlation coefficient.