Increased expression of cathepsin L: a novel independent prognostic marker of worse outcome in hepatocellular carcinoma patients

Abstract

Objectives: To investigate the expression and role of Cathepsin L (CTSL) in Hepatocellular carcinoma (HCC) tissue and cell line (MHCC-97H), and to evaluate the clinical and prognostic significance of CTSL protein in patients with HCC.

Methods: The expression of CTSL was examined in HCC tissue and MHCC-97H cells by Western-blotting, Real-time PCR and immunohistochemical staining. Cell growth curve assay and colony formation assay were used to verify the effect of CTSL on the proliferation and tumor progression ability of MHCC-97H cells. Tumor formation assay in nude mice was used to analyze the effect of CTSL on the tumorigenicity of MHCC-97H cells.

Results: The status of CTSL protein in carcinoma tissues is much higher than that in paracarcinoma tissues. The overall survival of the patients with high CTSL expression was significantly shorter than the low CTSL expression group. high CTSL expression was significantly correlated with advanced clinical staging, histological grade and tumor recurrence. In vitro experiments demonstrated that over-expression of CTSL in MHCC-97H cells promoted cell proliferation and tumor progression ability. Down-regulation of CTSL showed the opposite effects. Over-expression of CTSL increase the tumorigenicity of MHCC-97H cells by in vivo experiments. Moreover, multivariate analysis suggested that CTSL expression might be an independent prognostic indicator for the survival of HCC patients after curative surgery.

Conclusions: CTSL might involve in the development and progression of HCC as a oncogene, and thereby may be a valuable prognostic marker for HCC patients.

Figure 1. Expression levels of CTSL in HCC tissues.

A. Expression levels of CTSL protein in 13 paired HCC tissues by Western blotting. N, paracarcinoma (normal) liver tissues. T, HCC tissues. B. Quantitative analysis of CTSL protein in 13 paired HCC tissues. C. mRNA levels of CTSL in 13 paired HCC tissues by real-time PCR.

Figure 2. Analysis of CTSL protein in tissues by immunohistochemistry.

A and B, CTSL expression is negative in normal liver cells. C and D, CTSL expression is weak in well-differentiated HCC cells. E and F, CTSL expression is moderate in moderately differentiated HCC cells. G and H, CTSL expression is strong in poorly differentiated HCC cells. (A, C, E, G ×200; B, D, F, H ×400).

Figure 3. Survival curves for patients with high CTSL expression versus low CTSL-expressing carcinoma.

The 5-year overall survival rate was 22.7% in the high CTSL protein expression group (green line), but it was only 41.4% in the low expression group (blue line), P = 0.032.

Figure 4. The effect of CTSL on the proliferation and tumor progression ability of MHCC-97H cells.

A. Western blotting analysis of CTSL protein expression in HCC cell line (MHCC-97H), colorectal carcinoma cell lines (CaCO2 and LoVo), stably CTSL-expressed MHCC-97H cell line, stably CTSL-expressed CaCO2 cell line, empty vector stable cell lines (MHCC-97H-Con or CaCO2-Con), and MHCC-97H cell line transfected by CTSL-shRNA or Con-shRNA. B. Colony formation assay and MTT assay of MHCC-97H cells and CaCO2 cells with over-expression of CTSL. (Colony formation assay: MHCC-97H-Con (vector) vs MHCC-97H-CTSL, P = 0.0042; CaCO2-Con (vector) vs CaCO2-CTSL, P = 0.0072. MTT: MHCC-97H-Con (vector) vs MHCC-97H-CTSL, P = 0.012; CaCO2-Con (vector) vs CaCO2-CTSL, P = 0.035). C. Colony formation assay and MTT assay of MHCC-97H cells with down-regulated CTSL. (Colony formation assay: MHCC-97H-Con-shRNA vs MHCC-97H-CTSL-shRNA, P = 0.003; MTT: MHCC-97H-Con-shRNA vs MHCC-97H-CTSL-shRNA, P = 0.001. (**P<0.01 as compared to parental groups, *P<0.05 as compared to parental groups).

Figure 5. Effect of CTSL knockdown on subcutaneous tumorigencity of MHCC-97H.

A. Tumor growth curve of after injection of nude mice with CTSL or control vector expressing MHCC-97H cells. (P<0.001) B. The picture of tumors from nude mice with CTSL or control vector expressing MHCC-97H cells. C. The weight of tumors from nude mice with CTSL or control vector expressing MHCC-97H cells (P = 0.005). (**P<0.01 as compared to control groups, *P<0.05 as compared to control groups).