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Comparative Study
. 2013 Apr 28;19(16):2456-65.
doi: 10.3748/wjg.v19.i16.2456.

Annexin A1: A new immunohistological marker of cholangiocarcinoma

Nuttanan Hongsrichan  1 , Rucksak Rucksaken, Yaovalux Chamgramol, Porntip Pinlaor, Anchalee Techasen, Puangrat Yongvanit, Narong Khuntikeo, Chawalit Pairojkul, Somchai Pinlaor
Affiliations
Comparative Study

Annexin A1: A new immunohistological marker of cholangiocarcinoma

Nuttanan Hongsrichan et al. World J Gastroenterol. .

Abstract

Aim: To evaluate a new immunohistological marker, annexin A1 (ANXA1), in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC).

Methods: Expression of ANXA1 protein was investigated in liver tissues from patients with CCA and HCC by immunohistochemistry. Its expression on differences stages of tumor development was investigated in hamster CCA tissues induced by Opisthorchis viverrini and N-nitrosodimethylamine. Moreover, mRNA expression of ANXA1 was assessed in CCA cell lines by quantitative real-time polymerase chain reaction and silencing of ANXA1 gene expression using small interfering RNA.

Results: In human CCA tissue arrays, immunohistochemical analysis revealed that the positive expression of ANXA1 was 94.1% (64/68 cases) consisting of a high expression (66.2%, 45/68 cases) and a low expression (33.8%, 23/68 cases). However, expression of ANXA1 protein was negative in all histologic patterns for HCC (46/46 cases) and healthy individuals (6/6 cases). In hamster with opisthorchiasis-associated CCA, the expression of ANXA1 was observed in the cytoplasm of inflammatory cells, bile duct epithelia and tumor cells. Grading scores of ANXA1 expression were significantly increased with tumor progression. In addition, mRNA expression of ANXA1 significantly increased in all of the various CCA cell lines tested compared to an immortalized human cholangiocyte cell line (MMNK1). Suppressing the ANXA1 gene significantly reduced the matrix metalloproteinase (MMP) 2 and MMP9, and transforming growth factor-β genes, but increased nuclear factor-κB gene expression.

Conclusion: ANXA1 is highly expressed in CCA, but low in HCC, suggesting it may serve as a new immunohistochemical marker of CCA. ANXA1 may play a role in opisthorchiasis-associated cholangiocarcinogenesis.

Keywords: Annexin A1; Biomarker; Cholangiocarcinoma; Hepatocellular carcinoma; Opisthorchis viverrini.

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Figures

Figure 1
Figure 1
Immunostaining of annexin A1 protein expression in cholangiocarcinoma tissue microarrays. A-D: Tissue microarray of cholangiocarcinoma samples was stained with anti-annexin A1 antibody and counter stained with Mayer's hematoxylin and represented by A for negative, B for +, C for ++, and D for +++ when expression was < 10%, 10%-25%, 26%-75% and > 75%, respectively (magnification, × 200).
Figure 2
Figure 2
Immunostaining of annexin A1 in hepatocellular carcinoma tissue microarrays. Tissue microarray of hepatocellular carcinoma (HCC) samples was stained with anti-annexin A1 (ANXA1) antibody and counter stained with Mayer's Hematoxylin. ANXA1 shows low expression in all histologic patterns of HCC. A: Trabecular pattern; B: Broad trabecular pattern; C: Trabecular with pseudoacinar pattern; D: Solid growth pattern (magnification, × 40).
Figure 3
Figure 3
Illustration of the comparative immunohistochemical stains in 3 representative cases. The 3 representative cases include tubular type cholangiocarcinoma (CCA) (Tub CCA), papillary type CCA (Pap CCA) and hepatocellular carcinoma (HCC). Immunopositivity of cytokeratin 7 (CK7), annexin A1 (ANXA1), hepatocyte paraffin 1 (HepPar1) and alpha-fetoprotein (AFP) appeared as brown cytoplasmic staining of tumor cells, while positivity to carbohydrate antigen 19-9 (CA19-9) appeared as cytoplasmic and luminal staining of tumor (magnification, × 40). Noted that CCA are CK7+/CA19-9+/ANXA1+ and HCC is HepPar1+/AFP+/ANXA1-. -: Negative result; +: Positive result.
Figure 4
Figure 4
Immunostaining of annexin A1 protein expression in hamster tissues. A-D: Hamster tissues were stained with anti-annexin A1 antibody and counterstained with Mayer's hematoxylin represented by negative for A (normal at 6 mo post-treatment), grade + for B (21 d post-treatment), grade ++ for C (3 mo post-treatment) and grade +++ for D (6 mo post-treatment) (magnification, × 200). Ov: Opisthorchis viverrini; BD: Bile duct lumen.
Figure 5
Figure 5
Expression of the annexin A1 gene in various cholangiocarcinoma cell lines. The mRNA expression level of annexin A1 (ANAX1) was evaluated by real-time reverse transcription-polymerase chain reaction in various cholangiocarcinoma (CCA) cell lines including M156, M055 and M214 moderately differentiated CCA, M213 adenosquamous cell carcinoma and compared to an immortalized human cholangiocyte cell line (MMNK1). Data are derived from duplicate independent experiments and presented as mean ± SD, P < 0.01, P < 0.001 vs MMNK1 is significantly different by the Student t-test.
Figure 6
Figure 6
Effect of annexin A1 knockdown on the M214 cholangiocarcinoma cell line. A, B: Suppression of annexin A1 (ANXA1) expression at the translational level (at 48 h) and transcriptional level (at 48 and 72 h) in knockdown M214 cells was evaluated by Western blotting (A) and real-time reverse transcription-polymerase chain reaction (B).
Figure 7
Figure 7
Effect of gene expression in the knockdown of the annexin A1 gene in the M214 cholangiocarcinoma cell line. A: Relative transforming growth factor-β (TGF-β) mRNA expression; B: Relative matrix metalloproteinase (MMP) 2 mRNA expression; C: Relative MMP9 mRNA expression; D: Relative nuclear factor (NF)-κB mRNA expression. Real-time reverse transcription-polymerase chain reaction was used to confirm the expression of TGF-β, MMP2, MMP9 and NF-κB in the knockdown of the annexin A1 (ANXA1) gene in the M214 cholangiocarcinoma cell line relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are derived from duplicate independent experiments and presented as mean ± SD, and P < 0.05 vs scramble is significantly different by the Student t-test.
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