Aromatase in human liver and its diseases

Abstract

Estrogens play important roles in the cell proliferation and invasion of estrogen-dependent human neoplasms. Aromatase overexpression has been also reported in hepatitis and hepatocellular carcinoma (HCC) compared with normal liver but its details in these hepatic disorders have remained unclear. Therefore, in this study, we first immunolocalized aromatase using immunohistochemistry in patients with liver cirrhosis, steatosis, hepatitis, HCC, and metastasis liver carcinoma (MLC) in order to study the detailed status of intrahepatic aromatase. Aromatase immunoreactivity was predominantly detected in nonneoplastic hepatocytes around tumor cells. We then evaluated the effects of an interaction between hepatocytes and carcinoma cells upon aromatase mRNA expression, using HepG2 as a substitute model of hepatocytes by coculture systems. Aromatase mRNA levels in HepG2 were significantly increased by coculture with all carcinoma cell lines examined. We also evaluated alternative splicing of aromatase exon 1 but the same splicing variant was used in HepG2 cells regardless of carcinoma cell lines employed in the coculture system. These findings obtained in HepG2 indicated that carcinoma cells, whether metastatic or primary, induced aromatase expression in adjacent normal hepatocytes possibly through the soluble aromatase inducible factors in human hepatic microenvironments.

Keywords: Liver cancer; metastasis; pathology.

Figure 1

Immunolocalization of aromatase in representative histological sections of the liver diseases. (A) Hematoxylin–eosin-stained tissue sections. (B) Immunohistochemistry for aromatase in liver diseases. 1A and 1B, cirrhosis; 2A–3B, steatosis; 4A–5B, hepatitis; 6A–7B, HCC.

Figure 2

Immunolocalization of aromatase in representative histological sections of the metastatic liver carcinoma. (A) Hematoxylin–eosin-stained tissue sections. (B) Immunohistochemistry for aromatase in metastatic liver carcinoma (tumor cells). (C) Immunohistochemistry for aromatase in metastatic liver carcinoma (hepatocytes). 1A–1C, lung carcinoma; 2A–2C, stomach carcinoma; 3A–3C, colon carcinoma; 4A–4C, ovary carcinoma.

Figure 3

Results of immunohistochemistry scoring of aromatase expression in patients with normal liver and liver diseases. *P = 0.0009; **P = 0.0007; ***P < 0.0001.

Figure 4

Immunolocalization of ERβ in metastatic liver carcinoma. (A) Immunolocalization of ERβ in representative histological sections of the metastatic liver carcinoma. (a) Hematoxylin–eosin-stained tissue sections. (b) Immunohistochemistry for ERβ in metastatic liver carcinoma. 1A and 2A, uterus carcinoma, High-expression case of ERβ; 2A and 2B, stomach carcinoma, Low-expression case of ERβ. (B) Results of immunohistochemistry scoring of ERβ expression in patients with metastatic liver carcinoma in aromatase scoring. *P = 0.011.

Figure 5

Expression of aromatase in coculture/LCM HepG2. (A) Coculture/LCM model system. (B) Results of LCM/qPCR for aromatase in cocultured HepG2 on the membrane. Coculture was examined by toluidine blue staining. a, before cutting boundary region of HepG2 and DLD-1; b, after cutting boundary region of HepG2 and DLD-1; c, before cutting boundary region of HepG2 and MCF-7; d, after cutting boundary region of HepG2 and MCF-7. H, HepG2 region; D, DLD-1 region; M, MCF-7 region. (C) Results of LCM/qPCR for aromatase expression in cocultured HepG2. (a) cocultured with DLD-1; (b) cocultured with MCF-7. H, only HepG2 region; C, only cancer cell line region; B, boundary region of HepG2.

Figure 6

Amplified products obtained by PCR of RNA extracted from coculture HepG2. PCR was performed using oligonucleotides hybridizing to regions of the exons indicated in each row (A–C). The estimated sizes of the amplified products are indicated on the left. Coculture were obtained from: lane P, positive control; lane N, negative control; lane 1, HepG2; lane 2, cocultured HepG2 by MCF-7; lane 3, cocultured HepG2 by A549; lane 4, cocultured HepG2 by DLD-1; lane 5, cocultured HepG2 by WiDr; lane 6, cocultured HepG2 by HCT-15; lane 7, cocultured HepG2 by COLO205; lane 8, cocultured HepG2 by SW480.