Papillary hyperplasia of the gallbladder in pancreaticobiliary maljunction represents a senescence-related lesion induced by lysolecithin

Abstract

Cellular senescence, an irreversible growth arrest, is considered to play as safeguard against malignant progression, though such a mechanism is speculative in human carcinogenesis. In gallbladder carcinoma, cholecystolithiasis and pancreaticobiliary maljunction (PBM) are major risk factors. Here, by using 113 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs) and gallbladder carcinoma cell line (TGBC2TKB), we examined carcinogenesis with respect to cellular senescence. Among 15 cases of PBM in which carcinoma was found in 4 cases, nonneoplastic gallbladder mucosa showed diffuse papillary hyperplasia (PHP). PHP was not found in gallbladders with cholecystolithiasis. Interestingly, PHP exhibited senescent features such as expression of p16INK4A and low cell proliferative activity. In contrast, EZH2, a polycomb group protein, was overexpressed in intraepithelial neoplasm and carcinoma in gallbladders with cholecystolithiasis. In PBM, EZH2 was expressed only in carcinoma foci but not in PHP. Cultured HGECs treated with lysolecithin, the level of which is elevated in gallbladder bile of PBM, showed increased expression of p16INK4A and senescence-associated b-galactosidase. Conversely, enforced overexpression of EZH2 in senescent HGECs reduced p16 INK4A expression. A knockdown of EZH2 in cultured TGBC2TKB cells increased p16 INK4aexpression. In conclusion, PHP in PBM may act as a barrier to malignant transformation for decades. EZH2 may be responsible for the escape from cellular senescence followed by malignant transformation in the gallbladder of PBM.

Histone deacetylase inhibitor (SAHA) and repression of EZH2 synergistically inhibit proliferation of gallbladder carcinoma

“…The expression of EZH2, HDAC1, and HDAC2 were assessed immunohistochemically using monoclonal antibodies: anti-EZH2 (clone 11, 1:100; BD Transduction Laboratories, San Jose, CA, USA); anti-HDAC1 (clone 3E1, 1:250; Sigma, St Louis, MO, USA); and anti-HDAC2 (ab12169, 1:500; Abcam, Cambridge, UK), as described previously. (6,24) As a negative control, similar dilution of the control mouse IgG (Dako, Glostrup, Denmark) was applied.…”

“…Human gallbladder epithelial cells were established from a surgically resected gallbladder with cholecystolithiasis as described, (24,26) and grown as monolayers. Epithelial nature of these cells was confirmed by the expression of CK7.…”

“…Western blotting was carried out using a standard method. (6,24) The membrane was incubated with primary antibodies against EZH2 (1:500), HDAC1 (1:500), HDAC2 (1:500), p21 (Cip1 ⁄ WAF1, clone 70, dilution 1:100; BD Transduction Laboratories), p16…”

“…RT-PCR was carried out using a standard method. (6,24) The annealing temperature was 54°C for EZH2, HDAC1, and HDAC2, and 56°C for G3PDH. The number of PCR cycles was 27 for EZH2, and 35 for HDAC1 and HDAC2.…”

“…(24,25) In this study using human gallbladders and cultures of gallbladder epithelial cells and the gallbladder carcinoma cell line, we assessed the expression pattern of EZH2 and class I HDACs (HDAC 1 and 2). We considered the possible mechanism by which carcinoma cells are sensitive to SAHA and the possible efficacy of this new anticancer agent in therapeutic treatment in gallbladder carcinoma.…”