Production of Monoclonal Antibodies Against ADAMTS13 Determining Its Expression in Different Human Tissues.

Abstract

The deficiency of ADAMTS13/von Willebrand factor-cleaving protease in cancer patients, which can lead to the accumulation of ultra-large von Willebrand factor (UL-vWF) in plasma, has been confirmed by several previous reports. Nevertheless, the results of them did not avoid the heterogeneity between different tumors and the reasons accounted for its deficiency still remain to be elucidated. Since ADAMTS13 is mainly synthesized and released from liver and tumors derived from liver often accompanies with metastasis, we have studied the expression levels of ADAMTS13 in patients with primary hepatocarcinoma and in different hepatic cancer cell lines. Firstly, we detected the ADAMTS13 mRNA and protein levels in paired primary cancer tissues and adjacent normal tissues using semi-quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry. It showed that most of the cancer tissues expressed lower ADAMTS13 levels compared with their paired adjacent normal tissues. Significant low expression levels of ADAMTS13 had a close relationship with distal metastasis of hepatocarcinoma, but the abnormal of hepatic function did not have an association with the deficiency of ADAMTS13 expression. Interestingly, most of the patients suffering with hepatitis B and cirrhosis did not show significantly decreased ADAMTS13 levels, and most of the cancer patients with distal metastasis while without hepatitis B and cirrhosis accompanied markedly deficiency of the protease. The hepatocarcinoma cell lines, HepG2 and BEL7702, also expressed lower ADAMTS13 mRNA levels compared with SMMC7721 and a normal hepatic cell line L02. However, when the hepatocarcinoma cell lines were treated with a demethylation drug 5-aza-2′-deoxycytidine, its ADAMTS13 mRNA expression levels elevated with the increase of treatment dosage, especially in HepG 2 (P<0.05). It seems that the deficiency of ADAMTS13 in hepatocarcinoma might be associated with DNA methylation. The data above revealed that the ADAMTS13 expression levels were lower in hepatocarcinoma tissues. Furthermore, the activity of ADAMTS13 was significantly decreased in aged people older than 65 years, and the extent of methylation of genes are closely related with age. Therefore, based on our results, the deficiency of ADAMTS13 in hepatocarcinoma patients might not be caused by the abnormal of hepatic function, but be related to the DNA methylation of the protease.