The growth and metastasis of human hepatocellular carcinoma xenografts are inhibited by small interfering RNA targeting to the subunit ATP6L of proton pump

• Published in: Cancer research (Chicago, Ill.) Ill.), 2005-08, Vol.65 (15), p.6843-6849
• Main Authors: Lu, Xiaodong, Qin, Wenxin, Li, Jinjun, Tan, Ning, Pan, Dongning, Zhang, Haitao, Xie, Li, Yao, Genfu, Shu, Huiqun, Yao, Ming, Wan, Dafang, Gu, Jianren, Yang, Shengli
• Format: Article
• Language: English
• Subjects: Animals; Antineoplastic agents; Biological and medical sciences; Carcinoma, Hepatocellular - enzymology; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - pathology; Cell Growth Processes - genetics; Cell Line, Tumor; Gastroenterology. Liver. Pancreas. Abdomen; Humans; Liver Neoplasms - enzymology; Liver Neoplasms - genetics; Liver Neoplasms - pathology; Liver. Biliary tract. Portal circulation. Exocrine pancreas; Lung Neoplasms - prevention & control; Lung Neoplasms - secondary; Male; Matrix Metalloproteinase 2 - metabolism; Medical sciences; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Pharmacology. Drug treatments; RNA, Small Interfering - genetics; Transfection; Transplantation, Heterologous; Tumors; Vacuolar Proton-Translocating ATPases - antagonists & inhibitors; Vacuolar Proton-Translocating ATPases - genetics; Vacuolar Proton-Translocating ATPases - metabolism; Xenograft Model Antitumor Assays
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-04-3822

Extracellular pH is usually low in solid tumors, in contrast to the approximately neutral intracellular pH. V-ATPase, which overly functions in some cancers with metastatic potential, plays an important role in maintaining neutral cytosolic pH, very acidic luminal pH, and acidic extracellular pH. ATP6L, the 16 kDa subunit of proton pump V-ATPase, can provide proton hydrophilic transmembrane path. In this study, ATP6L in a human hepatocellular carcinoma cell line with highly metastatic potential (HCCLM3) was knocked down using DNA vector-based small interfering RNA (siRNA) to suppress the metastasis. The expression of ATP6L in stable siRNA transfectants, designated as si-HCCLM3 cells, was inhibited by approximately 60%. The proton secretion and the intracellular pH recovery from NH4Cl-prepulsed acidification were inhibited in si-HCCLM3 cells. The invasion of the si-HCCLM3 cells was suppressed in vitro; simultaneously, the expressions of matrix metalloproteinase-2 and gelatinase activity were reduced. In vivo, at 35th day after implantation of the si-HCCLM3 xenografts into the livers in BalB/c (nu+/nu+) mice, the size of liver tumor tissues was dramatically smaller in siRNA group than in the controlled group. The most impressing effect of ATP6L siRNA is its striking reduction of the metastatic potential of HCCLM3 cells. In control, all eight mice had the intrahepatic metastasis and six of eight the pulmonary metastasis, whereas in ATP6L siRNA-treated group, three of eight had the intrahepatic metastasis and only one of eight the pulmonary metastasis. The results suggest that the inhibition of V-ATPase function via knockdown of ATP6L expression using RNA interfering technology can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity.