The Growth and Metastasis of Human Hepatocellular Carcinoma Xenografts Are Inhibited by Small Interfering RNA Targeting to the Subunit ATP6L of Proton Pump

Abstract

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.

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

“…more differentiated) tumor cells. Because a peculiarity of malignant tumor cells is their ability to grow in an acidic condition (17), depending on the activity of proton pumps, we used a proton pump inhibitor (38,39) to analyze the role of low pH in favoring exosome traffic in malignant tumor cells. We found that exosome entry into target cells was significantly reduced by preincubation with proton pump inhibitors, suggesting that inhibition of extracellular acidification may interfere with exosome traffic in malignant tumor cells.…”

Clinicopathological and Biological Significance of Human Voltage-gated Proton Channel Hv1 Protein Overexpression in Breast Cancer

“…The importance of V-ATPases in cancer malignancy has been repeatedly demonstrated in several human cancer tumors and cell lines, including hepatocellular carcinoma, B-cell tumors, and melanoma (37)(38)(39). Inhibition of V-ATPase function via knockdown of the protein subunit ATP6L expression using RNAinterfering technology can effectively retard the growth and metastasis of human hepatocellular carcinoma xenografts (37). Inhibition of V-ATPase activity can also be achieved by treat- ment with proton pump inhibitors.…”

Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer

“…In addition, 4T1 cells with 90% V 1 C1 depletion had significantly less metastatic potential and reduced osteolytic lesions 27. Alternatively, siRNA knockdown of the ATP6L (V o c) subunit in HCCLM3 hepatocellular carcinoma cells resulted in significantly reduced invasion, decreased MMP‐2 expression, reduced average xenograft size, and a dramatic reduction in intrahepatic metastases 56. In support of this, siRNA‐mediated knockdown of the V o c subunit led to a reduction in both SK‐N‐MC and A‐673 Ewing sarcoma cell number 47…”