Inhibition of Cell Proliferation and Growth of Pancreatic Cancer by Silencing of Carbohydrate Sulfotransferase 15 In Vitro and in a Xenograft Model

Abstract

Chondroitin sulfate E (CS-E), a highly sulfated glycosaminoglycan, is known to promote tumor invasion and metastasis. Because the presence of CS-E is detected in both tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC), multistage involvement of CS-E in the development of PDAC has been considered. However, its involvement in the early stage of PDAC progression is still not fully understood. In this study, to clarify the direct role of CS-E in tumor, but not stromal, cells of PDAC, we focused on carbohydrate sulfotransferase 15 (CHST15), a specific enzyme that biosynthesizes CS-E, and investigated the effects of the CHST15 siRNA on tumor cell proliferation in vitro and growth in vivo. CHST15 mRNA is highly expressed in the human pancreatic cancer cell lines PANC-1, MIA PaCa-2, Capan-1 and Capan-2. CHST15 siRNA significantly inhibited the expression of CHST15 mRNA in these four cells in vitro. Silencing of the CHST15 gene in the cells was associated with significant reduction of proliferation and up-regulation of the cell cycle inhibitor-related gene p21CIP1/WAF1. In a subcutaneous xenograft tumor model of PANC-1 in nude mice, a single intratumoral injection of CHST15 siRNA almost completely suppressed tumor growth. Reduced CHST15 protein signals associated with tumor necrosis were observed with the treatment with CHST15 siRNA. These results provide evidence of the direct action of CHST15 on the proliferation of pancreatic tumor cells partly through the p21CIP1/WAF1 pathway. Thus, CHST15-CS-E axis-mediated tumor cell proliferation could be a novel therapeutic target in the early stage of PDAC progression.

Prognostic impact of carbohydrate sulfotransferase 15 in patients with pancreatic ductal adenocarcinoma

“…Human PDA tissue has been successfully treated in nude mice using small interfering RNA-based selective silencing of the CHST15 gene to selectively inhibit CS-E expression (16). Therefore, CHST15/CS-E axis-mediated PDA cell proliferation may be a therapeutic target for the early progression of PDA in humans.…”

Silencing of Carbohydrate Sulfotransferase 15 Hinders Murine Pulmonary Fibrosis Development

“…RNA interference was performed using the reverse transfection method and siRNA for CHST15, and negative control siRNA was designed to target the coding sequence of human CHST15 as described previously 33 . HEL299 cells and HFL1 cells, CHST15 siRNA (6.25, 12.5, 25, or 50 nM) or negative control siRNA, Lipofectamine 2000, and Opti-MEM medium (Invitrogen) were mixed and incubated according to the manufacturer’s instructions.…”

“…Total RNA was extracted from culture cells by homogenization using RNAiso Plus (Takara Bio) and the SV Total RNA Isolation kit (Promega) and was reverse transcribed as previously described 33 . Real-time PCR was performed using real-time PCR DICE and SYBR Premix Taq (Takara Bio).…”

“…To calculate the relative mRNA expression level, the expression of each gene was normalized to that of the reference gene. The primers for real-time RT-PCR were designed as previously reported 33 . The supernatant of the cell culture was measured using a Sircol Collagen Assay kit (Biocolor) according to the manufacturer’s instructions.…”

Long noncoding RNA HOTAIR promotes invasion of breast cancer cells through chondroitin sulfotransferase CHST15

“…Elevated CHST15 expression was associated with poor disease‐free survival and overall survival in patients with pancreatic adenocarcinoma (PDA) . Consistently, downregulation of CHST15 resulted in proliferation inhibition in vitro and in xenograft animal models of PDA . While these results indicate that CHST15 plays an important role in cancer cell growth, its function in breast cancer is not clear.…”