Can Antisense Oligonucleotides Specific to K-ras Point Mutation Inhibit the Tumor Growth, Invasion and MMP-2 and MMP-9 Expression in Hamster Pancreatic Cancer Model In Vitro and In Vivo?

Abstract

Background: Matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, are thought to play major roles in pancreatic cancer growth and metastasis. Ras activates a multitude of downstream activities with roles in cellularprocessing,includinginvasionandmetastasis.Therefore,antisense oligonucleotides (ASO) targeting this K-ras gene may be a therapeutic approach. Aim: To elucidate the effectiveness of this gene therapy in hamster experimental cancer model. Materials and Methods: HaP-T1, a cell line derived from BHP-induced pancreatic cancer was used. Transfection with ASO were performed. MTT and MTT agarose assays were done. Chemoinvasion assay was performed.MMP-2andMMP-9productionbythecelllineswasdetermined by gelatin zymography. For in vivo experiments, subcutaneously resected tumors were implanted orthotopically in Syrian golden hamsters, which were divided in 3 groups: (A) Positive control (PC), (B) Sense treated ham- sters (STH), and (C) Antisense treated hamsters (ATH). Oligonucleotides were administered for 2 weeks. Follow up was done. Five animals of each group were sacrificed at Days 10, 17, 24, 31, and 38, to study the local responseandmetastaticsites.Fiveanimalsofeachgroupwerelefttostudy the survival time. Specimens were studied histopathologically. Orthotopic pancreatic tumor MMP production was measured by gelatin zymography. Results: ASO inhibited the tumoral growth and invasiveness. They downregulated active forms of MMP-2 and MMP-9 in a dose dependent manner in vitro. Positive controls, STH, and ATH survived in average 72.7, 74.3, and 82.7 days, respectively. Spontaneous lymph node metastases were found from 31 days in ATH group, while PC and STH groups showed metastases and direct invasion to adjacent organs from 17 days. After death, metastatic sites were similar in the 3 groups. ASO downregulated the activation of MMP-9, more than MMP-2 in vivo. Conclusions: These experiments suggest that ASO targeted K-ras gene may be a good choice in the management of pancreatic cancer because of the suppression of tumor growth and invasiveness in vitro and in vivo. ASO also downregulated the activation of MMP-9 and MMP-2 in vivo.