Volume 122, Issue 2, Pages 308-317 (February 2002) 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors reduce human pancreatic cancer cell invasion and metastasis  Toshiyuki Kusama, Mutsuko Mukai, Teruo Iwasaki, Masaharu Tatsuta, Yoshirou Matsumoto, Hitoshi Akedo, Masahiro Inoue, Hiroyuki Nakamura  Gastroenterology  Volume 122, Issue 2, Pages 308-317 (February 2002) DOI: 10.1053/gast.2002.31093 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of HMG-CoA reductase inhibitor on ASPC-1 cell morphology. Cells were incubated for 24 hours in the medium containing 0.1% BSA with 15 μmol/L fluvastatin in the absence or presence of FOL or GGOL and photographed by phase-contrast microscopy. (A) Untreated cells, (B) cells treated with fluvastatin only, (C) cells treated with fluvastatin and 10 μmol/L FOL, and (D) cells treated with fluvastatin and 10 μmol/L GGOL. (E) Western blot analysis of the effect of GGOL on fluvastatin-induced translocation of RhoA. Cells were pretreated with fluvastatin in the absence or presence of FOL or GGOL for 24 hours, as described for A–D. Proteins were extracted and separated into cytosolic (c) and membrane (m) fractions, then RhoA was detected by immunoblotting as described in Materials and Methods. Gastroenterology 2002 122, 308-317DOI: (10.1053/gast.2002.31093) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Western blot analysis of the effect of fluvastatin on EGF-induced translocation of RhoA. (A) Time course of translocation of RhoA from cytosolic fraction to membrane fraction in ASPC-1 cells, induced by EGF (1 nmol/L). (B) Effect of fluvastatin on EGF-induced translocation of RhoA. ASPC-1 cells were pretreated with fluvastatin for 24 hours and stimulated with EGF (1 nmol/L) for 15 minutes. Proteins were extracted and separated into cytosolic (c) and membrane (m) fractions, then RhoA was detected by immunoblotting as described in Materials and Methods. Data points and columns represent the means of 3 independent experiments; bars = SD. *P < 0.05 vs. control; †P < 0.05 vs. control with EGF stimulation. Gastroenterology 2002 122, 308-317DOI: (10.1053/gast.2002.31093) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Inhibitory effect of HMG-CoA reductase inhibitors on EGF-induced growth of pancreatic cancer cells. ASPC-1 cells (1 × 104 cells/well) were seeded in 96-well plates and incubated for 24 hours in culture medium, then for 24 hours in serum-free medium containing 0.1% BSA without or with the indicated concentration of lovastatin or fluvastatin in the absence or presence of EGF. Cell growth was then determined using the MTT assay. Results are expressed as percentage of cell growth compared with that of untreated controls and are means ± SEM of quadruplicate determinations from 3 separate experiments. *P < 0.05, **P < 0.05 vs. control with EGF stimulation. (B) Effect of fluvastatin on EGF-stimulated phosphorylation of MAPK in ASPC-1 cells. The top panel represents MAPK phosphorylation detected using an antibody specific for the phosphorylated form of MAPK. The bottom panel represents a similar Western blot probed with an anti-ERK–specific antibody to demonstrate the specificity of the effects on phosphorylation and equal loading. Gastroenterology 2002 122, 308-317DOI: (10.1053/gast.2002.31093) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 The antimetastatic effect of fluvastatin. Nude mice were injected intrasplenically with 1 × 106 ASPC-1 cells. Mice were treated with (A) vehicle, (B) fluvastatin (0.6 mg/kg, orally) daily from days −2 to 42, or (C) fluvastatin (0.6 mg/kg, orally) daily from days 28 to 42. Gastroenterology 2002 122, 308-317DOI: (10.1053/gast.2002.31093) Copyright © 2002 American Gastroenterological Association Terms and Conditions