Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice

Abstract

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

Figure 1

Structure of rasfonin

Figure 2

Inhibition growth, contact-independent proliferation, migration and invasion of BxPC-3 and Panc-1 cells by rasfonin. (a) The dynamic effects of rasfonin on adhesion and proliferation of BxPC-3 and Panc-1 cells using the xCELLigence system. BxPC-3 and Panc-1 cells were seeded at a density of 8000 cells/well in 96-well E-Plates, then treated with different concentrations of rasfonin for 40 h. (b) The effects of rasfonin on survival of BxPC-3 and Panc-1 cells using the MTS assay. Cells were treated with different concentrations of rasfonin for 24 h. The percentage of surviving cell was calculated by normalizing the absorbance value of the treated cells with the absorbance value of the control cells within each cell line. Data are representative of three independent experiments. **P<0.01 versus control, n=3. (c) Rasfonin reduced contact-independent proliferation in Panc-1 cells. Panc-1 cells were seeded at 1000 cells per well containing vehicle or various concentrations of rasfonin (0.25, 0.5, 1, 5 μM). After 8 days of culture, live colonies were counted and photographed, stained with 0.05% crystal violet. Bars represent the mean numbers of colonies per field. **P<0.01 versus control, n=3. Insets are representative wells. (d) The effects of rasfonin on the migration of Panc-1 cells. Cells with rasfonin or vehicle were seeded on a CIM-plate as described in Materials and Methods. Migration was monitored for 24 h with the xCELLigence DP system. (e) The effects of rasfonin on the invasion kinetics of Panc-1 cells in the indicated dilutions of Matrigel using FBS as the chemoattractant. Panc-1 cells were seeded at 8 × 10⁴ cells per well in either uncoated wells (migration) or Matrigel-coated wells (invasion). Precoating was done at 37 °C for 4 h

Figure 3

Effects of rasfonin on Ras–MAPK protein expression and activity in Panc-1 cells. (a) Effects of rasfonin on Ras protein expression in Panc-1 cells. Panc-1 cells were cultured in DMEM containing 10% FBS for 18 h, starved for 9 h in 0.1% FBS and then treated with rasfonin at different concentrations (0, 1, 5 and 10 μM) for 12 h. After that, the cells were stimulated with EGF (50 ng/ml) for 5 min. Total proteins were extracted and analyzed for Ras protein by immunoblotting. (b) Effects of rasfonin on Ras activity in Panc-1 cells. Cells were handled similarly as mentioned in a. A Ras Activation ELISA assay kit was employed to determine the level of Ras–GTP. Data are representative of one to three independent experiments. Results shown are mean±S.D., *P<0.05, versus control; ^#P<0.05, ^##P<0.01, versus model. (c) Effects of rasfonin on expression of protein kinases involved in the Ras–MAPK signaling pathway in Panc-1 cells. Panc-1 cells were handled similarly as mentioned in a. Total proteins were extracted and analyzed for phosphorylated c-Raf, MEK1/2 and ERK44/42 by immunoblotting. Total MEK1/2 and ERK44/42were also detected as controls

Figure 4

Effects of rasfonin on expression and activity of protein kinases in Ras upstream pathway in Panc-1 cells. (a) Effects of rasfonin on protein kinase expression. Panc-1 cells were cultured in DMEM containing 10% FBS for 18 h, starved for 9 h in 0.1% FBS and then treated with rasfonin at different concentrations (0, 1, 5 and 10 μM) for 12 h. Total proteins (40 μg) were analyzed for Sos1, Grb2 and GAP by immunoblotting. (b) Effects of rasfonin on GEF activity (the dissociation of labeled [³H]-GDP from Ras). Ras was preloaded with 2 μCi [³H]-GDP, then GEF with or without rasfonin (10 μM) was added. The amount of [³H]-GDP bound to Ras was measured by scintillation spectrometry. **P<0.01, versus control. (c) Effects of rasfonin on GAP activity (the competitive binding of labeled [³H]-GDP to Ras). Ras was preloaded with 2 μCi [³H]-GDP and GTP, then GAP with or without rasfonin (10 μM) was added. The amount of [³H]-GDP bound to Ras was measured by scintillation spectrometry. *P<0.05, versus control

Figure 5

Effects of rasfonin on tumor volume and tumor weight in Panc-1 xenograft nude mice. Subcutaneous xenografts were established by injecting 1 × 10⁷ Panc-1 cells into the right flank of 6- to 7-week-old female nude mice. Rasfonin treatment was initiated when the tumors reached around 100–300 mm³. Three desired concentrations of rasfonin were administered once every 2 days for up to 20 days by intraperitoneal injection. FTS (15 mg/kg/day) was administered once daily for up to 20 days by intraperitoneal injection. Tumor sizes were measured at least three times a week and the changes in individual mice were plotted against the time of treatment. The graph of each 5-day treatment indicated both the control and rasfonin-treated mice. (a) Tumor volume. (b) After 20 days of treatment, mice bearing Panc-1 tumors were killed and the tumor weight in each mouse was measured. *P<0.05, n=6–8