Article Figures & Data
Figures
- Fig. 1.
Total and activated (phosphorylated on serine 473) AKT protein levels in human pancreatic cancer cells. A, paired samples of serum-starved cells at 75–80% confluence were incubated with or without 1 μm wortmannin for 1 h and lysed in a buffer containing 1% Triton, 150 mm NaCl, and 25 mm Tris-acetate (pH 7.4) supplemented with protease and phosphatase inhibitors. Proteins were resolved by 10% SDS-PAGE and detected with specific primary antibodies. Results are representative of three independent experiments. B, analysis of PTEN protein expression. Lysates were resolved by 10% SDS-PAGE, and PTEN was detected by immunoblotting. Blots were then stripped and reprobed with a rabbit polyclonal antiactin antibody (from Sigma) to confirm equal protein loading. The human HT-29 colon adenocarcinoma line contains wild-type PTEN and served as a positive control. Although PTEN protein was detected in all of the cell lines, note that the lowest levels were observed in the line (Hs766T) that demonstrated low constitutive AKT phosphorylation.
- Fig. 2.
Sensitivity of human pancreatic carcinoma cells to the treatment with PI3k inhibitors. Cells maintained in MEM with 5% FBS were incubated with or without wortmannin and LY294002 for 48 h. The cells were collected with 0.25% trypsin/0.02% EDTA, and apoptosis was measured by propidium iodide staining and flow cytometry. All experiments were performed in triplicate. Data were expressed as mean; bars, ± SD. A, treatment with wortmannin and LY294002 resulted in a dose-depended apoptotic cell death in the Colo-357-L3.6pl and Panc-1 cell lines. B, PI3k inhibitors were less effective in the treatment of the CFPAC-1, HPAF, CAPAN-2, and MiaPaCa-2 cells, and failed to induce apoptosis in the AsPc-1, Hs766T, and BxPc-3 pancreatic carcinoma cells.
- Fig. 3.
Analysis of ERK phosphorylation in pancreatic cancer cell lines. A, ERK phosphorylation in AsPC-1 and BxPC-3. Cells were either maintained in complete MEM or preincubated in serum-free MEM for 24 h. Cells were then incubated with or without 10 mm PD98059 for 1 h. Finally, one pair of serum-starved plates was incubated with 100 ng/ml EGF for 10 min. Cells were harvested and lysed, and levels of total and phosphorylated ERKs were measured by immunoblotting as described in “Materials and Methods.” B, effects of PD98059 on ERK and AKT phosphorylation in L3.6pl and Mia PaCa-2. Cells were preincubated in serum-free MEM before incubation with PD98059 and/or EGF as described above. Cells were harvested and lysed, and levels of total and phosphorylated ERKs and AKT were measured by immunoblotting as described in “Materials and Methods.”
- Fig. 4.
Effects of PD98059 on DNA fragmentation associated with apoptosis. AsPC-1, BxPC-3, and Mia PaCa-2 cells (75–80% confluent) were exposed to the indicated concentrations of PD98059 for 48 h, and DNA fragmentation was measured by propidium iodide staining and FACS analysis as described in “Materials and Methods.” Similar results were obtained when these cell lines were exposed to drug in serum-free medium. L3.6pl cells were grown to 80% confluence in complete medium. The medium was then replaced with serum-free complete MEM, and cells were exposed to the indicated concentrations of PD98059 in this medium for 48 h before analysis of DNA fragmentation by propidium iodide staining and FACS analysis. Similar results were obtained in two independent experiments.
- Fig. 5.
Proliferation and apoptosis in orthotopic tumor xenografts. Mice bearing established (7-day) orthotopic L3.6pl tumors were treated biweekly with 100 mg/kg LY294002 via i.p. injection. After 4 weeks of therapy, animals were sacrificed, and excised tumors were analyzed by H&E staining, expression of Ki-67 antigen, or apoptosis as described in “Materials and Methods.”
- Fig. 6.
Determination of AKT and activated AKT in pancreatic cancer xenografts. Mice bearing s.c. L3.6pl tumors were treated with 100 mg/kg LY294002 via biweekly i.p. injection. Mice were sacrificed after 4 weeks, and levels of total and phosphorylated (S473) AKT were measured by immunohistochemistry. Note that LY294002 therapy reduced levels of phosphorylated AKT to background levels, whereas the effects of therapy on total AKT levels were much less pronounced, confirming drug targeting in vivo.
Tables
- Table 1
Therapy of human pancreatic carcinoma growing in the pancreas of nude mice
One million Colo-357-L3.6pl cells were injected into the pancreas of nude mice. Seven days later, the mice were randomly assigned to four treatment groups of 10 mice each. The first group received twice-weekly i.p. injections of 5% DMSO in HBSS as control. The second, third, and fourth groups received twice-weekly i.p. injections of LY294002 at 10, 25, and 100 mg/kg. All mice were sacrificed after 4 weeks of treatment.
Treatment groups Tumor weight in milligrams Mean ± SD Lymph nodes metastases Liver metastases Peritoneal carcinomatosis Body weight in grams Mean ± SD Before After Control 921.7 ± 266.8 10/10 9/10 4/10 26.54 ± 2.62 26.45 ± 2.36 LY294002 10 mg/kg 826.1 ± 221.5 9/9 8/9 3/9 26.13 ± 2.45 27.98 ± 2.54 LY294002 25 mg/kg 740.8 ± 214.6 9/10 8/10 3/10 26.87 ± 2.98 29.08 ± 3.04 LY294002 100 mg/kg 615.3 ± 184.5a 8/10 5/10 1/10 26.64 ± 2.43 29.04 ± 2.89 -
↵a P < 0.005 versus control.
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- Table 2
Therapy of human pancreatic carcinoma growing subcutaneously in nude mice
One million of Colo-357-L3.6pl cells were mixed with Matrigel (3.5 mg of Matrigel in 0.5 ml of HBSS). Each mouse underwent two s.c. injections in bilateral flank regions. Seven days later, the mice were randomly assigned to four treatment groups of 5 mice each. The first group received twice-weekly i.p. injections of LY294002 at 25 mg/kg, the second group received gemcitabine at 62 mg/kg, the third group received combination of 25 mg/kg LY294002 and 62 mg/kg gemcitabine, and the last group received 5% DMSO in HBSS as control. All mice were sacrificed after 3 weeks of treatment.
Treatment groups Tumor volume in mm3 Mean ± SD Body weight in grams Mean ± SD Before After Before After Control 191.8 ± 76.9 2314.1 ± 874.8 23.12 ± 3.18 27.52 ± 3.26 Gemcitabine 62 mg/kg 183.9 ± 67.6 1596 ± 350.1a 23.66 ± 2.87 28.18 ± 2.54 LY294002 25 mg/kg 175 ± 41.1 1738.1 ± 387.4 23.09 ± 3.08 28.27 ± 3.34 LY294002 25 mg/kg + Gemcitabine 62 mg/kg 181.3 ± 51.3 1071.4 ± 224.3b 22.92 ± 2.78 29.3 ± 3.01
Volume 1, Issue 12
