Article Figures & Data
Figures
- Fig. 1.
A, the three-dimensional structures of a peptide constructed by a computer from the amino acid sequence 1127–1138 of the insulin-like growth factor (IGF) -1R (activation loop of the tyrosine kinase domain) using the Internal Coordinate Mechanics software compared with the structures of the cyclolignans podophyllotoxin (PPT) and picropodophyllin (PPP). The tyrosines 1131, 1135, and 1136 are indicated. B, the chemical structures of PPT, PPP, and etoposide.
- Fig. 2.
Effects of podophyllotoxin (PPT) and congeners on basal receptor tyrosine phosphorylation (pTyr) in intact cells. Cells were incubated with fresh complete medium containing the indicated compounds. Phosphorylation of the indicated receptors was determined by Western blotting. A, effects of different concentrations of picropodophyllin (PPP) and indicated congeners on insulin-like growth factor (IGF) -1R phosphorylation in FM 55 cells. B, effects of PPT on IGF-1R phosphorylation in SK-MEL-28, MCF 7, RD-ES, and P6 cells. C, effects of the derivatives deoxypodophyllotoxin, PPP, and DPPP (0.5 μm) on IGF-1R phosphorylation in FM55 cells. The effects of PPT (0.5 μm) and etoposide (E; 15 μm) are also shown (C = control). D, effects of various concentrations of PPT and PPP on phosphorylation of the indicated growth factor receptors and insulin receptor substrate-1 in FM 55 cells. Actin is loading control. The experiments were repeated three times with similar results.
- Fig. 3.
Dose-response on insulin-like growth factor (IGF) -1R tyrosine phosphorylation (IGF-1R pTyr) in intact cells. FM-55 cells were incubated with fresh complete medium containing the indicated concentrations of PPT, after which the levels of IGF-1R phosphorylation were assayed by Western blotting (A), and quantified by determining absorbances (OD; means and SE of means) of the signals (B). The expression of the IGF-1R β-subunit is shown as a loading control in the bottom panel of A. C, P6 cells were serum-depleted for 20 h. The cells were then treated with picropodophyllin (PPP) at various concentrations for 1 h, and finally stimulated with IGF-1 (20 ng/ml) for 5 min. After purification of IGF-1R with immunoprecipitation, Western blotting was performed for detection of phosphorylated IGF-1R. Phosphorylation of Akt (serine 473; pAkt) and Erk1/2 (pErk1/2) was assayed directly by Western blotting of the cell lysates. As loading controls the IGF-1R β-subunit, Akt and Erk1/2 were detected. D, P6 cells were serum-depleted for 20 h. The cells were then treated with podophyllotoxin (PPT), PPP, or tamoxifen at various concentrations for 1 h, and finally stimulated with IGF-1 (100 ng/ml) for 5 min. Western blotting was then performed directly on the cell lysate (without immunoprecipitation) for detection of phosphorylated IGF-1R using the PY antibody. As loading controls both the IGF-1R α- and β-subunit were detected. The experiments were repeated three to five times with similar results.
- Fig. 4.
Effects of cyclolignans on receptor tyrosine kinases in vitro. A, effects of the indicated concentrations of podophyllotoxin (PPT) on insulin-like growth factor (IGF) -1R-, insulin receptor (IR)- and epidermal growth factor receptor-catalyzed tyrosine phosphorylation of the poly Tyr Glu (pTG) substrate in vitro. Non-IGF-1R kinases represent IGF-1R immunodepleted activity. The reactions were quantified by densitometry, and the relative tyrosine kinase activity is shown. Mean values of three experiments are shown; bars, ±SE. B, dose-response of picropodophyllin (PPP) on IGF-1R autophosphorylation in vitro at different ATP concentrations. For all indicated values the SEs were <5%. The experiments were repeated two to five times with similar results.
- Fig. 5.
Effects of IGF-1R inhibitors on malignant cell growth in vitro. A, dose-response of 48-h treatments with picropodophyllin (PPP) and podophyllotoxin (PPT) on cell viability of cultures of the FM 55, SK-MEL-28, P6, and RD-ES cells as assayed by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt. The cell density at start of the experiments was in all experiments 25,000 cells/well. The values represent means of three experiments; bars, ±SE. B, FM 55, P6, and IGF-1R negative R- cells were either untreated (control = C) or treated with PPP (0.5 μm) for 6 h. Cells were then subjected to simultaneous Annexin V and propidium iodide staining followed by fluorescence-activated cell sorter analysis. In a parallel experiment the same cell lines, treated with PPP (0.5 μm), were analyzed for pAkt (serine473) and Akt (shown in the Annexin V graphs).
- Fig. 6.
Effects of insulin-like growth factor (IGF) -1R inhibitors on malignant cell growth in vivo. A, effect of picropodophyllin (PPP) on tumor growth of IGF-1R positive (P12) and IGF-1R negative (R- v-src) R- cells allografted in SCID mice (top panels). When the tumors were established (on days 16 and 9, respectively), mice were treated with PPP (20 mg/kg/12 h) or drug-free solvent. Tumor growth was measured every second day after start of treatments. In a separate experiment a tumor sample from PPP-treated P12-allografted mouse was taken 4 h after PPP injection, and the effects on phosphorylation of IGF-1R, insulin receptor (IR), Akt, and Erk1/2. B, effect of PPP on human cancer growth of ES-1, BE, and PC3 cells xenografted in SCID mice. When the tumors were established (arrows), mice were treated with PPP (20 mg/kg/12 h) or drug-free solvent. Three mice were treated in each experiment. Means are shown; bars, ±SD. C, effect of PPP on tumor growth of murine breast cancer cells (JC) allografted in immunocompetent mice. When the tumors were established (arrow), mice were treated with PPP (20 mg/kg/12 h) or drug-free solvent. The experiments were repeated 5–10 times with similar results.
Tables
- Table 1
Effects of podophyllotoxin (PPT) and picropodophyllin (PPP) on cell viability and basal insulin-like growth factor (IGF)-1R tyrosine phosphorylation in various cell lines
Cell viability and IGF-1R phosphorylation of intact cells were determined after 48 h and 1 h, respectively. The concentration required for IC50 is shown. Each value is based on at least three different experiments. The experiments were repeated three to eight times with similar results.
Origin Cell line PPT (μm) PPP (μm) Viability IC50 (48 h) pIGF-1R IC50 (1 h) Viability IC50 (48 h) pIGF-1R IC50 (1 h) Melanoma FM 55 <0.05 <0.05 0.06 <0.05 SK-MEL-28 <0.05 <0.05 <0.05 <0.05 SK-MEL-5 <0.05 <0.05 nda nd C8161 <0.05 <0.05 <0.05 <0.05 DFB 0.05 <0.05 0.05 <0.05 DFW 0.05 <0.05 0.05 <0.05 AA <0.05 <0.05 <0.05 nd Sarcoma RD-ES <0.05 <0.05 0.08 <0.05 Breast carcinoma MCF 7 0.25 <0.05 0.05 <0.05 Prostate carcinoma PC3 0.1 nd 0.1 nd Hepatoma HepG2 >15 —b >15 —b Embryonic mouse fibroblasts P6 <0.05 <0.05 <0.05 <0.05 R- >15 —b >15 —b -
a nd, not determined.
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b No IGF-1R activity detected.
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Volume 64, Issue 1
