GEO and CBS cell lines used in this study were originally developed from primary CRC tumors and have been extensively characterized (19). Cells were maintained at 37°C in humidified atmosphere of 5% CO₂ in a chemically defined serum-free medium consisting of McCoy's 5A medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with amino acids, pyruvate, vitamins, antibiotics and growth factors transferring (4 μg/ml; Sigma-Aldrich), insulin (20 μg/ml; Sigma-Aldrich), and EGF (10 ng/ml; R&D Systems) as previously described (20). Supplemented McCoy's medium (‘SM’) is McCoy's 5A medium supplemented with antibiotics and nutrients but lacking any growth factors. Cells were routinely subcultured with a 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) in Joklik's medium (Invitrogen) containing 0.1% EDTA. When cells were under growth factor deprivation status (GFDS), they were cultured in SM medium without growth factor or serum supplements for the indicated time periods without medium change in between.

IGF-1Rβ, pIGF-1Rβ (Y¹¹³⁵) and p21 antibodies were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). XIAP antibody was obtained from abcam. β-actin and GAPDH antibodies were from Sigma-Aldrich (St. Louis, MO, USA).

MK-0646 was provided by Merck & Co. (Whitehouse Station, NJ, USA) and OSI-906 was purchased from Chemitek, Indianapolis, IN, USA.

All experiments involving animals were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. The GEO and CBS cells were transfected with green fluorescence protein (GFP). Exponentially growing GFP-labeled GEO and CBS cells (~7 million cells/ml SF media) were inoculated subcutaneously onto the dorsal surfaces of athymic nude male mice and the growth of the tumor was monitored by biweekly measurements using a caliper. Once xenografts were established (~50–100 mm³), MK-0646 or OSI-906 treatment was initiated and continued for two weeks. MK-0646 was given by intraperitoneal (IP) injection weekly (20 mg/kg) on both GEO and CBS xenografted mice for three doses and formulation buffer was the vehicle. OSI-906 was given by daily oral gavage (40 mg/kg) on GEO xenografted mice and tartaric acid was the vehicle. Xenografts were harvested after 14 days of treatment for assessment of molecular effects by the two agents.

Xenografts were harvested and snap frozen in liquid nitrogen and stored at −80°C. Xenografts were first washed in cold 5% PBS and collected in lysis buffer [50 mmol/l Tris (pH 7.4), 100 mmol/l NaCl, 1% NP40, 2 mmol/l EDTA, 0.1% SDS, 50 mmol/l NaF, 10 mmol/l Na₃VO₄, 1 mmol/l phenylmethylsulfonyl fluoride, 25 μg/ml β-glycerophosphate, and one protease inhibitor cocktail tablet from Roche]. Crude xenograft lysates were homogenized to shear DNA and lysed for 30 min on ice. Xenograft lysates were then cleared by centrifugation at 13000 rpm for 20 min at 4°C. Protein concentrations were determined by the Pierce bicinchonimic acid protein assay (Pierce Biotechnology, Inc., Rockford, IL, USA).

GEO CRC cells were allowed to grow until 70–80% confluent in 100-mm culture plates and were treated with different concentrations of either MK-0646 or OSI-906 under growth factor deprivation status (GFDS) for 48 h. Cells were washed in cold 5% PBS and collected in lysis buffer. Crude cell lysates were homogenized using a 21-gauge needle to shear DNA and lysed for 30 min on ice. Cell lysates were then cleared by centrifugation at 13000 rpm for 20 min at 4°C. Protein concentrations were determined by the Pierce bicinchonimic acid protein assay (Pierce Biotechnology, Inc.).

Protein (30–100 μg) was fractionated on an acrylamide denaturing gel and transferred onto a nitrocellulose membrane (Amersham Biosciences) by electroblotting. The membrane was blocked with 5% nonfat dry milk in 1X TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h at room temperature or overnight at 4°C. The membrane was then incubated with primary antibodies for 1 h at room temperature or overnight at 4°C with 5% nonfat dry milk in 1X TBST or 5% bovine serum albumin (BSA) in 1X TBST according to the manufacturer's instructions. After washing three times with 1X TBST for 10 min each, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) for 1 h at room temperature. After further washing in 1X TBST three times for 10 min each, the proteins were detected by the enhanced chemiluminescence system (Amersham Biosciences).

DNA fragmentation assays were performed on cells treated with either MK-0646 or OSI-906 under GFDS. Cells were seeded in 96-well plates and allowed to grow to 70–80% confluence. The cells were then changed to Supplemented McCoy's medium (‘SM’) and treated with various concentrations of either MK-0646 or OSI-906 for 48 h. Assays were then performed using a cell death ELISA kit (Roche Applied Science) according to the manufacturer's protocol as previously described (21). The plate was read at 405 nm. Inhibition of proliferation was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as previously described (22).

GEO CRC cells were allowed to grow until 70–80% confluent in 100-mm culture plates and were treated with different concentrations of either MK-0646 or OSI-906 under growth factor deprivation status (GFDS) for 48 h and cell cycle analysis was performed as previously described (23).

Xenografts obtained from subcutaneous injection of GEO cells were harvested and placed in 10% neutral buffer formalin fixative for 12–24 h and then embedded in paraffin. Sections (4 μm) were cut from paraffin-embedded blocks using a microtome and were used for hematoxylin and eosin stains and immunohistochemical characterizations. Serial sections were cut to complement the hematoxylin and eosin sections and were stained with the Apo-tag (Millipore) terminal nucleotidyl transferase mediated nick end-labeling (TUNEL) kit following the manufacturer's protocol. The apoptotic rate was determined semi-quantitatively by counting the number of positively stained apoptotic bodies per 75-μm² field at a magnification of ×20. Approximately 1000 total cells were counted and the percentages of positively stained cells were calculated. Four control and four treated slides for MK-0646 and OSI-906 were analyzed, respectively. Staining was also performed with IgG₁ rabbit polyclonal antibody for Ki67 (Dako Corp.). Ki67 is a non-histone nuclear antigen present in late G₁, G₂, and S phases of the cell cycle but not in G₀. A 1:20 dilution was used and staining was performed following the manufacturer's protocol. The proliferation rate was determined semi-quantitatively by counting the number of positively stained proliferative cells per 75-μm² field at a magnification of ×20. Approximately 1000 total cells were counted and the percentages of positively stained cells were calculated. Four control and four treated slides for MK-0646 and OSI-906 were analyzed, respectively.

Sections (4 μm) were cut from the paraffin-embedded xenograft tumor blocks, deparaffinized in histoclear, and rehydrated in descending grades of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in water. Immunostaining was performed for XIAP using an indirect detection method (24). The staining was accompanied by a negative control in which slides were incubated with a matching blocking peptide to the primary antibody. Slides were counterstained with hematoxylin. Specimens were processed on the same day to eliminate any variability in conditions. Slides were digitally photographed using the same settings.

Statistical significance was determined using two-tailed Student's t-test with a p-value <0.05. All the experiments were repeated three times independently to determine consistency in the results. The results were expressed as mean ± SE for three replicates for each treatment.

The antitumor effects were determined in the IGF-1R-dependent CRC sub-cutaneous xenograft tumors following treatment with IGF-1R antagonist MK-0646 or OSI-906. MK-0646 treatment for three weeks at 20 mg/kg dose once weekly inhibited the growth of the GEO xenograft tumors (Fig. 1A and B). Similar results were obtained in MK-0646 treated CBS xenograft tumor (data not shown). The final tumor volume and weight were significantly reduced in MK-0646 treated xenografts compared with the control (Fig. 1C and D). Results obtained by daily treatment of OSI-906 (40 mg/kg) for two weeks orally (Fig. 2A and B) were comparable to the MK-0646 treated tumor xenografts showing decrease in tumor growth. However, we observed ~10% body weight reduction in OSI-906 treated mice compared with the MK-0646 treated mice (data not shown), which may be attributed to inhibition of insulin receptor by the dual kinase inhibitor.

We assessed the apoptosis level of control and drug-treated xenografts using TUNEL assays. Comparable results were obtained for both IGF-1R antagonists. Both MK-0646 and OSI-906 treated GEO xenografts had statistically significant increase in apoptosis (p<0.05) as compared with the control tumors (Fig. 3A–D). Based on the pro-apoptotic effects of both MK-0646 and OSI-906 in vivo, DNA fragmentation was performed in vitro on GEO CRC cells to determine cell death following IGF-1R antagonist treatment (Fig. 3E and F). In accordance with the in vivo study, both MK-0646 and OSI-906 treatment showed significant increases in apoptosis demonstrating that IGF-1R inhibition elicits pro-apoptotic effects on CRC cells.

Ki67 staining was performed to assess the proliferation level on MK-0646 and OSI-906 treated GEO xenografts. MK-0646 treated GEO xenografts showed no change in the cell proliferation compared with the control (Fig. 4A and B). However, OSI-906 treated GEO xenografts showed a statistically significant reduction (p<0.05) in cell proliferation compared with the control (Fig. 4C and D). We also assessed cell proliferation of GEO CRC cells in vitro by MTT assay after treating with different concentrations of MK-0646 or OSI-906. OSI-906 treatment showed a decrease in cell proliferation (Fig. 4E), altered cell cycle in G₀/G₁ phase (Fig. 5A) and showed a decrease in the 4N DNA content (Fig. 5B and C). However, MK-0646 treatment showed no effect on cell cycle (data not shown). OSI-906 treatment also led to increase in p21 expression (Fig. 5D).

Previous studies have shown that IGF-1R signaling exerts its anti-apoptotic effect through the IRS1/IRS2/PI3K/Akt pathway (25–28). We determined the effects of MK-0646 and OSI-906 on the IGF-1R and its downstream signaling pathways on control and treated GEO CRC xenografts. The xenograft tumor samples were analyzed for the expression of molecules associated with the IGF-1R signaling pathway, including IGF-1Rβ (Y¹¹³⁵), Akt and pAkt (S⁴⁷³). Inhibition of IGF-1R by MK-0646 and OSI-906 led to the downregulation of IGF-1R and its phosphorylation (Fig. 6A and B), confirming the inhibitory effect of both antagonists on IGF-1R signaling. IGF-1R inhibition by MK-0646 or OSI-906 showed dephosphorylation of Akt at S⁴⁷³ site (data not shown). We next determined the effects of IGF-1R inhibition in vitro, using GEO cells following treatment with either MK-0646 or OSI-906. GEO cells were treated with MK-0646 or OSI-906 under GFDS conditions. Both MK-0646 and OSI-906 showed similar response on IGF-1R and p-IGF-1R in vitro (data not shown). Additionally, marked reduction in pAkt (S⁴⁷³) was also observed in MK-0646 (20 μg/ml) or OSI-906 (0.5 μM) treated cells (data not shown). These results showed that both antagonists MK-0646 and OSI-906 effectively inhibited IGF-1R and its downstream signaling.

XIAP, an IAP (inhibitor of apoptosis) molecule and a key cell survival protein for inhibition of caspases, is a physiological substrate of Akt (29). Akt phosphorylates XIAP at Ser⁸⁷ and regulates its autoubiquitination and degradation, and thereby stabilizes XIAP (29). Chowdhury et al have shown that XIAP is associated with pAKT and this association is disrupted following TGFβ treatment leading to XIAP degradation (30). Moreover, XIAP and survivin form a complex in the cytosol and this complex inhibits caspase activity as well as cell death and promotes tumor growth in vivo(31). Inhibition of the aberrant cell survival signaling of XIAP through destabilization of XIAP/survivin complexes leads to caspase reactivation and cell death (30,32). We demonstrated that IGF-1R signaling pathway inhibition either by MK-0646 or OSI-906 both in vivo and in vitro downregulated Akt signaling, and this inhibition in turn leads to the downregulation of XIAP by immunohistochemical and western blot analysis (Fig. 7A–D). Next, we treated GEO cells with the IGF-1R antagonist in vitro for 48 h. Similar to the in vivo results, MK-0646 and OSI-906 both showed XIAP downregulation in the treated lysates compared to the control (Fig. 7E and F). These results showed that both antagonists exhibited their pro-apoptotic mechanism through inhibition of XIAP, an important downstream cell survival pathway molecule required for the survival of the IGF-1R-dependent CRC cells.