Tumor size was measured twice weekly using a caliper, and tumor volume was calculated with the for- mula: volume (V) ¼ 0.5 a b2, where a and b are the long and short diameters (in millimeters) of the tumor, respec- tively. SNP analysis with human gastric cancer tissues using an Illumina cancer SNP panel was conducted by Illumina, Inc. Cell-cycle analysis After incubation with PF00299804 under various con- centrations for 48 hours, the cells were centrifuged at 1,500 rpm for 5 minutes and Trichostatin A HDAC inhibitor then fixed in 70% alcohol and stored at À20 C. The samples were then dissolved in 10 mL RNAse and subsequently incubated at 37 C for 10 minutes. Next, the samples were treated with propidium iodide, after which the DNA contents of the cells were determined using an FACS Caliber flow cytometer (BD Biosciences) equipped with a ModFit LT program, as previously described. Western blot and immunoprecipitation Cells were incubated with PF00299804 in 10% FBS media. After 48 hours, the cells were treated with a lysis buffer. The same amount of protein was then obtained from each suspension and subjected to SDS- PAGE, after which it was transferred to nitrocellulose membranes. After blocking with a buffer, the membrane was incubated with primary antibodies at 4 C overnight.
Antibodies against p-EGFR (pY1068), p-HER2 (pY1221/ 1222), p-HER3 (pY1289), p-STAT3 (pY-705), p-AKT (pS- 473), p-ERK (p44/p42), EGFR, HER2, HER3, HER4, STAT3, AKT, ERK, caspase-3, caspase-7, PARP, Bcl-2, Bim, cyclin D, p27kip1, and PI3-Kinase p85 were purchased from Cell Signaling Technology. Antibodies against MCL-1, cyclin E, CDK2,Vorinostat HDAC inhibitor and actin were obtained from Santa Cruz Biotechnology. Anti- a-tubulin antibody was acquired from Sigma-Aldrich. For immunoprecipitation, 1 mg of total protein from cell lysates using lysis buf- fer was incubated with anti- EGFR or anti-HER3 antibodies and Protein A/G plus agarose (Santa Cruz Biotechnology) and gently shaken. The precipitates were washed twice with ice-cold lysis buffer and resolved by SDS-PAGE, after which they were subjected to Western blot analysis. Xenograft mouse model To determine the in vivo activity of PF00199804, 7- to 8- week-old female severe combined immunodeficient (SCID) mice were used. All studies were conducted in accordance with the recommendations of the Guide for Care and Use of Laboratory Animals. Mice were injected subcutaneously with N87 cells, and tumor growth was monitored as tumor volumes approached 180 to 220 mm3. Tumors were measured using Vernier calipers twice weekly. Twelve days later [designated as day 1 (D1) of the study], mice were sorted into treatment groups with group mean tumor volumes of 190 to 193 mm3. Volume was calculated using the follow- ing formula: tumor volume width length/2 measured in millimeters for an N87 tumor. Mice bearing subcutaneous N87 tumors of approxi- mately 200 mm3 were orally administered PF00299804 daily at 5 mg/kg/day as a single agent Gefitinib or in combination with cisplatin administered intraperitoneally weekly cancer cell lines. C, left, HuPrime gastric cancer xenograft were treated with vehicle or PF00299804.
Then, tumor growth inhibition values were calculated from the median tumor size of the treatment group divided by that of the control group on day 27. Right, each data point represents a given SNP of a tissue sample. Norm Theta represents genotype. Norm R represents the intensity value of the SNP signal. The data points in red, blue, and purple represent SNPs of normal human tissues,GDC-0449 Vismodegib displaying normal genotypes. The data points in other colors are SNPs of tumor tissues from a panel of HuPrime gastric cancer models. D, tumor growth-inhibitory effects in HuPrime gastric cancer xenograft model. Significant tumor growth inhibition was observed in the PF00299804-treated group from day 6 through the end of the study, achieving complete regression. Data are shown as mean. Downloaded from mct.aacrjournals.org on February 13, 2012 Copyright © 2012 American Association for Cancer Research 443 values and significantly inhibited the growth of N87 cells, even at the 0.001 mmol/L concentration. To confirm the growth-inhibitory effects of PF00299804 in HER2- amplified cells and see if additional sensitive markers for PF00299804 exist, a cell viability assay was conducted in another panel of 18 gastric cancer cell lines, of which molecular profiling of about 27 genes was included. As expected, HER2-amplified N87 cell lines were most sensitive to PF00299804. In an effort to further identify predictive markers for drug response, the antitumor effects of PF00299804 were tested using primary human gastric cancer xeno- graft models.