The human colorectal cancer cell lines HCT116, SW620 and HT29 were obtained from the American Type Culture Collection (Manassas, VA). The human gastric cancer cell line TMK-1 was obtained from Eiichi Tahara (University of Hiroshima, Hiroshima, Japan). The metastatic human pancreatic cancer cell line L3.6pl was kindly provided by Dr. I.J. Fidler (The University of Texas, MD Anderson Cancer Center, Houston, TX). HCT116 and SW620 cells were cultured in RPMI 1640, whereas TMK-1, HT29 and L3.6pl were grown in DMEM supplemented with 20% FCS (HCT116 and SW620), 15% FCS (L3.6pl), or 10% FCS (HT29, TMK-1). All in vitro experiments were performed at 60 - 70% cell density to reduce effects of confluence. Cell growth rates of transfected cells were assessed by MTT assays, as previously described .
HCT116 cells were stable transfected with either an ATF3-shRNA (TIB Molbiol; Berlin, Germany) or a luciferase-shRNA (Luc-shRNA) expression plasmid (TIB Molbiol; Berlin, Germany) by using the Lipofectamine transfection reagent (Invitrogen; Karlsruhe, Germany). Cells were grown and expanded in selective medium containing neomycin (G418, Sigma Aldrich, Deisenhofen, Germany). Successful transfection was verified by Western blotting and semi-quantitative PCR for ATF3.
Reagents and antibodies
The water-soluble Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxy-geldanamycin (17-DMAG) was purchased from Invivogen (Cayla-Invivogen) and was applied as previously published . Antibodies against ATF3 and anti-β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin served as a loading control in Western blotting.
Western blot analysis
Protein was extracted from whole-cell lysates with RIPA buffer as described before and 50-μg protein samples were subjected to Western blotting on a denaturing 10% sodium dodecyl sulfate-polyacrylamide gel . Membranes were probed for ATF3 and β-actin. For induction of ATF3 in vitro, the Hsp90 inhibitor 17-DMAG (100 ng/ml) was added to cell cultures for indicated times and ATF3 protein analysis was performed thereafter. Expression of ATF3 in 17-DMAG treated tumors was similarly determined by lysis of snap frozen tumor tissues and subsequent Western blotting, as described .
Real-time PCR was performed as we have previously described . Primer pairs were as follows: ATF3 forward 5-'ctgcagaaagagtcggag-3' and reverse 5'-tgagcccggacaatacac-3'; VEGF-A forward 5'-gcacccatggcagaaggaggag-3' and reverse 5'-agcccccgcatcgcatcag-3'; HIF-1α forward taccatgccccagattcaggat and reverse tcagtggtggcagtggtagtgg; GLUT-1 forward 5'-aactcttcagccagggtccac-3' and reverse 5'-cacagtgaagatgatgaagac-3'. Real-time PCR was done using the LightCycler system and Roche fast-Start Light Cycler-Master Hybridization Probes master mix (Roche Diagnostics) .
Migration assays were performed using modified Boyden chambers, as described elsewhere . Briefly, 105 cells were resuspended in 1% FCS medium and seeded into 8-μm filter pores inserts (Becton Dickinson Bioscience). 10% FCS-enriched medium ± 17-DMAG (100 nM) served as chemoattractant. After incubation, migrated cells were stained (Diff-Quick reagent, Dade Behring) and counted in four random fields.
Eight-week-old male nude mice (Charles River, Sulzfeld, Germany) were used. Experiments were approved by the Institutional Animal Care and Use Committee of the University of Regensburg and the regional authorities (Regierung der Oberpfalz, reference number HIF1_2004) and in accordance to the "Guidelines for the Welfare of Animals in Experimental Neoplasia" published by The United Kingdom Coordinating Committee on Cancer Research. In experiments, animals were weighed daily and monitored for weight-loss and other signs of distress.
(1) One-million human cancer cells (TMK-1, L3.6pl) were implanted into the subcutis of nude mice, as described . After implantation, tumors were allowed to grow to a volume of 400 mm3 until treatment with either the Hsp90 inhibitor 17-DMAG (3 × 25 mg/kg/week; i.p.), or PBS (control) was started. This dose has proven antineoplastic potential in previous models [8, 20]. Tumors were harvested after 14 days of treatment to determine ATF3 protein expression (n = 3 per group).
(2) One-million ATF3-shRNA, or Luc-shRNA transfected HCT116 human colorectal cancer cells were injected into the subcutis of nude mice (n = 9-10 per group). Tumor diameters were measured every other day, and volumes calculated using the estimation: width2 × length × 0.5.
(3) One-million ATF3-shRNA or Luc-shRNA transfected HCT116 cells were injected into the right lower liver lobe of mice to determine hepatic growth, as previously described . Animals were monitored daily and sacrificed on day 28 (n = 9-10/group). Following necropsy, liver weight was measured and all tumor nodules counted and weighed.
(4) For testing peritoneal carcinomatosis, stable transfected HCT116 cells (3 × 106) were implanted into the abdominal cavity by intraperitoneal injection, as previously described . Mice were monitored for 28 days and sacrificed; animals were evaluated for the presence of ascites and tumor nodules were counted.
Cryosections (7 μm) and paraffin-embedded sections (5 μm) were cut from xenograft tumors for immunohistochemical analyses. CD31-positive vessel area was analyzed by converting images to grey scale and setting a consistent threshold for all slides, as described [20, 24]. Vessel area is expressed as pixels per high-power field .
For the analysis of ATF3 mRNA expression, snap frozen tissue samples of primary human colon carcinomas (n = 5) and corresponding non-neoplastic colon tissues (n = 5) were obtained from the anonymized tumor tissue bank of the Department of Pathology (University of Regensburg), as approved by clinical ethics committee . Tumor characteristics were as follows: #1 sigmoid colon: pT3, L0, V0, pN0, R0; #2 cecum: pT4, pN2 (5/12), M1 (per), G2, R1, L1, V1; #3 sigmoid colon: pT3, pN0 (0/15), G2, R0, L0, V0; #4 cecum: pT3, pN2 (4/24), G3, L1, V0, R0; #5 sigmoid colon: pT3, pN2, G2, R0, L1, V0. Patients did not receive neoadjuvant therapy or chemotherapy before surgery.
Results from in vivo experiments were analyzed for significant outliers using Grubb's test http://www.graphpad.com. Tumor-associated variables in in vivo experiments were tested for statistical significance using the Mann-Whitney U test for non-parametric data. The two-sided Student t test was applied for analysis of in vitro data. All results are expressed as the mean ± SEM.