This study was approved by the Research Ethics Committee of the National Taiwan University Hospital (201107063RC).
Human 293 T cells and colon cancer cell lines SW480, HCT116, HT29, and LoVo were obtained from the American Type Culture Collection (ATCC, Manassas, VA). They were cultured in DMEM with 10% fetal bovine serum and 1% Penicillin/Streptomycin. Cells were grown at 37°C in a 5% CO2 atmosphere within a humidified incubator.
Human CRC tissue
Colorectal cryosections were prepared from colorectal cancer surgical samples which were collected from September 2000 to June 2003 after obtaining the written informed consent, following the guidelines set forth by the Research Ethics Committee of the National Taiwan University Hospital. All tissues were freshly frozen or immersed in optimal cutting temperature compound (OCT) (Ames Company, Elkhart, IN), and kept at −80°C until use. Clinical staging of cancers was determined based on the UICC-TNM classification. Stages I and II were collectively termed the localized disease group (n = 47) and stages III and IV as advanced disease group (n = 33).
Reagents and antibodies
The p38 MAPK inhibitor SB203580 and the MMP9 inhibitor were purchased from Merck KGaA (Darmstadt, Germany), and Zeocin from Invitrogen (Carlsbad, CA). Polyclonal rabbit antibodies against phospho-JNK, phospho-ERK 1/2, phospho-p38, and total JNK, ERK1/2, and p38 were purchased from Cell Signaling Technology (Danvers, MA). Other antibodies used were mouse monoclonal antibodies to FLAG M2 (α-FLAG, Sigma Chemical Co., St. Louis, MO), rat polyclonal anti-CD31 (BD Biosciences, San Jose, CA), rabbit polyclonal anti-PlGF (C-20) (Santa Cruz, CA), anti-Factor VIII (Biomeda Corporation, Foster, CA), anti-cleaved Caspase-3 from Cell Signaling Technology (Danvers, MA) and anti-MMP-9 (Abcam, Cambridge, UK). Recombinant PlGF was purchased from R&D Systems, Inc. (Minneapolis, MN).
Plasmids, small interfering RNA, and transfection
The human PlGF expression vector was generated by PCR amplification of full length of PlGF cDNA and inserted into the multiple cloning site of pcDNA4/TO-flag-strepII N1 Vector (Flag-StrepII tandem tag at the N-terminus, derived from Invitrogen pcDNATM4/TO vector). HA-tagged human Flt-1 expression vector, pCMV-TAG-Flt-1 (HA-Flt-1), was generated by PCR amplification of Flt-1 cDNA and insertion into the multiple cloning sites of pCMV-TAG vector (Invitrogen, Carlsbad, CA). Negative control siRNA, siFlt-1, sip38 (p38α), and siPlGF were purchased from Ambion Inc. (Austin, TX). LoVo cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). For generating the stable cell lines (LoVo, SW480, HT29 and HCT116), selection with Zeocin (100 μg/ml) began one day after transfection and maintained under same Zeocin condition.
RNA extraction and quantitative PCR
Total RNA from cell lines and tissue was isolated using an RNA extraction kit (Qiagen Inc., Valencia, CA), according to the manufacturer’s instructions. For reverse transcription, 1 μg of total RNA was transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative PCR was performed in a DNA Engine Opticon 2 (Bio-Rad, Hercules, CA) using iQ SYBR Green supermix (Bio-Rad, Hercules, CA) with human GAPDH as an internal control. Primers sequences used for RT-PCR were as the following, PlGF: Forward-5′-TGCGGCGATGAGAATCTGC-3′, Reverse-5′-AGCGAACGTGCTGAGAGAAC-3′; Flt-1: Forward-5′-AGCAGGTGCTTGAAACCGTAG-3′, Reverse-5′-GTCGCAGGTAACCCATCTTTT-3; MMP9: Forward-5′-GGGACGCAGACATCGTCATC-3′, Reverse-5′-TCGTCATCGTCGAAATGGGC-3′.
Western blot analysis
Cells were lysed with NP-40 lysis buffer and centrifuged at 15,000 rpm for 20 minutes at 4°C. The supernatant was assayed for protein concentration (Bradford). Equal amounts of protein (150-250 μg/lane) was added to Tris-Glycine SDS sample buffer (Invitrogen, Carlsbad, CA) and separated on 4-12% gradient Tris-Glycine gels (Invitrogen, Carlsbad, CA). Following electrophoresis, proteins were electro-transferred to polyvinylidene difluoride membranes and blocked with 5% bovine serum albumin in TBST (Tris-Buffered Saline and Tween 20). Membranes were then incubated with specific primary antibody overnight, washed, then incubated with appropriate secondary antibody conjugated to horseradish peroxidase, and developed using ECL (PerkinElmer Life Sciences). Membranes were stripped and re-probed with anti-total MAPK (for MAPK antibodies) or anti-actin to confirm equal protein loading.
Frozen sections (8 μm thick) were stained by using the NoVo Link Polymer Detection System (Leica, Biosystems Newcastle Ltd, UK), followed by AEC substrate kit (Vector Laboratories Inc. Burlingame, CA), according to the manufacturer’s instruction. Tissues were counterstained with Mayer’s hematoxylin. Isotype antibody was used as the staining negative control.
ELISA (enzyme linked immunoabsorbent assay)
Concentrations of PlGF in cell culture medium were quantified using a Quantikine PlGF immunoassay (R&D Systems, Inc., Minneapolis, MN), as previously described . Concentrations of PlGF were expressed as pg/ml of protein.
Apoptosis was quantified by staining with the Annexin V-FITC kit (Strong Biotech Corporation, Taipei, Taiwan), data collection by flow cytometer (Becton Dickinson) and analyzed by FlowJo 7.2 software. For tissue sections, immunohistochemical staining with cleaved caspase 3 antibody was used for apoptosis analysis.
In vitro invasion and migration assay
The invasive activity of the cancer cells was examined using a membrane invasion culture system in which a polycarbonate membrane with 8-μm pores (Millipore., Billerica, MA) coated with Matrigel (R&D Systems, Inc., Minneapolis, MN) at 5 mg/mL was placed between the upper and lower wells of a membrane invasion culture system chamber. 5 × 104 cells were placed into each upper well of the chamber. After incubating for 48 hours at 37°C, cells that had migrated through the coated membrane were removed from the lower chamber with 1 mM EDTA in PBS and dot blotted onto a polycarbonate membrane with 3-μm pores. Blotted cells were stained with Giemsa (Sigma Chemical Co., St. Louis, MO), and the number of cells on each blot was counted under a microscope at a magnification of × 50. Each experiment was performed three times, and each sample was assayed in triplicate. The migration assay was also performed using the same procedure, except without the Matrigel coating.
Cell growth was measured using MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] in the form of the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, UK), according to the manufacturer’s instructions. Briefly, 1,500 cells/well were plated in 96 well plates and allowed to proliferate for two days. The OD 490 correlated with cell density. All assays were repeated thrice.
Tumor xenograft growth assay
LoVo PlGF expression and mock (vector only) stable cells in log phase were trypsinized and washed twice with 137 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 0.5 mM EDTA, 0.1% (w/v) glucose. 106 cells in 100 μl PBS were injected subcutaneously into the backs of 8-week-old SCID mice. The body weight and tumor size were recorded twice per week. After 14 weeks, the mice were sacrificed. Tumor size was measured and further studies were performed on the tissue.
Microvessel density measurement
Using light microscopy at 200X magnification, the vascular counts were measured for the tissue section staining with CD31. The three areas with the highest number of discrete microvessels were chosen for analysis and the region with the highest microvessel counts was selected as the final result for that case . Any immunoreactive endothelial cells that were separate from adjacent microvessels were considered to be countable vessels.
Gene expression dataset from the Gene Expression Omnibus (GEO) database analysis
The colorectal cancer patient gene expression data was available on Gene Expression Omnibus (GEO) with accession number GSE17536. Expression data were analyzed using GeneSpring GX software (Agilent Technologies). High and low PlGF/Flt-1 expression was defined according to the median expression level in each group.
Statistical differences between groups were analyzed by Student’s t test or Mann–Whitney U test. Data was expressed as means ± standard errors (SE). Correlations between PlGF and MMP-9 expression levels were analyzed by Spearman’s correlation coefficient. A p value of 0.05 was considered to be statistically significant. The body weight difference along with the time between the LoVo-PlGF and control group, in terms of group effect, time effect, and their interactive effect, were analyzed using the mixed model. Based on fit statistics for Akaike information criterion (AIC) and Bayesian information criterion (BIC) criteria, the repeated measures were modeled using the first-order ante dependence for the covariance structure .