Reagents and antibody
Recombinant human PLAC1 and anti-human PLAC1 antibodies were provided by R&D System Inc. (Minneapolis, MN, USA). LY294002 (PI3K inhibitor) was ordered from Cell Signaling Technology (Beverly, MA, USA). The Akt inhibitor was purchased from BioVision (Mountain View, CA, USA). The Akt, phospho-Akt (Ser473), PI3K p85, phospho-PI3K p85 (Tyr 458)/p55 (Tyr199), NF-κB p65, and phospho-NF-κB p65 (Ser536) antibodies were purchased from Cell Signaling Technology (Mountain View, CA).
Cell culture
Human colorectal cancer cells (HT-29, WiDr, CaCo-2 and Colo320) were obtained from American Type Culture Collection (Rockville, MD, USA). HT-29 was incubated in McCoy’s medium containing 10% FBS (Gibco, Grand Island, NY, USA). WiDr and CaCo-2 cells were maintained in minimum essential medium eagle (MEME) with 10% FBS. RPMI-1640 medium with 10% FBS was used to culture Colo320 cells. HUVECs were obtained from Kurabo Co., and incubated in HuMedia-EB2 medium containing 2% FBS, 5 ng/ml bFGF, 10 mg/ml heparin, 10 ng/ml epidermal growth factor, and 1 mg/ml hydrocortisone.
RT-PCR Analysis
Total RNA from colorectal cancer cells was extracted by using an Isogen Kit, and the concentration of RNA was measured spectrophotometrically. Five microliters of total RNA was mixed with random hexamers and dNTP mix, incubated at 65°C for 5 min, chilled on ice, and then reverse-transcribed into cDNA using a cDNA Synthesis Mix, which included 10× RT buffer, 25 mM MgCl2, 0.1 MDTT, RNaseOUT and 200 U SuperScript ІІІ RT (Invitrogen, San Diego, CA, USA) at 50°C for 50 min. The reaction was discontinued at 85°C for 5 min. One microliter of each reaction mixture was used as a template for PCR. The following forward and reverse primers were used: PLAC1, 5’-TTCACCAGTGAGCACAA AGC-3’ and 5’-CCAGTCTATGG AGCACAGCA-3’. Amplification reactions were performed using a DNA Thermal Cycler.
Western blot analysis
Colorectal cancer cells and HUVECs (1×106/mL) were lysed in protein lysis buffer. Then, the protein concentrations were measured with a BCA protein assay kit (Pierce, Rockford, USA). The lysates (30 µg per lane) were separated by 10% SDS- polyacrylamide gel electrophoresis and transferred to polyvinylidene membranes. The membranes were incubated in blocking buffer for 60 min at RT. The blocking buffer included 5% nonfat dry milk solubilized into Tris-buffered saline comprising 0.1% Tween 20 (TBS-T). After three 5 min washes with TBS-T, the cells were immunoblotted with each primary antibody, diluted 500- to 1,000-fold by primary antibody dilution buffer and incubated overnight at 4ºC. Then, the membrane was washed with TBS-T three more times and subjected to incubation with the HRP-conjugated secondary antibody for 1 hr at RT. The antibody complex was detected by using the ECL Western blotting observation and analysis system. The grayscale values of the strips were measured by Image J software. The relative expression level of the proteins was expressed as the ratio of the target protein to the internal reference protein.
Real-Time quantitative RT-PCR
The qPCR was performed using a LightCycler apparatus. Freshly isolated RNA was converted to cDNA using the PrimeScriptTM TR Regent kit (Takara Bio Inc., Shiga, Japan), and the PCR was performed using the TaqMan® Gene Expression Assay Kit (Applied Biosystems, Foster City, CA, USA). In brief, a mixture of 1 µl of total RNA and 1 µl of oligo dT primer was incubated at 37ºC for 15 min and 85ºC for 5 sec for reverse transcription. The PCR was then performed in a 20 µl final volume containing the following: up to 20 µl of H2O, 10 µl of TaqMan® Universal PCR Master Mix, No AmpErase® UNG (2×)2–ordered separately, 1 µl of 20× TaqMan® Gene Expression Assay Mix, and 9 µl of cDNA diluted in RNase-free water. After an initial incubation at 94ºC for 15 sec, temperature cycling was initiated with each cycle (a total of 45-50 cycles) as follows: denaturation at 95ºC for 10 sec, hybridization at 60ºC for 30 sec, and elongation at 72ºC for 30 sec. The fluorescence signal was acquired at the final step of hybridization. Melting curves were obtained with a temperature range of 65-95ºC, read every 0.2ºC, held for 5 sec, and then incubated at 65ºC for 1 min. Cycling conditions of GAPDH were consistent with the above steps. A standard curve for each run was generated from serial dilutions of cDNA of the HT-29 cell line. The mRNA expression level of PLAC1 was normalized to that of GAPDH and shown as the mean ± standard deviation (s.d.). Relative mRNA expression of PLAC1 was calculated using the following formula: A/G÷A0/G0, in which A and G are the relative mRNA copy numbers of PLAC1 and GAPDH, while A0 and G0 are the relative mRNA levels of PLAC1 and GAPDH from the standard cDNA dilutions as control.
Proliferation assay
The colorectal cancer cells and HUVECs were planted at a density of 3×103 cells/100 µl into 96-well flat-bottomed plates and cultured overnight. The medium was exchanged, and the cells then cultured in the medium alone (control) or in the medium that included different concentrations of PLAC1 and anti-PLAC1 antibody. After 72 hr of incubation, 10 µl of WST-1 reagent was added to each well, and the cells were incubated for another 4 hr. Then, the cell proliferation was measured by the WST-1 Cell Proliferation Assay System (Takara Bio Inc., Shiga, Japan). The absorbance was determined using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm.
Invasion assay
The effect of PLAC1 on the invasive capability of human colorectal cancer cell lines and HUVECs was measured by using Matrigel-coated invasion chambers. The transwell chambers are separated by a PET membrane coated with Matrigel Matrix containing 8-μm pore-size polycarbonate membranes such that only invasive cells can migrate through the membrane to the reverse side. After rehydration for 2 hr in a humidified incubator at 37ºC. The cells were seeded at a density of 1×105 cells/well into the inner chambers of a cell culture insert and incubated at 37ºC for 24 hr with various concentrations of PLAC1. After 24 hr of incubation, cells that did not pass through were removed from the upper surface of the membrane by scrubbing gently with cotton-tipped applicators. The cells that invaded the reverse side of the membrane were fixed with 70% ethanol, stained with Giemsa solution, and counted in five random fields of the low filter surface under a microscope at 200× magnification.
Angiogenesis assay
To explore the effect of PLAC1 on tubule formation by HUVECs, HUVECs and fibroblasts were coincubated in basal medium using an angiogenesis kit according to the manufacturer’s protocols. First, HUVECs and fibroblasts were cocultured in 24-well plates with basal medium. The media were exchanged every two days with media containing various concentrations of PLAC1, with coincubation continuing for a total of 11 days. The coculturing system was stained with anti-CD31 antibody. The area of angiogenesis was measured quantitatively over ten different microscope fields for each well using an image analyzer (Kurabo Co.).
Angiogenic activity during cocultivation with colorectal cancer cells
To further investigate the influence of different colon cancer cells (HT-29 secreted PLAC1 or Colo320 not secreted PLAC1) on tubule formation by HUVEC. HT-29 or Colo320, HUVECs, and fibroblasts were co-cultured adopting a double chamber method. First, HUVEC/fibroblasts were cultured in a 24-well plate for 3 days, and then HT-29 or Colo320 cells (5×104 cells/mL) were incubated in trans-well chambers in which the bottom of chamber consisted of polycarbonate membrane with 0.45-μm pores, and then trans-well chambers were placed in HUVEC/fibroblast cultured 24-well plate. Approximately 100 ng/mL of PLAC1, or anti-PLAC1 Ab was added to the cultured medium, and the medium was changed every 2 days. All the cells were totally incubated for 12 days, then the upper trans-wells were removed, and the 24-well plate chambers were washed with 100 mL of 1×PBS three times. Then the HUVEC tubular formation was stained with anti-CD31 antibody by the protocols of manufacturer, and the total area of tubular formation was measured as described above.
Statistical analysis
Using the t-test for paired observations or one-way ANOVA with a post hoc test (Dunnett's multiple comparison) for multiple group comparisons, comparisons between groups were made. Statistical significance was provided, P<0.05. Data are presented as the mean ±s.d. Each experiment was performed in triplicate.