The Role of Notch and Gamma-secretase Inhibition in an Ovarian Cancer Model

Materials and Methods

Cell culture. Seven ovarian cancer cells lines were obtained commercially (SKOV3, OVCAR3, TOV 112D, TOV 21G, OV90, CaOV3, OVCAR8) from the American Type Culture Collection (ATCC, Manassas, VA, USA). Immortalized normal ovarian surface epithelium (IOSE 364, immortalized with SV40 large T-antigen) was a kind gift from the Canadian Cancer Bank (Vancouver, British Columbia, Canada). The commercially-obtained lines were plated in the ATCC-recommended media supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/ml penicillin (Gibco) and 100 μg/ml streptomycin (Gibco). IOSE 364 was maintained in MCDB 105:199 supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100μg/ml streptomycin. All lines were maintained in a humidified incubator at 37°C in 5% CO₂.

qPCR. Relative mRNA expression of the Notch receptors 1-3, the ligands JAG1 and DLL-4, and the downstream targets HES1 and HEY1 was measured via qPCR using an Applied Biosystem (Carlsbad, CA, USA) 7300 RT-PCR system. Threshold cycle numbers (Ct) were obtained via the 7300 SDS software. Commercially available PCR primer-probes were obtained from Applied Biosystem for the above listed genes. The mean Ct of the genes of interest was calculated from triplicate measurements, then normalized using the mean Ct of the housekeeping gene human hypoxanthine ribosyltransferase (HPRT) (12). The Ct for each gene was further normalized to the respective Ct from IOSE 364.

Proliferation assays. Each cell line was plated at a density of 5×10⁴ cells/ml in a 96-well plate and allowed to attach overnight. Four experimental treatments were examined: gamma secretase inhibitor (GSI) (compound-E) (Seoul, Korea), cisplatin (Sigma, St. Louis, MO, USA), compound-E plus cisplatin, and placebo. Compound-E was serially diluted from 1 μM to 25 nM in media. Cisplatin was serially diluted from 128 μM to 2 μM in media. The combination treatment arm employed serial dilutions of both compound-E and cisplatin from 1 μM to 25 nM and 128 μM to 4 μM, respectively. The placebo arm included corresponding volumes of DMSO (Sigma) (placebo for compound-E) and saline (placebo for cisplatin) in media. Each treatment arm had a negative control without the addition of drug or placebo. Treatments were added 24 h after plating. Plates were maintained at 37°C in 5% CO₂ for 48 h. The media were then removed and replaced with media combined with CK-8 dye (Dojindo Molecular Technologies, Rockville, MD, USA) at a 1:10 concentration. The plates were incubated for 45 min, then the media was transferred to a clean 96-well plate and the absorbance read with a Bio-Rad (Philadelphia, PA, USA) microplate reader at 450nm. The mean absorbance was calculated from triplicate measurements. This same protocol was followed using N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and dibenzazepine (DBZ) (both obtained from Tocris Bioscience, Minneapolis, MN, USA), two other commercially available GSIs.

Matrigel assay. Preliminary studies revealed that two lines, TOV 112D and TOV 21G, formed colonies in Matrigel (BD Biosciences, San Jose, CA, USA). These lines were plated in duplicate in Matrigel with 200 nM compound E or an equal volume of DMSO. Plates were incubated for 72 h at 37°C in 5% CO₂. Subsequently, 200 μL of MTT dye were added to each well. Plates were incubated for 3 h, then colonies were counted with a Nikon inverted microscope (Melville, NY, USA).

Drug efficacy assay. A total of 9×10⁵ cells of each cancer line were plated in a 10-cm tissue culture dish in duplicate. Cells were allowed to attach overnight; the media were then replaced in both plates. Compound-E at 200 nM was added to one plate and an equal volume of DMSO was added to the second plate. These were incubated at 37°C in 5% CO₂ for 48 h, then harvested for RNA isolation. RNA was extracted from these cells and made into cDNA. qPCR was performed on these cDNA samples to assess efficacy of GSI-induced down-regulation Notch downstream targets HES1 and HEY1.

In vivo experiment. 6-week-old nude female mice (average weight 20 g) were obtained from Taconic Farms (Germantown, NY, USA) and were allowed to acclimatize for one week. They were then injected with 10⁶ OVCAR3 cells in the right flank. Tumors were allowed to develop for one week. Mice were then randomized into four groups of six mice each. Mice in group one received daily weight-based compound-E at 30 μmol/kg intraperitoneally. Compound-E was dissolved first in DMSO and then further diluted in 0.5% methylcellulose/0.1% Tween 80. Mice in group two received 160 μg cisplatin intraperitoneally once weekly. Mice in group three received both compound-E daily and cisplatin weekly as described above. Mice in group four received DMSO diluted in vehicle daily and saline weekly as placebo treatments. The total treatment time was four weeks, after which 100 μg of bromodeoxyuridine [BrdU] (Roche Applied Bioscience, Mannheim, Germany) was injected into all mice intraperitoneally one hour prior to sacrifice. After sacrifice, tumors were harvested from the flank and weighed, then submerged in 4% paraformaldehyde for preservation and paraffin embedding.

Immunohistochemistry. Immunohistochemistry (IHC) was performed on serial sections of the tumors. Cluster of differentiation-31 (CD31) staining was performed per standard protocol for paraffin embedded slides (CD31 antibody 1:200, Angio-Proteomie [Boston, MA, USA) (13). Slides were stained for BrdU (Roche Applied Bioscience) and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Slides were examined using a Nikon Eclipse E800 microscope and 10 representative images from each section were taken at 20× magnification with a Nikon digital camera (DXM 1200). Microvessel density was calculated by counting the number of vessels contained in each field, then averaging those values for each specimen. The same method was used to quantify the number of BrdU-positive and TUNEL-positive cells.

Statistical analysis. qPCR results for the Notch receptors and ligands are reported as relative expression levels compared to normal ovarian surface epithelium. All data are reported descriptively. Mean mouse weights, tumor weights, and results from IHC staining from the in vivo studies were compared across groups using Student's t-tests. A p-value of <0.05 was considered statistically significant.