Material
5-FU, dimethyl sulfoxide (DMSO), and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-t-butyl ester (DAPT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). αM was provided by professor SY Seo (College of Pharmacy, Gachon University, Republic of Korea) (Figure 1A). 5-FU and αM were dissolved in DMSO. The following antibodies were used for Western blotting and flow cytometry: anti-β-actin (1:1000, Gene Tex, Irvine, USA), anti-HES1 (1:1500, Cell Signaling, Danvers, MA, USA), anti-Notch1, anti-NICD 1 (1:100, Santa Cruz, TX, USA), anti-Hey1 (1:500, abcam, Cambridge, UK), fluorescein (FITC)-conjugated anti-CD44 (1:20, BD bioscience, Franklin Lakes, NJ), and phycoerythrin (PE)-conjugated anti-CD133 (1:50, Miltenyi Biotec, Bergisch Gladbach, Germany).
Cell culture
Human colon cancer cell lines SW620 and HT29 were purchased from Korea Cell Line Bank (Seoul, Republic of Korea). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, MP Biomedicals, France) and 1% antibiotic antimycotic solution (10,000units/ml penicillin and 10 mg/ml streptomycin, Welgene, Daegu, Republic of Korea) in plastic tissue culture flasks under 37℃, 5% CO2, and 95% humidity.
Cell viability assay
Cell viability was measured by using Cell Counting Kit-8 (CCK-8, Enzo Life Sciences, Farmingdale, NY, USA). Cells were seeded in a 96-well plate (1× 104cells/well, 200μl/well, SPL, Republic of Korea) in an increasing gradient. SW620 cells were treated with 0, 2.5, 5, 10, 20, and 40μM αM for 72 h, and HT29 cells were treated with 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0μM αM. In each well, the medium was removed, and 90μl plus 10μl CCK-8 solution was added. Thereafter, the plate was incubated for 1h at 37℃. Absorbance was measured at 450 nm on a 96-well microplate reader (Spectra Max M5, MD, USA).
Colosphere forming assay
SW620 and HT29 cells (1000 cells/well) were seeded in 24-well ultralow adherence plates (Corning, NY, USA) in 1ml of CSC media, DMEM/F12 supplemented with B27 (Gibco, Invitrogen, Carlsbad, CA, USA), 2mM L-glutamine (Hyclone), 10ng/μl bFGF (Prospec, East Brunswick, NJ, USA), 20ng/μl EGF (Prospec), and 1% antibiotic antimycotic solution (10,000 units/ml penicillin and 10mg/ml streptomycin, Welgene). Cells were cultured for 14d, and CSC medium was changed every 72h. SW620 cells were treated with 0, 1.25, 2.5μM αM, and HT29 cells were treated with 0, 1.25, 2.5μM αM during the sphere forming assay. The spheres were examined using a microscope at 14 d (Zeiss Axiophot, Carl Zeiss Microscopy LLC, Thornwood, NY, USA). Quantitative real-time PCR and Western blot analyses were conducted with these cells.
3D spheroid invasion assay
The 3D spheroid invasion assay was conducted with the aforementioned HT29 cells. HT29 cells were trypsinized, and 1× 105 cells were resuspended in 5mL DMEM with 20% methocel solution (methylcellulose, Sigma-Aldrich) and 1% Matrigel (Corning). Hanging drops (25μl) were suspended on petri dishes (SPL), and cells were harvested after 2d. Harvested spheroid cells were embedded in collagen gels (rat tail collagen, BD bioscience), which were polymerized at 37℃. These spheroids were incubated for 5d, and invasion ratios were calculated using ImageJ software (version 1.51J8; National Institutes of Health, Bethesda, MD, USA).
Quantitative real-time PCR analysis
Total cellular RNA was extracted from HT29 cells by using Trizol reagent (Invitrogen) and the RNeasy Mini Kit (Invitrogen) in accordance with the manufacturer’s protocol. The total RNA concentration was measured using a Nanodrop spectrophotometer (Nabi UV/Vis Nano spectrophotometer, Microdigital, Gyeonggi, Republic of Korea) with an A260/280 cut-off of approximately 2.0. Purified RNA (2μg) was reverse-transcribed (with the Reverse Transcription Kit, Applied Biosystems, Framingham, MA, USA). Quantitative real-time PCR was performed with power SYBR Green master mix (Applied Biosystems) on Quant studio 3. The cycling conditions were as follows: denaturation for 2min at 50°C, 10min at 95°C, followed by 40 cycles at 95°C for 5 s and 60°C for 60s, followed by dissociation for 15s at 95°C and annealing and extension at 60°C for 20 s. The relative mRNA levels were normalized to those of β-actin mRNA using the 2-ΔΔCt method. Primers for quantitative real-time PCR are listed in Supplementary Table 1.
Western blot assay
The Western blot assay was conducted to determine the expression levels of Notch1, NICD1, Hes1, Hey1, and β-actin, under 4 experimental conditions. Proteins were extracted from cells by using radioimmunoprecipitation assay (RIPA) lysis buffer (iNtRON Biotechnology, Gyeonggi, Republic of Korea). The concentration of the isolated proteins was determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific-Pierce, Waltham, MA, USA). Proteins (20μg) were separated through 8%, 10%, and 12% SDS-PAGE (Hoefer, San Francisco, CA, USA) and transferred to polyvinylidene fluoride membranes (PVDF, Merck). The membranes were blocked using 3% bovine serum albumin (BSA, Sigma-Aldrich) for 30min at room temperature (RT). Protein extracts were incubated with primary antibodies overnight at 4℃ and with secondary antibodies for 1h at RT. Proteins were detected using the enhanced chemiluminescence (ECL) Western blotting Luminol reagent (Santa Cruz). Images were obtained using a Lumino image analyzer (LAS-4000 Mini, Fujifilm, Tokyo, Japan).
Flow cytometry
For flow cytometry, cells were washed with PBS and incubated with Accutase (Gibco) for 10 min. After adding flow cytometry buffer (2.5g BSA [Sigma-Aldrich] and 0.372g EDTA [Sigma-Aldrich] in 500ml PBS [Biosesang, Seongnam, Republic of Korea]), cells were incubated with primary antibodies at 4℃ in the dark for 45min. CD133 was conjugated with PE and CD44 was conjugated with FITC for labeling cells. Labeled cells were resuspended in flow cytometry buffer. All samples were analyzed using the Novo-Cyte flow cytometer (ACEA Biosciences, San Diego, CA, USA).
Assessment of in vivo anticancer efficacy
Six-week-old male Balb/c athymic mice were purchased from Orient Bio (Seongnam, Republic of Korea) and acclimated for 1 week. All mouse experiments were conducted under approved guidelines of the Animal Care and Use Committee of Ewha Womans University (EUM17-0368). HT29 cells (1× 106 cells) were suspended in DMEM with Matrigel matrix (1:1 ratio). The mixed cells were injected subcutaneously into the right rear flank of each mouse. After 11 days of injection, mice were divided into 4 treatment-based groups (5 mice per group): control, 5-FU only, αM only, and 5-FU and αM. 5-FU (30 mg/kg body weight) or/and αM (5 mg/kg) were administered intraperitoneally thrice a week for 18 d. Tumor volume was calculated (volume = length × width × width/2), and body weight was measured thrice a week. All mice were euthanized through CO2 asphyxiation, and the weight and volume of the excised tumor were measured on day 29.
Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM) or mean ± standard deviation (SD) values. All analyses were performed using Graph Pad Prism 8.0 software (Graph Pad Software, La Jolla, CA, USA) and SPSS software (version 22.0, Chicago, IL, USA). A P value of <0.05 was considered significant. Statistical significance was determined using the Mann–Whitney U test for nonparametric data and a two-tailed Student t test for parametric data.