A83-01 Cancer Letters br Patient derived colorectal cancer P
Cancer Letters 447 (2019) 12–23
2.14. Patient-derived colorectal cancer (PDC) cells
These A83-01 were derived from tissue samples donated by colorectal cancer patients. The Institutional Review Board of Guro Hospital (KUGH16275) provided an approval to the Korea University Guro Hospital Tissue Bank to obtain such tissue samples. The tissue samples were first chopped with a disposable knife and then stored in the tissue grinder tube, filled with DMEM. Collagenase (final concentration to 0.5%) and accutase (final concentration to 10%) were then added to the medium, which contained the tissue and the tissue was then ground. After incubation at 37 °C for 30 min, the ground tissue was filtered with a strainer and then centrifuged for 2 min at 2000 rpm. Pellets were then suspended with the medium and then seeded to the dish.
IncuCyte® Caspase-3/7 Reagent (Essen BioScience, MI, USA) was used to measure caspase 3/7 activities. When the PDC cells were seeded into the three-dimensional 96-well plate, the medium that contained the caspase 3/7 reagent, was then added to the cells. After 30 min of incubation, images of the cells were captured using the IncuCyte® system followed by further analysis.
2.16. Animal experiments
Animal experiments were performed in accordance with the guidelines approved by the Korea University Institutional Animal Care and Use Committee (IACUC). Four-week-old female BALB/c nude mice were purchased from Orient Bio (Seong-Nam, Republic of Korea), and grown in a specific pathogen-free environment. HCT116 Luc+ cells (1 × 107 cells in 100 μL of PBS) were injected and housed for 1 week. CBD was intraperitoneally injected for every 3 days. The tumor size was also calculated at the same time. The tumor size was calculated using the formula: length × width; the volume was calculated as 0.5 × length × (width)2. Five mice were examined in each treatment group.
2.17. Immunohistochemistry assay
The harvested tissue, maintained in 4% paraformaldehyde, was processed for embedding. After the tissue was inserted into the cas-settes, it was drained (70% EtOH, for 1 h; 95% EtOH, twice for 30 min; 100% EtOH, three times for 30 min; and Xylene, three times for 30 min). Then, the cassettes were maintained overnight in molten paraﬃn. The paraﬃn-embedded and sections were used for the im-munohistochemical assay. The tissues were then hydrated (Xylene, three times for 10 min; 100% EtOH, twice for 10 min; 95% EtOH, twice for 10 min; 70% EtOH, for 5 min, and distilled water (DW), three times for 10 min). The tissue was then treated with 30% H2O2 (diluted with DW) for 15 min, followed by washing with DW three times for 10 min. The retrieval solution (Dako REAL Target Retrieval Solution (10X); Dako, Glostrup, Denmark), prepared using a microwave, was used to retrieve the tissue. After washing the tissue with DW three times for
10 min, the Dako pen was used to draw the border of the tissue. Then, the blocking solution (Universal Blocking Reagent; Biogenex, CA, USA) was added to the boundary, and the tissue was blocked for 15 min at RT. The tissue was incubated with the primary antibody (diluted 1:200), overnight at 4 °C. To wash the primary antibody, PBS with Tween 20 (PBST) was used three times for 10 min, and the bubbles were removed with DW. Then, the secondary antibody was incubated with the tissue for 1 h at RT, and then washed with PBST three times for 10 min. After removing the bubbles with DW, the mounting solution containing DAPI was used for mounting. The tissue was observed by confocal microscopy.
2.18. Statistical analysis
Each experiment was conducted independently and repeated a minimum of three times. Statistical analyses were conducted using GraphPad InStat 6 Software. These included the unpaired Student's t-test. In all analyses, the level of statistical significance was more than the 95% confidence level (P < 0.05). P < 0.05 was considered sta-tistically significant.
3.1. CBD inhibits cell viability and induces apoptosis in human CRC cells
To investigate the ability of CBD to induce apoptotic cell death in CRC cells, various human CRC cells were cultured with diﬀerent con-centrations (0–8 μM) of CBD for 24 h, and the cell viability was mea-sured by WST-1 assay. As shown in Fig. 1A, CBD decreased the viability of CRC cells in a dose-dependent manner, but not of normal primary colorectal CCD-18Co cells and normal primary lung Beas2B cells (Fig. 1A). To examine the long-term eﬀect on clonogenic survival treated cells, colony formation assay was performed. This ability was reduced by CBD treatment (Fig. 1B).
To determine whether the decreased viability of CBD-treated cells could be attributed to apoptosis, HCT116 and DLD-1 cells were treated with CBD and tested for apoptotic changes. CBD caused a dose- and time-dependent increase in the expression of cleaved PARP, caspase-3, -8-and −9 (Fig. 1C and D), which are widely used apoptotic marker . Furthermore, CBD significantly elevated the number of Annexin V/ PI double-stained cells (Fig. 1E) and TUNEL-positive cells (Fig. 1F), indicating that CBD induces apoptosis.