br These authors contribute this work equally
1 These authors contribute this work equally.
underlying its actions is not fully known . CBD is a non-psy-choactive cannabinoids that exerts anticancer activity in diﬀerent types of tumors [2,5,33]. It is already a part of many clinical trials for the treatment of glioma . CBD induces autophagy as well as apoptosis in human breast and prostate cancer cells by activating extracellular signal-regulated kinases and inhibiting AKT/mammalian target of ra-pamycin signaling [25,27]. However, little is known about the me-chanisms underlying CBD-induced apoptosis in CRC.
In this study, we investigated the apoptotic cell death induced by CBD in CRC cells, and identified the relationship between Noxa acti-vation and CBD-induced cytotoxicity. We suggest, for the first time, that CBD can cause Noxa-induced cell death. Our results show that CBD induced apoptotic cell death via ROS/endoplasmic reticulum (ER) stress-regulated Noxa activation, suggesting that CBD has important implications for the potential treatment of human CRC.
2. Materials and methods
2.1. Reagents and antibodies
CBD (Sigma, St. Louis, MO, USA) dissolved in absolute Ethanol (EtOH) was stored at 4 °C until use. The LOXL2-IN-1 used and their sources were as follows: Cleaved Poly (ADP-ribose) polymerase (PARP), Caspase-3, Caspase-9, Cleaved Caspase-8, Noxa, phospho-PKR-like ER-resistant kinase (PERK), p-PERK, phospho-inositol requiring enzyme-1α (IRE1α), p-IRE1α, Bip, Activating transcription factor 6 (ATF6), Glucose Regulated Protein 94 (GRP94) were purchased from Cell Signaling (Beverly, MA, USA). Anti-β-Actin was purchased from Sigma (St. Louis, MO, USA). The secondary antibodies, anti-mouse-IgG-HRP was purchased from Santa Cruz Biotechnology and anti-rabbit-IgG-HRP was purchased from Cell Signaling Technology.
Human CRC HCT116 and DLD-1 cells, and human colorectal normal CCD-18Co cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and HCT116 Luc+ cells were obtained from JCRB Cell Bank. HCT116 and HCT116 Luc+ cells were cultured in McCoy's 5A medium. DLD-1 cells were cultured in RPMI 1640 medium, and CCD-18Co cells were cultured in Eagle Minimum Essential Medium (EMEM, ATCC). All media contained 10% fetal bo-vine serum (HyClone, Logan, UT, USA) and 1% antibiotic-antimycotic solution (100X; GenDEPOT, TX, USA). All cell lines were incubated in a 5% CO2 incubator at 37 °C.
2.3. Microarray assay
RNA was isolated from the cultured cells using TRIzol and RNA purity and integrity were confirmed by ND-1000 Spectrophotometer (NanoDrop, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Next, the Aﬀymetrix Whole transcript Expression array method was proceeded according to the manufac-turer's manual (GeneChip Whole Transcript PLUS reagent Kit). The cDNA was synthesized by using the GeneChip Whole Transcript Amplification kit which described by the manufacturer. The sense strand of the cDNA was then fragmented. It was biotin-labeled with terminal deoxynucleotidyl transferase by using the GeneChip WT Terminal labeling kit. Approximately 5.5 μg of targeted DNA, which was labeled, was hybridized in the Aﬀymetrix GeneChip 2.0 ST Array at 45 °C for 16 h. Hybridized arrays were washed and scanned on a GCS3000 Scanner (Aﬀymetrix) after staining at a GeneChip Fluidics Station 450. Signal values were computed by using the Aﬀymetrix® GeneChip™ Command Console software. Cancer Letters 447 (2019) 12–23
Cell viability was measured using the Cell Viability Assay Kit (EZ-Cytox, DOGEN, Daejeon, Republic of Korea). HCT116 cells (1 × 104 cells per well) and DLD-1 cells (8 × 103 cells per well) were seeded in 96-well plates, and incubated in 5% CO2 incubator at 37 °C for 24 h. Then, the cells were treated with CBD in serum-free medium for 24 h in under identical condition. The cells were then treated with EZ-Cytox for additional 2 h, and then subjected to detection.
2.5. Colony formation assay
Colony formation assay was used to study the proliferation of cells. The cells were seeded in a 60 π (60 × 15 mm) cell culture dish. After 24 h of incubation, the cells were subjected to CBD treatment for an-other 24 h. Trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA, GenDEPOT) was used to detach the cells, and the cells were seeded in 6-well plates. After 2 weeks, the cells were stained with the colony for-mation assay solution.
2.6. Immunoblotting analysis
To prepare cell lysates, lysis buﬀer (containing protease inhibitor, phosphatase inhibitor, and RIPA buﬀer) was added, and the cells were lysed by sonication. The suspension was then centrifuged. The protein content of the supernatant was quantified using the bicinchoninic acid assay kit (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, MA, USA) and 5X sample buﬀer. Proteins were separated using 8–12% so-dium dodecyl sulfate-polyacrylamide gels, and then transferred to a nitrocellulose blotting membrane. About 10% bovine serum albumin (BSA) was used as a blocking buﬀer for 1 h at room temperature (RT). The membrane was then probed with primary antibodies, which were diluted in a primary antibody dilution solution (0.5% BSA with 0.1% sodium azide; 1:1000), overnight at 4 °C. The membranes were then washed with 1X Tris-buﬀered saline containing Tween 20 (TBST). Membranes were then probed with the specific secondary antibodies for 2 h at 4 °C, and then detected with the electrochemiluminescence so-lution (EZ-Western Lumi Pico; DOGEN).